【作者】 常青；

【导师】 吴本俨；

【作者基本信息】 中国人民解放军军医进修学院 ， 老年医学， 2009， 博士

【摘要】 研究背景:骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMMSCs)是骨髓中一种非造血类的干细胞。近些年来的研究发现,间充质干细胞(mesenchymal stem cells,MSCs)由于具有多向分化潜能、低免疫原性和易于体外扩增等特点,受到医学应用领域的广泛关注。应用骨髓MSCs移植进行细胞替代治疗,目前已被广泛应用于缺血或损伤性疾病以及器官移植、抗免疫排斥反应等研究领域,并取得了初步效果。因此,干细胞替代治疗有望成为临床上一种新的治疗手段。目的:体外培养和扩增的骨髓间充质干细胞对损伤胃黏膜的修复作用及其机制研究。方法:1、分别采用全骨髓贴壁法和密度梯度离心法体外培养和扩增Wistar大鼠的骨髓MSCs,应用MTT方法比较两种方法下骨髓MSCs的自然生长曲线,流式细胞仪检测骨髓MSCs表面标志物,对培养、纯化的骨髓MSCs进行鉴定。2、以活体染料羟基荧光素乙酰乙酸(5,6-carboxyfluoresceindiacetate succinimidyl ester,CFDA SE)体外标记骨髓MSCs,荧光显微镜下、488nm波长激发光下观察标记后细胞的生长状态;制备大鼠胃黏膜损伤组织冰冻切片,荧光显微镜下观察移植骨髓MSCs的定植。利用大鼠性别决定基因SRY对移植细胞进行体内示踪。3、选择健康成年Wistar大鼠(体重200g-250g)54只,8周龄,雌雄各半,随机分为对照组和实验组,实验组又分为移植组和非移植组,每组18只。实验组用消炎痛30mg/Kg皮下注射,造成胃损伤模型。造模后2h,移植组大鼠经尾静脉注射骨髓MSCs(1×10~7/只);非移植组大鼠经尾静脉注射与移植组同体积的PBS。对照组大鼠给予碳酸氢钠10ml/Kg皮下注射2h后,经尾静脉注射骨髓MSCs(1×10~7/只)进行干细胞移植。各组大鼠分别于移植后24h、48h、72h处死并留取胃组织标本。按Guth评分标准评价胃黏膜损伤程度。光镜下观察并比较实验组和对照组胃黏膜的损伤情况。免疫组化检测VEGF、EGFR在实验组和对照组的表达差异。结果:1、骨髓间充质干细胞具有贴壁生长特性,多数为成纤维样梭形细胞,原代细胞12-14天左右贴满培养瓶底,传代细胞的生长速度较原代快,传代后的细胞24h几乎全部贴壁。全骨髓贴壁法与密度梯度离心法相比,细胞自然生长速度较慢,但两者无明显差异(P＞0.05);第3代以后的骨髓MSCs具有良好的均一性,细胞表面抗原CD90,CD44表达为阳性,CD45,CD31表达为阴性;2、体外采用CFDA SE标记骨髓MSCs后,细胞增殖几乎不受影响,且绿色荧光可随着细胞分裂传给子代。移植后48h,72h,在胃黏膜损伤组大鼠的胃黏膜中检测到带有绿色荧光标记的骨髓MSCs,而在胃黏膜未损伤组则未检测到带有绿色荧光标记的骨髓MSCs;移植后48h和72h移植组雌性大鼠的损伤胃黏膜中大鼠性别决定基因SRY阳性。3、消炎痛诱导胃黏膜损伤后,实验组大鼠胃黏膜损伤程度较均一,接受骨髓MSCs移植组的大鼠胃黏膜损伤指数较未接受骨髓MSCs移植组有所降低,以移植后72h差异最显著(P＜0.05);4、免疫组织化学染色结果显示:胃黏膜损伤组大鼠接受骨髓MSCs移植后,胃黏膜内VEGF,EGFR的表达较未接受骨髓MSCs移植组升高,以移植后72h组差异最显著(P＜0.05);结论:1、大鼠骨髓间充质干细胞体外培养生长稳定,经过换液、传代后细胞逐渐纯化,全骨髓贴壁法方法简单、易于操作、掌握和运用。2、CFDASE体外标记大鼠骨髓MSCs后,可在体内对移植的骨髓MSCs进行示踪,本研究发现经尾静脉移植的骨髓MSCs可定植于损伤的胃黏膜。3、大鼠的骨髓MSCs能一定程度地参与损伤胃黏膜的修复。骨髓MSCs移植后不同时间点损伤大鼠胃黏膜组织中VEGF、EGFR的表达较对照组大鼠明显升高,提示定植于损伤胃黏膜组织的骨髓MSCs可能通过自身分化或分泌某些生物因子的旁分泌途径促进损伤胃黏膜的修复。更多还原

