全反式维甲酸联合奥沙利铂对人SMMC-7721肝癌细胞株的作用

目的:观察全反式维甲酸(ATRA)联合奥沙利铂(L-OHP)对人SMMC-7721肝癌细胞增殖及凋亡的影响.方法:选用ATRA(10-5mol/L)及不同浓度的L-OHP(10mg/L、20mg/L、40mg/L),并选用10-5mol/L的ATRA分别联合L-OHP(10mg/L、20mg/L、40mg/L)作用于SMMC-7721肝癌细胞24h、48h、72h;采用MTT比色法观察其对SMMC-7721肝癌细胞的生长抑制作用,倒置显微镜下观察细胞形态变化;采用流式细胞术分析药物作用48h时SMMC-7721细胞的周期分布和凋亡的情况.结果:ATRA及不同浓度L-OHP单药及联合均可显著抑制SMMC-7721肝癌细胞的生长,细胞形态改变,凋亡比例增加,并呈剂量-时间依赖性;两药联合较单药相比作用明显增强(P<0.01);ATRA联合20mg/LL-OHP与ATRA联合40mg/LL-OHP相比,作用细胞48h及72h时,对细胞生长抑制作用无统计学差异;两药联合较单药相比,细胞凋亡率明显增加,细胞阻滞S期增强(P<0.01).结论:ATRA可抑制肝癌SMMC-7721细胞的生长并诱导细胞凋亡,与L-OHP联用后作用增强并可减少奥沙利铂用量,具有协同作用.     

干细胞相关基因在肝癌细胞系中的表达

目的 了解Oct4、Sox2、Nanog、SMO、β-Catenin、Wnt5b等干细胞相关基因在4株人肝癌细胞系SMMC-7721、Bel-7402、HepG2、MHCC-97和正常人肝脏细胞系L02中的表达情况,并比较不同细胞系中各基因量的差异和对全反式维甲酸(tRA)的反应.方法 逆转录聚合酶链反应测定4株人肝癌细胞系中Oct4、Sox2、Nanog、SMO、β-Catenin、Wnt5b mRNA的表达,实时荧光定量PCR比较不同细胞中各基因量的差异以及对tRA的反应.单因素方差分析比较各基因在不同细胞系中的表达差异.结果 干细胞相关基因Oct4、Sox2、Nanog、SMO、β-Catenin和Wnt5b在4株人肝癌细胞系SMMC-7721、Bel-7402、HepG2、MHCC-97和正常人肝脏细胞系L02中有不同程度的表达(P<0.05);人肝癌细胞系HepG2和正常人肝脏细胞系L02对tRA的反应也存在明显差异(P<0.05).结论 干细胞相关基因在人肝癌细胞系中不同程度的表达,不同肝癌细胞系中这些基因表达有一定的差异,可能与其生物学特性不同有关,人肝癌细胞系HepG2中Oct4和Sox2的表达调控不同于胚胎干细胞.在肝癌细胞中可能存在着Oct4对于Wnt/β-catenin信号转导通路的调节作用.     

三氧化二砷诱导人肝癌SMMC-7721细胞凋亡及对OPN表达的影响

目的探讨三氧化二砷（As2O3）治疗肝癌的可行性及机制。方法将一定浓度梯度的As2O3与人肝癌细胞株SMMC-7721孵育后,采用MTT法、荧光显微镜及流式细胞仪检测细胞增殖与凋亡变化,逆转录聚合酶链反应（RT-PCR）检测肝癌细胞株中骨桥蛋白基因（OPN mRNA）表达水平。结果As2O3作用后SMMC-7721细胞生长明显受抑,且呈时间-浓度依赖性;荧光显微镜下细胞呈典型的凋亡形态学改变;在流式细胞仪上可见“凋亡峰”,细胞周期阻滞于G2／M期;细胞OPN mRNA表达阳性,OPN mRNA表达水平明显下调。结论As2O3体外能有效抑制肝癌细胞株生长;其机制可能为诱导细胞凋亡、下调OPN mRNA表达。     

