Xie Hongbin, Chang Caihong, Jiang Jinxing, Li Qing, Qi Hui, Deng Chunyan, Li Furong
Clinical Medical Research Center, the Second Clinical Medical College (Shenzhen People's Hospital) of Jinan University, Shenzhen Guangdong, 518020, PR China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2011 May;25(5):597-601.
The bone marrow mesenchymal stem cells (BMSCs) have the capacity to differentiate into insulin-producing cells (IPCs) in vitro. However, low differentiation efficiency and poor maturity are the main obstacles. To investigate the feasibility of BMSCs differentiation into IPCs in diabetic pancreatic microenvironment of pigs.
BMSCs were isolated and purified from the bone marrow of a 4-week-old male pig. Fifteen female pigs (aged 8 to 10 weeks, weighing 8 to 10 kg) were randomly divided into 3 groups: normal control group (group A, n=5), diabetic control group (group B, n=5), and BMSCs transplanted group (group C, n=5). The pigs of groups B and C were treated by auris vein injections of streptozocin and alloxan for 3 days to induce diabetes mellitus (DM) model, whose blood glucose level 2 days all greater than 17 mmol/L was successful DM model. A total of 1.1 mL of the 3rd passage BMSCs labeled with enhanced green fluorescent protein (EGFP), with cell density of 5 x 10(7)/ mL, were injected into subcapsular pancreas of group C at multiple points, normal saline at the same dosage into those of groups A and B. After 30 days of monitoring blood glucose, the histological analysis of islet number and size were done; the immunofluorescence staining was used to detect the protein expression of insulin in the new-formed islets. The EGFP+ cells were collected from the sections using laser-capture microdissection; RT-PCR was used to detect insulin mRNA and pancreatic and duodenal homeobox factor 1 (PDX1) mRNA expressions from EGFP+ cells, and the insulin and sex determining region of the Y chromosome (SRY) genes were detected by fluorescence in situ hybridization (FISH).
The blood glucose level decreased significantly in group C when compared with that in group B from 18 days and gradually decreased with time (P < 0.05). The histological observation showed that the number of islets was increased significantly in group C when compared with that in group B (10.9 +/- 2.2 vs. 4.6 +/- 1.4, P < 0.05), and there was no significant difference when compared with that in group A (10.9 +/- 2.2 vs.12.6 +/- 2.6, P > 0.05). The size of new-formed islets in group C was significantly smaller than that in group A [(47.2 +/- 19.6) microm vs. (119.6 +/- 27.7) microm, P < 0.05]. The immunofluorescence staining showed that new-formed islets of group C expressed insulin protein. RT-PCR showed that the microdissected EGFP+ cells of group C expressed insulin mRNA and PDX-1 mRNA. FISH showed that the new-formed islet cells of group C contained SRY gene in Y chromosome and insulin double positive cells.
BMSCs can differentiate into IPCs in diabetic pancreatic microenvironment of pigs.
骨髓间充质干细胞（BMSCs）在体外具有分化为胰岛素产生细胞（IPCs）的能力。然而，低分化效率和差的成熟度是主要障碍。本研究旨在探讨在猪糖尿病胰腺微环境中BMSCs分化为IPCs的可行性。
从4周龄雄性猪的骨髓中分离并纯化BMSCs。将15只雌性猪（8至10周龄，体重8至10 kg）随机分为3组：正常对照组（A组，n = 5）、糖尿病对照组（B组，n = 5）和BMSCs移植组（C组，n = 5）。B组和C组猪通过耳静脉注射链脲佐菌素和四氧嘧啶3天以诱导糖尿病（DM）模型，血糖水平2天均大于17 mmol/L者为成功的DM模型。将1.1 mL第3代用增强型绿色荧光蛋白（EGFP）标记的BMSCs，细胞密度为5×10(7)/mL，多点注射到C组猪的胰腺被膜下，A组和B组注射相同剂量的生理盐水。监测血糖30天后，进行胰岛数量和大小的组织学分析；采用免疫荧光染色检测新形成胰岛中胰岛素的蛋白表达。使用激光捕获显微切割从切片中收集EGFP+细胞；采用RT-PCR检测EGFP+细胞中胰岛素mRNA和胰腺十二指肠同源盒因子1（PDX1）mRNA的表达，并通过荧光原位杂交（FISH）检测胰岛素和Y染色体性别决定区（SRY）基因。
与B组相比，C组自第18天起血糖水平显著下降，并随时间逐渐降低（P < 0.05）。组织学观察显示，与B组相比，C组胰岛数量显著增加（10.9±2.2对4.6±1.4，P < 0.05），与A组相比无显著差异（10.9±2.2对12.6±2.6，P > 0.05）。C组新形成胰岛的大小显著小于A组[（47.2±19.6）μm对（119.6±27.7）μm，P < 0.05]。免疫荧光染色显示C组新形成的胰岛表达胰岛素蛋白。RT-PCR显示C组显微切割的EGFP+细胞表达胰岛素mRNA和PDX-1 mRNA。FISH显示C组新形成的胰岛细胞含有Y染色体上的SRY基因和胰岛素双阳性细胞。
在猪糖尿病胰腺微环境中，BMSCs可分化为IPCs。
