大鼠骨髓间充质干细胞体外诱导分化为胰岛素阳性细胞的潜能

Potency of bone marrow mesenchyme stem cells differentiating into insulin-positive cells in vitro in rats

目的:观察大鼠骨髓间充质干细胞体外诱导分化为胰岛素阳性细胞能力。方法:①实验于2004-09/2005-01在南京医科大学第一附属医院内分泌及代谢病研究室完成。选用清洁级雄性SD大鼠10只。②取大鼠骨髓,体外分离、培养骨髓间充质干细胞,流式细胞术检测CD45/CD90表达及细胞周期,明确骨髓间充质干细胞特性。③取第3代细胞,随机分为2组,低糖诱导组[先予体积分数0.1胎牛血清低糖Dulbecco改良的Eagle培养液(5.6mmol/L葡萄糖)]或高糖诱导组[高糖Dulbecco改良的Eagle培养液(25mmol/L葡萄糖)]培养14d,换予体积分数0.05胎牛血清低糖Dulbecco改良的Eagle培养液或高糖Dulbecco改良的Eagle培养液+尼克酰胺(10mmol/L)培养7d,再加Exendin-4(10nmol/L)诱导培养7d。④采用反转录聚合酶链反应法检测胰腺-十二指肠同源盒基因1、胰岛素原和胰岛素基因的表达,激光共聚焦显微镜观察胰岛素蛋白的表达,流式细胞术检测胰岛素阳性细胞数和平均荧光强度,电镜观察诱导后细胞的超微结构。⑤组间计量资料比较采用方差分析。结果:①骨髓间充质干细胞贴壁生长,呈长梭形。流式细胞术检测显示,CD90阳性率为(96.3±1.3)%,CD45阳性率为(0.3±0.4)%,细胞周期静止期-DNA合成前期占(76.8±4.8)%,DNA合成后期-有丝分裂期(11.3±3.7)%,DNA合成期(11.9±5.7)%。②骨髓间充质干细胞诱导培养过程中,细胞形成团簇状分布,少数聚集成团,直径80～200μm,半悬浮于培养瓶中。电镜观察此类细胞胞浆内有较多分泌颗粒。③低糖诱导组和高糖诱导组均表达胰腺-十二指肠同源盒基因1、胰岛素原和胰岛素基因。④流式细胞术检测显示低糖诱导组和高糖诱导组胰岛素阳性细胞数和平均荧光强度均明显高于骨髓间充质干细胞诱导前[(21.9±11.1)%,(19.8±7.8)%,(1.4±1.2)%;21.0±7.6,22.5±14.5,8.7±3.5,P<0.05]。结论:大鼠骨髓间充质干细胞在体外可以诱导分化为胰岛素阳性细胞。

AIM： To observe the ability of bone marrow mesenchyme stem cells （BMMSCs） traMs-differentiation into insulin-positive cells in vitro. METHODS： ① The experiment was conducted in the Laboratory of Endocrinology and Metabolism Disease, First Affiliated Hospital of Nanjing Medical University from September 2004 to January 2005. Ten clean-grade male SD rats were sdected. ② Bone marrow of the rats were separated in vitro, and the BM-MSCs were cultured. The CD45/CD90 expressions and eell cycles were detected by flow cytometry to observe the features of BM-MSCs. ③ The 3-passage cells were gained and divided randomly into 2 groups, low glucose induced group [foetus ox serum low glucose with the volume fraction of 0.1, Dulbecco＇s Modified Eagle Medium （DMEM） （5.6 mmol/L glucose）] or high glucose induced group [high glucose DMEM （25 mmol/L glucose）] cultured for 14 days. Changed with foetus ox serum low glucose with the volume fraction of 0.05, DMEM ＋ nicotinamide （10 mmol/L） for 7 days, adding Exendin-4 （10 nmol/L）induced for 7 days. ④ Pancreas-pa-ncreatic and duodenal homeobox 1 （PDX-1）, the expression of proinsulin and insulin gene were detected with reverse transcription-polymerase chain reaction （RT-PCR） method. The expression of insulin protein was observed with laser confocal microscopy. The number of insulin positive cells and average fluorescent intensity were detected with flow cytometry. The uhrastructure of cells after inducing was observed with the electromicroscope. ⑤ The measurement data were compared with analysis of variance among groups. RESULTS： ① BM-MSCs grew adherently to the wall, showing long fusiform. The detection of flow eytometry showed the CD90 positive rate was （96.3±1.3）%, and the CD45 positive rate was （0.3±0.4）%; it accounted for （76.8±4.8）% in GO-G1 phase, （11.3±3.7）% in G2 phase-M phase, （11.9±5.7）% in synthesis phase. ② The cells distributed in mass- cluster shape in the process of BM-MSCs, and small amount of cells collected as a mass with the diameter of 80-200 μm, semi-suspending in the culture bottle. The effective multiple secretory granule in this kind of cell plasm was observed with electromicroscope.③ There were PDX-1, proinsulin and insulin gene in the low glucose induced group and high glucose induced group. ④ The detection of flow cytometry indicated that the number of insulin positive cells and average fluorescent intensity in the low glucose induced group and the high glucose induced group were both significantly higher than those before induction of BM-MSCs [（21.9±11.1）%, （19.8±7.8）%, （1.4±1.2）%; 21.0±7.6, 22.5±14.5, 8.7±3.5, P 〈0.05]. CONCLUSION： The BM-MSCs in rats can trans-differentiation into insulin-positive cells in vitro.