犬外周血基质细胞成骨诱导分化后的超微结构观察

miR-21诱导犬BMSCs骨向分化的体外实验研究王颖;张雷;周咏;张凯;王元银;何家才;邹多宏【摘要】目的体外研究检测miR-21诱导拉布拉多犬骨髓间充质干细胞(BMSCs)成骨分化的作用.方法构建LentimiR-21-Luciferase与Lenti-LacZ-Luciferase慢病毒载体,分别转染犬BMSCs,将研究分为miR-21组与LacZ组.利用体外细胞划痕实验来比较转染后BMSCs的爬行迁移能力.在转染后的第0、1、4、7、14天分别提取miR-21组与LacZ组BM-SCs的mRNAs和蛋白.通过Real time-PCR技术和Westernblot检测成骨分化关键因子骨钙素(OCN)和骨桥蛋白(OPN)的表达.结果 Lenti-miR-21-Luciferase与Lenti-LacZ-Luciferase慢病毒载体成功转入BMSCs后,miR-21组的BMSCs细胞爬行迁移能力显著增强(P＜0.001);Real time-PCR和Western blot结果显示,miR-21可以显著上调BMSCs成骨分化关键因子OCN和OPN的表达(P＜0.05).结论 miR-21可以显著促进BMSCs的成骨分化,为进一步的体内实验奠定基础.%Objective To explore the effect of miR-21 on the osteogenic differentiation of Labrador dog bone marrow mesenchymal stem cells (BMSCs) in vitro.Methods Lenti-miR-21-Luciferase and Lenti-LacZ-Luciferase lentiviral vector were constructed and then were transfected into canine BMSCs.The study was divided into miR-21 group and LacZ group.The cell scratch test was used to compare the migration ability of BMSCs after lentiviral vector transfection.The mRNA and protein of BMSCs were extracted on 0,1,4,7,and 14 days.The expression of osteocalcin (OCN) and osteopontin (OPN),the key factors of osteogenic differentiation of BMSCs,were detected by Real time-PCR and Western blot.Results After Lenti-miR-21-Luciferase and Lenti-LacZ-Luciferase weresuccessfully transfected into BMSCs,the migration of BMSCs in miR-21 group was enhanced significantly (P ＜ 0.001).Expression of OCN and OPN at the mRNA and protein levels were significantly increased (P ＜0.05).Conclusion MiR-21 can significantly promote the osteogenic differentiation of BMSCs,lay the foundation for further in vivo experiments.【期刊名称】《安徽医科大学学报》【年(卷),期】2017(052)009【总页数】6页(P1318-1323)【关键词】miR-21;骨髓间充质干细胞;慢病毒转染;成骨分化【作者】王颖;张雷;周咏;张凯;王元银;何家才;邹多宏【作者单位】安徽医科大学口腔医学院,合肥230032;安徽医科大学附属口腔医院,安徽省口腔疾病研究中心实验室,合肥230032;安徽医科大学口腔医学院,合肥230032;安徽医科大学附属口腔医院,安徽省口腔疾病研究中心实验室,合肥230032;安徽医科大学口腔医学院,合肥230032;安徽医科大学附属口腔医院,安徽省口腔疾病研究中心实验室,合肥230032;蚌埠医学院第一附属医院口腔科,蚌埠233000;安徽医科大学口腔医学院,合肥230032;安徽医科大学附属口腔医院,安徽省口腔疾病研究中心实验室,合肥230032;安徽医科大学口腔医学院,合肥230032;安徽医科大学附属口腔医院,安徽省口腔疾病研究中心实验室,合肥230032;安徽医科大学口腔医学院,合肥230032;安徽医科大学附属口腔医院,安徽省口腔疾病研究中心实验室,合肥230032【正文语种】中文【中图分类】Q7;R34干细胞治疗是组织再生中发展迅速的领域之一，有强大的组织再生治疗效果[1-2]。

