Bcl-2核酶对SMMC7721细胞的促凋亡机制

目的 观察Bel-2核酶对SMMC-7721细胞的作用,探讨bcl-2抑制细胞凋亡的机制。方法 经脂质体介导的方法将PMTr-neo(正向Bcl-2核酶真核表达载体)导入SMMC 7721细胞中．细胞克隆转移扩大培养后,采用TUNEL,TRAP结合ELISA流式细胞仪,免疫组化技术检测SMMC 7721／PMTr-neo细胞增殖及细胞凋亡。结果 较对照组SMMC 7721／PMTr-neo细胞bcl-2表达水平显著下降,伴有显著细胞凋亡现象及端粒酶活性下降,结论 Bcl-2核酶可促进SMMC 7721细胞发生凋亡,并降低细胞端粒酶活性,为反义技术在肝癌治疗中应用提供理论依据。     

人端粒酶逆转录酶干扰增强肿瘤坏死因子相关的凋亡诱导配体诱导肝癌细胞凋亡的机制

目的探讨人端粒酶逆转录酶(hTERT)干扰增加肿瘤坏死因子相关的凋亡诱导配体(TRAIL)诱导肝癌HepG2和SMMC 7721细胞凋亡的分子机制。方法采用膜联蛋白V/碘化丙锭染色的流式细胞术方法检测细胞凋亡;采用Western blot方法检测凋亡相关蛋白Procaspase-8、9、-3及Bax、Bcl-2和hTERT表达;采用端粒重复扩增法和端粒数量和长度测定法检测端粒酶活陛和端粒长度。结果hTERT干扰显著增加TRAIL诱导的肝癌细胞凋亡。100 ng/ml TRAIL作用24 h后,HepG2细胞凋亡率由5.53%增加至10.35%;SMMC 7721细胞凋亡率由14.73%增加至77.24%。hTERT干扰明显增加Procaspase-8、-9和Bcl-2表达,显著降低Bax表达,明显促进TRAIL作用后Procaspase-8、-9、-3活化,并且hTERT干扰后端粒酶活性显著降低,端粒长度明显缩短,然而对照细胞与未转染细胞相比各指标均无明显变化。结论hTERT干扰明显增加TRAIL诱导的肝癌细胞凋亡,其机制可能与Procaspase-8、-9表达增加,端粒酶活性降低和端粒长度缩短有关,而与Bcl-2和Bax表达无关。     

c-myc 调节人端粒酶逆转录酶启动子活性的体外研究

目的研究c-myc对人端粒酶逆转录酶(hTERT)启动子的调节作用.方法采用Lipofect脂质体转染法将DNA质粒分别转染至肿瘤细胞、猴肾COS-7细胞和NIH3T3细胞,孵育48 h后,分别检测其报告基因萤虫素酶活性.结果载有hTERT 800 bp启动子的质粒TERTLuc(800)在肿瘤细胞HepG2中活性显著增强,比对照质粒pXP高11.7倍,而在正常细胞3T3中未见表达.外源性转录因子c-myc可显著上调hTERT启动子的表达,其激活作用与剂量呈正相关.人工突变c-myc结合位点明显降低hTERT启动子的活性,下调作用达46%;外源性c-myc对其结合位点突变的hTERT启动子无明显上调作用.结论 c-myc可直接激活hTERT启动子的表达,是调节端粒酶活性的重要转录因子.     

