Cell Transplantation,Vol.19,pp.279–289,20100963-6897/10$90.00+.00 Printed in the USA.All rights reserved.DOI:10.3727/096368909X481764 Copyright 2010Cognizant Comm.Corp.E-ISSN1555-3892
https://www.doczj.com/doc/7e4027685.html, Isolation,Characterization,and Differentiation Potential
of Canine Adipose-Derived Stem Cells
N.M.Vieira,*V.Brandalise,*E.Zucconi,*M.Secco,*B.E.Strauss,?and M.Zatz* *Human Genome Research Center,Biosciences Institute,University of Sa?o Paulo,Sa?o Paulo,Brazil
?Viral Vector Group,Heart Institute,InCor,University of Sa?o Paulo,Sa?o Paulo,Brazil
Adipose tissue may represent a potential source of adult stem cells for tissue engineering applications in
veterinary medicine.It can be obtained in large quantities,under local anesthesia,and with minimal discom-
fort.In this study,canine adipose tissue was obtained by biopsy from subcutaneous adipose tissue or by
suction-assisted lipectomy(i.e.,liposuction).Adipose tissue was processed to obtain a fibroblast-like popula-
tion of cells similar to human adipose-derived stem cells(hASCs).These canine adipose-derived stem cells
(cASCs)can be maintained in vitro for extended periods with stable population doubling and low levels of
senescence.Immunofluorescence and flow cytometry show that the majority of cASCs are of mesodermal
or mesenchymal origin.cASCs are able to differentiate in vitro into adipogenic,chondrogenic,myogenic,
and osteogenic cells in the presence of lineage-specific induction factors.In conclusion,like human lipoaspir-
ate,canine adipose tissue may also contain multipotent cells and represent an important stem cell source
both for veterinary cell therapy as well as preclinical studies.
Key words:Canine adipose-derived stem cells;Tissue engineering;Veterinary cell therapy
INTRODUCTION Successful transplantation of canine adipose-derived
stem cells(cACSs)in dogs was reported by Li et al.
(12)and Black et al.(1).However,these manuscripts
A promising application in the emerging field of vet-
erinary regenerative medicine and surgery is cell ther-lacked the full characterization of the administered cell
apy,rendering the isolation and characterization of stem
population.Here we report the isolation,characteriza-
cells from a variety of sources areas of great interest.tion,and multilineage differentiation potential of cASCs
An abundant and accessible source of stem cells is
from subcutaneous adipose tissue by liposuction and bi-
adipose tissue.These cells,called adipose-derived stro-opsy procedures.
mal cells(ASCs),are fibroblast-like cells capable of
MATERIALS AND METHODS multipotential differentiation,which have been found in
different species(4,27,29,35).Several groups have All experimental protocols were approved by the eth-
demonstrated that human mesenchymal cells within the
ics committee on animal use from the Institute of Biosci-
stromal-vascular fraction(SVF)of subcutaneous adipose ences,University of Sa?o Paulo.For this study,adipose
tissue[processed lipoaspirate(PLA)cells]are capable
tissue was collected from normal golden retriever dogs
of differentiation in multiple lineages,including myo-from the Brazilian Colony of Golden Retriever Muscular
cytes,in the presence of lineage-specific inductive me-
Dystrophy,Faculty of Veterinary Medicine and Zoo-
dia(2,5,8–10,15,16,19,20,22,23,25,34,35).tecny,University of Sa?o Paulo.Subcutaneous adipose
In humans,ASCs for autologous transplantation are
tissue was collected from the area over the dorsal gluteal
isolated relatively quickly from adipose tissue by colla-muscles of10dogs(aged4months to4years).
genase digestion(6).We have recently shown that ASCs
Adipose Tissue Harvesting
from human subcutaneous fat were able to differentiate
in adipogenic,osteogenic,chondrogenic,and myogenic Dogs were sedated upon intramuscular(IM)injection
with meperidine(2mg/kg)and acetylpromazine(0.05 lineages and produce human muscle proteins in vitro
and in vivo(30,31).mg/kg).The area over the dorsal gluteal muscles was
Received March27,2009;final acceptance December2,2009.Online prepub date:December8,2009.
Address correspondence to Dr.Mayana Zatz,Ph.D.,Human Genome Research Center,Institute of Biosciences,University of Sa?o Paulo,Rua do Mata?o,n.106Cidade Universita′ria,Sa?o Paulo-SP,Brasil-CEP:05508-090.Tel/Fax:(55)(11)3091-7966;E-mail:mayazatz@usp.br
279
280VIEIRA ET AL. asceptically prepared,and skin and subcutaneous tissues debris and seeded in tissue culture plates(NUNC)at were desensitized by local infiltration of2%lidocaine
1,000–3,500cells/cm2in DMEM-HG10%FBS.Cul-(Fig.1A).A0.5–1.0-cm incision was made parallel to tures were washed with PBS24–48h after plating to the vertebral column.The liposuction procedure was
remove unattached cells and fed with fresh media. performed by injecting infiltrate containing the vasocon-The cultures were maintained at37°C with5%CO2 strictor epinephrine.Then adipose tissue was removed
in growth media(GM-DMEM-HG10%FBS).When from the subcutaneous space by means of blunt-tip hol-they achieved about70%confluence,the cells were low cannula attached to a syringe at negative pressure
trypsinised(0.025%,Invitrogen)and plated at a density (Fig.1B).About15ml of adipose tissue was harvested of5,000/cm2.Cultures were passaged repeatedly after over the superficial gluteal fascia for immediate cASC
achieving a density of70–80%.The remaining cells isolation and the skin incision apposed with nylon su-were cryopreserved in cryopreservation media(10%di-tures(Fig.1C).Adipose tissue biopsies were performed
methylsulfoxide,10%DMEM-HG,80%FBS),frozen at under local anesthesia.A1–2-cm incision was made and?80°C in an isopropanol-jacketed closed container,and the subcutaneous adipose tissue was collected(Fig.1D)
stored in liquid nitrogen the next day.
and the incision was closed with nylon sutures(Fig.1E).
Multilineage Differentiation
cASC Isolation and Expansion Cells were analyzed for their capacity to differentiate
into adipogenic,chondrogenic,osteogenic,and myo-Cells were isolated using modified methods pre-
genic lineages as described in Zuk et al.(35).
viously described(7).Briefly,the adipose tissue was
washed extensively with equal volumes of PBS contain-Adipogenic Differentiation.Subconfluent cells were ing antibiotics(100U/ml of penicillin and100g/ml of
cultured in GM supplemented with1μM dexametha-streptomycin).The infranatant containing hemopoietic sone(Sigma),500μM3-isobutyl-1-methyl-xanthine cells suspended in PBS was removed.Then the tissue
(IBMX,Sigma),60μM indomethacin(Sigma),and5 was dissociated with0.075%collagenase(Sigma)for15μg/ml insulin(Sigma).Adipogenic differentiation was min.Enzyme activity was neutralized with Dulbecco’s
confirmed on day21by intracellular accumulation of modified Eagle’s media-high glucose(DMEM-HG;lipid-rich vacuoles stainable with Oil Red O(Sigma). Gibco)containing10%FBS(Gibco).The infranatant
For the Oil Red O stain,cells were fixed with4%para-was centrifuged at1200×g for5min to pellet the cells.formaldehyde for30min,washed,and stained with a The cells from the pellet SVF were filtered to remove
working solution of0.16%Oil Red O for20min.
Figure1.Adipose tissue harvest.(A)The area over the dorsal gluteal muscles prepared for the procedure.(B)Liposuction proce-dure.(C)Skin incision after the liposuction.(D)Subcutaneous adipose tissue biopsy.(E)Skin incision after the biopsy.
CANINE ADIPOSE-DERIVED STEM CELLS281 Chondrogenic Differentiation.Subconfluent cells were temperature.After incubation,cells were washed three cultured in chondrogenic differentiation medium con-
times with PBS and resuspended in0.25ml of cold PBS. sisting of DMEM-low glucose supplemented with100Cell viability was accessed with Guava ViaCount re-
agent(Guava Technologies).
nM dexamethasone,50μM ascorbic acid-2phosphate
(Sigma),1mM sodium pyruvate(Gibco),10ng/ml cASCs were incubated with the following primary TGF-β1(R&D Systems),and1%ITS-Premix(Becton
antibodies:CD13-PE,CD29-PECy5,CD31-PE,CD34-Dickinson).Medium was changed every3–4days,and PE,CD44-FITC,CD45,CD73,CD90-PE,CD105e cells were fixed on day21with4%paraformaldehyde
CD117-PECy5(Becton Dickinson).The following anti-(PFA).Chondrogenesis was demonstrated by staining bodies have been raised against human cells:CD13,
CD71,and CD105.Unconjugated markers were treated with toluidine blue and immunofluorescence using anti-
collagen type II antibody(1:100,Abcam).with anti-mouse PE secondary antibody(Guava Tech-
nologies).
