CT26结肠癌耐药动物模型

Novel Murine Model for Colon Cancer:Non-OperativeTrans-Anal Rectal Injection 1Melissa Donigan,B.S.,*,†Laurie S.Norcross,M.D.,‡,§John Aversa,D.O.,‡,§Jimmie Colon,M.S.,*Joshua Smith,*Rafael Madero-Visbal,M.D.,*Shuan Li,M.D.,*,§Neal McCollum,M.D.,*,§Andrea Ferrara,M.D.,‡,§Joseph T.Gallagher,M.D.,‡,§and Cheryl H.Baker,Ph.D.*,†,2*Cancer Research Institute of M.D.Anderson Cancer Center Orlando,Orlando,Florida;†Burnett School of Biomedical Sciences,University of Central Florida,Orlando,Florida;‡Colon and Rectal Clinic,Orlando,Florida;and §Orlando Regional Medical Center,Orlando,FloridaSubmitted for publication April 11,2008Background.This study was conducted to develop a modified murine model of colon cancer that is non-operative.Currently,the most accurate orthotopic murine model of colon cancer requires an invasive procedure involving cecal injection of colon cancer cells and therefore limits the ability to perform immu-nological studies subsequent to cecal resections.Materials and methods.Murine colon cancer (CT26)cells were injected submucosally into the distal,pos-terior rectum of BALB/c mice.Care was taken not to pass transmurally into the pelvic cavity.Different magnifications (10؋versus 100؋)were used for injec-tion,and primary tumor growth and metastatic dis-ease were studied.Results.In the initial study,3/7mice injected using 10؋magnifications had notable,large tumor originat-ing from the rectal wall,and histology revealed that all excised tumors were poorly differentiated adeno-carcinoma.In the second study,8/10mice injected us-ing 100؋magnifications had notable tumor originat-ing from the rectal well,and 4/8mice had abnormal lung tissue with pathological evidence of hemorrhagic pulmonary edema.The use of 10؋magnification re-sulted in 43%tumor take.In sharp contrast,80%tumor take was observed with 100؋magnification.The over-all success of tumor take was 65%using the trans-anal rectal injection model.Conclusions.Our modified orthotopic murine model of colon cancer offers an alternative non-operativemurine model for colon cancer and is less invasive than the traditional orthotopic model (i.e.,cecal injec-tion).This model may allow for more accurate inves-tigations of inflammation and immune responses to surgical intervention without the influence of previ-ous abdominal surgery.©2009Elsevier Inc.All rights reserved.Key Words:colon cancer;orthotopic murine model;non-operative injection of cancer cells.INTRODUCTIONIt was estimated that in 2007over 1,500Americans would die of cancer each day.As the third most common cancer in both men and women,colon cancer represents 10%of these cancer-related deaths [1].Despite earlier detection and dropping death rates in colon cancer,112,340new cases were estimated for 2007[1].The most common treatment for colon and rectal cancer is surgical resection,followed by adjuvant therapy with oxaliplatin,5-fluorouricil,and leucovorin.Early detection can provide a 5-y survival rate of up to 90%,and surgery is most often curative.However,if patients present with distant me-tastasis at the time of diagnosis,the 5-y survival rate drops to only 10%[1].Since its inception in the early 1990s,laparoscopy has been an alternative technique to the more tradi-tional open laparotomy for colorectal pa-roscopic surgery incorporates a much smaller incision (ϳ2cm)than either hand-assisted laparoscopic (ϳ5–8cm)or open surgery (ϳ30cm).Some of the benefits of minimally invasive surgery include a shorter hospital stay,less pain,and a quicker return of bowel function [2].However,concerns for safety and adequate onco-logic resection slowed the acceptance of laparoscopy as1Melissa Donigan and Laurie S.Norcross contributed equally to this manuscript.2To whom correspondence and reprint requests should be ad-dressed at Cancer Research Institute of M.D.Anderson Cancer Center Orlando,110Bonnie Loch Court,Orlando,FL 32806.E-mail:cheryl.baker@ .Journal of Surgical Research 154,299–303(2009)doi:10.1016/j.jss.2008.05.0282990022-4804/09$36.00©2009Elsevier Inc.All rights reserved.a safe option in most colorectal settings,most notably in colorectal cancer.Since then,several large-scale ex-periments comparing traditional open surgery(open laparotomy)to minimally invasive surgery(laparo-scopic and hand-assisted laparoscopic)have been con-ducted.With the publication of the COST(Clinical Outcomes of Surgical Therapy)trial[3]published in the New England Journal of Medicine in2004and the “Barcelona