人永生化胰腺星形细胞系的构建及其功能的验证

Construction and functional verification of immortalized human pancreatic stellate cells

目的构建人永生化胰腺星形细胞(PSCs)系并探讨其生物学功能。方法采用outgrowth方法提取人原代胰腺星形细胞(hPSCs),慢病毒感染方法导入SV40LT基因和hTERT基因,嘌呤霉素筛选稳定高表达两个基因的细胞(im PSCs),qRT-PCR和Western bolt方法检测表达水平;CCK8法、核型分析、免疫荧光、体内成瘤实验检测im PSCs生长曲线,染色体数目,胶原基质蛋白表达和有无恶性表型转化;共培养方法、CCK8法、细胞划痕、迁移侵袭方法检测imPSCs促瘤作用。结果hPSCs呈多角形和梭形,慢病毒感染的im PSCs,显微镜下有亮绿色荧光;im PSCs细胞高表达SV40LT基因和hTERT基因;im PSCs生长曲线呈典型的“S”型,染色体数目增多,体内不能成瘤,表达α-SMA、Collagen I、Fibronectin,具有PSCs的生物学功能和特点;其与胰腺癌细胞共培养后,明显促进肿瘤生长,与人原代PSCs功能相当。结论采用慢病毒转染SV40LT基因和hTERT基因可成功构建人胰腺星形细胞永生化细胞系,为胰腺星形细胞本身及胰腺癌微环境相关研究提供实验材料和模型。

Objective To construct the human pancreatic stellate cell line immortalization and discuss their biological function.Methods We extracted human primary pancreatic stellate cells(hPSCs)by outgrowth and then infected the cells with SV40LT and hTERT genes using lentivirus infection.The cells(named imPSCs)with stable and high expression of SV40LT and hTERT genes were screened with puromycin.The next,qRT-PCR and Western Bolt methods were used to detect the expression levels of the two genes.CCK8 method,karyotype analysis and immunofluorescence method were conducted respectively to detect the growth curve,chromosome number,protein expression ofα-SMA,collagen I and fibronectin of the imPSCs.We also performed tumor formation assay in vivo to detect whether the imPSCs had malignant phenotypic transformation.The tumorigenesis of imPSCs was detected by transwell cell co-culture,CCK8 methods,wound healing assay,migration and invasion methods.Results Human primary PSCs were polygonal and spindle shaped,and bright green fluorescence was observed under the microscope after successfully infected lentivirus.In imPSCs,SV40LT and hTERT genes were stably and highly expressed in mRNA and protein levels.The imPSCs had typed“S”growth curve and increased number of chromosomes,but they could not grow into tumors in vivo.The imPSCs showed high expression ofα-SMA,collagen I and fibronectin,which had the biological functions and characteristics of PSCs.After co-culture of imPSCs and pancreatic cancer cells,tumor growth was significantly promoted.Conclusion After transfected with SV40LT and hTERT genes,an immortalized human pancreatic stellate cell line was successfully constructed,which could provide experimental materials and models for the study of pancreatic astrocytes themselves and the microenvironment of pancreatic cancer.