【作者】 周乐杜；

【导师】 王志明；

【作者基本信息】 中南大学 ， 外科学， 2010， 博士

【摘要】 目的：通过特异性阻断PI3K和mTOR,观察HepG2和Hep3B细胞株PI3K/Akt/mTOR信号通路活性及生物学行为的改变,探讨相关的分子机制。方法：在培养的HepG2、Hep3B人肝癌细胞株和人正常肝细胞株QSG-7701上,以免疫印迹方法(Western blot)检测各细胞株中PI3K(p110α亚单位)、PTEN、pAkt (S473,T308)和p-mTOR(S2448)的表达情况；分别用PI3K抑制剂LY294002 (50μmol/ml)和mTOR抑制剂Rapamycin (RAPA,50 nmol/ml)孵育HepG2和Hep3B细胞,以MTT比色法检测细胞的增殖能力,以流式细胞术(Flow cytometry)检测细胞周期和凋亡情况,以Western blot法检测细胞中pAkt (S473, T308)和p-mTOR (S2448)的表达改变。结果.PTEN在HepG2和Hep3B细胞中基本无表达,在QSG-7701细胞株中高表达,pAkt和p-mTOR在HepG2和Hep3B细胞中的表达较QSG-7701细胞均显著升高；LY294002和RAPA均呈剂量-时间依赖的抑制HepG2和Hep3B细胞生长。饱和效应浓度的LY294002和RAPA作用24小时后,HepG2和Hep3B细胞均呈现明显的G0/G1期阻滞,处于S期的细胞比例较对照组显著减少(P<0.01)；两给药组中HepG2细胞和Hep3B细胞的凋亡率与对照组比较均显著增加(P<0.01)；两给药组HepG2细胞的凋亡率显著高于Hep3B细胞(P<0.01或P<0.05),并且HepG2细胞的凋亡率在RAPA给药组显著高于LY294002给药组(P＜0.01),但Hep3B细胞的凋亡率在两组间无显著差异。饱和效应浓度的LY294002作用48小时后,HepG2和Hep3B细胞中pAkt (T308, S473)和p-mTOR (S2448)的表达水平较对照组均显著降低(P<0.01),饱和效应浓度的RAPA作用48小时后,HepG2和Hep3B细胞中p-mTOR (S2448)的表达水平较对照组均显著降低(P<0.01),而pAkt (T308, S473)的表达水平较对照组均显著升高(分别P<0.01)。结论：(1)HepG2和Hep3B细胞存在PI3K/Akt/mTOR信号通路的组成性激活；(2)LY294002和RAPA能有效阻断HepG2和Hep3B细胞PI3K/Akt/mTOR信号通路,抑制HCC细胞的生长增殖,诱导细胞周期阻滞,促进细胞凋亡,且阻断效应与HCC细胞P53的表达有关；(3)一定浓度范围内,HepG2细胞对PI3K/Akt/mTOR信号通路阻断剂的敏感性高于Hep3B细胞,RAPA对PI3K/Akt/mTOR信号通路的部分阻断效应优于LY294002。目的：观察阿霉素(Doxorubicin, DOX)单用或与RAPA联合用药对HepG2和Hep3B细胞生物学行为及PI3K/Akt/mTOR信号通路活性的影响,探讨mTOR抑制剂增强HCC化疗药物疗效的相关作用机制。方法：分别取对数生长期的HepG2和Hep3B细胞,以不同浓度的DOX单用或与RAPA (20nmol/ml)联合应用培养48h或24h,以MTT法测定药物对HepG2和Hep3B细胞的增殖抑制作用,以流式细胞技术检测二者的凋亡情况,以Western blot法检测二者P-p70S6K (T389)、P-4E-BP1 (S65)和pS6(S235/236)的表达改变。结果：0.156-2.5 mg/L浓度范围内,DOX能抑制HepG2和Hep3B细胞的增殖,且呈浓度依赖性；DOX在0.625-10mg/L浓度范围内,Hep3B细胞的相对存活率显著大于HepG2细胞(P<0.01)；与DOX单用比较,DOX (0.156-2.5mg/L)与RAPA联用显著降低了HepG2和Hep3B细胞的存活率(P<0.01或P<0.05),DOX (0.156-10mg/L)与RAPA联用,Hep3B细胞存活率显著大于HepG2细胞(P<0.01)DOX (0.156-10mg/L)单用或与RAPA联用24h均可诱导HepG2和Hep3B细胞凋亡发生,且随DOX浓度增加,凋亡率升高；与DOX单用比较,DOX (1.25-10mg/L)与RAPA联用,HepG2细胞凋亡率较显著升高(P<0.01或P<0.05),DOX (2.5-10 mg/L)与RAPA联用,Hep3B细胞凋亡率显著升高(分别P<0.05)；DOX (5.0-1 Omg/L)单用,HepG2细胞凋亡率显著大于Hep3B细胞(分别P<0.05),DOX(2.5-10mg/L)与RAPA联用,HepG2细胞凋亡率显著大于Hep3B细胞(分别P<0.01)。RAPA(20nmol/L)、DOX (2.5mg/L)以及二者联用均可导致HepG2或Hep3B细胞P-p70S6K(T389)、P-4E-BP1(S65)和pS6(S235/236)表达减少,且以DOX与RAPA联用效应最为明显。结论：(1)DOX可抑制HepG2和Hep3B细胞生长增殖,促进细胞凋亡,下调PI3K/Akt/mTOR信号通路的活化程度；(2)DOX与RAPA联合用药能显著抑制HepG2和Hep3B细胞的增殖,促进细胞凋亡,下调PI3K/Akt/mTOR信号通路的活化程度,并在一定浓度范围内表现出协同效应；(3)在一定浓度范围内,HepG2细胞对DOX单用或与RAPA联合用药的敏感性显著高于Hep3B细胞,可能与HCC细胞p53的表达差异有关。目的：分析人HCC组织中PI3K/Akt/mTOR信号通路的活化水平与p53的表达及HCC临床病理参数之间的关系,评估PI3K/Akt/mTOR信号通路在HCC诊断和预后评价中的作用。方法：76例HCC组织样本经临床病理分期分级后,以免疫组织化学方法检测HCC组织和癌旁组织中p53、pAkt、PTEN、p-mTOR和pS6的表达情况,分析上述指标在不同HCC病理特征条件下的阳性表达率,并与正常肝组织比较。