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目的探讨大鼠损伤胰腺组织提取液对诱导大鼠BMSCs分化为胰岛素分泌细胞(in sulin producing c ells,IPCs)的效果和机制。方法取6周龄SD大鼠80只,将其中40只大鼠胰腺切除60%,48 h后取残存胰腺组织制备损伤胰腺组织提取液;余40只大鼠用于制备正常胰腺组织提取液。取4周龄SD大鼠的胫骨及股骨分离培养BMSCs,取第3代细胞分为实验组、正常对照组和空白对照组,分别加入损伤胰腺组织提取液、正常胰腺组织提取液和普通细胞培养液诱导培养。培养14 d过程中,观测细胞形态学改变以及胰腺发育相关基因和蛋白的表达;14 d时,流式细胞仪检测分化细胞C肽表达阳性率,葡萄糖刺激实验评价分化细胞释放胰岛素的水平。结果损伤胰腺组织提取液可诱导大鼠BMSCs分化为IPCs,在分化过程中表达与胰腺发育相关的基因:胰岛素促进因子1(pancreatic duodenal homeobox 1,PDX-1)、胰岛因子1、葡萄糖转运蛋白2、前蛋白转化酶2、神经元素3、Nkx6.1、生长抑素;细胞免疫荧光染色示其表达成熟胰腺β细胞标志性蛋白PDX-1、胰岛素、C肽、Nkx6.1。而正常对照组和空白对照组未能检测到相关基因和蛋白的表达。实验组C肽表达阳性率为13.8%±1.8%,显著高于正常对照组和空白对照组的1.6%±0.4%(P<0.05)。实验组在高、低糖刺激下胰岛素释放水平明显高于正常对照组和空白对照组(P<0.05),但仅为天然胰岛的1/40和1/47(P<0.05)。结论胰腺损伤后,损伤胰腺组织将分泌与胰腺修复和发育分化相关的转录蛋白,这些蛋白可诱导大鼠BMSCs向IPCs分化。
Objective To investigate the effect and mechanism of rat pancreatic tissue extracts on the induction of BMSCs differentiation into insulin-producing cells (IPCs) in rats. Methods Eighty male Sprague-Dawley rats aged 6 weeks were randomly divided into four groups. Forty of them were excised 60% of the pancreas. Forty-eight hours later, the remaining pancreatic tissue was harvested to prepare the pancreatic tissue extract. The remaining 40 rats were used to prepare normal pancreatic tissue extract. BMSCs were isolated from the tibia and femur of 4-week-old SD rats. The third generation of cells were divided into experimental group, normal control group and blank control group. The pancreatic tissue extract, normal pancreatic tissue extract and normal cell culture Liquid induction culture. During 14 days of culture, the morphological changes of cells and the expression of genes and proteins related to the development of pancreas were observed. On the 14th day, the positive rate of C-peptide expression in differentiated cells was detected by flow cytometry, and the release of insulin by differentiated cells was evaluated by glucose stimulation test. Results Injured pancreatic tissue extracts induced the differentiation of rat BMSCs into IPCs. The expression of pancreatic ductal homeobox 1 (PDX-1), islet 1, glucose transporter 2 , Proprotein convertase 2, neuron 3, Nkx6.1, and somatostatin. Immunofluorescence staining showed that it expresses mature pancreatic β-cell marker protein PDX-1, insulin, C-peptide and Nkx6.1. The normal control group and blank control group failed to detect the expression of related genes and proteins. The positive expression rate of C-peptide in experimental group was 13.8% ± 1.8%, which was significantly higher than 1.6% ± 0.4% (P <0.05) in normal control group and blank control group. The experimental group under high and low sugar stimulated insulin release was significantly higher than the normal control group and the blank control group (P <0.05), but only the native islets of 1/40 and 1/47 (P <0.05). Conclusion After pancreatic injury, the injured pancreas secrete transcriptional proteins that are involved in the repair and development of pancreas. These proteins can induce the differentiation of rat BMSCs into IPCs.