短发夹状RNA抑制survivin基因在肝癌细胞中的表达

目的 构建抗凋亡因子survivin的短发夹状RNA(shRNA)，观察其在肝癌细胞株HepG2、SMMC—7721中对survivin基因表达的抑制作用。方法 设计，合成两对survivin编码基因的反向重复序列，中间分别间隔4和8个nt，分别定向克隆至载体pTZU6 1的U6转录启动子下，构建pshRNA—survivin1和pshRNA—survivin2重组质粒，与表达survivin基因的质粒pEGFP—Cl—survivin共转染肝癌细胞株HepG2和SMMC—7721，荧光显微镜下观察融合蛋白中GFP的表达以分析pshRNA—survivin对survivin基因表达的抑制作用。结果 酶切分析和测序证实pshRNA—survivin1和pshRNA—survivin2构建成功；两组shRNA对survivin的表达均有明显的抑制作用，抑制率达80％以上；两重组质粒在HepG2和SMMC—7721两种细胞中抑制目的基因的作用无明显差异；反向重复序列中间隔4或8个nt构建的shRNA，对抑制目的基因的表达无显著差异。结论 构建的pshRNA—survivin重组质粒能有效抑制survivin基因在肝癌细胞株HepG2、SMMC—7721中的表达。     

重组人肿瘤坏死因子调节端粒酶活性的体外研究

目的：研究重组人肿瘤坏死因子（rhTNF)对端粒酶活性的调节作用,方法：将不同浓度rhTNF加入肝癌细胞株HepG2和HepG1-6细胞,以TRAP－ELISA方法测定其端粒酶活性,采用Liofect脂质体转染法将携带端粒酶催化亚单位一端酶逆转录酶（hTERT)启动力质粒转染至肝癌细胞株HepG2细胞,2h后加入不同浓度的rhTNF,孵育48h后,分别检测其报告基因萤虫素酶活性,结果：在10－1000IU／ml浓度范围内,rhTNF可抑制端粒酶活性,同时在浓度范围内,rhTNF可显著抑hTERT启动子的表达,其抑制作用与rhTNF剂量呈正相关,结论：rhTNF杀伤肿瘤细胞的机制可能与其抑hTERT的表达有关。     

茜草蒽醌对SMMC-7721肝癌细胞的抑制作用及分子机制

目的探讨茜草提取物蒽醌单体对人SMMC-7721肝癌细胞的抑制作用及分子机制。方法将茜草蒽醌作用于体外培养的人肝癌SMMC-7721细胞，采用MTY法检测其对肝癌细胞生长的抑制作用，应用TRAP-PAGE银染半定量法和RT—PCR半定量法检测端粒酶活性及端粒酶亚单位即端粒酶相关蛋白1（TP1）基因、端粒酶RNA（hTR）基因、端粒酶催化亚单位（hTERT）基因变化。结果茜草蒽醌能抑制肝癌细胞的生长，呈现与浓度、时间的依赖趋势；降低端粒酶活性，使hTERT基因的表达量明显下降（P〈0．01），而hTR、TP1基因的表达无显著变化（P〉0．05）。结论茜草蒽醌能显著抑制肝癌细胞的生长并抑制端粒酶的活性，可能与其下调hTERT基因的表达有关。     

丙型肝炎病毒核心区蛋白对 HepG2细胞周期、细胞凋亡和细胞端粒酶活性的影响

目的　研究丙型肝炎病毒核心区 (HCV C)蛋白对肝癌细胞HepG2细胞周期、细胞凋亡和细胞端粒酶活性的影响。方法　首先运用基因重组技术构建含有HCV C基因的真核表达质粒pcDNA3.1( ) ,然后利用脂质体介导将重组真核表达质粒转染HepG2 ,经G4 18筛选获得稳定转染HepG2细胞 (HCV C转染HepG2细胞 ) ,经逆转录 聚合酶链反应技术 (RT PCR)和间接免疫荧光法证实其中有HCV C蛋白表达。然后进行如下实验 :( 1)利用四甲基偶氮唑蓝比色 (MTT)法检测HCV C转染HepG2细胞、空白质粒转染HepG2细胞和未转染HepG2细胞的生长增殖率 ;经流式细胞术(FACS)检测 3组细胞的细胞周期 ;( 2 )经流式细胞术检测细胞凋亡率 ;( 3)经端粒重复扩增 酶联免疫吸附试验 (TRAP ELISA)法检测上述 3组细胞端粒酶活性表达情况。结果　 ( 1)HCV C转染HepG2细胞增殖率显著高于空白质粒转染HepG2和未转染HepG2细胞增殖率 ;HCV C转染HepG2细胞S期所占百分率高于未转染HepG2细胞S期所占百分率 ;( 2 )HCV C转染HepG2细胞凋亡率显著低于无HCV C转染细胞凋亡率 ;( 3)上述 3组细胞端粒酶活性之间差异无显著性。结论　 ( 1)HCV C蛋白具有抑制细胞凋亡的作用 ;( 2 )HCV C蛋白促进HepG2从G0 /1期进入S期 ,从而可能促进细胞生长增殖 ,抑制细胞凋亡 ;( 3)HCV     