Osteogenic Differentiation.To promote osteogenic
Flow cytometer settings were established using un-differentiation,subconfluent cells were treated with GM
stained cells.Cells were gated by forward scatter to supplemented with50μM ascorbate-2phosphate,10
eliminate debris.To eliminate the possible autofluores-mMβ-glycerophosphate(Sigma),and0.1μM dexa-
cence of cASCs,we removed the contribution of uns-methasone for21days.Osteogenesis was demonstrated
tained cells in the measurement channel.A minimum of by accumulation of mineralized calcium phosphate as-
10,000events was counted for each analysis.
sessed by von Kossa stain.Briefly,cells were stained
with1%silver nitrate(Sigma)for45min under ultravio-
RNA Isolation and Reverse Transcriptase-Polymerase let light,followed by3%sodium thiosulphate(Sigma)
Chain Reaction(RT-PCR)
for5min,and then counterstained with van Gieson.
Total RNA was harvested from cultured cells using Myogenic Differentiation.For myogenic differentia-Tryzol(Invitrogen)following manufacturer’s instruc-tion,cASCs cells were cultured in GM supplemented tions.The RNA was treated with DNase(Invitrogen).A with0.1μM dexamethasone(Sigma),50μM hydrocor-total of1μg of total RNA was reverse-transcribed with tisone(Sigma),and5%horse serum(Gibco)for45SuperScript TM III First-Strand Synthesis System(In-days.After that cells were labeled with anti-myosin(1:vitrogen).All amplifications were performed in an MJ 100,Sigma).Research PTC-200thermocycler(MJ Research)for24
cycles after the initial2-min denaturation at94°C.The Immunofluorescence
PCR primers are listed in Table1.The PCR products Cells were fixed in4%paraformaldehyde in PBS for were separated on6%polyacrylamide gel by electropho-20min at4°C,permeabilized in0.05%Triton X-100in resis,stained with ethidium bromide,and visualized un-PBS for5min.Nonspecific binding was blocked with der UV light.Digital images were captured with Image-10%FBS in PBS for1h at room temperature.Cells Quant(GE Healthcare).
were incubated with primary antibody(1:100)overnight
cASCs Transduction With Lentivirus Vector
at4°C.After several washes,cells were incubated with
secondary(1:100,Sigma)antibodies against mouse IgG The visualization of cASCs cells for in vitro and in tagged with Cyanine3(Cy3;red)for2h at room tem-vivo studies can be improved if done with GFP-positive perature.Slides were counterstained with DAPI(4′-6-cells.For this purpose we transducted cASCs with GFP diamidino-2-phenylindole,Sigma).All images in the lentivirus.
same set(samples and controls)were obtained using the Supernatant containing the FUGW lentivirus(13) same photographic parameters of exposition and speed.was produced as described previously by Strauss et al. Images were captured using the Axiovision3.0image(26)and concentrated by ultracentrifugation.Undiffer-analysis system(Carl Zeiss).entiated cASCs at passage2were incubated at37°C,in
a six-well plate(Nunc),using a minimal volume of GM Flow Cytometry in the presence of vector particles(20PFU/cell)and8 Cells were evaluated for cell surface protein expres-
μg/ml Polybrene(Sigma).After4h,2ml of GM was sion using flow cytometry.The flow cytometry was per-added and the media was changed the next day. formed on Guava EasyCyte System(Guava Technolo-
Karyotype Analysis
gies)using a blue laser(488nm).Cells were pelleted,
resuspended in PBS at a concentration of1×105cells/For evaluation of any chromosomal abnormality at μl,and stained with saturating concentration of antibod-
latter passages,chromosome preparations were per-ies.Cells were incubated in the dark for45min at room formed in cASC cultures.Briefly,metaphase cells were
282VIEIRA ET AL.
Table1.PCR Primers
Amplicon Ann.
Markers/Gene Primer Sequence(5′–3′)Size Temp.(°C)Reference
Myogenic
MyoD Forward GACGGCATGATGGACTACAG11860
Reverse ACACCGCAGCACTCTTCC
Dystrophin Forward AAACACAGTGGTAGCCCACAAGAT11660
Reverse TGGTGACAGCCTGTGAAATC
Myogenin Forward GACGGCATGATGGACTACAG10260
Reverse ACACCGCAGCACTCTTCC
Adipocytes
FABP4Forward ATCAGTGTAAACGGGGATGTG1176017
Reverse GACTTTTCTGTCATCCGCAGTA
Leptin Forward CTATCTGTCCTGTGTTGAAGCTG1026017
Reverse GTGTGTGAAATGTCATTGATCCTG
LPL Forward ACACATTCACAAGAGGGTCAC1326017
Reverse CTCTGCAATCACACGGATG
Chondrocytes
COL2A Forward GAAACTCTGCCACCCTGAATG1566417
Reverse GCTCCACCAGTTCTTCTTGG
SOX9Forward GCTCGCAGTACGACTACACTGAC1016017
Reverse GTTCATGTAGGTGAAGGTGGAG
Aggrecan Forward ATCAACAGTGCTTACCAAGACA1225817
Reverse ATAACCTCACAGCGATAGATCC
Osteocytes
Osteopontin Forward CATATGATGGCCGAGGTGATAG11460
Reverse CAAGTGATGTGAAGTCCTCCTC
COL1A1Forward GTAGACACCACCCTCAAGAGC11862
Reverse CCAGTCGGAGTGGCACAT
BSP Forward TTGCTCAGCATTTTGGGAAT29560
Reverse AACGTGGCCGATACTTAAAGAC
Housekeeping
GAPDH Forward CCATCTTCCAGGAGCGAGAT9760
Reverse TTCTCCATGGTGGTGAAGAC
arrested with0.1μg/ml colchicine(Sigma)for20min.are easy to expand in vitro and show a fibroblast-like Then,cASCs were detached from cultures flasks using
morphology,consistent with that of human ASCs(Fig. TrypLE(Gibco),resuspended in a hypotonic solution2A–D).At both early and late passages,cells main-
tained a diploid karyotype of78chromosomes(Fig.2E).
(0.075M KCl),and incubated for20min at37°C.Cells
were pelleted at1000rpm for10min and fixed by wash-cASCs from four unrelated dogs were characterized ing three times in methanol/glacial acetic acid(3:1).
by flow cytometry for the expression of10cell surface Chromosome spreads were obtained by pipetting sus-proteins(CD13,CD29,CD31,CD34,CD44,CD45, pension drops onto clean glass slides and air dried.The
CD73,CD90,CD105,and CD117).Cell viability was best metaphases were captured with Axioplan2micro-above96%by Guava ViaCount reagent(Guava Tech-scope(Zeiss)and analyzed with Ikaros3software
nologies).
(Zeiss).At passage4,the majority of cASCs expressed
CD44,CD29(β1integrin)and CD90(Thy1)adhesion RESULTS
molecules.Other markers,including CD14,CD34, Characterization of cASCs CD45,and CD117,were consistently absent or ex-cASC cultures were maintained in DMEM supple-
pressed in few cells(Fig.3).Interestingly,CD13, mented with10%FBS.Supplementation with FBS has CD105,and CD73,known to be positively expressed in
human ASCs,were negative in the canine ASC popula-been shown to be important for human ASC attachment
and proliferation in vitro(35).We observed that cASCs tion,which might be explained by the nonspecific stain-
CANINE ADIPOSE-DERIVED STEM CELLS283 ing of human antibodies in canine cells.As surface the mesenchymal nature of the isolated cells and their markers are not sufficient for the identification or defini-
multipotent potential.
tion of mesenchymal stem cell(MSC),cASCs were sub-
jected to differentiation studies for further confirmation Adipogenesis.cASCs showed a rounder shape after of their MSC property.
7days in adipogenic medium.Two weeks after initial The plasticity of cASCs was assessed after lineage induction,the adipogenic differentiation was confirmed induction.Myogenic,adipogenic,chondrogenic,and os-
by Oil Red O staining of lipid droplets present through-teogenic differentiation was demonstrated by the expres-out the cytoplasm(Fig.4A).Expression of FABP4and sion of myogenic markers(myosin),lipid vacuoles,mu-
LPL was seen only in adipo-induced cells(Fig.5B).On copolysaccharide-rich extracellular matrix,and calcium the other hand,basal level of leptin mRNA was ob-deposits,respectively(Fig.4)and by the expression of
served in noninduced control cells,and the expression tissue-specific mRNAs(Fig.5).These results confirmed level was increased following adipogenic induction.
