Tet-on基因表达系统调控HIF-1α表达对肝癌细胞凋亡的影响

目的探讨HIF-1α表达在体外对肝癌细胞凋亡的影响。方法利用Tet—on基因表达系统调控HePG2细胞HIF-1α的表达，观察细胞凋亡。结果酶切和DNA测序证实Tet—on基因表达系统反应质粒P^tre-hif-1α构建成功。获得了受强力霉素调控、稳定表达HIF-1α的肝癌细胞。经阿霉素诱导凋亡后，强力霉素终浓度为0μg／ml、0．02μg／ml、0．2μg／ml、1μg／ml、2μg／ml、5μg／ml时的细胞凋亡率分别为59．6％、50．9％、38．1％、30．5％、23．9％、18．3％；强力霉素0．02μg／ml、0．2μg／ml、1μg／ml、2μg／ml、5μg／ml处理细胞后，Casepase-3活性分别降低了2．3、5．5、8．7、12．6、18．8倍。RT—PCR检测结果显示，随着强力霉素浓度的增加，HIF-1α表达水平增加，Survivin和Bcl-2基因表达强度逐渐增加，差异有统计学意义（P〈0．001）。结论HIF-1α基因在体外可以抑制肝癌细胞凋亡，而且随着HIF-1α表达水平的增加，对肝癌细胞的凋亡抑制作用进一步加强，可能与HIF-1α诱导survivin和Bcl-2的表达及抑制casepase-3的活性有关。     

The Effect of HBx Gene on the Apoptosis of Hepatic Cells

To study the effect of HBx gene on the apoptosis of the cell lines (L02, HepG2) and the interaction between HBx and X-linked inhibitor of apoptosis protein (XIAP), the apoptosis of pcDNA3.1-HBx transiently transfected cell lines (L02, HepG2) was detected by flow cytometry and the mRNA expression of XIAP was assayed by real-time RT-PCR. Our study showed (1) the mor- phology of L02/pcDNA3.1-HBx was changed and the appearance of the cells mimicked that of HepG2 cells; (2) HBx gene could be detected in L02/pcDNA3.1-HBx and HepG2/ pcDNA3.1-HBx; (3) the apoptosis rate of L02/pcDNA 3.1-HBx was higher than that of L02 cells (P<0.01) and the apoptosis rate of HepG2/pcDNA3.1-HBx was lower than that of HepG2 cells (P<0.05); (4) the XIAP expression in L02 was about 3 times that in L02/pcDNA3.1-HBx cells (P<0.01), and the expression of XIAP in HepG2/pcDNA3.1-HBx was about 4 times that in HepG2 (P<0.01). It is concluded that HBx gene may promote the apoptosis of normal hepatocytes and inhibit the apoptosis of cells of he- patic carcinoma by regulating the expression of XIAP.     

碘油羟基磷灰石纳米微粒对兔VX2肝肿瘤细胞凋亡的影响及机制探讨

目的探讨碘油羟基磷灰石纳米微粒(nHAP)经肝动脉治疗对兔VX2肝肿瘤细胞凋亡的影响及机制。方法100只新西兰白兔肝内肿瘤种植后2周,随机分为5组,每组20只,经肝动脉灌注给药,实验设生理盐水组(A组)、nHAP组(B组)、单纯碘油组(C组)、阿霉素碘油组(D组)及碘油nHAP组(E组)。治疗2周后,流式细胞术检测肝肿瘤组织中细胞凋亡,并利用Western blot检测凋亡抑制相关蛋白XIAP及Survivin的表达。结果流式细胞术检测肝肿瘤组织细胞凋亡结果显示,A组和B组比较无明显差异,C组和D组残余肿瘤区域细胞凋亡率较A组升高(P0.05),而E组细胞凋亡率较C、D两组明显升高(P0.05)。Westernblot结果提示与A组相比,C组和D组残余肿瘤区的XIAP和Survivin表达降低(P0.05),E组XIAP和Survivin蛋白表达较C、D两组降低(P0.05)。结论碘油羟基磷灰石纳米微粒可进一步促进肝脏肿瘤细胞凋亡,这种作用可能与碘油nHAP降低凋亡抑制蛋白XIAP和Survivin表达有关。     