端粒酶逆转录酶基因反义寡核苷酸抑制肺癌细胞端粒酶活性和诱导细胞凋亡

目的 研究端粒酶逆转录酶（hTERT)基因反义寡核苷酸（ASODN）对肺癌细胞端粒酶活性和细胞凋亡的影响。方法 实验分为ASODN组、正义寡核苷酸（SODN）组和空白对照组,所用ASODN和SODN的浓度分别为10μmol/L,脂质体为16mg/L,采用端粒重复扩增法、逆转录－聚合酶链反应、Western Blot及流式细胞术分别观察各组端粒酶活性、hTERP mRNA和蛋白质表达以及细胞凋亡。结果 ASODN组显著下调或抑制肺癌细胞端粒酶活性和hTERT表达,但直到第21天才出现细胞凋亡增多。结论 端粒酶活性与hTERT表达密切相关。     

生存素反义核酸诱导肝癌细胞株SMMC-7721凋亡和增加其对抗肿瘤药物敏感性的研究

背景：生存素是凋亡抑制蛋白家族的重要成员，生存素基因有可能成为肿瘤反义基因治疗的理想靶基因。目的：研究生存素反义核酸诱导肝癌细胞株SMMC-7721凋亡和增加其对常用抗肿瘤药物敏感性的作用，探讨生存素反义核酸用于肿瘤基因治疗的可能性。方法：应用基因重组技术构建pEGFP-C1-生存素反义核酸重组质粒，以脂质体转染法转染SMMC．7721细胞，用逆转录聚合酶链反应(RT-PCR)检测生存素mRNA的表达，用流式细胞仪检测细胞凋亡。将生存素反义核酸分别与7种常用抗肿瘤药物共同作用于SMMC-7721细胞，用四唑蓝(MTT)比色法测定细胞杀伤率。结果：生存素反义核酸可抑制SMMC-7721细胞中生存素mRNA的表达，从而导致细胞凋亡增加，其作用呈剂量依赖性。生存素反义核酸可增加SMMC-7721细胞对7种常用抗肿瘤药物的敏感性，明显增强药物的杀伤作用。结论：生存素反义核酸能靶向抑制野生型生存素基因的表达，提高肝癌细胞对常用抗肿瘤药物的敏感性，有可能成为肿瘤基因治疗的新方法。     

Small interfering RNA (siRNA) inhibited human liver cancer cell line SMMC7721 proliferation and tumorigenesis

BACKGROUND/AIMS: The purpose of this study is to understand how AKR1C2 small interfering RNA (siRNA) influences human liver cancer cell line SMMC7721 proliferation and tumorigenesis in vitro and in vivo. METHODOLOGY: We constructed AKR1C2 small interfering RNA (siRNA) expression vector pGenesil-1/AKR1C2 RNAi and then transfected it into liver cancer cell line SMMC7721. We analyzed SMMC7721 cells proliferation by MTT method, cell cycle distribution determined by measuring the cellular DNA content using flow cytometry, colony-formation efficiency, tumorigenesis in vivo. RESULTS: SMMC7721 cells stably transfected with pGenesil-1/AKR1C2 RNAi showed a significant increase in cells of the G0/G1 phase and a significant decrease in cells of the S phase and G2/M phase than in mock cells transfected with pGenesil-1. AKR1C2 siRNA significantly inhibited the cell proliferation (p < 0.01) and resulted in a lower colony-formation efficiency in soft agar P < 0.01), and decreased tumor formation ability in vivo. CONCLUSIONS: AKR1C2 RNAi could lead to alteration of SMMC7721 cell cycle and inhibit turmorigenesis in vivo and in vitro, which strongly suggests that AKR1C2 siRNA might play a critical role in blocking hepatocarcinogenesis.     