Trial”[4]published in Lancet in2002,lapa-roscopic surgery became accepted as a safe alternative to traditional open laparotomy for colorectal resection. Both trials demonstrated that patients undergoing laparoscopy had either equivalent or better cancer-related survival compared with patients with major open laparotomy.Some believe that the possible bene-fits of minimally invasive surgery(i.e.,shorter recovery period,less pain,and earlier return of bowel function) may be related to the patient’s immune and cytological responses,such as serum protein concentration,cyto-kinefluctuations,as well as tumor growth,metastasis, and recurrence.Although minimally invasive surgical techniques have been widely accepted by the medical community,there is still active debate concerning the possible immunological benefit of limited surgical trauma with minimally invasive surgery.Preclinical evaluation and comparison of traditional open laparotomy and minimally invasive surgical tech-niques on the effects on colon cancer and metastasis require animal models.To date,there are multiple mu-rine models for the study of colon cancer[5,6].The orthotopic murine model,which involves injection of tu-mor cells into the cecum of mice,is one of the most accurate representation of human colon cancer[6].This orthotopic model requires a surgical incision for injection of cancer cells,and therefore,an initial immune response following this surgical model would limit the feasibility and reliability of future research goals of measuring im-mune responses following open or minimally invasive cecal resections.Therefore,the traditional cecal injection model must be modified to involve a non-operative ap-proach for injection of colon cancer cells(to establish a primary tumor).Previous studies have demonstrated that colon can-cer can be established orthotopically after the induc-tion of colitis[7].This enema model,however,requires initial induction of colitis to allow invasion of intralu-minal cells,leading to a significant inflammatory re-sponse.In addition,other laboratories developed a mu-rine model of rectal cancer using an intra-rectal injection,which resulted in minor lymph node metas-tasis noted in18%of animals[8].Despite their avail-ability,these models of colon cancer are not widely used and are not reliable models for preclinical evalu-ation of future immunological studies subsequent to cecal resection,most often performed clinically.In this article,we describe a trans-anal injection of colon cancer cells in BALB/c mice.The method is similar to the intra-rectal injection[8]but is performed with a few modifications.This modified orthotopic murine model that does not require abdominal surgery for injec-tion of colon cancer cells makes it a reliable and clinical relevant technique to study the inflammation and im-mune response subsequent to cecal resection.Further-more,in contrast to the colitis and intra-rectal murine models of colon cancer,this novel technique has the po-tential to allow for the study of metastasis and possible tumor recurrence following open laparotomy or the much debated minimally invasive surgical techniques.MATERIALS AND METHODSCell Line and Culture ConditionsThe murine colon cancer CT26cells were obtained from the Amer-ican Type Culture Collection(Manassas,VA).In brief,these cells were initially induced in a BALB/c mouse by chemical carcinogen, and stable cell lines were established[9].The murine colon cancer CT26cells have been demonstrated to be highly metastatic to the liver and lungs[10].Cells for injection were obtained from frozen stocks and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM)supplemented with10%fetal bovine serum(FBS),sodium bicarbonate,and a penicillin–streptomycin mixture(Invitrogen, Carlsbad,CA)for no more than12weeks.To account for the influ-ence of inflammatory cytokine(i.e.,IL-6,IL-1␤)production by the FBS in cell culture,enzyme-linked immunosorbent assays were car-ried out on different FBS concentrations.Results show(unpublished data)that FBS cytokine levels are below the detection limit,indicat-ing that the levels of cytokines present in the FBS are negligible. Adherent monolayer cultures were maintained on plastic at37°C in 5%CO2and95%air.All cultures were free of Mycoplasma and the following pathogenic murine viruses:retrovirus type3,pneumonia virus,K virus,Theiler’s encephalitis virus,Sendai virus,min virus, mouse adenovirus,mouse hepatitis virus,lymphocytic choriomenigi-tis