结果：p53、pAkt、PTEN、p-mTOR和pS6在人HCC组织有不同程度的表达,HCC组织p53 (60.5%,46/76)、pAkt (92.1%,70/76)、p-mTOR (84.2%,64/76)和pS6(88.2%,67/76)的阳性表达比例较癌旁肝组织(分别为10.5%,8/76、63.2%,48/76、46.1%,35/76、52.6%,40/76)和正常肝组织(分别为0%、55.0%,11/20、50.0%,10/20、45.0%,9/20)显著升高(P<0.01),而PTEN (65.8%,50/76)的阳性表达比例较癌旁肝组织(89.5%,68/76)和正常肝组织(85.0%,17/20)显著减少(P<0.05)；p53阳性表达HCC组织pAkt、p-mTOR和pS6的阳性表达率较p53阴性表达组织显著升高(P<0.01),而PTEN的阳性表达率较p53阴性表达组织显著降低(P<0.01)。低分化、存在血管侵袭、TNM分期高的HCC组织p53、pAkt、p-mTOR和pS6的阳性表达率较相对应的分化较好、无血管侵袭、TNM分期低的HCC组织显著升高(分别P<0.01或P<0.05)；而PTEN的阳性表达率显著降低(分别P<0.01或P<0.05)结论：(1)人HCC组织存在PI3K/Akt/mTOR信号通路的激活；(2)人HCC组织PI3K/Akt/mTOR信号通路的活化程度与p53的表达可能有相关性；(3)人HCC组织PI3K/Akt/mTOR信号通路的活化程度与HCC的临床病理特征有关,活化程度越高的HCC其分化程度越低、血管侵袭多发、TNM分期高。更多还原

【Abstract】 Objective:The purpose of this study is to investigate the molecular mechanisms associated with PI3K/Akt/mTOR pathway in hepatocellular carcinoma (HCC).Method:We firstly observed the expression of PI3K (subunit of p110α), PTEN, pAkt (S473, T308) and p-mTOR (S2448) in HepG2, Hep3B and QSG-7701 by Western Blot analysis; And then, the cell proliferation, cell cycle, apoptosis and expression of pAkt (S473, T308) and p-mTOR (S2448) were determined in HepG2 and Hep3B after incubation with rapamycin (50nmol/ml), the mTOR inhibitor and LY294002 (50 umol/ml), the PI3K inhibitor.Result:There was absence or low of PTEN expression in HepG2 and Hep3B, but it is highly expressed in QSG-7701; The pAkt and p-mTOR levels were significantly higher in HepG2 and Hep3B than in QSG-7701; LY294002 and RAPA inhibit the proliferation of HepG2 and Hep3B in both dose-and time-dependent manner; HepG2 and Hep3B in G0/G1 phase were both inhibited remarkably after treated by saturation concentration of LY294002 and Rapamycin for 24 hours, while the proportion of cells in S phase is largely less than the control group (P<0.01), and the apoptotic rates of HepG2 and Hep3B in both group are significantly higher than that of the control group (P<0.01), the apoptotic rate of HepG2 is obvious higher than that of Hep3B in the both group(P<0.01 or 0.05), and the apoptotic rate of HepG2 treated by saturation concentration of RAPA is obvious higher than that of the HepG2 treated by saturation concentration of LY294002 (P<0.01),but the apoptotic rates of Hep3B between the both group have no significant difference. The expressions of pAkt (T308, S473) and p-mTOR (S2448) in HepG2 and Hep3B both down-regulated significantly compared with the control group (P<0.01) after saturation concentration of LY294002 treatment for 48 hours, while the results of p-mTOR (S2448) expression in both kinds of cells which treated with RAPA for 48 hours were almost the same, but the expression of pAkt (T308, S473) was up-regulated (P<0.01).Conclusion:(1) there is a constitutive activation of PI3K/Akt/mTOR pathway in HCC