目的探讨沉默转录因子7类似物2(TCF7L2)对胰岛素抵抗(IR)Hep G2细胞胰岛素降解酶(IDE)表达的调控作用及可能机制。方法将Hep G2细胞分为空白组、TCF7L2干扰组、空载体组、IR组、IR+TCF7L2干扰组、IR+空载体组。采用高浓度胰岛素(5×...目的探讨沉默转录因子7类似物2(TCF7L2)对胰岛素抵抗(IR)Hep G2细胞胰岛素降解酶(IDE)表达的调控作用及可能机制。方法将Hep G2细胞分为空白组、TCF7L2干扰组、空载体组、IR组、IR+TCF7L2干扰组、IR+空载体组。采用高浓度胰岛素(5×10-6mol/L)持续作用24h诱导IR模型(IR-Hep G2细胞)生成。以人TCF7L2 m RNA编码序列为干扰靶点构建TCF7L2特异性小干扰RNA慢病毒载体(LV-TCF7L2-si RNA)转染空白组及IR组细胞,空载体病毒转染空载体组及IR+空载体组细胞。q RT-PCR法检测各组细胞TCF7L2及IDE m RNA的表达,Western blotting检测各组细胞TCF7L2、IDE、胰岛素刺激后蛋白激酶B(AKT)、磷酸化蛋白激酶B(p-AKT)蛋白表达的变化,流式细胞术检测各组2-脱氧-D-葡萄糖(2-NBDG)荧光葡萄糖摄取率。结果与空白组比较,IR组细胞葡萄糖消耗量及2-NBDG摄取率均明显降低(P<0.01),证明IR细胞模型建立成功。q RT-PCR及Western blotting结果显示,IR组TCF7L2及IDE m RNA种蛋白表达水平均明显低于空白组(P<0.05),TCF7L2干扰组TCF7L2、IDE m RNA和蛋白表达水平较空白组、空载体组明显下降,IR+TCF7L2干扰组TCF7L2、IDE m RNA和蛋白表达水平较IR组、IR+空载体组均明显下降(P<0.05)。生理剂量胰岛素刺激后,IR组、IR+TCF7L2干扰组p-AKT蛋白水平较空白组明显下降(P<0.01),各组总AKT水平差异无统计学意义。TCF7L2干扰组2-NBDG荧光葡萄糖摄取率较空白组和空载体组明显下降,IR+TCF7L2干扰组2-NBDG荧光葡萄糖摄取率较IR组、IR+空载体组明显下降(P<0.01)。结论 TCF7L2联合IDE致肝细胞IR,其机制可能与减少胰岛素信号通路关键酶p-AKT蛋白的表达有关。展开更多
Aim To evaluate the inhibitory effect of chitosan-cysteine conjugate onenzymatic degradation and hypogly-cemic enhancement effect of insulin. Methods Chitosan-cysteineconjugate was synthesized. The protective effect o...Aim To evaluate the inhibitory effect of chitosan-cysteine conjugate onenzymatic degradation and hypogly-cemic enhancement effect of insulin. Methods Chitosan-cysteineconjugate was synthesized. The protective effect of the conjugate against degradation of insulin byα-chymotrypsin and trypsin was evaluated in vitro. Insulin enteric- microspheres were prepared byusing O_1 /Q_2 emulsion solvent evaporation method. The hypoglycemic enhancement effect of theconjugate was studied by oral administration of insulin solution or enteric-microspheres to rats.Results The thiol group content of the synthesized conjugate was about 200 μmol·g^(-1) polymer,which showed a strong protective effect on insulin from enzymatic degradation in vitro. Almost allthe insulin incubated in a-chymotrypsin solution or trypsin solution without chitosan-cysteineconjugate was degraded entirely within 1 h and 5 h respectively, whereas above 75% of insulinremained in the same content of the enzymatic solution containing 4 mg·mL^(-1) conjugate. The drugloading of insulin enteric-microspheres was about 7% . In vivo experiment, chitosan-cysteineconjugate (85 μg·kg^(-1)) prolonged the hypoglycemic time of insulin solution orenteric-microspheres when administered simultaneously with the absorption enhancer SNAC. ConclusionChitosan-cysteine conjugate has a marked inhibitory effect on the enzymatic degradation of insulinin vitro, and it displays a significant hypoglycemic enhancement effect on insulin oral formulationin vivo.展开更多