virus,ectromelia virus,and lactate dehydrogenase virus(assayed by Microbiological Associates,Bethesda,MD).Animals and Animal CareMale BALB/c mice were purchased from the National Cancer Institute(NCI)Animal Program of Charles River Laboratory(Fred-erick,MD).The mice were housed and maintained in specific pathogen-free conditions and facilities approved by the American Association for Accreditation of Laboratory Animal Care,and in accordance with current regulations and standards of the United States Department of Agriculture,United States Department of Health and Human Services.The mice were used in accordance with institutional guidelines when they were8–12wk old.Preparation of Cell Suspension for Injection Murine colon cancer CT26cells were harvested from near-confluent cultures by a brief(3-min)exposure to0.5%trypsin and0.02%EDTA (Invitrogen).Trypsinization was stopped with DMEM containing10% FBS,and the cells were concentrated with centrifugation and resus-pended in DMEM containing10%FBS.Trypan blue staining was used to assess cell viability,and only cell suspensions consisting of single cells withϾ90%viability were used for the injections.300JOURNAL OF SURGICAL RESEARCH:VOL.154,NO.2,JUNE15,2009Trans-Anal InjectionMale BALB/c mice were anesthesized with100mg/kg ketamine and50mg/kg xylazine.Mice then received a gentle anal dilation using blunt-tipped forceps at the anal opening.A29-gauge syringe was used to inject2.5ϫ104CT26cells,suspended in DMEM with 10%FBS,submucosally into the distal,posterior rectum.The injec-tion was performed approximately1–2mm beyond the anal canal and into the rectal mucosa,which minimizes the chance of establish-ing anal tumors.In addition,care was taken not to pass transmu-rally into the pelvic cavity.Mice were observed for1h until fully recovered and were then monitored three times weekly for tumor burden.In thefirst experiment,seven mice were injected by using operating loupes(Designs for Vision,Inc.,Ronkonkoma,NY)with 10ϫmagnification and sacrificed on post-injection day17.In a sec-ond experiment,10mice were injected using a100ϫmagnification (Leica MC16F microscope;Leica Microsystems,Bannockburn,IL) and then sacrificed on post-injection day20.Necropsy,Tissue Preparation,and Hemotoxylinand Eosin StainingMice were killed by CO2inhalation on day17(experiment#1)or day20(experiment#2).After dissection and removal of the tumor (rectum),rectal wall,and lungs,the tissue samples were photo-graphed and prepared for histological analysis.For hematoxylin and eosin(H&E)staining procedures,the tissue samples werefixed in formalin(rectum and rectal wall)or bouins(lungs)and embedded in paraffin.All slides were reviewed by a board-certified pathologist.A normal BALB/c mouse,which had not received prior injection or surgical intervention,was dissected for normal anatomy description of the injection site.Images were collected using the100ϫmagnifi-cation(Leica MC16F microscope).RESULTSFormation of Colon Cancer in Distal Posterior RectumIn afirst set of preliminary in vivo injection studies, the investigators performed trans-anal injections on BALB/c mice using trypan blue as an indicator of in-jection location.Due to systemic dissemination of the trypan blue dye,the investigators carried out another set of preliminary in vivo studies,using India Ink.The success of injection was apparent on dissection.Subse-quent to these studies,murine colon cancer CT26cells were injected submucosally into the distal,posteriorrectum of another set of BALB/c mice(Fig.1A).As illustrated in Fig.1B,the injections were performed approximately1–2mm beyond the anal canal and into the rectal mucosa.The short,smooth,white epithelial tissue of the anal canal can clearly be identified as a separate tissue plane from the pink mucosa of the rectum.The submucosal injections were performed into the rectum proximal to the anal canal.In thisfirst set of experiments,7mice were injected with CT26 cells using10ϫmagnification(operating loupes)and killed on post-injection day17.Three of7mice(43%) had notable large tumor originating from the rectal wall.In the second set of experiments,10mice were injected with CT26cells using100ϫmagnification (Leica microscope)and killed on post-injection day20. Eight of10mice(80%)had gross tumor originating from the rectal wall(Fig.2A and B).Even more strik-ing,4of these8mice had the presence of abnormal lung tissue.There was an overall65%successful tumor take using the trans-anal,rectal colon cancer injection model. The discrepancy between successful tumor take in exper-iment#1(3/7)and successful tumor take in experiment #2(8/10)is believed to be due to the limitation of the10ϫmagnification used in experiment#1.Therefore,the use of100ϫmagnification is recommended.Histology Analysis of Rectal Tumors and LungsThe rectal walls and lungs from both experiments were examined by a pathologist for the presence of primary tumor and metastasis,respectively.Rectal tu-mors in both experiments showed poorlydifferentiated FIG.1.Non-operative trans-anal injection of murine colon can-cer CT26cells into BALB/c mice.