【作者】 范子扬；

【导师】 宋振顺；

【作者基本信息】 第四军医大学 ， 外科学， 2011， 硕士

【摘要】 目的:有研究表明骨髓间充质干细胞(MSCs)能够分泌基质细胞衍生因子(SDF-1),而SDF-1与其特异性受体CXCR4结合后形成具有修复受损组织,参与血管生成等多种生物学功能的单位。本实验通过分离、纯化大鼠的骨髓间充质干细胞,并在体外一定条件下培养、扩增。并将培养的第3代细胞移植入链脲佐菌素(STZ)诱导的糖尿病大鼠体内,观察同种异体MSCs移植入受体后,SDF-1/CXCR4轴在促进残存胰岛及其周围新生血管增殖中的作用。方法:将SD大鼠处死后,无菌条件下取出股骨和胫骨, DMEM培养液冲洗髓腔,200μm尼龙网过滤除去大的组织团块。细胞悬液离心(1000rpm,10min)后,收集细胞接种于含有10%胎牛血清的DMEM培养液中培养。通过腹腔注射链脲佐菌素(STZ,65mg/kg)构建糖尿病大鼠模型。诱导成功的糖尿病大鼠随机分为A组(MSCs移植组)、B组(MSCs移植+SDF-1/CXCR4轴阻断剂AMD组)和C组(糖尿病对照组),另设D组(正常大鼠对照组)。将传至3代的MSCs移植入糖尿病大鼠体内,观察各组大鼠的生存状态,每3天采鼠尾静脉血用血糖仪检测各组血糖水平,采用放免法检测血清中空腹和餐后不同时间点的胰岛素水平、Elisa法检测SDF-1水平。并于移植MSCs后第30天取出各组大鼠胰腺和血清,胰腺组织采用H-E染色和免疫组织化学染色法观察CD31、PCNA、PDX-1在胰腺组织的表达水平。结果:1、A组残存胰岛周围可见新生血管,CD31、PCNA、PDX-1染色阳性率分别为(71.2±5.3)%、(76.5±4.5)%、(69.8±6.7)%;B组和C组残存胰岛周围基本未见新生血管,CD31、PCNA、PDX-1染色阳性率B组为(7.4±2.1)%、(5.5±3.7)%、(8.8±2.9)%,C组为(5.7±3.1)%、(6.2±1.4)%、(3.5±1.6)%,A组与B组比较有统计学差异(p<0.05),B组与C组比较无统计学差异(p>0.05)。2、移植后第25天,A组糖尿病大鼠血糖水平基本正常,低于B组和C组,而胰岛素水平明显高于B组和C组(p<0.05)。3、A组与B组血清SDF-1水平差异无统计学意义(p>0.05),但都明显高于C组(p<0.05)。结论:通过分离、纯化、培养、扩增可以获得大量的MSCs,MSCs移植后明显的促进了糖尿病受体残存胰岛再生及其周围新生血管的形成,SDF-1/CXCR4轴特异性阻断剂-AMD3100能抑制MSCs逆转高血糖,促进胰岛再生的作用,进而提示SDF-1/CXCR4轴在胰岛再生和及其周围新生血管形成中具有重要作用。更多还原

【Abstract】 Objective:There is controversy about the mechanism how decreasing hyperglycemia and promoting islets regeneration after transplanted MSCs. Recent research suggest that MSCs can secrete stromal cell-derived factor 1(SDF-1). SDF-1 is associated with revascularization and tissue repairing, which might play an important role on revascularization and regeneration of damaged remnant islets of the recipients.To investigate the role of SDF-1/CXCR4 axis on recipients’remnant islets regeneration and neovascularization after the transplantation of allogeneic bone marrow mesenchymal stem cells(MSCs).Methods:MSCs were isolated from SD rat, cultured, Proliferated and purified in vitro under certain condition and then passaged 3 was transplanted in the diabetic rats.The diabetic rats was induced by peritoneal streptozotocin injection (65mg/kg).SD rats were anesthetized with intraperitoneal sodium pentobarbital (30mg/kg). Femurs and tibiae of rats were collected and adherent soft tissue was carefully removed. Bone marrow cells were flushed from the medullary cavities of femurs and tibiae and disaggregated into a single-cell suspension by sequential passage through a 25-gauge needle. MSCs were separated by centrifugation(1000rpm,10min). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM/F12) containing10% fetal bovine serum (FBS). The diabetic rats induced by peritoneal injection of streptozotozin were randomly divided into group A (MSCs transplant group),group B(MSCs transplant+AMD group),group C(DM control group) ;Group D is control group.Each group contains 10 SD rats. The pancreas were removed and blood samples were retrieved from each group simultaneously at the 30th day after cells transplant.Detect the expression of CD31,PCNA and PDX-1 in each group of pancreas tissue by immunohistochemistry and observe the islets shape by Hematoxylin and Eosin(H-E) . Blood glucose and insulin in serum were detected by blood glucose monitor, radioimmunoscintigraphy and respectively SDF-1 in serum was detected by enzyme linked immunosorbent assay(ELISA).Results:Neovascularization were observed in the remnant islets of the recipient pancreatic tissue and CD31-positive cells(71.2±5.3)%,PCNA-positive cells(76.5±4.5)%,PDX-1-positive cells(69.8±6.7)% were observed in group A. Compared with group A, seldom-positive cells[CD31(7.4±2.1)%, PCNA(5.5±3.7)% and PDX-1(8.8±2.9)%] and rarely neovascularization were observed in group B and group C(p<0.05). Blood glucose level in serum of group A was lower than that of group B and group C,but insulin level in serum of group A was significantly higher than that of group B and group C(p<0.05). There are no significant difference between group A and group B in SDF-1 level in serum(p>0.05),but both were higher than that of group C(p<0.05).Conclusion:Obviously,MSCs promote recipient neovascularization surrounding the islets,which enhanced the proliferation and regeneration remnant of islet cells.AMD 3100 has the function of interrupting SDF-1/CXCR4 axis which inhibits the effect of MSCs on promoting islets regeneration.Therefore we concluded that SDF-1/CXCR4 axis play an important role in vascularization and islets regeneration.更多还原