【作者】 李烨；

【导师】 邓存良；

【作者基本信息】 泸州医学院 ， 内科学， 2011， 硕士

【摘要】 目的：观察不同浓度的灵芪蠲肝胶囊含药血清对血小板衍化生长因子(platelet-derived growth factor, PDGF)诱导的大鼠肝星状细胞-T6(hepatic stellate cell-T6, HSC-T6)增殖和两面神激酶-2(janus kinase-2, JAK-2)、信号传递与转录激活因子-3(signal transducer and activator of transcription-3, STAT-3)、组织金属蛋白酶抑制剂-1(tissue inhibitor of metalloproteinase-1, TIMP-1)蛋白量表达的影响,探讨其可能的抗肝纤维化机制。方法：1.制备含药血清：SD大鼠25只,随机分为5组,每组5只,分别用生理盐水(A组,10ml/kg)、复方鳖甲软肝片药液(B组,1.5g/kg)、灵芪蠲肝胶囊药液小剂量(C1组,2.125g/kg)、中剂量(C2组,4.25g/kg)、大剂量(C3组,8.5g/kg)灌胃7天,经腹主动脉采血,静置、离心、灭活、过滤,取得含药血清。2.将各组含药血清按200ml/L分别作用于lOng/ml PDGF刺激的HSC-T6,24、48、72h后用CCK-8法测定各组HSC-T6的吸光度值并绘制生长曲线,透射电镜观察各组细胞超微结构的变化,免疫印迹法检测24h时各组细胞中JAK-2、STAT-3和TIMP-1蛋白量的表达。3.统计学处理：计量资料以均数±标准差((?)±s)表示,各组间比较用单因素方差分析,多样本均数两两比较用SNK-q检验,应用SPSS16.0统计软件进行统计学分析。检验水准：双侧a=0.05,P<0.05表示有统计学意义。结果：1.对HSC-T6增殖的影响：共培养24h时,与A组比较,B、C2、C3各组药物血清均可明显抑制HSC-T6的增殖,有显著统计学差异(均P<0.01)；C1组无抑制作用(P>0.05)；B、C3组较C2组抑制明显,有统计学差异(均P<0.05)；B、C3组间抑制无统计学差异(P>0.05)。48h时,与A组比较,B、C1、C2、C3各组药物血清均可抑制HSC-T6的增殖,有统计学差异(均P<0.05)；B、C3组较C1、C2组抑制明显,有统计学差异(均P<0.05)；B、C3组间抑制无统计学差异(P>0.05)；C1、C2组间抑制无统计学差异(P>0.05)；72h时,与A组比较,B、C1、C2、C3各组药物血清均可抑制HSC-T6的增殖,有统计学差异(均P<0.05)；但B、C1、C2、C3组各组间抑制均无统计学差异(P>0.05)。C1、C2、C3各组药物血清在各时间点对HSC-T6的抑制作用随血清中药物浓度增加呈逐渐增强趋势。2.透射电镜下HSC-T6超微结构的改变：共培养24h后,A组细胞的细胞核形态正常,核膜结构清晰,染色质分布正常,细胞器丰富,可见大脂滴。B、C1、C2、C3组可见大量不同程度的坏死和凋亡细胞,其细胞体积变小,核膜消失,胞质内空泡增多,细胞器较少,细胞核染色质固缩,形态不规则,可见核碎裂。同一时间点,上述改变在B、C3组最明显,C2组次之,C1组最轻；且培养时间越长,改变越显著。3.共培养24h时JAK-2、STAT-3、TIMP-1蛋白表达量的变化：免疫印迹法显示,与A组相比,B、C1、C2、C3组大鼠HSC-T6中JAK-2、STAT-3和TIMP-1蛋白表达量均明显减少(均p<0.01);C1组JAK-2、STAT-3、TIMP-1蛋白表达量均较B组高,有显著统计学差异(均P<0.01)；C2组JAK-2、STAT-3、TIMP-1蛋白表达量均较B组低,但无统计学差异(均P>0.05)；C3组JAK-2、TIMP-1蛋白表达量较B、C2组均降低,有显著统计学差异(均P<0.01), STAT-3蛋白表达量较B、C2组降低,但无统计学差异(均P>0.05)。结论：1.灵芪蠲肝胶囊可以抑制PDGF诱导的HSC-T6增殖,且作用具有剂量依赖性；2.灵芪蠲肝胶囊能降低JAK-2、STAT-3在活化的HSC-T6中的表达,从而阻断JAK/STAT信号通路的传导,发挥抗肝纤维化作用；3.灵芪蠲肝胶囊能降低TIMP-1在活化的HSC-T6中的表达,从而减少其对基质金属蛋白酶(matrix metalloproteinases, MMPs)的抑制,实现抗肝纤维化作用。更多还原