(A)To demonstrate a non-operative approach to an orthotopic murine model of colorectal cancer,a29-gauge syringe was used to inject2.5ϫ104CT26cells,suspended in DMEM with10%FBS,submucosally into the distal,posterior rec-tum of BALB/c mice.(B)A normal BALB/c mouse,which had not received prior injection or surgical intervention,was dissected for an anatomical description of the trans-anal injections.The short, smooth,white epithelial tissue of the anal canal can clearly be identified as a separate tissue plane from the pink mucosa of the rectum.The submucosal injections were performed into the rectum proximal to the anal canal.301DONIGAN ET AL.:NOVEL MURINE MODEL FOR COLON CANCERadenocarcinoma originating in the submucosal tissue (Fig.3).The lung tissue was grossly abnormal when compared with normal lung (Fig.4A),and pathology showed diffuse,severe,hemorrhagic pulmonary edema (Fig.4B).There were no gross changes noted in the livers of the mice,therefore no histological examina-tion of hepatic tissue was performed.DISCUSSIONMurine models are useful in evaluation of human colon cancer [5,6],and the importance of orthotopic murine models to study the biology and therapy of neoplasm has been demonstrated [6].Nonetheless,these models tradi-tionally involve abdominal surgery for injection of the cancer cells.The non-operative murine model of colon cancer described in this report compliments the most recently reported murine models and may overcome some limitations (induced by surgery)associated with them.First,we verified that the trans-anal injection of colon cancer cells produces large tumor originating from the rectal wall.Second,we determined that 100ϫmag-nification is necessary to produce a more successful tu-mor take than 10ϫmagnification (80%and 65%,respec-tively).The growth of colon cancer cells within the rectal wall is similar to that observed clinically in patients with colorectal cancer,suggesting that the model is clinically relevant.In addition,changes within the lung paren-chyma were observed in four of eight mice.The appear-ance of pulmonary edema in these specimens is concern-ing and may indicate systemic inflammatory changes (i.e.,systemic inflammatory response syndrome).None-theless,these mice did not clinically appear sicker than the others in the series.These findings are currently under investigation in additional trials using the trans-anal model in a larger cohort of mice.The absence of metastatic disease is most likely due to the number of primary colon cancer cells injected and the termination date of the experiment.To overcome this,studies using a larger number of injected cells and longer periods of time between injection and termination will be performed.The most common treatment for colon and rectal cancer is surgery followed by treatment with chemo-therapy agents (with or without neoadjuvant therapy)[1].Furthermore,if the cancer is confined and no met-astatic disease is present,surgery is most often cura-tive and patients may not have to receive chemother-apy.Surgical resection of colon and rectal cancer can be performed using two techniques:a traditionallaparot-FIG.3.Histology of primary tumor growing orthotopically in BALB/c mice.H&E stains were carried out on the submucosal rectal tumor established 17d after 2.5ϫ104CT26cells were injected submucosally into the distal,posterior rectum of BALB/c mice.All slides showed similar findings of poorly differentiated adenocarci-noma arising in the submucosallayers.FIG.2.Gross primary tumor growing orthotopically in BALB/c mice.(A)Evidence of gross tumor protruding from the rectum of mice 17d after a 29-gauge syringe was used to inject 2.5ϫ104CT26cells submucosally into the distal,posterior rectum.