Effects of adenovirus-mediated human cyclooxygenase-2 antisense RNA on the growth of hepatocellular carcinoma

AIM: To investigate the relation between the expression of cyclooxygenase-2 (COX-2) and liver cancer, to construct the recombinant adenovirus encoding human COX-2 antisense RNA, and to explore its effects on liver cancer cell proliferation. METHODS: We studied the expression of COX-2 in 34 cases of hepatocellular carcinoma (HCC) and SMMC7402 and SMMC7721 by immunohistochemical technique. Recombinant adenovirus Ad-AShcox-2 was constructed and transfected into human HCC cell lines SMMC7402 and SMMC7721, and its effects on COX-2 expression, cell apoptosis and cell cycle were analyzed by flow cytometry. Cell proliferation was determined by colony-forming efficiency. RESULTS: We observed COX-2 expression in 82.4% of HCC and SMMC7402 cells, but no COX-2 expression in SMMC7721 cells. In addition, recombinant adenovirus encoding antisense COX-2 fragment Ad-AShcox-2 was obtained with the titer of 1.06×1012PFU/mL. Ad-AShcox-2 could reduce the expression of COX-2 and enhance the percentage of cells in G1/G0 phase in SMMC7402 cell line. The difference of apoptotic index between the Ad-AShcox-2 group and control group was statistically significant (tcontrol group=32.62 and tAd-Lacz = 10.93, P<0.001) in SMMC7402 but not in SMMC7721. Similarly, colony-forming rates of SMMC7402 and SMMC7721 cell lines, after the transfer of Ad-AShcox-2, were (2.7±0.94)% and (33.6±4.24)%, respectively. CONCLUSION: Reduction in the expression of COX-2 can inhibit COX-2 expressing HCC cells.     

化疗药对人肝癌细胞SMMC-7721端粒酶活性的影响

目的研究常用化疗药物对人肝癌细胞ＳＭＭＣ７７２１端粒酶活性的影响．方法利用端粒重复扩增实验（ＴＲＡＰ）结合非变性聚丙烯酰胺凝胶电泳银染法,测定ＳＭＭＣ７７２１细胞经过几种化疗药物（顺铂、阿霉素、丝裂霉素、５ＦＵ）不同浓度和时间作用后端粒酶活性的变化．结果当各种药物大剂量处理细胞４ｈ后再祛除药物培养２０ｈ后,顺铂对酶的活性完全抑制,丝裂霉素有部分抑制,阿霉素和５ＦＵ无抑制作用;而未经２０ｈ再培养则无作用;小剂量药物处理细胞５ｄ,其结果同大剂量相似．结论顺铂和丝裂霉素对人肝癌ＳＭＭＣ７７２１细胞端粒酶活性有抑制作用,可能与药物的作用机制有关．     

miR-155在肝细胞癌中的表达及对肝癌细胞增殖的影响

目的:检测微小RNA-155(miR-155)在肝癌组织中的表达并分析其对肝癌细胞增殖和细胞凋亡的影响.方法:采用TagMan MGB探针法荧光定量P C R分析42例原发性肝癌及对应的癌旁组织miR-155的表达;利用miR-155反义寡核苷酸(ASO-miR-155)降低肝癌细胞HepG2和SMMC7721中miR-155的表达;利用MTT比色法检测肝癌细胞增殖的变化,并通过流式细胞技术检测肝癌细胞早期凋亡情况.结果:42例肝癌及癌旁组织标本中,miR-155在52%(22/42)肝癌组织中的表达明显高于癌旁组织(P<0.05);利用脂质体将ASO-miR-155转染肝癌细胞HepG2和SMMC7721后,miR-155的表达明显降低,肝癌细胞HepG2和SMMC7721生长受到明显抑制;并且细胞的早期凋亡明显增加.结论:miR-155在肝癌组织中过表达,降低其表达能明显抑制肝癌细胞的生长并诱导细胞早期凋亡,miR-155有可能成为肝癌治疗的新靶点.     