纳米颗粒介导的P53联合Rb基因靶向治疗对兔VX2肝转移癌多药耐药的影响及分子机制

目的联合P53及Rb抗癌基因,通过纳米基因靶向治疗技术,探索其对肝转移癌灶多药耐药的影响及其分子机制。方法以超液化碘油及多聚赖氨酸修饰的羟基磷灰石纳米颗粒乳剂为载体,将含有野生型P53、Rb基因的重组表达质粒经肝动脉共同或者分别转运至兔VX2肝转移癌灶局部,采用蛋白质印迹法及原位激光扫描共焦显微镜确定P53及Rb共表达蛋白在转移灶的表达。通过检测肿瘤对化疗药物丝裂霉素(MMC)、5-氟尿嘧啶(5-FU)和阿霉素(ADM)的敏感性来评价疗效。最后应用实时反转录聚合酶链反应(real time RT-PCR)及电化学发光法(ECL)蛋白杂交技术检测多药耐药蛋白(PGP),多药耐药相关蛋白1(MRP1),肺耐药蛋白(LRP),乳腺癌耐药蛋白(BCRP)的表达差异。结果 (1)与对照碘油组(A)和nanoplex/lipiodol组(B)相比,nanoplex-p53/lipiodol组(C),nanoplex-Rb/lipiodol组(D),nanoplex-(p53+Rb)/lipiodol组(E)PGP、BCRP、MRP1 mRNA表达均降低(P<0.05)。(2)C组及E组表现出比D组更低的PGP mRNA水平(P<0.05)。(3)E组BCRP和LRP表达水平低于C、D组(P<0.05)。(4)C、E组PGP蛋白表达显著低于A组,仅E组BCRP和LRP蛋白表达显著低于A组,C、D、E组MRP1蛋白表达均低于A组(P<0.05)。(5)C、D、E组表现出比A组更高的对化疗药ADM和MMC的敏感性;D、E组比A组更高的对化疗药5-FU的敏感性(P<0.05)。结论以Pll-n HAP为载体的P53基因协同Rb基因的纳米靶向治疗,通过调节肿瘤细胞多重耐药机制中重要基因的表达,增强了其对化疗药的敏感性。P53基因联合Rb基因的纳米基因靶向治疗有可能成为针对肝转移灶多药耐药的有效治疗方法。     

靶向药物联合应用对肝癌细胞SK-Hep-1增殖的影响及其机制

目的 探讨靶向药物联合应用对多驱动基因调控增殖的肝癌细胞SK-Hep-1的影响及作用机制。方法 利用对肝癌细胞SK-Hep-1敏感的靶向药物（HG6-64-1、dasatinib、crizotinib、sunitinib）绘制单靶点动力学分析曲线和双相分析曲线,对SK-Hep-1细胞进行动力学分析;Western blot法检测这些靶向药物对SK-Hep-1细胞关键信号通路的影响;MTT法检测这些药物单用和联合应用对SK-Hep-1细胞增殖的影响。结果 与单靶点动力学分析曲线比较,双相分析曲线可更好拟合靶向药物对肝癌细胞SK-Hep-1的影响,并且预测出HG6-64-1、dasatinib和MK-2206三药联合应用能有效抑制SK-Hep-1细胞增殖。结论 双相动力学分析可以更好地定量描述多驱动增殖的肝癌细胞SK-Hep-1对靶向治疗的反应,联合使用HG6-64-1、dasatinib和MK-2206是治疗肝癌潜在的药物组合。     

免疫细胞在特发性肺纤维化发病机制中的作用研究进展

特发性肺纤维化是极具破坏性,与年龄相关且病因不明的一种疾病,因此可选择的治疗方案十分有限。研究显示,固有免疫细胞和适应性免疫细胞在特发性肺纤维化发病过程中均发挥着十分重要的作用。在这里,我们总结并讨论了免疫细胞在特发性肺纤维化疾病中所扮演的角色,同时归纳了一些针对特发性肺纤维化的免疫治疗手段。     

ABC转运蛋白在大鼠肝脏卵圆细胞中的表达

目的研究ABC转运蛋白基因家族的三个主要成员MDR1、MRP1和Bcrp1基因在大鼠肝脏卵圆细胞中的表达及意义。方法建立大鼠2-乙酰氨基芴／三分之二肝切除模型，两步胶原酶灌注结合Percoll密度梯度离心分离大鼠肝脏卯圆细胞和肝细胞，采用免疫组织化学染色检测大鼠肝脏组织中MDR1、MRP1、Bcrp1转运蛋白的表达；采用荧光定量PCR方法检测MDR1、MRP1和Bcrp1基因mRNA在卵圆细胞和肝细胞中的表达水平。结果免疫组织化学染色显示大鼠肝脏组织中MDR1表达位于门静脉区附近，呈放射状分布，Bcrp1表达定位在细胞膜上。大鼠肝脏卵圆细胞MDR1、MRP1和Bcrp1基因mRNA的表达水平分别是肝细胞的9倍、1．5倍和13．8倍。结论卵圆细胞表达高水平的ABC转运蛋白，后者参与卵圆细胞免受外源性化学物质损伤的自我保护机制。     