(B)Anatomical dissection of gross tumor originating from the rectal wall of mice on day 17after injection of 2.5ϫ104CT26cells submucosally into the distal,posterior rectum of BALB/c mice.The growth of colon cancer cells within the rectal wall is representative of that observed clinically in patients with colorectal cancer,suggesting that the model is clinically relevant.302JOURNAL OF SURGICAL RESEARCH:VOL.154,NO.2,JUNE 15,2009omy with a large abdominal incision or newer mini-mally invasive techniques with significantly smaller incisions and laparoscopic instruments.The clinical benefits of laparoscopic surgery have been repeatedly proven throughout surgical literature.It is accepted that colorectal cancer patients undergoing minimally invasive surgical techniques have a shortened length of hospital stay,require less postoperative pain medica-tions,and have a shorter time to return of bowel func-tion and tolerating a diet.Additionally,some studies have suggested a potential benefit in cancer-free sur-vival,disease progression,recurrence,and metastasis in certain patients undergoing laparoscopic colon re-section for colon cancer,specifically in advanced onco-logic stages [3,4].The proven benefits of these mini-mally invasive surgeries may be related to the patient’s immune response.Therefore,these observed clinical differences need further evaluation,including assess-ment of the immunological differences in patient re-sponse to open and laparoscopic colon resection.Previously reported murine studies have shown that,in mice receiving an abdominal incision,there was a reduced cell mediated immune response as compared with mice receiving anesthesia alone [11].In addition,a significant increase in tumor growth was observed in mice receiving surgical intervention as compared with control [12,13].We have developed a murine model of colon cancer that involves a trans-anal injection of colon cancer cells which does not require abdominal surgery for injection.This model will provide a more accurate,unaltered in vivo tumor growth pattern for colon and rectal cancer.In the end,future in vivo studies involving cecal resections (i.e.,open laparot-omy or minimally invasive)and their associated im-munological benefits are now possible.ACKNOWLEDGMENTSWe thank Donna Schade (M.D.Anderson Cancer Center Orlando)for helping with preparation of this manuscript.REFERENCES1.American Cancer Society.Anonymous Cancer Facts and Fig-ures 2007.2.Lee SW,Whelan RL.Immunologic and oncologic implications of laparoscopic surgery:What is the latest?Clin Colon Rectal Surg 2006;19:5.3.Nelson H,Sargent DJ,Wieand S,et al.A comparison of lapa-roscopically assisted and open colectomy for colon cancer.N Engl J Med 2004;350:2050.4.Lacy AM,Garcia-Valdecasas JC,Delgado S,et paroscopy-assisted colectomy versus open colectomy for the treatment of non-metastatic colon cancer:A randomized ncet 2002;359:2224.5.Rashidi B,Gamagami R,Sasson A,et al.An orthotopic mouse model of remetastasis of human colon cancer liver metastasis.Clin Cancer Res 2000;6:2556.6.Heijstek MW,Kranenburg O,Borel Rinkes IH.Mouse models of colorectal cancer and liver metastases.Dig Surg 2005;22:16.7.Takahashi T,Morotomi M,Nomoto K.A novel mouse model of rectal cancer established by orthotopic implantation of colon cancer cells.Cancer Sci 2004;95:514.8.Kashtan H,Rabau M,Mullen JB,et al.Intra-rectal injection of tumour cells:A novel animal model of rectal cancer.Surg Oncol 1992;1:251.9.Corbett TH,Griswold DP,Roberts BJ,et al.Tumor induction relationships in development of transplantable cancers of the colon in mice for chemotherapy assays,with note of carcinogen structure.Cancer Res 1975;34:2434.10.Schackert HK,Fidler IJ.Development of an animal model to study the biology of recurrent colorectal cancer originating from mesenteric lymph system metastases.Int J Cancer 1989;44:177.11.Allendorf JD,Bessler M,Whelan RL,et al.Better preservation of immune function after laparoscopic-assisted vs.open bowel resection in a murine model.Dis Colon Rectum 1996;39:S67.12.Whelan RL,Allendorf JD,Gutt CN,et al.General oncologic effects of the laparoscopic surgical approach.1997Frankfurt International Meeting of Animal Laparoscopic Researchers.Surg Endosc 1998;12:1092.13.Allendorf JD,Bessler M,Kayton ML,et al.Increased tumor establishment and growth after laparotomy vs laparoscopy in a murine model.Arch Surg1995;130:649.FIG. 4.Histology of murine lungs.(A)H&E-stained slide of normal lung.(B)H&E of lung presenting with diffuse,severe,hem-orrhagic pulmonary edema,consistent with systemic effects.303DONIGAN ET AL.:NOVEL MURINE MODEL FOR COLON CANCER。