肝癌细胞的诱导分化及凋亡的研究

本文把肝癌细胞（SMMC-7721）作为靶细胞采用TRAP银染法、Westernblot法、电镜等方法,分别检测对肝癌细胞的全反式维甲酸（ATRA）的诱导凋亡及奥曲肽的诱导分化作用。ATRA在TRAP银染中未见梯状条带,然而在Westernblot分别于32KD、20KD处显示较强的Caspase-3的表达。提示,ATRA有较强的诱导凋亡的作用。在奥曲肽的刺激下可见肝细胞核浓缩、微绒毛减少、线粒体空泡化等变化,而且促进Caspase-3的表达并使其激活,从而诱导肝癌细胞的分化及凋亡。结果显示,上述两种制剂分别对肝癌细胞具有较强的诱导分化及促进凋亡的作用。     

Expression of angiostatin cDNA in human hepatocellular carcinoma cell line SMMC-7721 and its effect on implanted carcinoma in nude mice

AIM: To transfect murine angiostatin cDNA into human hepatocellular carcinoma cell line SMMC-7721 and to investigate its effects on implanted carcinoma in nude mice. METHODS: A eukaryotic expression vector of pcDNA3.1-mAST containing murine angiostatin was constructed. Then pcDNA3.1-mAST plasmid was transfected into cell line SMMC-7721 by Lipofectamine. The resistant clone was screened by G418 filtration and identified by RT-PCR and Western blotting. Nude mice were divided into three groups of 10 each. Mice in blank control group were only injected with SMMC-7721 cells. Mice in vector control group were injected with SMMC-7721 cells transfected with pcDNA3.1 (+) vector, whereas mice in angiostatin group were injected with SMMC-7721 cells transfected with pcDNA3.1-mAST plasmid. Volume, mass and microvessel density (MVD) of the tumors in different groups were measured and compared. RESULTS: Murine angiostatin cDNA was successfully cloned into the eukaryotic expression vector pcDNA3.1 (+). pcDNA3.1-mAST was successfully transfected into SMMC-7721 cell line and showed stable expression in this cell line. No significant difference was observed in the growth speed of SMMC-7721 cells between groups transfected with and without angiostatin cDNA. Tumor volume, mass and MVD in the angiostatin group were significantly lower than those in the blank control group and vector control group (P<0.01). The inhibitory rate of tumor reached 78.6%. Mass and MVD of the tumors only accounted for 34.6% and 48.9% respectively of those in the blank control group. CONCLUSION: Angiostatin cDNA could be stably expressed in human hepatocellular carcinoma cell line SMMC-7721 without obvious inhibitory effects on the growth of SMMC-7721 cells. When implanted into nude mice, SMMC-7721 cells transfected with angiostatin cDNA show a decreased tumorigenic capability. It suggests that angiostatin can inhibit tumor growth through its inhibition on angiogenesis in tumors.     

黄芩苷对肝癌细胞SMMC-7721 JAK-STAT信号通路STAT3的影响

目的:探讨黄芩苷对肝癌细胞SMMC-7721JAK-STAT信号通路STAT3的影响.方法:将肝癌细胞SMMC-7721分为4组:对照组、黄芩苷组、AG490组、黄芩苷+AG490组.应用RT-PCR法检测各组肝癌细胞SMMC-7721中STAT3mRNA表达,Westernblot法检测肝癌细胞SMMC-7721中STAT3、P-STAT3蛋白表达.结果:黄芩苷可以下调肝癌细胞SMMC-7721STAT3mRNA表达,与对照组比较明显下降(0.505±0.111vs0.697±0.145,P<0.05);并可以降低STAT3蛋白的表达量(0.879±0.012vs1.087±0.015,P<0.05);还可以抑制STAT3向活化形式P-STAT3转化,与对照组比较P-STAT3表达明显下降(0.983±0.085vs1.103±0.074,P<0.05),而与AG490联合应用后P-STAT3蛋白表达量较单用黄芩苷下降明显(0.756±0.103vs0.983±0.085,P<0.05).结论:黄芩苷能下调STAT3mRNA表达水平,降低STAT3蛋白表达,还可以抑制STAT3向活化形式P-STAT3转化,...     