#论著#组织工程技术修复犬牙槽骨缺损的实验研究王敏翁雨来胡晓洁张艳柴岗祝联刘伟崔磊冯希平曹谊林=摘要>目的研究以犬骨髓基质细胞(bone marrow stromal cells,BM SC)为种子细胞,利用组织工程技术修复水平型牙槽骨缺损的可行性。

方法抽取成年犬骨髓,用梯度离心法获取单个核细胞,经条件培养液体外诱导培养后,进行组织化学、免疫细胞化学、扫描电镜检测,并将培养的第3代细胞与藻酸钙形成复合物。

在犬双侧下颌第3、4前磨牙和第1磨牙颊侧制备冠根向深5mm的水平型牙槽骨缺损,用以下方法进行治疗:(1)细胞-藻酸钙复合物修复组;(2)藻酸钙对照组;(3)空白对照组。

4、12、24周后经组织学检查骨缺损修复情况。

并比较12周时实验组与对照组的修复效果。

结果在体外经诱导培养的BM SC可表达AK P、cbfa1、Ñ型胶原等,表现出成骨细胞活性;4、12、24周细胞-藻酸钙复合物修复部位显示逐渐有骨形成;第12周时,实验组牙槽嵴高度可增高214mm?019mm,达到原来的48159%,空白对照组增高018mm?018mm,达到原来的15176%,材料对照组增高110mm?019mm,达到原来的19174%,实验组的牙槽嵴增高度和修复率与两对照组均有显著性差异(P<0101)。

结论能以BM SC为种子细胞,以藻酸钙为支架材料,利用组织工程技术部分修复水平型牙槽骨缺损。

=关键词>生物医学工程;骨髓细胞;藻酸盐Repair of alveolar bone defect with tissue engineered bone:an experim ental study of dogs WA N GM in*,W EN G Y u-lai,H U Xiao-j ie,ZH A N G Yan,CH A I Gang,ZH U Lian,L I U Wei,C UI Lei,FEN G X i-p ing,CA O Yi-lin.*D ep ar tment of Plastic Surgery of the9th H osp ital,Shanghai SecondM edical University.Shanghai T issue Engineer ing Resear ch Center,Shanghai200011,ChinaCorresponding author:CA O Y-i lin=Abstract>Objective T o study the feasibility of repairing ex perimental horizontal alveolar bonedefects by t i ssue engineer ing based on bone marrow stromal cells(BM SC).Methods Dog bone mar rowmononuclear cells were iso lated from the bo ne marrow by g radient centrifug ation and then cultured inconditional medium to be induced to become osteogenic.I mmuohistochenistry w as used to ex amine theex pression of co re-binding factor alpha subunit1(Cbfa1),osteocalcin(OCN),and typeÑcollagen in thecultured BM SCs.Histochemical technique w as used to examine the expression of alkaline phosphatase(AK P)in the BM SCs.Inversed phase-contrast microscopy and electron microscopy were used to observ et he morpholog y and proliferation of t he BM SCs.Induced BM SCs at passage3w er e harv ested and mixedw ith calcium alginate to form a gelatin form cel-l scaffold construct.A hor izontal alveolar bone defect(5mmhig h)w as created surgically in each buccal side o f the mandibular premolars3and4and molar1of11dogs.T he defects was randomly repaired w ith a cel-l scaffold construct(exper imental group,20teeth),calcium alg inate alone(contr ol g roup A,15teeth),or left untreated(control group B,12teeth).At four,t welve,and twenty-four w eeks after oper ation,2,7,2dogs were killed respectively and block sections o fmandibular bo nes at the defects w er e collected and processed for g ross and histolo gical observation as well asX-ray ex amination.T he status of bone r epair12weeks after oper atio n in the3groups w as compared.Result In v itro induced BM SCs exhibited an osteo genic pheno type.Since the passage3calcium saltsedimentation could be seen in the extr acellular stroma of BM SCs.Cbfa1,ty peÑcollag en,and A KP w er eex pressed in the BM SCs in every passage.OCN was ex pr essed since the second passage.Histolog ically,bone nodule structur e was observed in the exper imental group4w eeks after oper at ion.T he eng ineered bonebecame more mature,similar to the normal bone,12weeks after oper at ion.T welve w eeks after operation,t he alveolar ridge reg eneration amounted to a repair heig ht of2.43?0.93mm,0.98?0.87mm,and0.78?0.75mm and r eached48.59%,19.74%,and15.76%of the orig inal height in the exper imental group,control group A,and control group B respectively,w ith a significant difference between the ex perimental基金项目:九七三国家重点基础研究发展规划基金资助项目(G1999054300)作者单位:200011上海第二医科大学附属第九人民医院整形外科上海组织工程研究中心(王敏、胡晓洁、张艳、柴岗、祝联、刘伟、崔磊、曹谊林),口腔内科(翁雨来),口腔预防科(冯希平)通讯作者:曹谊林and contro l groups A and B(all P<0.01).C onclusion BM SCs can be induced to become osteogenic and be used as seed cells to eng ineer bone tissue and repair ex perimental alv eolar bone defect.=Key words>Biomedical engineering;Bone marrow cells;Alginates牙周病是一种慢性破坏性疾病,损坏牙周支持组织,尤其是牙槽骨的破坏和丧失可导致牙齿脱落,严重影响患者生活质量。

