体外诱导糖尿病鼠骨髓间充质干细胞向血管内皮样细胞分化的研究

目的 观察链脲佐菌素(STZ)诱导的糖尿病大鼠骨髓间充质干细胞(BMSCs)的体外培养方法和向血管内皮样细胞分化的能力.方法 利用密度梯度离心分离和贴壁培养相结合的方法从STZ诱导的糖尿病大鼠骨髓中提纯单个核细胞,体外扩增3代,倒置显微镜观察细胞生长及形态,流式细胞仪检测BMSCs表面抗原表达.取扩增第3代的BMSCs,分为2组,诱导组加入诱导培养剂含10％胎牛血清、10μg/L血管内皮生长因子(VEGF)、2μg/L碱性成纤维细胞生长因子(bFGF)、100 U/ml青霉素、100 mg/L链霉素的M199培养基]中诱导分化,对照组则不加任何细胞因子.2周后行细胞形态学观察,免疫细胞化学法检测VEGF受体(VEGFR)-2表达,流式细胞仪测定CD34表达量,紫外线分光光度法测定一氧化碳(NO)含量,电镜观测胞质WeibelPalade小体.结果 STZ腹腔注射可诱导糖尿病大鼠模型.流式细胞仪显示第3代BMSCs表达表面抗原:CD44和CD90阳性细胞表达率分别为(97.8±0.9)％和(96.8±1.4)％,而CD11 b/c和CD34阳性细胞表达率分别为(13.2±0.6)％和(1.2±0.5)％.诱导组大部分细胞形态变化不明显,呈长梭形、杆状、多角形,诱导组VEGFR-2、CD34阳性表达率分别为(97.1±1.0)％和(65.0±3.9)％,而对照组则为(7.0±1.0)％和(0.9±0.3)％,两组比较差异有统计学意义(P＜0.05)；诱导组胞外NO含量(94.14±3.25) μmol/L亦明显高于对照组(70.37±2.10) μmol/L(P ＜0.05)；电镜两组均未观察到胞质Weibel Palade小体.结论 经密度梯度离心分离和贴壁培养相结合的方法可从骨髓中提纯BMSCs.糖尿病鼠BMSCs可在体外诱导向血管内皮样细胞方向分化,BMSCs有望作为治疗糖尿病下肢缺血性疾病的种子细胞.

In vitro differentiation into endothelial-like cells from bone marrow mesenchymal stem calls in diabetic rats

Objective To investigate the methods for separation and culture of bone marrow mesenchymal stem cells (BMSCs) in vitro in diabetic rats and the capability of differentiation into endotheliallike cells.Methods Rat diabetic model (DM) was induced by injection of stretopzin (STZ) in SD rats.After 12 weeks of DM duration,BMSCs were separated from bone marrow with density gradient centrifugation,wall sticking screening and amplified in vitro.The surface markers of BMSCs were evaluated with fluorescence-activated cell sorter (FACS).BMSCs of the third passage were obtained and divided into two groups.In the induction group,the cells were induced to differentiate in M199 medium containing 20％ fatal bovine serun ( FBS),vascular endothelial growth factor ( VEGF,10 μg/L) and basic fibroblast growth factor (bFGF,2 μg/L).No cytokine was added in the blank control group.Two weeks later,the cell morphology was observed under the phase contrast microscopy.The expression levels of VEGFR-2 were detected by using immunohistochemistry.EPC-specific marker CD34 was evaluated with FACS,and release of nitrous oxide (NO) from culture cells was also detected by NO detection reagent.Weibel-Palade (W-P) bodies in the cytoplasm were observed under the transmission electron microscopy.Results Rat diabetic model in SD rats that were injected with STZ intraperitoneally was established.Cultured BMSCs at the third passage were positive for CD44 and CD90 (97.8 ± 0.9 ) ％ and (96.8 ± 1.4) ％,respectively ],but negative for CD11 b/c and CD34 ( 13.2 ± 0.6 ) ％ and ( 1.2 ± 0.5 ) ％,respectively ].On the 14 day in the induction group,the cell morphology observed under the phase contrast microscopy was not distinctly different.Differentiation antigens VEGFR2 and CD34 in the induction group were higher (97.1 ± 1.0) ％,(65.0 ± 3.9) ％ ] than in the blank control group (7.0 ± 1.0 ) ％,( 0.9 ± 0.3 ) ％ ] ( P ＜ 0.05 ).The content of nitrous oxide in the induction group was also obviously increased as compared with the blank control group (94.14 ± 3.25 ) vs.( 70.37 ± 2.1 0) μmol/L,P ＜ 0.05 ).W-P bodies in the cytoplasm of the cultured cells were not found in the two groups.Conclusion BMSCs in diabetic rats can be isolated by density gradient centrifugation and adherent culture.VEGF and bFGF can induce differentiation of BMSCs in diabetic rats into endothelial-like cells in vitro.It is suggested that BMSCs may be used as a new type of seed cells for the diabetic lower limb ischemia disease.