CT26细胞系移植瘤模型、高淋巴转移肿瘤模型的建立及评价的开题报告一、研究背景癌症是一种常见的疾病，其中部分癌症具有高度的转移性。

目前，对于高淋巴转移肿瘤的研究尚不够深入。

CT26细胞系是一种分化程度低、高度转移的结直肠癌细胞系，具有广泛的应用价值。

因此，建立CT26细胞系移植瘤模型以及高淋巴转移肿瘤模型，对于研究高淋巴转移肿瘤的机制以及建立有效的治疗方案具有重要意义。

二、研究目的本研究的主要目的是建立CT26细胞系移植瘤模型以及高淋巴转移肿瘤模型，并评价其模型的可靠性和适用性。

其中，移植瘤模型用于研究CT26细胞系在体内形成转移瘤的过程，而高淋巴转移肿瘤模型则用于深入探究高淋巴转移肿瘤的生物学特性以及制定有效的治疗方案。

三、研究内容1.建立CT26细胞系移植瘤模型将CT26细胞根据细胞密度的需要，注射入小鼠皮下或肌肉内，观察移植瘤的生长情况，记录生长曲线，并测定体积和重量。

2.建立高淋巴转移肿瘤模型将CT26细胞注射到小鼠尾静脉中，随后观察淋巴结转移和肝、肺等远处转移的情况。

通过对小鼠血清和淋巴液中肿瘤标志物的检测，评估肿瘤转移的程度。

3.模型评价对模型进行数据统计和分析，评价模型的可靠性和适用性，并比较两种模型的差异。

同时，利用CT检查和病理学检测对模型进行确认，确保结果的准确性。

四、研究意义建立CT26细胞系移植瘤模型以及高淋巴转移肿瘤模型，有利于研究高淋巴转移肿瘤的机制，探究转移及其发生机制，同时有助于筛选新的治疗方法和药物。

该研究有望为临床治疗提供新的思路和手段，为结直肠癌及其他高淋巴转移性肿瘤的治疗和治疗监测提供新的方法和参考依据。

凋亡大肠癌CT26细胞对小鼠血清免疫因子水平以及免疫细胞增殖、活性的影响孙朝文;张广钰;赵辰;成怀福;钟漓;戴凌【摘要】目的探讨凋亡大肠癌CT26细胞对小鼠血清免疫因子水平以及免疫细胞增殖、活性的影响.方法取对数生长期大肠癌CT26细胞制备凋亡、坏死肿瘤细胞.取Balb/c小鼠20只,分离T淋巴细胞亚群及自然杀伤(NK)细胞、获取巨噬细胞,并制备细胞毒性T淋巴细胞(CTL).将小鼠CTL、NK细胞、巨噬细胞分别经相应肿瘤细胞处理后,均分为凋亡肿瘤细胞组、坏死肿瘤细胞组、肿瘤细胞对照组,应用51Cr释放实验测定各组CTL、NK细胞、巨噬细胞的活性,细胞计数(CCK-8)法检测各组CTL、NK细胞和巨噬细胞的增殖情况.另选取Balb/c小鼠分为凋亡肿瘤细胞组、坏死肿瘤细胞组、肿瘤细胞对照组各10只,分别将凋亡、坏死、对数生长期CT26细胞按通过皮下及静脉输注小鼠,采用酶联免疫吸附试验(ELISA)检测各组小鼠血清可溶性Fas(sFas)、γ干扰素(IFN-γ)、白细胞介素(IL)4、IL-10、IL-12、转化生长因子-β(TGF-β)的表达水平.结果与坏死肿瘤细胞组及肿瘤细胞对照组比较,凋亡肿瘤细胞组中不同效靶比的CTL、NK细胞及巨噬细胞活性均降低(P＜0.05).CCK-8法检测结果显示,凋亡肿瘤细胞组的CTL、NK细胞及巨噬细胞的A450值均低于坏死肿瘤细胞组、肿瘤细胞对照组(P＜0.05).ELISA检测显示凋亡肿瘤细胞组sFas相对表达量低于坏死肿瘤细胞组和肿瘤细胞对照组(P＜0.05).凋亡肿瘤细胞组IL-4表达水平高于肿瘤细胞对照组(P＜0.05),IL-10、TGF-β表达水平则高于其他两组(P＜0.05),IL-12表达水平低于其他两组(P＜0.05).结论凋亡大肠癌CT26细胞能够抑制小鼠特异性CTL、NK细胞、巨噬细胞增殖及其活性,影响大肠癌小鼠正常免疫功能.【期刊名称】《广西医学》【年(卷),期】2016(038)010【总页数】6页(P1337-1342)【关键词】大肠癌;CT26细胞;细胞毒性T细胞;自然杀伤细胞;巨噬细胞;可溶性Fas 【作者】孙朝文;张广钰;赵辰;成怀福;钟漓;戴凌【作者单位】桂林医学院附属医院胃肠外科,桂林市541001;桂林医学院附属医院胃肠外科,桂林市541001;桂林医学院附属医院胃肠外科,桂林市541001;桂林医学院附属医院胃肠外科,桂林市541001;桂林医学院附属医院胃肠外科,桂林市541001;桂林医学院附属医院胃肠外科,桂林市541001【正文语种】中文【中图分类】R735.3大肠癌是最为常见的消化道恶性肿瘤之一，其发病率位居全球恶性肿瘤第3位[1]。