Figure2.Typical morphology of cASCs.(A)Forty-five minutes after the establishment of the
culture.Some cells remain in the supernatant.Scale bar:200μm.(B)cASCs at passage3.Scale
bar:200μm.(C)cASCs at passage4:cASCs morphology is similar to that found in human ASCs.
Scale bar:100μm.(D)High-density ASCs culture at passage4.Scale bar:200μm.(E)Karyotype
of cASC cell lineage after10passages,showing an euploid number of chromosomes.
284VIEIRA ET AL.
Figure3.Immunophenotyping of ASCs at passage4.Values represent the mean percentage of positively stained cells as analyzed by flow cytometry.Graphs show forward scatter versus fluorescence intensity of the indicated antigen.
Osteogenesis.cASCs exposed to osteogenic me-Chondrogenic Differentiation.After21days cul-
tured in chondrogenic medium,cASCs cells were dium exhibited changes in cell morphology after5
days in culture,showing a polygonal form.Mineral-stained with toluidine blue,showing the typical metach-ized nodular structures appeared in1or2weeks and
romasia of cartilage.Chondrogenic differentiation was were assessed by von Kossa Stain,which localized demonstrated by the mucopolyssaccharide-rich extracel-the calcium deposits(Fig.4C).Expression of osteo-
lular matrix(Fig.4E,G).Chondrogenic treatment re-pontin,COL1A1,and BSP was observed only in in-sulted in specific expression of COL2A,SOX9,and ag-
grecan,all of which were undetected or had basal duced cells,with no basal expression in control cells
(Fig.5C).expression in noninduced cells(Fig.5D).
CANINE ADIPOSE-DERIVED STEM CELLS285
Figure4.Differentiation potential of cASCs at passage4.(A)The adipogenic differentiation was
detected by the formation of intracytoplasmic lipid droplets stained with Oil Red O.Scale bar:200
μm.(B)Undifferentiated ASCs stained with Oil Red O.Scale bar:200μm.(C)cASCs after
induction with adipogenic media still show GFP expression.Scale bar:200μm.(D)Osteogenic
differentiation was demonstrated by calcium deposition shown by von Kossa stain.Scale bar:200
μm.(E)Undifferentiated ASCs stained with Von Kossa.Scale bar:200μm.(F)cASCs after
induction with osteogenic media still show GFP expression.Scale bar:200μm.(G)Chondrogenic
differentiation in monolayer culture was demonstrated by staining with toluidine blue.Scale bar:
200μm.(H)Undifferentiated ASCs stained with toluidine blue.Scale bar:200μm.(I)cASCs
after induction with chondrogenic media still show GFP expression.Scale bar:200μm.(J)Chon-
drogenic differentiated cells labeled with anti-collagen type II antibody.Scale bar,200μm.(K)
Undifferentiated ASCs labeled with anti-collagen type II antibody.Scale bar:200μm.(L)Myo-
genic differentiation was assessed by immunofluorescence.Induced cells were labeled with anti-
myosin monoclonal antibody.Scale bar:50μm.(M)Undifferentiated ASCs labeled with anti-
human myosin monoclonal antibody.Scale bar:50μm.(N)cASCs after induction with myogenic
media still show GFP expression.Scale bar:50μm.
286VIEIRA ET AL.
Figure5.mRNA expression of specific differentiation markers.(A)Myogenic markers:myogenin
(Myog),dystrophin(Dyst)and MyoD.(B)Adipogenic markers;FABP4,leptil,and LPL.(C)
Osteogenic markers:osteopontin(OPN),COL1A1,bone sialoprotein(BSP).(D)Chondrogenic
markers;COL2A,SOX9,and aggrecan(AGC).The expression of glyceraldehyde-3-phosphate
dehydrogenase(GAPDH)was used as reference for evaluating the quality of mRNA.
Myogenesis.After10days in myogenic medium,GFP Transduction of cASCs
cASCs formed multinucleated structures.Controls main-
tained only with GM did not contain any multinucleated Transgene expression was examined by flow cy-structures.To confirm the myogenic differentiation,the
tometry72h posttransduction.About75%of cells expression of myosin by immunofluorescence was as-were GFP positive and GFP expression did not de-sessed after45days(Fig.4H).The specificity of this
cline during culture passages(Fig.6).To evaluate if assay was corroborated by the absence of staining in GFP interfered with the multipotent capacity of cASCs.Expression of myogenin,dystrophin,and MyoD
cASCs,both GFP-positive and-negative cells at suc-was observed only in induced cells,with no basal ex-cessive passages were analyzed by flow cytometry pression in control cells(Fig.5A).
and multilineage differentiation,revealing no influ-
CANINE ADIPOSE-DERIVED STEM CELLS287
Figure6.GFP-positive cASCs.(A)GFP-positive cASCs at passage8.(B)Percentage of positively GFP cells as analyzed by flow cytometry.
ence of GFP on the cellular response to inductive me-isolation of adipose stem cells from other mammals, dia(Fig.4).
such as rabbit,mice,horse,and pig(27,29,32,33).
We observed that the plastic adherent cells obtained DISCUSSION after isolation can be expanded in vitro,reaching num-Zuk et al.(35)were the first to describe the isolation
bers that would be sufficient for a therapeutic assay, and characterization of human stem cells derived from without any numeric chromosome alteration.In addi-adipose tissue.These cells were able to differentiate into
tion,cASCs can be stored frozen in liquid nitrogen with-adipogenic,osteogenic,chondrogenic,and myogenic out cell death.
lineages when exposed to inductive media.
During the first days in culture,endothelial cell popu-Human ASCs are usually obtained from fat tissue lations were found in the plates;however,these cells that is discarded after liposuction cosmetic surgery(35).
were not seen after passage4.These data are in accor-Adipose tissue can be harvested in large quantities with dance with Rodriguez et al.(21),where the isolation of
hASCs by adherence properties was reported.At pas-minimal morbidity in several regions of the body and,
on average,100ml of human adipose tissue yields about sage4,cASCs show a fibroblast-like morphology com-1×106stem cells(14).In dogs,the adipose tissue can
monly found in mesenchymal stem cell(MSCs).The be collected by a simple adapted liposuction surgery,analysis of the cell surface markers showed that the through biopsies or in routine veterinary surgery proce-
cASCs cell population expresses the known immnophe-dures because we could isolate cASCs from just100μl notype of MSCs(35).At passage4the majority of the of adipose tissue.
cells are positive for CD29,CD44,and CD90.cASCs In the present study we show the isolation and char-do not express the hematopoietic marker CD45,but10% acterization of the canine adipose-derived stromal cell
of the cells are CD34positive.Traktuev et al.(28)de-(cASC)population.While this manuscript was in prepa-scribed that the population of CD34-positive cells that
are found in human adipose stromal-vascular fraction ration,Neupane et al.(17)published an article reporting
the isolation of canine adipose stem cells.However,are reside in a periendothelial location.The authors their article lacked important characteristics of the iso-
showed that these cells are CD31negative.This result lated cell population such as the immunophenotype,the is in accordance with our finding with cASCs,because myogenic and chondrogenic potential,and karyotype
we found CD34-positive cells but not CD31-positive analysis at late passages.These characteristics are im-cells.This adherent cASCs cell population(CD34+/
CD31?)may also interact with endothelial cells at the portant for veterinary cell therapy and preclinical studies.
Our results show that cASCs can be harvested by a perivascular niche.However,further studies will be es-rapid process,an important step towards preclinical
sential to identify the localization of these cells at the studies of cell https://www.doczj.com/doc/7e4027685.html,ing this methodology,we were canine adipose tissue.In order to evaluate the MSC able to harvest cells from10canine subcutaneous fat
property of cASCs,we subjected them to differentiation samples(2from liposuction and8from biopsy)with a studies.