【Abstract】 Ojective:To investigate the effects of Ling Qi Juan Gan Capsule drug-containing serum at different concentrations on the proliferation and the expression of janus kinase-2 (JAK-2), signal transducer and activator of transcription-3 (STAT-3) and tissue inhibitor of metalloproteinase-1 (TIMP-1) proteins in hepatic stellate cell-T6 (HSC-T6) induced by platelet-derived growth factor (PDGF). Methods:1. Drug-containing serum preparation:25 SD rats were randomly divided into 5 groups with 5 rats in each group. The drug-containing serum was obtained by intragastric administration to the SD rats respectively with physiological saline (lOml/kg, group A), Fufang Biejia Ruangan Tablet solution (1.5g/kg, group B), low dose (2.125g/kg, group C1), middle dose (4.25g/kg, group C2) and high dose (8.5g/kg, group C3) of Ling Qi Juan Gan Capsule solution.2. HSC-T6 induced by 10ng/ml PDGF was incubated at 200ml/L of the each group drug-containing serum. After 24,48,72 hours, the proliferation of HSC-T6 was measured by Cell Counting Kit-8 (CCK-8) and the growth curve was drew. The HSC-T6 ultrastructure changes were observed by transmission electron microscopy. After 24 hours, the expression of JAK-2, STAT-3 and TIMP-1 of HSC-T6 in each group was measured by western blot.3. Statistic Analysis: Measurement data was expressed by x±s. Multiple groups comparisons were made by one-way ANOVA analysis with Student-Newman-Keuls post hoc analysis. Significance level:Statistical significance was accepted at P<0.05. Results:1. Inhibiting proliferation of HSC-T6:Compared with group A, groups B, C2, C3 had a significantly higher proliferation inhibition rate at 24 hours (all P<0.01), while group C1 had no proliferation inhibition rate (P>0.05). The HSC-T6 proliferation inhibition rate of groups B and C3 were higher than that of group C2 (P>0.05), but there was no difference between B and C3 (P>0.05). Compared with group A, the other four groups B, C1, C2, C3 had a higher proliferation inhibition rate at 48 hours (P<0.05). The HSC-T6 proliferation inhibition rate of groups B and C3 were higher than that of groups C1 and C2 (P>0.05), but neither groups B and C3 nor C1 and C2 had difference (P>0.05). Compared with group A, the other four groups B, C1, C2, C3 had a higher proliferation inhibition rate at 72 hours (P<0.05), but there was no difference between each two groups (P>0.05). With increasing concentration of the Ling Qi Juan Gan Capsule drug-containingl serum, proliferation inhibition rate for HSC-T6 increased.2. Transmission electron microscopy showed:After 24 hours, there were no significant abnormalities of HSC-T6 indicated in group A. The shape of the nucleus was normal with clear nuclear membrane. The chromatin was in normal distribution. Abundant organelles and the lipid droplet could be seen. While in groups B,C1,C2 and C3, the varying degrees of necrotic and apoptotic cells were there. The cell volume was smaller and the nuclear membrane disappeared, the vacuoles in cytoplasm increased, the organelles were less, the chromatin became pyknotic and irregular, karyorrhexis was visible. These changes were more obvious in groups B and C3 than others at the each same time point and aggravated as the time proceeded.3. The expression of JAK-2, STAT-3 and TIMP-1 was detected by western blot after 24 hours:Compared with group A, JAK-2, STAT-3 and TIMP-1 in the other four groups were significantly decreased (all P<0.01). The expression of JAK-2, STAT-3 and TIMP-1 in group C1 was increased (P<0.01), while that was decreased in group C2 compared with group B,but there was no significant difference (P>0.05). JAK-2, TIMP-1 and STAT-3 in group C3 were all decreased compared with group B or C2, but the former two had a significant difference (P<0.01), and the latter didn’t have (P>0.05). Conclusion:1. Ling Qi Juan Gan Capsule can inhibit the proliferation of HSC-T6 with a dose-dependent manner.2. Ling Qi Juan Gan Capsule can decrease the expression of JAK-2 and STAT-3 in activated HSC, and thus block JAK/STAT signal transduction pathway.3. Ling Qi Juan Gan Capsule can decrease the expression of TIMP-1 in activated HSC, alleviate the inhibition to MMPs. So Ling Qi Juan Gan Capsule had therapeutic effect on hepatic fibrosis.更多还原