腺病毒介导的环氧合酶-2反义RNA对肝癌细胞株生长的影响

目的探讨环氧合酶-2(COX-2)的表达与肝癌的关系,并构建表达人COX-2反义RNA的腺病毒载体,研究其对人肝癌细胞生长的抑制作用。方法采用免疫组织化学法探讨34例肝癌组织COX-2 的表达与肝癌病理特征的关系。采用基因重组法把人COX-2的cDNA片段反向克隆于穿梭质粒pHCMVSP1A,获得pAd-AShcox-2,通过脂质体与pJM17共转染293细胞,经同源重组产生编码COX-2反义RNA的重组腺病毒--Ad-AShcox-2。经聚合酶链反应法鉴定为阳性克隆者大量扩增、纯化,转染人肝癌细胞株SMMC-7402和SMMC-7721,采用免疫细胞化学、细胞集落形成率及流式细胞术检测其对肝癌细胞生长、凋亡及细胞周期分布的影响。结果34例肝癌组织中有28例COX-2高度表达,阳性率达82.4%; COX-2的表达水平与肝癌的病理分级有关,与甲胎蛋白、细胞类型、有无肝内转移无关。成功构建、扩增、纯化得到编码COX-2反义RNA的重组腺病毒Ad-AShcox-2,滴度达1.06×1012PFU/ml;Ad-AShcox- 2转染两种肝癌细胞株后,发现高度表达COX-2的SMMC-7402 COX-2表达水平明显降低,细胞凋亡率明显增加,出现G1期阻滞,与Ad-LacZ组及空白对照组比较差异有统计学意义(P<0.05);而不表达COX- 2的SMMC-7721变化不明显。细胞集落形成实验显示SMMC-7402细胞集落形成率较低(2.7%±0.94%); 而SMMC-7721     

Signal transduction of gap junctional genes,connexin32, connexin43 in human hepatocarcinogenesis

AIM: To investigate gap junctional intercellular communication (GJIC) in hepatocellular carcinoma cell lines, and signal transduction mechanism of gap junction genes connexin32(cx32),connexin43(cx43) in human hepatocarcinogenesis. METHODS: Scarped loading and dye transfer (SLDT) was employed with Lucifer Yellow (LY) to detect GJIC function in hepatocellular carcinoma cell lines HHCC, SMMC-7721 and normal control liver cell line QZG. After Fluo-3AM loading, laser scanning confocal microscope (LSCM) was used to measure concentrations of intracellular calcium (Ca(2+))i in the cells. The phosphorylation on tyrosine of connexin proteins was examined by immunoblot. RESULTS: SLDT showed that ability of GJIC function was higher in QZG cell than that in HHCC and SMMC-7721 cell lines. By laser scanning confocal microscopy, concentrations of intracellular free calcium (Ca(2+))i was much higher in QZG cell line (108.37 nmol/L) than those in HHCC (35.13 nmol/L) and SMMC-7721 (47.08 nmol/L) cells. Western blot suggested that only QZG cells had unphosphorylated tyrosine in Cx32 protein of 32 ku and Cx43 protein of 43 ku; SMMC-7721 cells showed phosphorylated tyrosine Cx43 protein. CONCLUSION: The results indicated that carcinogenesis and development of human hepatocellular carcinoma related with the abnormal expression of cx genes and disorder of its signal transduction pathway, such as decrease of (Ca(2+))i, post-translation phosphorylation on tyrosine of Cx proteins which led to a dramatic disruption of GJIC.     

Down-modulation of heat shock protein 70 and up-modulation of Caspase-3 during schisandrin B-induced apoptosis in human hepatoma SMMC-7721 cells