100%rate of success.Other groups reported successful
MSCs are defined by their ability to self-renew and
288
VIEIRA ET AL.their capacity to generate committed cells in vitro and potential relevance to future canine veterinary tissue en-gineering and regenerative veterinary medical therapies.in vivo.Human ACSs can be induced to differentiate along the adipogenic,chondrogenic,and osteogenic lin-ACKNOWLEDGMENTS:We gratefully acknowledge our col-eages using specific culture medium (32).Even plated leagues Tatiana Jazedje,Marcos Valadares,Maria Denise Carvalho,Amanda Assoni,Camila Almeida,Mayra Pellati,into scaffolds they survive long-term culture and could Bruno Lima,Heloisa Caetano,Constancia Urbani,Dr.Mariz be terminally differentiated into adipocytes and osteo-Vainzof,and Dr.Maria Rita Passos-Bueno for helpful sugges-blasts (18).In all 3of our 10lineages obtained we dem-tions as well as the earlier support of the veterinarians.This onstrated the multipotency and plasticity of cASCs by research was supported by FAPESP-CEPID (Fundac ?a ?o de their differentiation in adipogenic,chondrogenic,myo-Amparo a `Pesquisa do Estado de Sa ?o Paulo-Centro de Pes-quisa,Inovac ?a ?o e Difusa ?o),CNPq (Conselho Nacional de De-genic,and osteogenic lineages.The differentiation was senvolvimento Cient?′fico e Tecnolo ′gico),and INCT (Instituto confirmed by the appearance of lipid vacuoles,muco-Nacional de Cie ?ncia e Tecnologia:ce ′lulas-tronco em doenc ?as polysaccharide-rich extracellular matrix,myosin label-gene ′ticas).ing,and calcium deposits,respectively.Although we ob-served a morphological change in cells submitted to REFERENCES adipogenic differentiation,we found poor lipid vacuoles in cASCs when compared to hASCs submitted to the 1.Black,L.L.;Gaynor,J.;Gahring,D.;Adams,C.;Aron,same conditions.
D.;Harman,S.;Gingerich,D.A.;Harman,R.Effect of The stable GFP expression after cASC transduction adipose-derived mesenchymal stem and regenerative cells on lameness in dogs with chronic osteoarthritis of the cox-represents a great advantage in preclinical studies.GFP-ofemoral joints:A randomized,double-blinded,multicen-positive cells could be screened though fluorescence ter,controlled trial.Vet.Ther.8:272–284;2007.microscopic imaging and flow cytometry,and no signif- 2.Brzoska,M.;Geiger,H.;Gauer,S.;Baer,P.Epithelial icant decline of GFP expression during cASC differenti-differentiation of human adipose tissue-derived adult stem ation was found.This property allows their potential use https://www.doczj.com/doc/7e4027685.html,mun.330:142–150;2005.for in vitro (29)and in vivo assays (3),where cell mark- 3.Darabi,R.;Gehlbach,K.;Bachoo,R.M.;Kamath,S.;ing with GFP was used to track stem cells seeded on a Osawa,M.;Kamm,K.
E.;Kyba,M.;Perlingeiro,R.C.scaffold,fusing to other cells and engraftment into dif-Functional skeletal muscle regeneration from differentiat-ferent tissues.
ing embryonic stem cells.Nat.Med.14:134–143;2008.Successful canine stem cell transplantation was re- 4.Di Rocco,G.;Iachininoto,M.G.;Tritarelli,A.;Straino,S.;Zacheo,A.;Germani,A.;Crea,F.;Capogrossi,M.C.ported by Sampaolesi et al.(24).According to these Myogenic potential of adipose-tissue-derived cells.J.Cell authors,the intra-arterial delivery of canine mesoangi-Sci.119:2945–2952;2006.oblasts (vessel-associated stem cells)resulted in an ex- 5.Erickson,G.R.;Gimble,J.M.;Franklin,D.M.;Rice,tensive recovery of muscle morphology and function in H.E.;Awad,H.;Guilak,F.Chondrogenic potential of golden retriever muscular dystrophy (GRMD)dogs.Be-adipose tissue-derived stromal cells in vitro and in https://www.doczj.com/doc/7e4027685.html,mun.290:763–769;2002.cause ASCs are much easier to be obtained and can be 6.Garcia-Olmo, D.;Garcia-Arranz,M.;Herreros, D.;injected without immunosuppression (11,31),they might Pascual,I.;Peiro,C.;Rodriguez-Montes,J.A.A phase I represent a promising alternative to canine mesoangi-clinical trial of the treatment of Crohn’s fistula by adipose oblasts for GRMD stem cell therapeutic trials.
mesenchymal stem cell transplantation.Dis.Colon Rec-In short,results from the present study demonstrate tum 48:1416–1423;2005.7.Gimble,J.;Guilak,F.Adipose-derived adult stem cells:that adherent cells isolated from canine adipose tissue Isolation;characterization;and differentiation potential.can be defined as multipotent MSC with the ability to Cytotherapy 5:362–369;2003.differentiate into at least four mesodermal lineages.8.Halvorsen,Y.D.;Bond,A.;Sen,A.;Franklin,D.M.;Mouse models of human diseases have been extensively Lea-Currie,Y.R.;Sujkowski,D.;Ellis,P.N.;Wilkison,used in preclinical studies.However,mouse models W.O.;Gimble,J.M.Thiazolidinediones and glucocorti-coids synergistically induce differentiation of human adi-have several limitations when extrapolating to humans:pose tissue stromal cells:Biochemical,cellular,and mo-the small size of the mouse limits the proliferative de-lecular analysis.Metabolism 50:407–413;2001.mand placed on transplanted tissue and the short life 9.Halvorsen,Y.D.;Bond,A.;Sen,A.;Franklin,D.M.;span of the mouse also prevents long-term follow-up.Lea-Currie,Y.R.;Sujkowski,D.;Ellis,P.N.;Wilkison,Large-animal models,such as the dog,more faithfully W.O.;Gimble,J.M.Extracellular matrix mineralization and osteoblast gene expression by human adipose tissue-mimic human pathologies.There are many canine ani-derived stromal cells.Tissue Eng.7:729–741;2001.mal models of human genetic diseases that are well 10.Justesen,J.;Pedersen,S.B.;Stenderup,K.;Kassem,M.characterized,making them ideal for in vivo studies.Subcutaneous adipocytes can differentiate into bone-form-The characterization of cASCs represents a valuable tool ing cells in vitro and in vivo.Tissue Eng.10:381–391;for in vitro and in vivo preclinical evaluation and for the 2004.
11.Keyser,K.A.;Beagles,K.E.;Kiem,https://www.doczj.com/doc/7e4027685.html,parison
screening of therapeutic drugs.These findings also have
CANINE ADIPOSE-DERIVED STEM CELLS289 of mesenchymal stem cells from different tissues to sup-Tonlorenzi,R.;Innocenzi,A.;Mognol,P.;Thibaud,J.;
press T-cell activation.Cell Transplant.16:555–562;
Galvez,B.G.;Barthelemy,I.;Perani,L.;Mantero,S.;
2007.Guttinger,M.;Pansarasa,O.;Rinaldi,C.;Angelis,M.G.C.;
12.Li,H.;Dai,K.;Tang,T.;Zhang,X.;Yan,M.;Lou,J.
Torrente,Y.;Bordignon, C.;Bottinelli,R.;Cossu,G.
Bone regeneration by implantation of adipose-derived Mesoangioblast stem cells ameliorate muscle function in stromal cells expressing BMP-2.Biochem.Biophys.Res.
dystrophic dogs.Nature444:574–579;2006.
Commun.356:836–842;2007.25.Seo,M.J.;Suh,S.Y.;Bae,Y.C.;Jung,J.S.Differentia-13.Lois,C.;Hong,E.J.;Pease,S.;Brown,E.J.;Baltimore,
tion of human adipose stromal cells into hepatic lineage
D.Germline transmission and tissue-specific expression in vitro and in https://www.doczj.com/doc/7e4027685.html,mun.
of transgenes delivered by lentiviral vectors.Science95:
328:258–264;2005.
868–872;2002.26.Strauss,B.E.;Patricio,J.R.;de Carvalho,A.C.;Bajgel-14.Meliga,E.;Strem,B.M.;Duckers,H.J.;Serruys,P.W.
man,M.C.A.A lentiviral vector with expression con-Adipose-derived cells.Cell Transplant.16:963–970;trolled by E2F-1:A potential tool for the study and treat-2007.
ment of proliferative diseases.Biochem.Biophys.Res.
15.Miranville,A.;Heeschen,C.;Sengenes,C.;Curat,C.A.;Commun.348:1411–1418;2006.
Busse,R.;Bouloumie,A.Improvement of postnatal neo-
27.Torres,F.C.;Rodrigues,C.J.;Stocchero,I.N.;Ferreira,
vascularization by human adipose tissue-derived stem M.C.Stem cells from the fat tissue of rabbits:An easy-cells.Circulation110:349–355;2002.
to-find experimental source.Aesthetic Plast.Surg.31: 16.Mizuno,H.;Zuk,P.A.;Zhu,M.;Lorenz,H.P.;Benhaim,574–578;2007.