cells of HepG2 and Hep3B; (2) LY294002 and RAPA could effectively block PI3K/Akt/mTOR pathway in HepG2 and Hep3B, inhibit the growth of HCC cells, induce cell cycle arrest and promote apoptosis, while the effect of blocking is probably related to the expression of P53 in HCC cells; (3) Within certain concentration, HepG2 is more sensitive to inhibitors of PI3K/Akt/mTOR pathway than Hep3B, while RAPA has a more effective impact in partly blocking PI3K/Akt/mTOR pathway than LY294002.Objective:The purpose of this study is to observe the influences of Doxorubicin (DOX) alone or DOX combined with RAPA on the biological actions and the functions of PI3K/Akt/mTOR pathway in HepG2 and Hep3B, and explore the mechanism of mTOR inhibitor which enhancing the effect of chemotherapy on HCC.Method:HepG2 and Hep3B in logarithmic phase were treated with DOX in different concentration alone or with different concentration of DOX combined with RAPA (20 nmol/ml) for 24 or 48 hours respectively. The growth inhibition of HepG2 and Hep3B were assessed by MTT; the apoptosis of both cells were analysed by flow cytometry; and expression changes of P-p20S6K (T389), P-4E-BP1 (S65) and pS6 (S235/236) were assayed by Western blot.Result:Within the concentration of 0.156 to 2.5 mg/L, the proliferation of HepG2 and Hep3B were inhibited by DOX in a dose-dependent manner; within the concentration of 0.625 to 10 mg/L the relative survival rate of Hep3B were higher than HepG2 (P<0.01), the survival rate was decreased significantly when HepG2 was treated with DOX (0.312-5mg/L) plus RAPA compared with DOX alone (P＜0.01 or P<0.05), the survival rate of Hep3B was also decreased after exposure to DOX (2.5-10mg/L) plus RAPA (P＜0.01 or P＜0.05), and the survival rate of Hep3B was largely higher than HepG2 after the treatment with DOX (0.312-5mg/L) plus RAPA (P＜0.01 or P＜0.05). Treat cell HepG2 and Hep3B with DOX(0.156-10mg/L) alone or DOX combined with RAPA for 24 hours could both induce apoptosis in a dose-dependent manner, DOX (1.25-10mg/L) combined with RAPA induces an obvious increase in apoptotic HepG2 cell number compared with DOX alone (P＜0.01 or P＜0.05), DOX (2.5-10mg/L) combined with RAPA induces a significant increase in apoptotic Hep3B cell number compared with DOX alone (P＜0.05), DOX alone causes more apoptotic cell number in cell HepG2 than cell Hep3B(P＜0.05), while DOX (2.5-10mg/L) combined with RAPA induces higher apoptosis rate in cell HepG2 than cell Hep3B (P＜0.01). RAPA (20nmol/ml) alone, DOX (2.5mg/L) alone and both of them could all induce the decreased expression of P-p20S6K (T389), P-4E-BP1(S65) and pS6 (S235/236) in HepG2 and Hep3B, and this effect was most obvious when DOX and RAPA