摘要
目的 利用基因工程的方法 ,建立受胰岛素负调控、地塞米松正调控的人工 β细胞株。方法 将已经突变的含有蛋白酶furin酶切位点的胰岛素原cDNA置于PEPCK启动子控制之下 ,构建表达胰岛素的逆转录病毒载体 pN PEPCK mINS。通过脂质体法用该表达载体转染体外培养的大鼠Hepa1 6细胞 ,经G418(80 0mg/L)筛选两月后 ,得到表达胰岛素的细胞株。采用逆转录 聚合酶链反应 (RT PCR)和放射免疫分析法 ,从mRNA和蛋白水平观察细胞培养基中胰岛素和地塞米松浓度的变化对胰岛素基因表达的影响。结果 经转染和筛选后 ,在获得的 3 2个表达不同水平胰岛素 (0～ 10 .79μIU/10 6cells/d-1)的细胞株中挑选一株细胞Hepa1 6/INS2 1,其胰岛素的表达量最高 ,为 10 .79μIU /10 6cells/d-1。不同浓度的呋塞米和胰岛素培养Hepa1 6/INS2 1细胞 2 4h后 ,收集细胞培养液和细胞总RNA。检测胰岛素的结果发现 10 -7mol/L的地塞米松显著促进胰岛素的分泌 ,此时的表达量为基础水平的 3 .6倍 ;RT PCR也证实在转录水平上外源性胰岛素明显抑制胰岛素基因的表达 ,而地塞米松上调其表达。结论 基因工程改造肝细胞系能获得生理模式分泌胰岛素的细胞株 ,利用微囊包裹技术移植这类细胞可望为Ⅰ型糖尿病患者提供一种安全可行的治疗手段。
Objective To engineer an artificial β cell line by which insulin production is down regulated by insulin and up regulated by dexamethasone.Methods A regulatory secretion system was devised by placing proinsulin cDNA,containing genetically engineered furin endoprotease cleavage sites,under the regulatable promoter for PEPCK and constructed a retroviral insulin expression vector pN PEPCK mINS.The expression vector was transduced to Hepa1 6 cells by lipofectin mediated DNA transfection.Following G418 screening,the survived cells expressing insulin were selected and enriched.Then,at the levels of mRNA and protein,the regulating effect of insulin and dexamethasone in culture medium on the expression of insulin gene was estimated respectively with RT PCR and radioimmunoassay.ResultsAfter transfection and screening,one clone cells (Hepa1 6/INS21) were selected,which secreted the highest level of insulin (10.79 μIU/10 6 cells/ d -1 ),of 32 clones expressing insulin from 0 to 10.79 μIU/10 6 cells/d -1 .Following treating Hepa1 6/INS21 cells with various concentrations of dexamethasone and insulin,the culture media and the total RNA of these cells were collected 24 h later.Insulin production was augmented 3.6 fold by the addition of 10 -7 mol/L of dexamethasone than that in non treated Hepa1 6/INS21 cells.Addition of exogenous insulin to the culture decreased the insulin mRNA expression remarkably on RT PCR when dexamethasone increased the insulin gene expression at the transcriptional level.Conclusion Genetically engineered Hepa1 6 cells could synthesize,process,and secrete insulin in a physiological manner.The implantation of encapsulated engineered Hepa1 6 cells might provide a safe and practical therapeutic approach for IDDM treatment.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2005年第1期52-54,共3页
Chinese Journal of Experimental Surgery
基金
上海市科委基金资助项目 (0 1 4 1 1 90 2 9)
