Liu Hong-Sheng, Zhao Xiao-Dong, Su Qin, Wang Qiong, Yao Yong-Ming
Sichuan Da Xue Xue Bao Yi Xue Ban. 2014 Jul;45(4):557-62.
To evaluate expression of PAPP-A, IGF-I and the effect of TNF-alpha, IL-1beta on the cytological functions of hCASMCs with IGF-I gene silencing after inflammatory factor, in order to further study on the action of IGF axis hormone in the rupture of astable atheroxclerosis plaque.
A RNA interference (RNAi) aimed at the gene of IGF-I was carried out to have an eukaryon transfection to hCASMCs. When the IGF- I-shRNA-hCASMC were treated by TNF-alpha, IL-1beta, IGFBP4, the expression of PAPP-A and IGF-I were detected with Western blot and ELISA. And then, the effect of TNF-alpha, IL-1beta, IGFBP4 on the proliferation of IGF-I-shRNA- hCASMC were assessed by MTT assay and changing in cell cycle and apoptosis were evaluated by using flow cytometry.
Significant positive expression of PAPP-A in hCASMCs which were treated by TNF-alpha + IL-1beta or TNF-alpha + IL-1beta + IGFBP4 in Blank control (CON), Negative control (NC) and RNAi group were observed, but lower expression in the RNAi group than that in CON and NC groups. However, there was no positive expression of PAPP-A in hCASMCs of CON, NC, RNAi group treating without TNF-alpha + IL-1beta or TNF-alpha + IL-1beta + IGFBP4. The expression of IGF-I in hCASMCs of CON, NC, RNAi group treated with TNF-alpha + IL-1beta + IGFBP4 were greater than that only with or without TNF-alpha + IL-1beta treatment. In RNAi group, the A570 decreased when the hCASMCs treated with TNF-alpha + IL-1beta + IGFBP4 and significant lower than that in hCASMCs treated with TNF-alpha + IL-1beta. When the hCASMCs were treated with TNF-alpha + IL-1beta or TNF-alpha + IL-1beta + IGFBP4, the rate of apoptosis significantly increased in RNAi group, which was significantly higher than that in CON group and NC group. In addition, in RNAi group, the rate of apoptosis in hCASMCs treated with TNF-alpha + IL-1beta + IGFBP4 was significant higher than that in hCASMCs treated only with TNF-alpha + IL-1beta.
When the IGF-I-shRNA-hCASMCs were stimulated by some inflammation factors, its proliferation decreased but the apoptosis enhanced. So the activated IGF-I level in local microebvironnment increased, which may cause the descend of cell proliferation and the increasing of apoptosis.
评估妊娠相关血浆蛋白-A（PAPP-A）、胰岛素样生长因子-I（IGF-I）的表达以及肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）对炎症因子作用后IGF-I基因沉默的人冠状动脉平滑肌细胞（hCASMCs）细胞学功能的影响，以进一步研究IGF轴激素在不稳定动脉粥样硬化斑块破裂中的作用。
针对IGF-I基因进行RNA干扰（RNAi），对hCASMCs进行真核转染。当用TNF-α、IL-1β、胰岛素样生长因子结合蛋白4（IGFBP4）处理IGF-I短发夹RNA-hCASMCs时，采用蛋白质免疫印迹法和酶联免疫吸附测定法检测PAPP-A和IGF-I的表达。然后，采用MTT法评估TNF-α、IL-1β、IGFBP4对IGF-I短发夹RNA-hCASMCs增殖的影响，并使用流式细胞术评估细胞周期和细胞凋亡的变化。
在空白对照（CON）组、阴性对照（NC）组和RNAi组中，用TNF-α + IL-1β或TNF-α + IL-1β + IGFBP4处理的hCASMCs中观察到PAPP-A有显著阳性表达，但RNAi组中的表达低于CON组和NC组。然而，在未用TNF-α + IL-1β或TNF-α + IL-1β + IGFBP4处理的CON组、NC组、RNAi组的hCASMCs中未观察到PAPP-A的阳性表达。用TNF-α + IL-1β + IGFBP4处理的CON组、NC组、RNAi组的hCASMCs中IGF-I的表达高于仅用或不用TNF-α + IL-1β处理的情况。在RNAi组中，当用TNF-α + IL-1β + IGFBP4处理hCASMCs时，A570降低，且显著低于用TNF-α + IL-1β处理的hCASMCs。当用TNF-α + IL-1β或TNF-α + IL-1β + IGFBP4处理hCASMCs时，RNAi组的细胞凋亡率显著增加，显著高于CON组和NC组。此外，在RNAi组中，用TNF-α + IL-1β + IGFBP4处理的hCASMCs的细胞凋亡率显著高于仅用TNF-α + IL-1β处理的hCASMCs。
当IGF-I短发夹RNA-hCASMCs受到某些炎症因子刺激时，其增殖减少但细胞凋亡增强。因此，局部微环境中活化的IGF-I水平升高，这可能导致细胞增殖下降和细胞凋亡增加。
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