P.;Hedrick,M.H.Myogenic differentiation by human
28.Traktuev,D.O.;Merfeld-Clauss,S.;Li,J.;Kolonin,M.;
processed lipoaspirate cells.Plast.Reconstr.Surg.109:Arap,W.;Pasqualini,R.;Johnstone,B.H.;March,K.L.
199–209;2002.
A population of multipotent CD34-positive adipose stro-
17.Neupane,M.;Chang,C.C.;Kiupel,M.;Yuzbasiyan-Gur-mal cells share pericyte and mesenchymal surface mark-
kan,V.Isolation and characterization of canine adipose-
ers,reside in a periendothelial location,and stabilize en-derived mesenchymal stem cells.Tissue Eng.Part A14:dothelial networks.Circ.Res.102:77–85;2008.
1007–1015;2008.
29.Vidal,M.A.;Kilroy,G.E.;Lopez,M.J.;Johnson,J.R.;
18.Neuss,S.;Stainforth,R.;Salber,J.;Schenck,P.;Bovi,Moore,R.M.;Gimble,J.M.Characterization of equine
M.;Knu¨chel,R.;Perez-Bouza,A.Long-term survival and
adipose tissue-derived stromal cells:Adipogenic and os-bipotent terminal differentiation of human mesenchymal teogenic capacity and comparison with bone marrow-stem cells(hMSC)in combination with a commercially
derived mesenchymal stromal cells.Vet.Surg.36:613–available three-dimensional collagen scaffold.Cell Trans-622;2007.
plant.17:977–986;2008.
30.Vieira,N.M.;Brandalise,V.;Zucconi,E.;Jazedje,T.;
19.Planat-Benard,V.;Silvestre,J.S.;Cousin,B.;Andre,M.;Secco,M.;Nunes,V.A.;Strauss,B.E.;Vainzof,M.;
Nibbelink,M.;Tamarat,R.;Clergue,M.;Manneville,C.;
Zatz,M.Human multipotent adipose-derived stem cells Saillan-Barreau,C.;Duriez,M.;Tedgui,A.;Levy,B.;restore dystrophin expression of Duchenne skeletal-Penicaud,L.;Casteilla,L.Plasticity of human adipose lin-
muscle cells in vitro.Biol.Cell100:231–241;2008.
eage cells toward endothelial cells:Physiological and ther-31.Vieira,N.M.;Bueno,Jr.,C.R.;Brandalise,V.;Moraes, apeutic perspectives.Circulation109:656–663;2004.
L.V.;Zucconi,E.;Secco,M.;Suzuki,M.F.;Camargo, 20.Rehman,J.;Traktuev, D.;Li,J.;Merfeld-Clauss,S.;M.M.;Bartolini,P.;Brum,P.C.;Vainzof,M.;Zatz,M.
Temm-Grove,C.J.;Bovenkerk,J.E.;Pell,C.L.;John-
SJL dystrophic mice express a significant amount of hu-stone,B.H.;Considine,R.V.;March,K.L.Secretion man muscle proteins following systemic delivery of hu-of angiogenic and antiapoptotic factors by human adipose
man adipose-derived stromal cells without immunosup-stromal cells.Circulation109:1292–1298;2004.pression.Stem Cells26:2391–2398;2008.
21.Rodriguez, A.M.;Pisani,D.;Dechesne,C. A.;Turc-
32.Williams,K.J.;Picou,A.A.;Kish,S.L.;Giraldo,A.M.;
Carel,C.;Kurzenne,J.Y.;Wdziekonski,B.;Villageois,Godke,R.A.;Bondioli,K.R.Isolation and characteriza-
A.;Bagnis,C.;Breittmayer,J.P.;Groux,H.;Ailhaud,G.;
tion of porcine adipose tissue-derived adult stem cells.
Dani,C.Transplantation of a multipotent cell population Cells Tissues Organs188:251–258;2008.
from human adipose tissue induces dystrophin expression
33.Yamamoto,N.;Akamatsu,H.;Hasegawa,S.;Yamada,T.;
in the immunocompetent mdx mouse.J.Exp.Med.201:Nakata,S.;Ohkuma,M.;Miyachi,E.;Marunouchi,T.;
1397–1405;2005.
Matsunaga,K.Isolation of multipotent stem cells from
22.Rodriguez,L.V.;Alfonso,Z.;Zhang,R.;Leung,J.;Wu,mouse adipose tissue.J.Dermatol.Sci.48:43–52;2007.
B.;Ignarro,L.J.Clonogenic multipotent stem cells in hu-
34.Zuk,P. A.;Zhu,M.;Ashjian,P.;De Ugarte, D. A.;
man adipose tissue differentiate into functional smooth Huang,J.I.;Mizuno,H.;Alfonso,Z.C.;Fraser,J.K.;
muscle https://www.doczj.com/doc/7e4027685.html,A103:12167–
Benhaim,P.;Hedrick,M.H.Human adipose tissue is a 12172;2006.source of multipotent stem cells.Mol.Biol.Cell13:4279–23.Safford,K.M.;Hicok,K.C.;Safford,S.D.;Halvorsen,
4295;2002.
Y.D.;Wilkison,W.O.;Gimble,J.M.;Rice,H.E.Neuro-35.Zuk,P. A.;Zhu,M.;Mizuno,H.;Huang,J.;Futrell, genic differentiation of murine and human adipose-derived
J.W.;Katz,A.J.;Benhaim,P.;Lorenz,H.P.;Hedrick, stromal https://www.doczj.com/doc/7e4027685.html,mun.294:M.H.Multilineage cells from human adipose tissue:Im-371–379;2002.
plications for cell-based therapies.Tissue Eng.7:211–24.Sampaolesi,M.;Blot,S.;D’Antona,G.;Granger,N.;228;2001.
启动： ●BIOS中必须添加保护卡BIN File，否则无法安装，添加完Bin File会在开机时加载保 护卡。 1.开机左上角会出现保护卡黄色版本号“Rom Star (Build:xxxxxx) ”xx为bin file建 立日期 2.之后会出现搜索服务器界面，BIOS中PXE开启，网线连接时提示“Searching Server F2/F3 forced waiting,ESC to skip” 3.BIOS中PXE关闭或网线无连接时则提示“Network failure, press any key boot form disk! Time left:10” ●点击光盘根目录，驱动自动判断BIOS中保护卡的版本号，并弹出相匹配的驱动安装程 序。 安装： 保护卡分为底层（DOS）驱动安装和windows驱动安装，故不支持静默安装，请按照安装向导提示进行。安装前先装好系统补丁和主板设备驱动再安装保护卡驱动（如装有杀毒软件，安装保护卡驱动前请先退出） 1.进入windows系统，点安装程序中的，,弹出软件安装界面，点击检查版本是否匹配。 2.版本匹配成功，点击下一步继续 选择全新安装 3.弹出修正硬盘大小
安装需要占用磁盘尾部的部分空间来存放保护卡数据，需要用户手动删除最后一个分区 点击确定，弹出保护卡分区界面，新建分区。选择保护类型
分区规划完，选择添加系统，点确定。 ，系统和分区规划完毕，点安装后重启，出现保护卡开机界面。把光标移到第二个系统按钮处，放入系统光盘，安装第二个操作系统。 进入系统，继续安装保护卡安装第二步，安装window保护卡驱动，点击
