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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。结论 突变的胰岛素原cDNA能成功转染非胰岛β细胞并表达胰岛素
Objective To study the expression of mature insulin in non-β cells and to explore alternative methods of insulin injection or islet transplantation for the treatment of type 1 diabetes. Methods The cDNA of proinsulin was amplified from the proinsulin genomic gene by overlap gene amplification (geneSOEing) and Arg Xaa Lys / Arg Arg was introduced into both ends of the C peptide to recognize and cleave the protease furin. The recombinant plasmid pcDNA3 .1 / C.mINS was transfected into Hela, 293 and L0 2 cells by lipofectamine 2000. The transfected L02 cells were treated with G41 The cells stably expressing insulin were screened at 8 (450mg / L) for 2 months. The expression of insulin in cells was monitored by radioimmunoassay and immunohistochemistry. Results Three sites of the proinsulin cDNA were successfully mutated and the empty vector transfected cells had no insulin expression. However, the expression of insulin in the cell culture medium of the proinsulin gene cDNA after the mutation was transfected varied from day to day 8.45 ~ 1 88.0 0 μU / 2.0 × 10 6 HeLa cells, 1 59.88 ~ 2 4 2 .1 4 μU / 2.0 × 10 6 2 93 cells per day, 2.56 ~ 61 .95 μIU / 2 .0 × 10 6L0 2 cells; immunohistochemistry showed vesicular secreting granules in the cytoplasm. Conclusion Mutant proinsulin cDNA can successfully transfect non-islet β cells and express insulin