BMSCs在糖尿病胰腺微环境下分化的可行性研究

FEASIBILITY OF BONE MARROW MESENCHYMAL STEM CELLS DIFFERENTIATION IN DIABETIC PANCREATIC MICROENVIRONMENT

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。

Objective 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.Methods BMSCs were isolated and purifi ed 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 styeptozocin 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 5×107/ 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 ofinsulin 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 sexdetermining region of the Y chromosome（SRY） genes were detected by fluorescence in situ hybridization（FISH）.Results The blood glucose level decreased significantly in group C when compared with that in group B from 18 days and gradually decreased with time（P0.05）.The histological observation showed that the number ofislets was increased significantly in group C when compared with that in group B（10.9±2.2 vs.4.6±1.4,P0.05）,and there was no significant difference when compared with that in group A（10.9±2.2 vs.12.6±2.6,P0.05）.The size of new-formed islets in group C was significantly smaller than that in group A [（47.2±19.6） μm vs.（119.6±27.7） μm,P0.05].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.Conclusion BMSCs can differentiate into IPCs in diabetic pancreatic microenvironment of pigs.