As China is speeding up its modernization drive, and due to its accelerating urbanization process, many people feel isolated from each other. Write a composition of about 400 words on this issue. We are living in a much more crowded world, and the fast development of information technology is making this world unimaginably smaller. Despite all these, a sense of isolation has been growing among people, esp. among the urban dwellers. Here are some of the reasons. First, modern people experience greater tensions so that they have less time to communicate with each other. As more and more people move to the cities, the pace of life is getting exhaustingly fast. Due to the fierce competition, urban residents have to work hard to as to keep up with the steps of city life. Moreover, living in apartments in different blocks, people find it virtually inconvenient to meet and chat with each other as freely as before. Second, the wide use of telephone and the internet prevent people from seeing each other quite often. Face-to-face contact used to be fundamental way to get information. However, technologies today have enormously reduced the necessity of meeting each other, and a felling of isolation arises accordingly. In the past, when a festival came, the way people send congratulations was to meet each other, to get together or write letters and send postcards. Nowadays, people can simply send an E-mail or make a phone call to express good wishes in a few minutes. There are some solutions to the present situation. First of all, people need to be fully aware of the fact that the modern ways of communication cannot replace the traditional ones. Sending #-mails cannot have the same effect as writing or face-to-face talk. Traditional ways should be encouraged and people can write letters as much as possible. Secondly, activities held in one neighborhood can create a warm atmosphere among people. Urbanization refers to a process in which an increasing proportion of an entire population lives in cities and the suburbs of cities. I Structure: -- logical: (cause and effect) 1 Thesis: problem/phenomenon 2 Body: analysis (reasons, examples
清华同方电脑还原卡破解的几个方法 进入bios,清华同方BIOS的通用口令:thtfpc,依次进入--INTEGRATED PERIPHERALS---ONBOARD DEVICE----ONBOARD LAN：CONTROLLER 此项设为ENABLED(集成网卡生效）----ONBOARD LAN：BOOT ROM 此项设为DISABLED（取消还原功能）测试，有效 清华同方还原卡的安装： 第一次安装 1 确定硬盘的type (cmos资料)设定是否正确 2 请将驱动盘放入驱动器中并按enter... * 按[esc]不安装 * 按f1进行自动网络搜寻安装功能. hard disk type size:38162mb cyls:4865 head:255 sector:63 解决这个问题建议最好是挂上个光驱,在出现此画面的时候把同方易教那张光盘放到光驱中,然后按回车键.如果想保存原有的硬盘数据,就选择"简易安装",可以根据屏幕提示操作,后面的就很简单了 1、开启还原卡。进入bios,使用CTRL+F1组合键进入工程模式BIOS图形配置界面。找到Integrated Peripherals选项，使用ONBOARD LAN BOOT ROM选项打开/关闭保护卡功能。保存重启。 2、安装还原卡底层驱动程序（还原驱动程序在附送的绿色光碟里面）。 重启后，会出现“第一次安装”的界面，插入光盘，回车，等搜索完成后会出现“安装设定”的界面，选择“简单安装”，回车，确定。完成后，还原底层驱动安装成功。注：“简易安装”可以保留先有分区信息；“自定义安装”需要格式化所有硬盘分区，保护效果比较好。“简易安装”可以先安装系统，后装还原卡；“自定义安装”必须先装好还原卡再装系统。一般我们选择“简易安装”，安装完成后，重启。 3、安装还原卡上层驱动程序。开机后按F10，进去还原卡设定界面，进入“分区信息”，选择系统分区不还原，保存，重启。进入windows后，在光盘里面找到SETUP文件夹，运行SETUP.EXE。完成后，所有安装完成。 4、如何使用：开机后按F10，可以进行“密码设定”、“参数设定”、“分区信息”、“工具”、“重新分割”等选项目。去除还原是进去“分区信息”，将不还原的分区设置为“不还原”，然后保存，重启。各种功能，各位慢慢研究。 同方易教保护卡网络拷贝、增量拷贝常见问题 1同方易教网络拷贝设置中为什么有3种网络拷贝模式，各有什么差别？ 答：A. 同方易教支持三种网络拷贝模式,模式1和模式3为可靠拷贝模式,模式2为快速拷贝模式，模式3属于增强型数据校验网络拷贝(针对某些特殊环境，比如说发现网络拷贝过去接收端的数据有错误或者操作系统起不来)，模式2和模式3必须工作在所有机器都安装了同方易教底层驱动的基础上！ B. 网络安装接收端请选用模式1
总RNA 提取 （SV Total RNA Isolation System Kit，Promega） 1.低于30mg的样品中加入175μl裂解缓冲液(SV RNA Lysis Buffer＋ BME)，充分匀浆。 2.加入350μl蓝色的RNA 稀释缓冲液（SV RNA Dilution Buffer）， 颠倒3-4次使样品充分混合。 3.放入70℃水浴中裂解3min。 注：温育时间不要超过3分钟，否则可能破坏RNA的完整性。 4.14，000g 离心10 min。小心用移液器将上清液转移到新的Eppendorf 管中。 注：①若是组织中脂肪含量较高，需重复几次此步骤；②避免吸到颗粒物。 5.加入95%的乙醇200μl于上清液，用枪头冲吸3-4 次。 6.将其全部转移到带有滤膜的微型柱中，14,000g, 离心1min，弃滤液。 7.加入600μl冲洗缓冲液（SV RNA Wash Solution）, 14,000g, 离心1 min，弃滤液。 注：确保SV RNA Wash Solution已用乙醇稀释过。 8.按样品数量准备DNA 酶Ⅰ混合液，用于降解DNA。每样需40μl黄 色缓冲液, 5μl 0.09M的MnCl2和5μl DNA 酶Ⅰ配制的DNA 酶Ⅰ混 合液。 注：①DNA 酶Ⅰ必须在冰上解冻；②温柔用移液器混合，不可涡旋。 9.确保加50μl DNA 酶Ⅰ混合液到滤膜中间，室温保持15 min。 10.加入200μl终止反应缓冲液（SV DNase Stop Solution），14,000g, 离 心1 min，不必弃滤液。 注：确保SV DNase Stop Solution中已加入乙醇。 11.加入600μl 冲洗缓冲液（SV RNA Wash Solution），14,000g，离心 1 分钟，弃滤液。 12.再加入250μl冲洗缓冲液洗一次（SV RNA Wash Solution）， 14,000g，离心2 min，弃滤液，转移微型柱到新的洗脱管中。 13.用100μl无RNA酶的水（Nuclease－Free Water）洗膜，14,000g， 离心1min，-70℃保存洗脱下来的总RNA。 注：确保无RNA酶的水覆盖膜表面。
利用同方原卡安装教室网络的方法及微机房的日常维护技巧 马恩辉储成俊 2009.6 摘要：安徽省滁州市2005年与“农远工程”配套的“校校通工程”使用的学生电脑是清华同方超越E220，它主板上集成有同方还原卡。目前校园和教室网络的组建和维护技术虽然很成熟，但是针对“农远工程”项目学校网络环境建设而言大量的书籍和网络文章则显得庞杂、更缺乏针对性强的实战技巧，为了学校“农远管理员”能更好地维护网络教室，本文将详细介绍利用同方还原卡快速合理地安装网络教室的方案和网络教室的日常维护技巧。 关键词：同方还原卡网络教室维护技巧 本文之所以要讲?利用同方原卡安装教室网络的方法以及微机房的日常维护技巧?，是因为?农远项目?学校网络管理中存在以下问题： （1）目前校园和教室网络的组建和维护技术虽然很成熟，但是针对农远工程项目学校网络环境建设而言大量的书籍和网络文章则显得庞杂、更缺乏针对性强的实战技巧。 （2）农远管理员虽也经过培训上岗管理，但是培训中却有很多实际问题没有讲到。例如未讲到在安装有同方还原卡的电脑上如何确保成功地安装操作系统、在无光驱和软驱的情况下如何给网络更换操作系统、用什么方法能简洁快速地开通网上邻居等。 （3）各种硬软件的安装说明书往往讲解得很杂乱，这页见那页绕得你头发晕，甚至该说清的未说清，或者就没给出最合理的解决方案，使你产生误解或费解。例如，同方易教说明书中就没说到在还原卡不移出情况下更换操作系统时还原卡参数设定中开机选择项应选择BIOS项；再如，同方易教说明书中说到：?在windows安装完成后，请以总管模式进入系统，将同方还原卡驱动（即《同方易教》光盘）插入光驱中，参考《同方易教》光盘TOOL目录下的readme.txt文件来制作驱动软盘。双击新制作出来的那张软盘里的SETUP.EXE来安装系统保护程序和IP自动修改选项。?这里又是用光驱又是制作驱动软盘，现在软盘几乎不用了，甚至在安装维护电脑时连光驱都不想用，显然说明书中的这种做法太繁琐太不切实际，是不可取的；又如，在没有安装C盘保护驱动程序时安装系统或软件不须在总管模式下进
同方易教增量版使用指南 同方股份有限公司 thtfpc
前言 ◎欢迎使用同方易教增量版 ◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆ ※本手册所有的产品商标与产品名称均属于同方股 份有限公司。 ※本手册所有图形仅供参考，请您以实际软件界面为 准。 ※请您在做安装、移除、修改同方易教增量版操作时， 备份好您的硬盘数据，如果数据丢失，本公司不予 找回。 ※软件版本如有变更恕不另行通知。 ◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆ 同方易教增量版广泛应用于学校机房或网吧等局域网环境，成为广大机房管理者的得力助手。它以方便、安全的优势备受系统管理者的青睐。
目录 1.产品介绍 (1) 1.1产品说明 (1) 1.2功能简介 (1) 1.3最低硬件配置 (2) 1.4支持的操作系统 (2) 1.5支持的文件系统 (3) 2.快速开始指南 (4) 2.1安装同方易教增量版 (4) 2.2安装流程图 (5) 2.2.1 安装发送端流程图 (5) 2.2.2 网络克隆接收端流程图 (6) 2.3安装发送端（以﹤全新安装﹥为例） (7) 2.3.1 选择安装方式——全新安装 (7) 2.3.2 安装操作系统及应用软件 (9) 2.3.3 安装同方易教增量版操作系统驱动 (10) 2.3.4 安装完成 (11) 2.3.5 设置发送端网络拷贝信息 (11) 2.4网络克隆接收端 (12) 2.4.1 网络安装接收端底层驱动 (12) 2.4.2 配置接收端信息（IP地址/计算机名） .. 15 2.4.3 传送操作系统数据 (17) 3.增量拷贝—安装、卸载软件，修改系统设置 (20) 3.1增量拷贝的流程图 (20) 3.2实现增量拷贝的前提条件 (21) 3.3增量拷贝全过程 (21) 3.3.1 准备增量数据 (21) 3.3.2 执行增量拷贝 (22)
