Resistin基因过表达对大鼠胰岛β细胞株RINm5F基础胰岛素分泌的影响

Effect of Overexpression Resistin Gene on Insulin Secretion of Insulinoma β-Cell RINm5F in Rats

目的探讨Resistin基因过表达对大鼠胰岛β细胞株RINm5F基础胰岛素分泌的影响。方法体外培养大鼠胰岛β细胞株RINm5F,采用PCR法扩增大鼠Resistin基因全编码区cDNA片段,BamH和Xho双酶切后插入pcDNA3.1B表达载体,经测序验证正确后,采用脂质体转染pcDNA3.1-Resistin真核表达质粒入RINm5F细胞,筛选稳定表达株,并采用RT-PCR验证过表达Resistin的效率。在RINm5F细胞密度达到80%,缓冲液洗3遍,继续于缓冲液中生长2 h,收集上清液,采用ELISA法检测细胞培养上清中的胰岛素水平。同时Trizol一步法提取RINm5F细胞总RNA,采用RT-PCR法检测葡萄糖转运子2(Glut2)mRNA水平,并用紫外吸收剂凝胶密度扫描仪分析条带吸光度值。采用SPSS11.0软件进行统计学分析。结果观察过表达Resistin的RINm5F中ResistinmRNA表达,发现过表达Resistin的细胞株中Resistin mRNA水平为对照组的2.46倍(P<0.001)。过表达Resistin的RINm5F细胞的基础胰岛素分泌低于转染空载质粒的细胞,较对照组下降约32%(P<0.001)。过表达Resistin的RINm5F细胞中Glut2表达明显低于转染空载质粒的细胞,较对照组下降约38%(P<0.05)。结论Resistin基因过表达显著下调胰岛β细胞RINm5F的基础胰岛素分泌,其机制可能与Glut2有关。

Objective To explore the effect of overexpression Resistin gene on insulin secretion of insulinoma β- cell RINm5F in rats. Methods Full - length rat Resistin were amplified by PCR contained BamH I and Xho I sites. PCR products were then subeloned into the eukaryotic expression vector pcDNA3. 1Myc/HisB. The pcDNA3. 1 - Resistin vectors were each transfeeted separately into RINm5F cells using Lipofectamine. The neomycin （G418） added to the medium to select for transfected cells. The reverse transcriptase - polymerase chain reaction（ RT- PCR） was used to verify the different Resistin mRNA level in Resistin transfected cells and controls. Samples were collected and centrifuged to remove any cells that might had detached from the culture flasks. The basal insulin secretion from RINm5F cells was assessed by ELISA kit. Total RNA was isolated from adipose tissue with Trizol. The glucose transporter 2 （Glut2） mRNA level was assessed by RT - PCR. Results Resistin mRNA level was upregulated in resistin transfected group compared with the controls,confirmed that the overexpression rate was effective （ Resistin mRNA level was 2.46 fold over control group P 〈 0. 001 ）. Compared with the controls, Resistin transfection down -regulted the supernatant insulin, confirmed that Resistin impaired the basal insulin secretion in RINm5F cells （the basal insulin secretion in RINm5F cells was attenuated by 32% by overexpression Resistin P 〈 0. 001 ）. The Glut2, key regulator of insulin secretion in β-cells was down - regulated by overexpression Resistin （The Glut2 mRNA level was attenuated by 38% by overexpression Resistin P 〈0.05 ）. Conclusion Resistin impaired the basal insulin secretion in insulinoma cell RINm5F of rats. The down - regulated Glut2 mRNA level mac involve in the Resistin impaired the insulin secrtion.