AIM: To investigate the effect of schisandrin B (Sch B) on proliferation and apoptosis of human hepatoma SMMC-7721 cells in vitro and regulation of Hsp70 and Caspases-3, 7, 9 expression by Sch B. METHODS: Human hepatoma cell line SMMC-7721 was cultured and treated with Sch B at various concentrations. Growth suppression was detected with MTT colorimetric assay. Cell apoptosis was confirmed by DNA ladder detection and flow cytometric analysis. The expression of Hsp70, Caspases-3, 7, 9 were analyzed by Western blot analysis. RESULTS: Sch B inhibited the growth of hepatoma SMMC-7721 cells in a dose-dependent manner, leading to a 50% decrease in cell number (LC50) value of 23.50 mg/L. Treatment with Sch B resulted in degradation of chromosomal DNA into small internucleosomal fragments, evidenced by the formation of a 180-200 bp DNA ladder on agarose gels. FCM analysis showed the peak areas of subdiploid at the increased concentration of Sch B. The results of Western bolt analysis showed that Hsp70 was down-regulated and Caspase-3 was up-regulated, while the activity of Caspases-7, -9 had no significant change. CONCLUSION: Sch B is able to inhibit the proliferation of human hepatoma SMMC-7721 cells and induce apoptosis, which goes through Caspase-3-dependent and Caspase-9-independent pathway accompanied with the down-regulation of Hsp70 protein expression at an early event.     

Expression of gap junction genes connexin32 and connexin43 mRNAs and proteins, and their role in hepatocarcinogenesis

AIM: To investigate the relationship between hepatocarcinogenesis and the expression of connexin32 (cx32), connexin43 (cx43) mRNAs and proteins in vitro.METHODS: Gap junction genes cx32 and cx43 mRNA in hepatocellular carcinoma cell lines HHCC, SMMC-7721 and normal liver cell line QZG were detected by in situ hybridization (ISH) with digoxin-labeled cx32, and cx43 cDNA probes. Expression of Cx32 and Cx43 proteins in the cell lines was revealed by indirect immuno-fluorescence and flow cytometry (FCM).RESULTS: Blue positive hybridization signals of cx32 and cx43 mRNAs detected by ISH with cx32 and cx43 cDNA probes respectively were located in cytoplasm of cells of HHCC, SMMC-7721 and QZG. No significant difference of either cx32 mRNA or cx43 mRNA was tested among HHCC, SMMC-7721 and QZG (P=2.673, HHCC vs QZG; P=1.375, SMMC-7721 vs QZG). FCM assay showed that the positive rates of Cx32 protein in HHCC, SMMC-7721 and QZG were 0.7%, 1.7% and 99.0%, and the positive rates of Cx43 protein in HHCC, SMMC-7721 and QZG were 7.3%, 26.5% and 99.1% respectively. Significant differences of both Cx32 and Cx43 protein expression existed between hepatocellular carcinoma cell lines and normal liver cell line (P=0.0069, HHCC vs QZG; P=0.0087, SMMC-7721 vs QZG). Moreover, the fluorescent intensities of Cx32 and Cx43 proteins in HHCC, SMMC-7721 were lower than that in QZG.CONCLUSIONS: Hepatocellular carcinoma cell lines HHCC and SMMC-7721 exhibited lower positive rates and fluorescent intensities of Cx32, Cx43 proteins compared with that of normal liver cell line QZG. It is suggested that lower expression of both Cx32 and Cx43 proteins in hepatocellular carcinoma cells could play pivotal roles in the hepatocarcinogenesis. Besides, genetic defects of cx32 and cx43 in post-translational processing should be considered.     

全反式维甲酸对卵巢癌SKOV3细胞增殖、分化的影响

目的 探讨全反式维甲酸（ATRA）对卵巢癌SKOV3细胞增殖、分化的影响.方法 以1×10^-6 mol/L的ATRA处理人卵巢癌SKOV3细胞为实验组,细胞不加ATRA处理为对照组.应用MTT法测定细胞增殖抑制率,流式细胞仪测定细胞周期分布,免疫组化法检测鼠抗人分化相关基因1（NDRG1）表达,显微镜观察细胞形态变化.结果 与对照组比较,实验组细胞增殖抑制率、NDRG1表达升高（P均＜0.05）;随着作用时间延长,实验组G/G0期细胞增多、S期细胞减少,显微镜下可见细胞生长差,增殖缓慢,分裂像少见.结论 ATRA有较强的抑制卵巢癌SKOV3细胞增殖和诱导其分化作用.     