同方易教常见问题解决 1安装操作系统，需要用开放模式进入安装吗？ 答： 不用。我们在安装系统时，将系统安装盘放入光驱，引导次序改为光驱引导，在同方易教操作系统引导选单上选择要安装的系统，直接回车即可安装。 2同方易教windows 驱动在何时安装合适？ 答： 1) 安装完系统后，装载计算机硬件驱动和应用软件后，再安装各种硬件的windows 驱动。设置好计算机名和IP,DNS等等….，此驱动一旦安装后，系统将会以此时的系统作为还原基准点。 2) 安装完同方易教操作系统驱动之后,重新启动后系统开始保护 3DOS操作系统什么时候开始保护 答： A. 操作系统必须以DOS(大小写均可)才能支持保护DOS B. 一旦DOS保护分区格式化,重启之后就开始保护 4已经安装了同方易教windows 驱动，想要增加软件，是要以开放模式进去安装吗？ 答：可以,同方易教可以保护模式下和开放模式下进行增量拷贝 5安装同方易教windows 驱动时提示资料分区未格式化，重启动后为不保护状态？ 答：安装同方易教windows 驱动是在所有安装操作及磁盘设定完成后再执行，故要先格式化后再安装。如上情况，用户只需要将资料盘格式化后，重启计算机即可正常。 6备份复原型安装的操作系统，需要安装同方易教windows 驱动吗？ 答：不用。因为备份复原性系统是通过手动备份进行的，直接将系统分区克隆到其对应的暂存区。 7为什么用同方易教分区时，可以设置FA T32文件系统，但在安装系统是却无法格式化成FA T32分区？答：WINDOWS系统只支持小于32G分区使用FA T32格式。 8为什么Windows2000看不见137GB以后的分区了 答：Windows2000磁盘驱动最大只能支持137GB的硬盘,如果超过137GB的地方,分区会不可见,所以对于有安装Windows2000操作系统,而且硬盘大于130G的时候,一定要注意引导分区,专属分区,共享分区都必须划分在137GB以前的磁盘空间 9为什么Windows2003在安装完操作系统以后进去只有一个分区呢 答：Windows2003的所有扩展分区需要重新分配盘符,具体操作如下 右键点击”我的电脑”---à选择”管理”-à在左边列表中的”存储”中选择”磁盘管理”-à给每个已分派的分区添加相应的盘符! ***注意:对”未指派”的空间不要去操作 10如何去除Windows登录时的密码验证呢 答： A. 对于Windows2000,选择”开始”-à选择”运行”à在命令行中输入 control userpasswords B. 对于Windows2003/XP,选择”开始”-à选择”运行”à在命令行中输入 control userpasswords2 11在同方易教使用时，如何重新安装其中的一个操作系统(针对保留安装和全新安装情况) 答： 1) 进入F10,点击“增量设置”，将所对应的操作系统的增量支持去掉 2) 到分区设置中将操作系统对应的引导分区的还原方式设置为“不使用” 3) 以开放模式(Ctrl+Enter)进入操作系统，进入控制面板中，点击“添加/删除程序”， 4) 将同方易教的驱动和卸载
Social Isolation-Definition Imagine what the life would be like if you lived in a place for 30 years and was then stranded completely alone on a deserted island for the rest of your life.(Robinson) And of course, the conclusion is that, social isolation would be horribly lonely for someone used to being around people. Socialization is such a basic part of our lives that it is easy to overlook its importance. But it is the reason we laugh, cry, talk and do many of the other things we think of as just a part of being human. Socialization doesn't always happen, though, and certainly can't happen in social isolation. This is a state that occurs when someone experiences a complete lack of contact with the social world. Further negative effects-cause more harmfull illnesses. So what would you do if you feel yourself in the state of isolation? More eating? Or more drinking? The way we deal with the isolation can become another problems such as overweight and alcohol abuse.
同方易教管理系统同方易教管理系统的的使用使用培训培训培训讲解讲解讲解方案方案 同方易教管理系统同方易教管理系统的安装的安装 同方易教安装分为两种类型：微软操作系统简易安装，和多操作系统隔离安装。 在简易安装的情况下，用户磁盘上应该已经安装了微软的操作系统，同方易教会在不破坏任何磁盘已有分区数据的情况下在该操作系统下安装同方易教管理系统。 在多操作系统隔离的模式下，同方易教需要对整个磁盘进行重新的分区规划，可能会破坏一些原有分区。多操作系统隔离安装后，用户可以在磁盘上安装多个操作系统，这些操作系统系统分区相互隔离不能互相访问。如果用户需要在同方易教下使用linux 系列操作系统，那么用户必须选择多操做系统隔离安装。因为同方易教版本不支持Linux 操作系统下的任何维护工具。 ---------在操作的同时叙述 同方易教管理系统同方易教管理系统的进度管理功能的进度管理功能 与传统保护卡不同的是，同方易教管理系统支持多进度。在安装了同方易教管理系统的windows 保护驱动后，用户可以在实模式（引导windwos 之前）或者windwos 之上为系统创建新的进度、删除旧的进度或者还原到以前的一个进度。 所谓进度，就是一个还原点，这个还原点也可以称之为一个磁盘快照，它记录了进度创建时刻当前操作系统的所有软件环境和文件系统状态。所有的进度都是相对独立的，这意味着，用户还原到一个进度，或者删除一个进度的时候都不会影响其他的进度。还原是可逆的。 操作操作：：在windwos 上为系统创建新的进度上为系统创建新的进度。。 ---------在操作的同时叙述 打开同方易教管理系统的主界面，右键点击托盘图标输入密码后，弹出易教主界面选择进度管理。 点击创建进度 输入进度名称和进度描述，点击创建，系统将为当前的磁盘软件环境创建一个新的进度。 可以进入恢复进度界面验证进度已经创建。 在windwos 上执行进度还原上执行进度还原。。 ---------在操作的同时叙述 打开同方易教的主界面，右键点击托盘图标输入密码后，弹出易教主界面选择进度管理。
Miltenyi Biotec GmbH Friedrich-Ebert-Stra?e 68, 51429 Bergisch Gladbach, Germany Phone +49 2204 8306-0, Fax +49 2204 85197macs @miltenyibiotec.de https://www.doczj.com/doc/7e4027685.html, Miltenyi Biotec Inc. 2303 Lindbergh Street, Auburn, CA 95602, USA Phone 800 FOR MACS , +1 530 888 8871, Fax +1 530 888 8925macs @https://www.doczj.com/doc/7e4027685.html, page 1/2 140-002-824.06 Components 1 mL Pan T Ce ll Biotin-Antibody Cocktail, mouse: Cocktail of biotin-conjugated monoclonal antibodies against CD11b, CD11c, CD19, CD45R (B220), CD49b (DX5), CD105, Anti-MHC class II, and Ter-119. 2 mL Anti-Biotin MicroBeads: MicroBeads conjugated to monoclonal anti-biotin antibodies (isotype: mouse IgG1). Capacity For 10? total cells, up to 100 separations. Product format All components are supplied in buffer containing stabilizer and 0.05% sodium azide.Storage Store protected from light at 2 ? 8 °C. Do not freeze. The expiration date is indicated on the vial label. Safety information For re se arch use only. Not inte nde d for any animal or human therapeutic or diagnostic use. Be fore use , ple ase consult the Mate rial Safe ty Data She e t for information regarding hazards and safe handling practices. Cell separation methods 1.Fully automated cell labeling and separation using the autoMACS? Pro Separator 2. Manual magnetic labeling 2.1 Subsequent automated cell separation using the autoMACS? Separators 2.2 Subsequent semi-automated cell separation using the MultiMACS? Cell24 Separator Plus 2.3 Subsequent manual cell separation General notes ▲ For an overview of the sample preparation procedure and recommendations for magnetic labeling and separation, refer to https://www.doczj.com/doc/7e4027685.html,/faq.▲ For product-specific background information and applications of this product, refer to the respective product page at https://www.doczj.com/doc/7e4027685.html,/130-095-130.Reagent and instrument requirements ● Buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA by diluting MACS? BSA Stock Solution (# 130-091-376) 1:20 with autoMACS? Rinsing Solution (# 130-091-222). Degas buffer before use, as air bubbles could block the column. ● (Optional) Pre-Separation Filters, 30 μm (# 130-041-407) to remove cell clumps. ● Choose the appropriate MACS Separator and MACS Columns. Column M ax. number of labeled cells Max. number of total cells Separator LS 10? 