【Abstract】 Background:Bone marrow mesenchymal stem cells（BMMSCs）are non-hematogenic stem cells isolated from marrow.They have been paid extensive attention in medical field due to their multi-directional differentiation potential, low immunogenicity and can be easily isolated and cultured in vitro. Transplantation of MSCs has already been used in treatment of ischemic and injury diseases.Therefore,stem cell replacement therapy is expected to become a new therapeutic means.Objective:To study the effect and mechanism of cultured mesenchymal stem cells（MSCs）on the repair of injured gastric mucosa.Methods:（1）Density gradient centrifugation and differential attachment methods were used to isolate of MSCs from adult Wistar rats and the proliferation ability of the cells was analyzed by MTT examination.The surface antigens of MSCs were detected by flow cytometry.（2）CFDA SE was used to label the MSCs in vitro,then the growth state of MSCs was observed by fluorescence microscope. In situ hybridization technique was used to trace the transplanted cells by detecting the sex-determining gene（SRY）of rats.（3）Fifty-four healthy Wistar rats of 8 weeks old（200g-250g,half of them were female）were randomly divided into three groups:the control group and two gastric mucosa injury groups.Each group had 18 rats.Gastric mucosa injury was induced by hypodermical injection of indomethacinand injected amount was 30mg/Kg.The control group received hypodermical injection of NaHCO₃ with 10ml/Kg.Two hours after injection,one group of rats with gastric mucosa injury and control group were received MSCs（1×10⁷/rat）by intravenous injection,while another group anamals were received PBS as the same way.The gastric tissues were dissected respectively 24-h,48-h, 72-h after transplanting of MSCs respectively.The degree of gastric injury was evaluated by the ulcer index（UI）.The patho-histological changes of gastric tissues corresponding to different time points were investigated through biological microscope.The difference expression of VEGF and EGFR in the experimental group and control group were detected by immunohistochemistry.Results:（1）MSCs have the characteristics of adherent growth,The morphology of cultured MSCs was fibroblast-like form.MSCs possessed intensive self-renewal potential and integrated in single layer in 12-14 days after planting.The growth ability of subcultured MSCs was greater than primary cultured MSCs.Nearly all of subcultured MSCs were adhesive in 24 hours. Compare with density gradient centrifugation,the growth rate of MSCs is slower in differential attachment method group.But there was no significant difference. Examination of MSCs revealed that in passage 3 of initial culture almost all adherent cells took on typical morphologies of fibroblastoid cells in vitro and maintained similar morphology with passages.They were positive for CD90, CD44 and were negative for CD31,CD45.（2）The cell multiplication of MSCs had not been affected after labelling with CFDA SE in vitro.The green fluorescence can be handed down to off-spring with cell division.On the 48-h and 72-h after MSCs transplantation,the CFDA SE labeled cells were found scattered in the gastric of the rats that underwent MSCs transplantation,while these were not detected in the control group at the corresponding points.Moreover,the SRY gene was detected in the injuried gastric mucosa of rats that underwent the transplantation of MSCs derived from male rats 2 weeks after transplantation,while it was not detected in the control group.（3）The UI was significantly decreased at 72-h after transplanting of MSCs（P＜0.05）.（4）The immunohistochemistry staining of VEGF and EGFR demonstrated that the positive expression of cells were increased in the injuried gastric mucosa of the rats underwent MSCs transplantion than that of control group,it was significantly on 72-h after transplantion. Conclusion:（1）MSCs can grow stablely in vitro and nonadherent hematopoietic cells were removed through replacing medium.Compared with density gradient centrifugation,the differential attachment method is simple,easy to operate and grasp.（2）The technique of CFDA SE labelling is applicable to be used to trace the transplanted cells in vivo.In present study,it revealed that MSCs could be located in the injured gastri mucosa through intravenous injection. （3）The MSCs of rats may accelerate gastric wound healing course and the mechanism maybe that MSCs could not only differentiate into gastric epithelium but also secreted some biological factors.更多还原