ByDONGCP2015-03-17C6-/-转基因小鼠肠癌脾肝转protocol研究目的以C6-/-转基因小鼠为受试动物，评价补体系统抑制后对鼠CT26.WT肠癌脾肝转移模型的影响。

试验动物C6-/-转基因小鼠。

小鼠以C6-/-转基因小鼠与C57BL6Crossbreed十代以上。

实验材料麻醉剂：水合氯醛（或者戊巴比妥钠替代），生理盐水注射器：1ml注射器，2-5ml注射器手术器械：固定班，胶带，止血钳，眼科镊，手术缝合针术后：青霉素，电热毯（也可以无），纱布试验方法1, 小鼠肠癌CT-26细胞悬液的制备。

取对数生长期细胞，用0.25% 胰蛋白酶消化收集细胞，PBS或者无血清培养基洗涤重悬制成单细胞悬液。

台盼蓝染色测定活细胞率≥95%，细胞浓度为：1X10^7/只。

个人感觉是70-80%细胞活力最强。

注意将收集好的细胞放置于冰盒内。

2，小鼠麻醉与固定。

麻醉剂水合氯醛，一次配制10ml（10ml生理盐水融入0.38g水合氯醛）。

注射时候按照0.01ml/g体重注射。

麻醉后用胶带固定在手术展板上。

20g可以考虑0.25ml腹腔注射。

3，脾脏注射切脾组小鼠左上腹行横切口约6mm，逐层剥离进腹后于腹外侧找到柳叶状脾脏，小心显露脾脏，使脾上托于切口外用1ml注射器5号针头从脾上极进针约3mm，注意进针与脾脏平行，将肿瘤细胞悬液注入至脾被膜下，每只缓慢注入细胞浓度为1X10^7/ml细胞悬液0.2ml,可见注射部位脾被膜发白肿胀，待白色肿胀被膜消退后拔出针头压迫止血2分钟（结扎脾蒂,切除脾脏）查无出血，逐层关腹。