肿瘤坏死因子相关凋亡诱导配体基因对肝癌细胞杀伤作用及旁观者效应研究

目的 探讨肿瘤坏死因子相关凋亡诱导配体(TRAIL)基因在诱导肝癌细胞凋亡时是否存在旁观者效应，以及旁观者效应的可能机制。方法 构建表达TRAIL基因的双腺病毒载体系统Ad／GT-TRAIL Ad／PGK-GV16，通过293包装细胞产生的病毒上清液将TRAIL基因转入肝癌细胞SMMC7721细胞中，RT-PCR检测TRAIL基因的表达，以MTT法检测细胞生长抑制率，流式细胞仪检测细胞凋亡率；不同比例混合培养SMMC7721／TRAIL和SMMC7721细胞，检测混合细胞生长抑制率来评价TRAIL基因的旁观者效应，并以滤去细胞成分的转染细胞培养液培养未转染细胞，观测可溶性因子在TRAIL基因旁观者效应中的作用。结果 TRAIL基因对SMMC7721细胞的生长抑制率与PBS、LacZ基因和Bax基因比较，差异有显著性(P＜0．05)；SMMC7721细胞的凋亡率，TRAIL基因与PBS、LacZ基因和Bax基因比较，差异有显著性(P＜0．05)；混合细胞的生长抑制率，以SMMC7721／TRAIL占0％时为0，占5％时为15．9％，占25％时为67．0％，占50％时为80．2％，占100％时为87．7％；以PBS对SMMC7721的生长抑制率为0，则滤去细胞成分的转染细胞培养液对SMMC7721的生长抑制率为4％，两者差异无显著性。结论 双腺病毒载体系统介导的TRAIL基因对肝癌细胞有明显的抑制生长和促凋亡作用，并存在旁观者效应，可溶性因子在TRAIL的旁观者效应中不起作用。     

人端粒酶逆转录酶干扰对肿瘤坏死因子相关凋亡诱导配体诱导肝癌细胞凋亡的影响

目的研究人端粒酶逆转录酶(hTERT)干扰对肝癌．HepG2、SMMC-7221细胞生物学形为的影响和对肿瘤坏死因子相关的凋亡诱导配体(TRAIL)诱导凋亡的影响。方法将HepG2细胞和SMMC-7721细胞分为转染组 (转染重组质粒真核表达载体)、对照组(转染空载体质粒)和未转染组。采用聚合酶链反应方法检测hTERT干扰序列, 逆转录聚合酶链反应方法检测hTERT表达,HE染色、生长曲线和流式细胞术方法分别检测细胞形态、增殖情况和细胞周期,β-半乳糖苷酶染色方法检测细胞状态,Armexin V／PI染色流式细胞术检测细胞凋亡。结果转染组细胞内均存在hTERT干扰序列,HepG2和SMMC 7221细胞hTERT干扰率分别为100％和43．3％;与未转染组细胞相比, 转染细胞核质比明显缩小,增殖率下降差异有统计学意义(P<0．05),老化细胞和G2-M期细胞明显增加(P<0．05)。细胞老化率分别由未转染组的0增加到转染组的20．4％,由3．60%,增加到10．O％;G2-M期分别由未转染组的7．1％、6．9％增加到转染组的10．6％、7．9％。hTERT干扰显著增加肝癌细胞凋亡和TRAIL诱导凋亡敏感性(P<0．05)。两株肝癌细胞凋亡率分别由未转染组的3．5％、4．8％增至转染组的5．2%、7．9％;100 ng／ml TRAIL作用24 h后两株肝癌细胞凋亡率分别由未转染组的5．3％、13．9％增加到转染组的10．4％、77．2％,而对照组细胞各指标均无显著变化。结论 hTERT干扰明显影响肝癌细胞的生物学行为,显著增加细胞凋亡和TRAIL诱导凋亡的敏感性。     