2 ×10?MidiMACS, QuadroMACS, VarioMACS, SuperMACS II autoMACS 2×10? 4 ×10?autoMACS Pro, autoMACS Multi-24 Column Block 10? 10? MultiMACS Cell24 Separator Plus ▲ Note: If using the MultiMACS Cell24 Separator Plus with the Single-Column Adapter, please refer to the user manual for column capacities. For additional requirements not included with the product, such as instruments or fluorochrome-conjugated antibodies, refer to https://www.doczj.com/doc/7e4027685.html,. 1. Fully automated cell labeling and separation using the autoMACS? Pro Separator ▲ All buffer temperatures should be ≥10 °C. ▲ Place tubes in the following Chill Rack positions:position A = sample, position B = negative fraction, position C = positive fraction.1. For appropriate resuspension volumes and cell concentrations, please visit https://www.doczj.com/doc/7e4027685.html,/autolabeling. 2. Turn on the instrument for automatic initialization. 3. Program autolabeling in the Reagent menu by selecting Read Reagent and scan the 2D barcode of each reagent vial with the barcode scanner on the autoMACS? Pro Separator. Place the reagent into the appropriate space on the reagent rack.4. Place sample and collection tubes into the Chill Rack. Sample tube should be in row A, and the collection tubes in rows B and C. 5. Go to Separation menu and select the reagent name for each sample from the Labeling submenu (the correct labeling, separation, and wash protocols will be selected automatically). 6. Enter sample volume into the Volume submenu. 7. Select run . For more details on complete walk away automation, please refer to the autoMACS Pro Separator user manual. Pan T Cell Isolation Kit II mouse Order no. 130-095-130
同方易教EGV1.0 使用指南 同方股份有限公司
前言 ◎欢迎使用同方易教 ◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆ ※本手册所有的产品商标与产品名称均属于同方股 份有限公司。 ※本手册所有图形仅供参考，请您以实际软件界面为 准。 ※请您在做安装、移除、修改同方易教操作时，备份 好您的硬盘数据，如果数据丢失，本公司不予找回。 ※软件版本如有变更恕不另行通知。 ◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆◆ 同方易教广泛应用于学校机房或网吧等局域网环境，成为广大机房管理者的得力助手。它以方便、安全的优势备受系统管理者的青睐。
目录 1.产品介绍 (1) 1.1产品说明 (1) 1.2功能简介 (1) 1.3最低硬件配置 (2) 1.4支持的操作系统 (2) 1.5支持的文件系统 (3) 2.快速开始指南 (4) 2.1安装同方易教 (4) 2.2安装流程图 (5) 2.2.1 安装发送端流程图 (5) 2.2.2 网络克隆接收端流程图 (6) 2.3安装发送端（以﹤全新安装﹥为例） (7) 2.3.1 选择安装方式——全新安装 (7) 2.3.2 安装操作系统及应用软件 (9) 2.3.3 安装同方易教操作系统驱动 (10) 2.3.4 安装完成 (11) 2.3.5 设置发送端网络拷贝信息 (11) 2.4网络克隆接收端 (12) 2.4.1 网络安装接收端底层驱动 (12) 2.4.2 配置接收端信息（IP地址/计算机名） .. 15 2.4.3 传送操作系统数据 (17) 3.已经安装好操作系统后安装同方易教 (20) 3.1安装同方易教 (20) 3.2选择安装方式 (20) 3.2.1 简易安装和保留安装 (20) 3.2.2 安装操作系统 (22) 3.2.3 安装同方易教系统驱动 (23)
Frequently Asked Questions About Vibration What does it mean to isolate vibration? 隔离振动的意思是？ Isolation refers to a reduction in transmitted vibratory forces. 隔离是指减少传递振动的力。 How is isolation achieved?隔离如何达到？ Isolation is achieved by placing an isolator (elastic element) between the unit vibrating and its support. This allows the inertia of the unit to oppose and thereby reduce the vibratory motion transmitted to the support. 隔离通过放置一个减震器（弹性装置）在振动单元和支撑物之间达到。这就让装置惯性反对从而减少振动传递到支撑物。 What characteristics must an isolator have?减震器有什么性能？ An isolator must be (and remain) elastic for the life of the installation. It must have the capacity to support the static weight of the unit as well as the unbalanced dynamic force. It must have a natural frequency lower than the offending unit's disturbing frequency. 减震器必须（并且保持）有弹性在使用寿命内。不但有能力必须支持装置的静重，而且能支持不稳定的动态力。它的自然频率必须比引起干扰装置的干扰频率低。 How do we determine the natural frequency of an isolator?如何确定减震 器的自然频率？ The natural frequency of an isolator is determined by the following mathematical relationship: 减震器的自然频率可以通过以下的数学关系式确定： Where: K D = Dynamic Spring Rate, lb/in动态弹性系数 W = Static Weight of the Isolated Unit, lb减震器的的静重 Isolator manufacturers have this information readily available in their publications.减震器的制造商可以容易获得现成的资料在他们的出版物中。What natural frequency should an isolator have?减震器的自然频率是多 少？ It depends on the desired percent reduction in transmitted vibration, referred to as transmissibility, and is governed by the ratio of disturbing frequency to isolator natural frequency. The larger this ratio, the greater the reduction. Isolation begins at a ratio of 1.414. 这个取决于在传递振动中期望衰减的百分比，称之为传递率，并且是由干扰频率对减震器自然频率的比值。比值越大，衰减的越大从比值1.414开始。
清华同方易教使用总结 最近教育局拨了20台电脑,10台带光驱的教师机,10台不带的光驱的学生机,目前教师机已经投入使用,但在一个星期后,其中一个教师的办公室电脑出现了问题,幸亏还有一台,这样我也就可以有足够的时间进行研究.开机按del进去,密码一般都是thtfpc,呵呵,OK了,然后进入bios里,将启动设置为restorecard,再重启,进入我们的还原卡设置界面,其中设置分区的各种参数"属性"说明：。 立即复原型引导盘（A）。 用于引导、安装操作系统，可瞬间复原分区数据。 备份复原型引导盘（B）。 用于引导、安装操作系统，需占用与该分区同样大小的硬盘空间备份该分区数据，以供复原时使用，并且复原时间较长。 一般引导盘（C）。 用于引导、安装操作系统，此种引导盘不具备复原功能。 共用资料盘（S）。 资料盘，该分区可被分区格式兼容的引导盘共享。 专属资料盘（P）。 资料盘，该分区只能被与其名称相同且分区格式相兼容的引导盘识别使用。例如:如果引导盘的名称叫做"Win2K"，那么它的专属资料盘的名称也必须叫做"Win2K"；如果引导盘的名称叫做"Win2000"，那么它的专属资料盘的名称也必须叫做"Win2000"。 "磁盘名称"说明：设定引导盘显示在开机选单界面的名称。(一般情况下，该磁盘装什么操作系统就命名为什么样的磁盘名称。如：如果装的是windows98,那么该磁盘就命名为"win98"，以便开机时识别)。 分割类别"说明：本分区的分区格式。（可分为FAT16、FAT32、NTFS 等格式）。 还原方式"说明：一般情况下，"立即复原型引导盘"设置为"每次"，"备份复原型引导盘"设置为"手动"，*盘"不使用"。 我现在就是将"立即复原型引导盘"设为每次,然后告诉其他老师要注意,不要将文件放在C盘或桌面,这样也能起到一个很好的作用,先试试看吧。 安装完以后，在操作系统上安装一次还原卡，具体方法为：将还原卡光盘放入光驱，点击"SETUP"，开始安装，（在安装过程中注意要选中"自动修改IP功能"，这样在网络同传的时候才能自动批量修改接收端电脑的IP地址）。 这样就OK了,先试验看看。