黄色字体步骤为切除脾脏方案的操作3，仔细缝合，两层缝合。

缝合后伤口用青霉素涂敷。

也可追加青霉素溶液注射，注射单位参考青霉素说明书。

注意：术中保持脾脏表面湿润，用5ml注射器滴加生理盐水。

术后小鼠的保暖工作对生存有一定影响。

可以用电热毯辅热。

小鼠结肠癌肝转移模型的构建杨扬;李乃卿【摘要】目的建立适合基因表述谱研究的小鼠结肠癌肝转移模型.方法将小鼠结肠癌细胞(C26)悬液接种于BALB/C小鼠脾内,分为切脾组和保脾组,观察小鼠生存时间及腹腔内肿瘤生长情况.结果切脾小鼠自然生存期11～15d,平均(13.05±0.71)d,保脾小鼠自然生存期9～15d,平均(11.64±1.49)d.小鼠肝脏转移率均为100%.切脾小鼠肝各叶完全被肿瘤占据,保脾小鼠脾内肿瘤巨大,肝内转移多为散在的米粒大小瘤结节转移.结论用于基因表达谱研究的小鼠结肠癌肝转移模型应采用脾内注射切脾法.【期刊名称】《中国医药指南》【年(卷),期】2010(008)017【总页数】2页(P53-54)【关键词】结肠癌;肝转移;动物模型【作者】杨扬;李乃卿【作者单位】广东省中山市人民医院,528400;北京中医药大学东直门属医院,100700【正文语种】中文【中图分类】R-332随着分子生物学的深入研究，基因芯片作为基因组及后基因组时代的主要研究技术，已成为揭示药物作用机制的重要方法。

基因芯片检测技术的前提是成功而适用的动物模型，首先需要获得足够的RNA检测量，所以成功的造模方法是研究课题成败的关键所在。

下面就我们近期进行的结肠癌肝转移的造模方法总结如下。

1 材料和方法1.1 材料1.1.1 实验动物4～6周龄体质量20～24g的BALB/C小鼠雄性（♂）81只，由北京维通利华实验动物技术有限公司提供，动物许可证号：京动许字（2000）第004号总049号，在Ⅱ级动物实验室饲养。

1.1.2 瘤株小鼠结肠癌C26细胞株（美国引进，广安门医院肿瘤实验室惠赠）。

经3次皮下接种传代后，无菌条件下摘取瘤体，生理盐水洗涤，剪成小块，匀浆，离心后取沉淀，反复3次，培养于10%新生牛血清和RPMI1640培养液（含青霉素100U/mL、链霉素100U/mL），置37℃、含5%CO2培养箱中3d，每日换液。

《CT26.WT结肠癌细胞对RAW264.7巨噬细胞增殖及分化的影响》篇一一、引言结肠癌作为一种常见的消化系统恶性肿瘤，其发生与发展往往伴随着多种复杂的生物学过程。

近年来，越来越多的研究开始关注肿瘤细胞与免疫细胞之间的相互作用。

在免疫系统中，巨噬细胞作为重要的免疫细胞之一，在肿瘤微环境中扮演着重要的角色。

因此，研究CT26.WT结肠癌细胞对RAW264.7巨噬细胞增殖及分化的影响，有助于我们更深入地理解肿瘤与免疫系统的相互作用机制。

二、材料与方法1. 细胞系本实验采用CT26.WT结肠癌细胞系和RAW264.7巨噬细胞系。

2. 实验方法（1）细胞培养：使用DMEM培养基培养CT26.WT结肠癌细胞和RAW264.7巨噬细胞，分别以适当的比例将两种细胞进行共培养。

（2）细胞增殖检测：采用MTT法检测共培养后巨噬细胞的增殖情况。

（3）流式细胞术：通过流式细胞术检测共培养后巨噬细胞的分化情况。

（4）免疫荧光染色：利用免疫荧光染色技术观察共培养后巨噬细胞的形态变化。

三、结果1. 细胞增殖情况共培养后，我们发现CT26.WT结肠癌细胞的存在对RAW264.7巨噬细胞的增殖具有显著影响。

与单独培养的巨噬细胞相比，共培养条件下巨噬细胞的增殖率明显降低。

这表明CT26.WT结肠癌细胞的分泌物或代谢产物可能对巨噬细胞的增殖产生了抑制作用。

2. 巨噬细胞分化情况流式细胞术检测结果显示，共培养后RAW264.7巨噬细胞的分化情况发生了明显变化。

与单独培养的巨噬细胞相比，共培养条件下巨噬细胞向M1型（经典活化型）和M2型（替代活化型）分化的比例均有所增加。

这表明CT26.WT结肠癌细胞可能通过某种机制诱导巨噬细胞的分化。

3. 形态学观察通过免疫荧光染色观察共培养后的巨噬细胞形态，我们发现共培养条件下巨噬细胞的形态发生了明显变化，表现为胞体变大、突起增多等现象。

这可能与巨噬细胞在肿瘤微环境中的活化状态有关。

四、讨论本研究表明，CT26.WT结肠癌细胞对RAW264.7巨噬细胞的增殖及分化具有显著影响。