胰岛素基因在非β细胞中的调节表达

目的　应用四环素 (Tet)可调控性表达系统 ,建立条件控制性人工 β细胞株 ,定量调节胰岛素基因在tet2 93细胞中的表达。方法　构建含四环素反应元件和表达胰岛素原基因的重组载体pTRE2mINS。再将此载体与具有潮霉素抗性的质粒pTK Hyg共转染能稳定表达反义四环素激活因子的细胞株tet2 93,然后用潮霉素筛选表达胰岛素的细胞株 ,通过四环素的衍生物强力霉素 (Dox)调节胰岛素在此细胞株中的表达。采用Northern印迹、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强力霉素浓度的变化对胰岛素基因表达的影响。结果　从2 8个单克隆细胞株中筛选 1株能自主分泌一定量的胰岛素 ,又能受强力霉素调控分泌的细胞株。当细胞培养液中加入或不加强力霉素时 ,培养基中胰岛素的表达量分别为每 2 4h 2 4 1 0U/1 0× 10 6细胞和每 2 4h 9 7U/1 0× 10 6细胞 ,加强力霉素组是不加强力霉素组的 2 5倍。而且随着强力霉素浓度的增加 ,tet2 93细胞内外胰岛素的表达水平逐步增高。结论　人胰岛素基因在tet2 93细胞中的成功转移和高效表达 ,受四环素及其衍生物定时、定量调节 ,从而提高糖尿病基因治疗的精确性 ,安全性和有效性。

Regulated expression of insulin gene in non-beta cell

OBJECTIVE: To engineer 293 cells to an artificial beta cell line which secrets insulin in response to stimulation by doxycycline. METHODS: A recombined expression vector, pTRE2mINS, which contained both of the tetracycline response element and proinsulin gene, was constructed. This vector was co-transfected with plasmid pTK-Hyg encoding hygromycin in the tet293 cells, which express the reverse tetracycline-controlled transactivator stably. Following hygromycin screening, the survived cells expressing insulin were selected and enriched. Then, doxycycline one of tetracycline derivatives, was used to control the expression of insulin in these cells. At the levels of mRNA and protein, the regulating effect of doxycycline in culture medium on the expression of proinsulin gene was estimated respectively with Northern blotting, RT-PCR, and radioimmunoassay. RESULTS: Of the 28 hygromycin resistant cell strains, we selected one cell strain secreting insulin not only automatically, but in response to stimulation by doxycycline was selected. The cells secreted insulin at the rate of 9.7 U/24 h/1.0 x10(6) cells without doxycy cline treatment, and the amount of secreted insulin increased to 241.0 U/24 h/1.0 x 10(6) cells (25-fold) in the presence of doxycycline(1 000 ng/ml). I insulin secretion was induced by doxycycline in a dose dependent manner. CONCLUSION: Human proinsulin gene was transfected successfully and expressed efficiently in 293 cells, and the expression was modulated by tetracycline and its derivatives, improving the accuracy, safety, and reliability for the gene therapy of diabetes mellitus.