摘要
目的 研究在非β细胞中表达成熟胰岛素 ,探讨替代胰岛素注射或胰岛移植治疗Ⅰ型糖尿病的方法。方法 采用重叠区扩增基因拼接法 (geneSOEing)自胰岛素原基因组基因中扩增胰岛素原的cDNA ,并于C肽的两端引入蛋白酶furin的识别和切割位点Arg Xaa Lys/Arg Arg ,将获得的突变体重组表达载体 pcDNA3 .1 /C .mINS通过脂质体法转染体外培养的Hela、2 93和L0 2细胞 ,转染后 48、72h取细胞培养液 ;并将L0 2细胞经G41 8(450mg/L)筛选 2个月后 ,得到稳定表达胰岛素的细胞株 ,同时用放射免疫和免疫组织化学的方法监测细胞内外胰岛素的表达水平。结果 成功地对胰岛素原cDNA的 3个位点进行了突变 ,空载体转染的细胞无胰岛素表达 ,而在转染突变后胰岛素原基因cDNA的细胞培养液中胰岛素的表达量各不相同 :每天 8.45～ 1 88.0 0 μIU/2 .0× 1 0 6 Hela细胞、每天 1 59.88～ 2 4 2 .1 4 μIU/ 2 .0× 1 0 6 2 93细胞、每天 2 .56～ 61 .95μIU/ 2 .0× 1 0 6L0 2细胞 ;免疫组织化学显示细胞浆内有囊泡状分泌颗粒。
Objective To study an insulin and islets transplantation replacement therapy for type Ⅰdiabetes based on engineered human non βcells,which secreted mature insulin.Methods Human proinsulin cDNA was cloned from its genomic gene and mutated by gene SOEing method,introducing furin consensus cleavage sequences (Arg Xaa Lys/Arg Arg).An expression vector encoding a genetically modified human proinsulin cDNA was generated,and transduced to Hela,293 and L02 cells by lipofectin mediated DNA transfection.48 and 72?h after the transfection,the culture media were collected for further assay.After screening with G418 (450?mg/L) for two months,the survived L02 cells secreting insulin stably were selected and enriched.Insulin levels in the supernatants and cells were evaluated respectively with radioimmunoassay and immunohistochemical analysis.Results Three sites were mutated simultaneously.Insulin gene modified cells were able to express insulin at different levels,8.45 to 188.00 μIU/(2.0×10 6 Hela cells·d -1 ),159.88 to 242.14?μIU/(2.0×10 6 293 cells·d -1 ),and 2.56 to 61.95?μIU/(2.0×10 6 L02 cells·d -1 ),while no insulin was released by control cells.Immunohistochemical analysis confirmed that (pro)insulin was stored as vacuoles in the cytoplasm of L02 cells.Conclusion A correctly mutated human proinsulin cDNA was obtained successfully,transfected,and expressed efficiently in non βcells.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2002年第3期267-269,T002,共4页
Chinese Journal of Experimental Surgery
基金
上海市"百人计划"基金资助项目(98BR0 18)
