【摘要】 目的 探究C1q肿瘤坏死因子相关蛋白3(CTRP3)调控仙台病毒(SeV)载体过表达Gata4、Mef2c和Tbx5(SeVGMT)重编程心脏纤维母细胞(CFs)为心肌样细胞(iCMs)的效率及作用机制。方法 将CFs分为Control组、NC-Lv组、CTRP3-Lv组、NC-sh组和CTRP3-sh组。使用Lipofectamine 3000试剂将NC-Lv、CTRP3-Lv、NC-sh和CTRP3-sh转染至CFs,转染48 h。然后将Lipofectamine 3000试剂与SeVGMT混合并在室温下孵育48 h,更换培养基后继续培养21 d。显微镜下观察不同培养时间点(0、3、7、14、21 d)的细胞形态。通过细胞免疫荧光、RT-qPCR和Western blot检测不同时间点的心肌特异性蛋白α-MHC、α-actin、cTnT、Cx43、Actc1和Myh6的表达，并计算不同时间点的跳动细胞比例。结果 随着培养时间的延长，各组CFs中α-MHC、α-actin、cTnT、Cx43、Actc1和Myh6的相对荧光强度、mRNA和蛋白水平均显著升高(P<0.05),并且培养14 d时的上述心肌特异性蛋白的表达水平较培养7 d时大幅度升高(P<0.05)。培养3、7、14和21 d时，与NC-Lv组比较，CTRP3-Lv组CFs中α-MHC、α-actin、cTnT、Cx43、Actc1和Myh6的相对荧光强度、mRNA和蛋白水平显著升高(P<0.05)。与NC-sh组比较，CTRP3-sh组CFs中α-MHC、α-actin、cTnT、Cx43、Actc1和Myh6的相对荧光强度、mRNA和蛋白水平均显著降低(P<0.05)。培养7 d时，各组CFs中出现跳动细胞，随着培养时间的延长，各组CFs中的跳动细胞比例显著升高(P<0.05),并且培养14 d时的跳动细胞比例较培养7 d时大幅度升高(P<0.05)。培养7、14和21 d时，与NC-Lv组比较，CTRP3-Lv组CFs中的跳动细胞比例显著升高(P<0.05)。与NC-sh组比较，CTRP3-sh组CFs中的跳动细胞比例显著降低(P<0.05)。结论 CTRP3可增强SeVGMT重编程CFs为iCMs。更多还原

【Abstract】 Objective To investigate the efficiency and mechanism of C1q tumor necrosis factor-related protein 3(CTRP3) on reprogramming of cardiac fibroblasts(CFs) into induced cardiomyocyte-like cells(iCMs) by Sendai virus(SeV) vector overexpressing Gata4, Mef2c and Tbx5(SeVGMT). Methods CFs were divided into Control, NC-Lv, CTRP3-Lv, NC-sh, and CTRP3-sh groups. NC-Lv, CTRP3-Lv, NC-sh, and CTRP3-sh were transfected into CFs using Lipofectamine 3000 reagent for 48 hours. Lipofectamine 3000 reagent was then mixed with SeVGMT and incubated at room temperature for 48 hours, the culture medium was then replaced, and cells were cultured for 21 days. Cell morphology was observed under a microscope at 0, 3, 7, 14, and 21 days. Expression levels of the myocardial-specific proteins α-myosin heavy chain(α-MHC), α-actin, cardiac troponin T(cTnT), connexin 43(Cx43), cardiac muscle α-actin(Actc1), and myosin heavy chain 6(Myh6) were detected at different time points by immunofluorescence, quantitative reverse transcription-polymerase chain reaction, and Western blot, and the proportions of beating cells at different time points were calculated. Results The relative fluorescence intensity and mRNA and protein levels of α-MHC, α-actin, cTnT, Cx43, Actc1, and Myh6 in CFs in each group increased with increasing culture time(P<0.05), with significantly higher expression levels of myocardial-specific proteins at 14 days of culture than at 7 days(P<0.05). The relative fluorescence intensities and mRNA and protein levels of α-MHC, α-actin, cTnT, Cx43, Actc1, and Myh6 in CFs at 3, 7, 14, and 21 days of culture were significantly increased in the CTRP3-Lv group compared with the NC-Lv group(P<0.05), but were significantly decreased in CFs in the CTRP3-sh group compared with the NC-sh group(P<0.05). Beating cells appeared in CFs in each group at 7 days of culture. The proportion of beating cells in each group increased with increasing culture time(P<0.05), and the proportion was significantly higher at 14 days than at 7 days(P<0.05). The proportion of beating cells among CFs was increased in the CTRP3-Lv group at 7, 14, and 21 days of culture compared with the NC-Lv group(P<0.05), while the proportion of beating cells in the CTRP3-sh group was decreased compared with the NC-sh group(P<0.05). Conclusions CTRP3 can enhance SeVGMT reprogramming of CFs into iCMs.更多还原