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目的探讨脂酰肌3激酶-蛋白激酶B-雷帕霉素靶蛋白(PI3K/AKT/mTOR)在华支睾吸虫感染小鼠致肝纤维化作用。方法 6周龄雌性C3H/HeN小鼠随机分为正常对照组和华支睾吸虫感染组,感染组每只小鼠经口灌胃45个华支睾吸虫囊蚴,对照组每只小鼠经口灌入等量的生理盐水,感染28d后取出小鼠肝脏组织,Masson染色,观察病理变化;免疫组织化学染色检测肝脏组织中α-SMA和p-mTOR蛋白的定位与表达;实时荧光定量PCR(qRT-PCR)检测小鼠肝脏组织中α-SMA、COL1和TGF-βmRNA表达水平;Western blot法检测小鼠肝脏组织中TGF-β、α-SMA、pPI3K、p-AKT和p-mTOR的蛋白水平。结果成功建立小鼠肝纤维化模型。与对照组相比,感染组小鼠肝脏的胶原沉积显著增加,α-SMA及p-mTOR阳性细胞数量显著增多(t=-8.374,P<0.01;t=-9.545,P<0.01),α-SMA、COL1、TGF-βmRNA(t=-3.553,P<0.01;t=-3.448,P<0.01;t=-3.052,P<0.01)以及α-SMA、TGF-β、p-PI3K、p-AKT和p-mTOR的蛋白表达水平显著升高(t=-7.076,P<0.01;t=-5.054,P<0.01;t=-6.941,P<0.01;t=-8.123,P<0.01;t=-7.445,P<0.01)。结论 PI3K/AKT/mTOR在华支睾吸虫感染小鼠肝脏高度表达,因此推测该信号通路在华支睾吸虫感染致肝纤维化中发挥重要作用。
Objective To investigate the effect of PI3K / AKT / mTOR on hepatic fibrosis induced by Clonorchis sinensis in mice. Methods Six-week-old female C3H / HeN mice were randomly divided into normal control group and Clonorchis sinensis infection group. Each mouse in the infected group was orally administered with 45 Clonorchis sinensis metacercaria, The rats were sacrificed and the same amount of saline was infused. After 28 days of infection, the liver tissues of mice were removed and stained with Masson to observe the pathological changes. The localization and expression of α-SMA and p-mTOR protein in liver tissues were detected by immunohistochemical staining. The expression of α-SMA, COL1 and TGF-βmRNA in the liver of mice were detected by qRT-PCR. The expressions of TGF-β, α-SMA, pPI3K, p-AKT and p-mTOR Protein level. Results Mouse hepatic fibrosis model was successfully established. Compared with the control group, collagen deposition in the liver of infected mice increased significantly, the number of α-SMA and p-mTOR positive cells increased significantly (t = -8.374, P <0.01; t = -9.545, P <0.01) The expression of α-SMA, TGF-β, p-PI3K, p-STAT3, The protein expression levels of -AKT and p-mTOR were significantly increased (t = -7.076, P <0.01; t = -5.054, P <0.01; t = -6.941, t = -7.445, P <0.01). Conclusions PI3K / AKT / mTOR is highly expressed in the liver of Clonorchis sinensis infected mice, suggesting that this signaling pathway plays an important role in hepatic fibrosis induced by Clonorchis sinensis infection.