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目的探讨由虫草多糖、苦杏仁苷、绞股蓝总皂苷组成的“CKJ方”药物血清对大鼠原代肝星状细胞(primary hepatic stellate cells,r HSCs)活化的影响及相关药理作用机制。方法分离并培养r HSCs,将培养4天的r HSCs分为正常组、模型组及CKJ组。模型组及CKJ组以2.5 ng/m L转化生长因子β1(TGF-β1)刺激造模24 h,正常组以不含TGF-β1的基质液(无血清的DMEM)作为对照。药物组以5%CKJ方药物血清继续孵育24 h,正常组及模型组以5%空白血清孵育24 h。采用细胞高内涵筛选技术(HCS)免疫荧光法及Western blot检测r HSCsα-平滑肌肌动蛋白(α-SMA)表达;实时荧光定量聚合酶链式反应(qRT-PCR)检测α-SMA、Ⅰ型胶原蛋白(Col-Ⅰ)、血小板源性生长因子β受体(PDGF-βR)、TGF-β1、转化生长因子β受体1(TGF-βR1)、转化生长因子β受体2(TGF-βR2)mRNA的表达水平。结果与正常组比较,模型组r HSCsα-SMA蛋白表达及α-SMA、Col-Ⅰ、PDGF-βR、TGF-β1、TGF-βR1、TGF-βR2 mRNA表达水平明显升高(P<0.05,P<0.01);与模型组比较,5%CKJ药物血清组r HSCsα-SMA蛋白表达及α-SMA、Col-Ⅰ、PDGF-βR、TGF-β1、TGF-βR1、TGF-βR2 mRNA表达水平明显降低(P<0.05,P<0.01)。结论组分复方“CKJ”药物血清可降低大鼠r HSCsα-SMA的蛋白表达,其主要作用机理可能与抑制TGF-β1及其相关受体有关。
OBJECTIVE: To investigate the effect of “CKJ” drug serum composed of Cordyceps polysaccharide, amygdalin and gypenosides on the activation of rat primary hepatic stellate cells (r HSCs) and related pharmacological mechanisms. Methods The r HSCs were isolated and cultured. The r HSCs cultured for 4 days were divided into normal group, model group and CKJ group. The model group and the CKJ group were stimulated with 2.5 ng / ml transforming growth factor-β1 (TGF-β1) for 24 hours. The normal group was treated with TGF-β1-free matrix fluid (serum-free DMEM). The drug group was incubated with serum of 5% CKJ for 24 hours, and normal group and model group were incubated with 5% blank serum for 24 hours. The expression of α-smooth muscle actin (α-SMA) in HSCs was detected by HCS immunofluorescence and Western blot. The expressions of α-SMA and α-SMA were detected by qRT-PCR and real-time fluorescence quantitative polymerase chain reaction Collagen-1, PDGF-βR, TGF-β1, TGF-βR1, TGF-βR2 ) mRNA expression level. Results Compared with the normal group, the expressions of α-SMA, α-SMA, Col-Ⅰ, PDGF-βR, TGF-β1, TGF-βR1 and TGF-βR2 mRNA in model group were significantly increased (P <0.05, P <0.01). Compared with the model group, the expression of α-SMA, α-SMA, Col-Ⅰ, PDGF-βR, TGF-β1, TGF-βR1 and TGF- (P <0.05, P <0.01). Conclusion The compound “CKJ” serum can decrease the protein expression of r HSCs α-SMA in rats. The main mechanism may be related to the inhibition of TGF-β1 and its related receptors.