Antisense hTERT inhibits thyroid cancer cell growth

Activation of telomerase represents an early step in carcinogenesis. Increased telomerase expression in malignant thyroid tumors suggests that inactivation of telomerase may represent a potential chemotherapeutic target. The purpose of this study was to inhibit the protein component of telomerase, hTERT, in a human thyroid cancer cell line in vitro and in vivo using an antisense strategy. A 235-bp fragment of hTERT cDNA was subcloned, and sense and antisense hTERT expression vectors were constructed. These vectors were transfected into a human thyroid carcinoma cell line (FRO). Tumorigenic potential was determined by cellular growth assay, rate of apoptosis, anchorage-independent growth, and tumor growth in a nude mouse model. Significant down-regulation of hTERT expression was seen in the antisense transfected cells, compared with control and those transfected with the sense vector. A decrease in telomerase activity by TRAP assay was observed in the antisense hTERT cells but not in cells transfected with the sense hTERT construct. Inhibition of cell growth was observed after approximately 20 population doublings in the antisense-hTERT clones and was associated with an increase in the rate of apoptosis and a change in cellular morphology. Moreover, anchorage-independent growth was reduced in vitro, and tumor growth rate was diminished in vivo in the antisense hTERT clones. Inhibition of telomerase activity with antisense hTERT in human thyroid cancer cells is achievable and may represent a novel target to inhibit tumor growth.     

Inhibitory effect of endostatin expressed by human liver carcinoma SMMC7721 on endothelial cell proliferation in vitro

AIM: To construct a stable transfectant of human liver carcinoma cell line SMMC7721 that could secret human endostatin and to explore the effect of human endostatin expressed by the transfectant on endothelial cell proliferation. METHODS: Recombinant retroviral plasmid pLncx-Endo containing the cDNA for human endostatin gene together with rat albumin signal peptide was engineered and transferred into SMMC7721 cell by lipofectamine. After selection with G418, endostatin-transfected SMMC7721 cells were chosen and expanded. Immunohistochemical staining and Western blot were used to detect the expression of human endostatin in transfected SMMC7721 cells and its medium. The conditioned medium of endostatin-transfected and control SMMC7721 cells were collected to cultivate with human umbilical vein endothelial cells for 72 hours. The inhibitory effect of endostatin, expressed by transfected SMMC7721 cells, on endothelial proliferation in vitro was observed by using MTT assay. RESULTS: A 550 bp specific fragment of endostatin gene was detected from the PCR product of endostatin-transfected SMMC7721 cells. Immunohistochemistry and Western blot analysis confirmed the expression and secretion of foreign human endostatin protein by endostatin-transfected SMMC7721 cells. In vitro endothelial proliferation assay showed that 72 hours after cultivation with human umbilical vein endothelial cells, the optical density (OD) in group using the medium from endostatin-transfected SMMC7721 cells was 0.51 +/- 0.06, lower than that from RPMI 1640 group (0.98 +/- 0.09) or that from control plasmid pLncx-transfected SMMC7721 cells (0.88 +/- 0.11). The inhibitory rate for medium from endostatin-transfected SMMC7721 cells was 48%, significantly higher than that from empty plasmid pLncx-transfected SMMC7721 cells (10.2%, P<0.01). CONCLUSION: Human endostatin can be stably expressed by SMMC7721 cell transferred with human endostatin gene and its product can significantly inhibit the proliferation of human umbilical vein endothelial cell in vitro.