文摘目的探讨沉默转录因子7类似物2(TCF7L2)对胰岛素抵抗(IR)Hep G2细胞胰岛素降解酶(IDE)表达的调控作用及可能机制。方法将Hep G2细胞分为空白组、TCF7L2干扰组、空载体组、IR组、IR+TCF7L2干扰组、IR+空载体组。采用高浓度胰岛素(5×10-6mol/L)持续作用24h诱导IR模型(IR-Hep G2细胞)生成。以人TCF7L2 m RNA编码序列为干扰靶点构建TCF7L2特异性小干扰RNA慢病毒载体(LV-TCF7L2-si RNA)转染空白组及IR组细胞,空载体病毒转染空载体组及IR+空载体组细胞。q RT-PCR法检测各组细胞TCF7L2及IDE m RNA的表达,Western blotting检测各组细胞TCF7L2、IDE、胰岛素刺激后蛋白激酶B(AKT)、磷酸化蛋白激酶B(p-AKT)蛋白表达的变化,流式细胞术检测各组2-脱氧-D-葡萄糖(2-NBDG)荧光葡萄糖摄取率。结果与空白组比较,IR组细胞葡萄糖消耗量及2-NBDG摄取率均明显降低(P<0.01),证明IR细胞模型建立成功。q RT-PCR及Western blotting结果显示,IR组TCF7L2及IDE m RNA种蛋白表达水平均明显低于空白组(P<0.05),TCF7L2干扰组TCF7L2、IDE m RNA和蛋白表达水平较空白组、空载体组明显下降,IR+TCF7L2干扰组TCF7L2、IDE m RNA和蛋白表达水平较IR组、IR+空载体组均明显下降(P<0.05)。生理剂量胰岛素刺激后,IR组、IR+TCF7L2干扰组p-AKT蛋白水平较空白组明显下降(P<0.01),各组总AKT水平差异无统计学意义。TCF7L2干扰组2-NBDG荧光葡萄糖摄取率较空白组和空载体组明显下降,IR+TCF7L2干扰组2-NBDG荧光葡萄糖摄取率较IR组、IR+空载体组明显下降(P<0.01)。结论 TCF7L2联合IDE致肝细胞IR,其机制可能与减少胰岛素信号通路关键酶p-AKT蛋白的表达有关。
文摘Aim To evaluate the inhibitory effect of chitosan-cysteine conjugate onenzymatic degradation and hypogly-cemic enhancement effect of insulin. Methods Chitosan-cysteineconjugate was synthesized. The protective effect of the conjugate against degradation of insulin byα-chymotrypsin and trypsin was evaluated in vitro. Insulin enteric- microspheres were prepared byusing O_1 /Q_2 emulsion solvent evaporation method. The hypoglycemic enhancement effect of theconjugate was studied by oral administration of insulin solution or enteric-microspheres to rats.Results The thiol group content of the synthesized conjugate was about 200 μmol·g^(-1) polymer,which showed a strong protective effect on insulin from enzymatic degradation in vitro. Almost allthe insulin incubated in a-chymotrypsin solution or trypsin solution without chitosan-cysteineconjugate was degraded entirely within 1 h and 5 h respectively, whereas above 75% of insulinremained in the same content of the enzymatic solution containing 4 mg·mL^(-1) conjugate. The drugloading of insulin enteric-microspheres was about 7% . In vivo experiment, chitosan-cysteineconjugate (85 μg·kg^(-1)) prolonged the hypoglycemic time of insulin solution orenteric-microspheres when administered simultaneously with the absorption enhancer SNAC. ConclusionChitosan-cysteine conjugate has a marked inhibitory effect on the enzymatic degradation of insulinin vitro, and it displays a significant hypoglycemic enhancement effect on insulin oral formulationin vivo.