3基金项目:陕西省社会发展攻关计划项目基金资助 (No :20042k162G 16)33通讯作者:赵淑贤:Tel :029********* E 2mail :zhaoshu @APBSC 与TEB 犬体内埋植诱导成骨的实验研究3赵淑贤,张　兰,王　敏,董　凯,胡小春(西安交通大学口腔医院口腔内科教研室陕西西安710004) [摘要]　目的:研究APBSC 2TEB 复合埋植于犬的腹部皮下组织,在TGF 2β1和BMP 的诱导下成骨,为牙周骨缺损骨修复作准备。

方法:新鲜猪髂骨脱蛋白、脱钙制成TEB ,浸入自犬提取的APBSC 种子细胞悬液中,制备成APBSC 2TEB 复合物,埋植于犬腹部皮下组织,在不同时间观察成骨情况。

结果:实验组术后1周APBSC 细胞与移植前没有变化;术后2周,APBSC 细胞壁触突向胶元纤维和成纤维细胞中伸展;术后1个月,TEB 被机体吸收,代之以少量的骨小梁形成,APBSC 已演变为成骨细胞。

3～5个月,骨小梁增多,其间也有大量的结缔组织成分。

对照组为结缔组织,没有成骨细胞形成。

结论:APBSC 2TEB 复合移植于非骨部位,在TGF 2β1和BMP 的诱导下可以形成骨样组织。

[关键词]　组织工程骨;外周血干细胞;转化生长因子2β1;骨形成蛋白[中图分类号]　R780.2 [文献标识码]　A [文章编号]　1003-1634(2006)08-0454-03 Experiment reseach of APBSC 2TEB in to bone in the bod y of the dog ZHA O S hu 2xian ,ZHA N G L an ,W A N GMin ,DON G Kai ,HU Xao 2chun.Depart ment of Oral Medicine of S tom atological Hospital of Xi ’an Jiao Tong U ni 2versity ,Xi ’an 710004,China[Abstract ]Objective :For observes generate bone ,we are trans planted APBSC 2TEB in to dog abdomen ,for the perpa 2ration transplantion bone.Method :TEB are manufactured through pig Frash ilium bone cleared off protein and calcium.TEB are immerged APBSC 2liquid for get to APBSC 2TEB.APBSC 2TEB are bury to dogs abdomen subcutaneous tissues.for observes generate bone in the different time.R esult :After operation 1week ,APBSC cell no changes in the experiment groups.After operation 2weeks ,APBSC cell 2point extend to between collagenous fiber and fibroblast ,APBSC cell changes to osteoblast.After operation 1month ,TEB are absorb in the dog body.there are few generate osteameba.After operation 3～5month ,there are better genrate osteameba ,But there are plentiful conjunctive tissues 。