combined.Conclusion:(1) DOX could inhibit cell proliferation, promote apoptosis, and reduce the activation of PI3K/Akt/MTOR pathway in HepG2 and Hep3B; (2) RAPA could enhance the sensitivity of HepG2 and Hep3B to DOX, which evidenced by higher inhibition of cell proliferation, apoptosis rate, and the activation of PI3K/Akt/MTOR pathway; (3) HepG2 appears more sensitive to DOX alone or DOX plus RAPA than Hep3B within certain concentration, which might relate to the differential expression of p53 in HCC cells.Objective:The purpose of this study is to analysis the activation level of PI3K/Akt/MTOR pathway and the expression of p53 in human HCC tissue, and also analysis the connection between them and clinical pathological parameters, and then assesses the value of PI3K/Akt/MTOR pathway in HCC diagnose and prognosis.Method:After classing the pathological stage and grade of HCC tissues of 76 cases, detecting the expression of p53, pAkt, PTEN, p-mTOR and pS6 in HCC tissue and adjacent tissue by immunohistochemistry, and analysis the positive expression frequency of all above indicators in different pathological condition of HCC, while also compare them with the normal hepatic tissue.Result:There were different levels of expression of p53, pAkt, PTEN, p-mTOR and pS6 in human HCC tissue, and the positive expression rates of p53 (60.5%,46/76), pAkt (92.1%,70/76), p-mTOR (84.2%,64/76) and pS6 (88.2%,67/76) in HCC tissue were significant higher than the expression rate in adjacent tissue (10.5%,8/76、 63.2%,48/76、46.1%,35/76、52.6%,40/76) and the rate in normal hepatic tissue (0%、55.0%,11/20、50.0%,10/20、45.0%,9/20) (P<0.01), while the positive expression rate of PTEN (65.8%,50/76) was lower than adjacent tissue (89.%,68/76) and normal hepatic tissue (85.0%,17/20) (P<0.05). The positive expression frequency of pAkt, p-mTOR and pS6 is higher in p53 positive HCC tissue than negative one, but the positive expression frequency of PTEN is greatly lower than the frequency of p53 negative tissue(P<0.01). HCC tissue with lower differentiation, vascular invasion and high stage of TNM demonstrated higher positive expression frequency of p53, pAkt, p-mTOR and pS6 compared with HCC tissue well differentiated, without vascular invasion and low stage of TNM (P＜0.01 or P<0.05).Conclusion:(1) It does exist the activation of PI3K/Akt/MTOR pathway in human HCC; (2) The level of activation of PI3K/Akt/MTOR pathway in human HCC may be related to the expression of p53; (3) The level of activation of PI3K/Akt/mTOR pathway is related to the clinical pathological stage of HCC, the higher activation of HCC cells, appeared to be the poorer differentiation, more vascular invasion and higher stage of TNM.更多还原