碘油羟基磷灰石纳米粒对兔VX2肝肿瘤生长及血管新生的作用

目的 观察碘油羟基磷灰石纳米粒(nHAP)经肝动脉灌注对兔VX2肝肿瘤生长、微血管密度(MVD)及血管内皮生长因子(VEGF)表达的影响.方法 100只新西兰白兔肝内肿瘤种植后2周,随机分为5组,每组20只,兔上腹正中开腹,胃十二指肠动脉插管固定,经肝动脉灌注给药,实验设生理盐水组(A组)、nHAP组(B组)、单纯碘油组(C组)、阿霉素碘油组(D组)及碘油nHAP组(E组).治疗后1周及2周,采用CT检测并计算肿瘤的生长率,治疗后2周,病理观察肿瘤坏死率,免疫组化方法测定瘤区的MVD及VEGF表达强度.记录各组实验兔治疗后的存活期.结果 治疗后1、2周,碘油nHAP组肿瘤体积及生长率明显小于其它各组(均P<0.05).治疗后2周,碘油nHAP组肿瘤坏死率大于单纯碘油组及阿霉素碘油组(均P<0.05),分别为(80.8±12.5)%、(68.2±10.4)%、(75.3±11.6)%.单纯碘油组及阿霉素碘油组栓塞后,残余肿瘤区的MVD升高,两者分别为(33.6±7.3)和(34.9±7.7),高于生理盐水组的(22.6±6.5)(均P<0.05);两组的VEGF表达强度分别为(0.184±0.018)和(0.180±0.017),高于生理盐水组的(0.140±0.008)(均P<0.05);碘油nHAP组残余瘤区的MVD减低,VEGF表达减弱,两者分别为(16.5±3.6)和(0.104±0.003).和其它组相比,碘油nHAP组治疗后兔的生存期明显延长(均P<0.05).结论 碘油nHAP可抑制肿瘤的生长,增加肿瘤的坏死率,抑制肿瘤血管新生,降低栓塞后残瘤VEGF表达,延长瘤兔的生存时间.     

超声引导射频消融治疗肝癌的一些难点及其解决方案研究进展

原发性肝癌是世界范围内最多见且高死亡率的恶性肿瘤之一,全世界每年新发肝癌超过150万例,其中约45%在我国大陆地区[1-2].由于大多数患者仅在肿瘤晚期才表现出症状,使早期诊断和建立在其基础上的手术彻底根治无法开展.     

The Roles of Four Multi-drug Resistance Proteins in Hepatocellular Carcinoma Multidrug Resistance

The roles of multi-drug resistance protein 1 (MDR1), multi-drug resistance related pro- tein 1 (MRP1), lung resistance protein (LRP) and breast cancer resistance protein (BCRP) in the multi-drug resistance (MDR) of hepatocellular carcinoma (HCC) were studied. By exposing HepG2 cell line to progressively increased concentrations of adriamycin (ADM), HepG2 multi-drug resistant subline (HepG2/ADM) was induced. The MDR index of HepG2/ADM was detected by using MTT. The expressions of the four MDR proteins in the three cell lines (L02, HepG2, HepG2/ADM) were investigated at mRNA and protein levels by real-time RT-PCR and Western blot respectively. Our re- sults showed that when the ADM concentration was under 100 μg/L, HepG2 could easily be induced to be drug-resistant. The IC50 of the HepG2/ADM to ADM was 282 times that of HepG2. The expres- sion of MDR1 and BCRP mRNA in HepG2/ADM cells were 400 and 9 times that of HepG2 cells re- spectively while there was no difference in the mRNA expressions of MRP1 and LRP. There was no difference between HepG2 and L02 cells in the mRNA expressions of the four genes. At the protein level, the expressions of MDR1, BCRP and LRP but MRP1 in HepG2/ADM were significantly higher than those of HepG2 and L02. Between HepG2 and L02, there was no difference in the ex- pressions of four genes at the protein level. HepG2/ADM is a good model for the study of MDR. The four genes are probably the normally expressed gene in liver. The expressions of MDR1 and BCRP could be up-regulated by anti-cancer agents in vitro. The MDR of HCC was mainly due to the up-regulation of MDR1 and BCRP but MRP1 and LRP. These findings suggest they may serve as targets for the reversal of MDR of HCC.