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目的 BMSCs在体外分化为胰岛素分泌细胞存在分化效率低、成熟度差的问题。研究BMSCs在糖尿病猪胰腺微环境下分化为胰岛素分泌细胞的可行性。方法取1只4周龄雄性贵州小香猪骨髓,采用贴壁法制备BMSCs。15只8～10周龄雌性贵州小香猪,体重8～10 kg,随机分为正常组(A组)、糖尿病组(B组)和BMSCs移植组(C组),每组5只。B、C组连续3 d从耳缘静脉推注链脲菌素加四氧嘧啶溶液,连续2 d血糖>17 mmol/L提示糖尿病造模成功。C组将标记增强型绿色荧光蛋白(enhanced green?uorescent portein,EGFP)的第3代BMSCs(细胞密度为5×107个/mL)1.1 mL多点注射移植至胰腺包膜下,A、B组注射等量生理盐水。监测血糖至30 d后,取胰腺组织行HE染色观察并检测胰岛数目及直径;免疫荧光组织化学染色检测新生胰岛的胰岛素表达;激光捕获显微切割技术获得EGFP+细胞,提取总RNA,采用RT-PCR检测胰岛素mRNA和胰腺十二指肠同源框蛋白1(pancreatic and duodenal homeobox factor 1,PDX1)mRNA的表达;荧光原位杂交(fluorescence in situ hybridization,FISH)检测胰岛素基因和SRY(sexdetermining region ofthe Y chromosome)基因的共表达。结果移植18 d后C组血糖开始较B组明显降低,且随时间延长逐渐下降(P<0.05)。组织学观察发现BMSCs移植30 d后C组胰岛数目(10.9±2.2)个较B组(4.6±1.4)个明显增加,差异有统计学意义(P<0.05),与A组(12.6±2.6)个比较差异无统计学意义(P>0.05);C组新生胰岛直径(47.2±19.6)μm,明显小于A组(119.6±27.7)μm,差异有统计学意义(P<0.05),B组未见新生胰岛。免疫荧光组织化学染色显示C组新生胰岛有胰岛素表达。RT-PCR检测示C组EGFP+细胞有胰岛素mRNA和PDX1 mRNA表达。FISH检测C组细胞中有SRY基因和胰岛素基因共表达。结论 BMSCs在糖尿病猪胰腺微环境条件下可分化为胰岛素分泌细胞。
Purpose BMSCs differentiate into insulin-secreting cells in vitro, which results in low differentiation efficiency and poor maturity. To investigate the feasibility of BMSCs differentiating into insulin-secreting cells in diabetic porcine pancreas microenvironment. Methods Bone marrow was collected from a Guizhou male Guizhou pig with 4 weeks of age. BMSCs were prepared by adherence method. Fifteen female Guizhou 8-10 weeks old, weighing 8-10 kg, were randomly divided into normal group (group A), diabetic group (group B) and BMSCs transplantation group (group C), with 5 in each group. Group B and C were injected streptozotocin plus alloxan solution continuously from the ear vein for 3 consecutive days, and blood glucose> 17 mmol / L for 2 consecutive days showed that diabetes was successfully established. In group C, the third generation BMSCs labeled with enhanced green fluorescent protein (5 × 107 cells / mL) labeled with enhanced green fluorescent protein (EGFP) The same amount of saline injection. After 30 days of blood glucose monitoring, the number of pancreatic islets was measured by HE staining and the number and diameter of islets were detected. Immunofluorescence staining was used to detect the insulin expression of islet cells. EGFP + cells were obtained by laser capture microdissection. Total RNA was extracted by RT- PCR was used to detect the expression of insulin mRNA and pancreatic and duodenal homeobox factor 1 (PDX1) mRNA; fluorescence in situ hybridization (FISH) was used to detect the insulin gene and SRY (sexdetermining region of the Y chromosome co-expression. Results After 18 days of transplantation, blood glucose in group C began to decrease significantly compared with that in group B, and gradually decreased with time (P <0.05). Histological observation showed that the numbers of islets in group C (10.9 ± 2.2) increased significantly after 30 days of transplantation compared with those in group B (4.6 ± 1.4) (P <0.05), which were significantly different from those in group A (12.6 ± 2.6) There was no significant difference between the two groups (P> 0.05). The diameter of newborn islets in group C was (47.2 ± 19.6) μm, which was significantly lower than that in group A (119.6 ± 27.7) μm, the difference was statistically significant (P <0.05) See nascent islets. Immunofluorescence histochemical staining showed that insulin secretion was observed in the newborn islets in group C. RT-PCR showed that EGFP + cells in group C had insulin mRNA and PDX1 mRNA expression. FISH detected group C cells with SRY gene and insulin gene co-expression. Conclusion BMSCs can differentiate into insulin-secreting cells in diabetic porcine pancreas microenvironment.