RNA干扰p21对肝癌细胞SMMC-7721增殖的影响

目的:探讨RNA干扰技术沉默p21基因对肝癌细胞SMMC-7721增殖及恶性表型变化的影响.方法:通过慢病毒载体将p21小干扰RNA片段稳定转染入SMMC-7721细胞,通过RT-PCR,Western blot分别检MJp21 Mrna和蛋白表达变化,流式细胞仪检测7721-p2l RNAi组(感染p21 siRNA...     

肝癌细胞蛋白质组在DTT诱导内质网应激条件下的表达

目的:研究内质网应激条件下肝癌细胞SMMC-7721蛋白质组的表达,为人类肝细胞癌的诊治提供新的靶点.方法:培养肝癌细胞株SMMC-7721,随机分为两组,即实验组和对照组.实验组加入二硫苏糖醇(DTT,2.5 mmol/L),对照组加入等量的培养基,裂解细胞,提取全细胞蛋白.双向电泳(2-DE)分离,Image Ma...     

Telomerase inhibition and telomere lossin BEL—7404 humen hepatoma cells treated with doxorubicin

AIM：To study the effects of doxorub icin on telomerase activity and telomere length in hepatocellular carcinoma.METHODS:Telomerase activity was assayed with a non-radioisotopic quantitative telomerase repeat amplification protocal-based method.The effect of doxorubicin(DOX) on the growth of BEL-7404 human hepatoma cells was the growth of BEL-7404human hepatoma cells was determined by microculture tetrazoloum assay.Mean telomere length(terminal restriction fragment)was detected by Southern blot method.The expression of telomerase subunits genes was investigated by RT-PCR,Cell apoptosis and cell cycle distribution were evaluated by flow cytometry.RESULTS:Telomerase activity was inhibited in a dose and time-depandent manner in BEL-7404human hepatoma cells treated with DOXfor24,48or72h in concentrations from 0.156to 2.5μMwhich was crrelated with the inhibition of cell growth.No changes were found in the mRNA expression of three telomerase subunits(hTERT,hTR and TP1)after drug exposure for 72h with indicated concentrations.The cells treated with DOX showed shortened mean telomer length and accumulated at he G2/M phase.However,there was almost no effects on cell apoptosis by DOX.CONCLUSION:The telomerase inhibition an d the telomere shortening by ODXmay contribute to its efficiency in the treatment in hepatocellular carcinoma.     

Knockdown of survivin gene expression by RNAi induces apoptosis in human hepatocellular carcinoma cell line SMMC-7721

AIM: To investigate the survivin gene expression in human hepatocellular carcinoma cell line SMMC-7721 and the effects of survivin gene RNA interference (RNAi) on cell apoptosis and biological behaviors of SMMC-7721 cells. METHODS: Eukaryotic expression vector of survivin gene RNAi and recombinant plasmid pSuppressorNeo-survivin (pSuNeo-SW), were constructed by ligating into the vector, pSupperssorNeo (pSuNeo) digested with restriction enzymes Xba I and Sail and the designed double-chain RNAi primers. A cell model of SMMC-7721 after treatment with RNAi was prepared by transfecting SMMC-7721 cells with the lipofectin transfection method. Strept-avidin-biotin-complex (SABC) immunohistochemical staining and RT-PCR were used to detect survivin gene expressions in SMMC-7721 cells. Flow cytometry was used for the cell cycle analysis. Transmission electron microscopy was performed to determine whether RNAi induced cell apoptosis, and the method of measuring the cell growth curve was utilized to study the growth of SMMC-7721 cells before and after treatment with RNAi. RESULTS: The eukaryotic expression vector of survivin gene RNAi and pSuNeo-SW, were constructed successfully. The expression level of survivin gene in SMMC-7721 cells was observed. After the treatment of RNAi, the expression of survivin gene in SMMC-7721 cells was almost absent, apoptosis index was increased by 15.6%, and the number of cells was decreased in G2/M phase and the cell growth was inhibited. CONCLUSION: RNAi can exert a knockdown of survivin gene expression in SMMC-7721 cells, and induce apoptosis and inhibit the growth of carcinoma cells.     

人参三醇组皂甙诱导分化人肝癌细胞的研究

观察人参三醇组皂甙对人肝癌细胞株SMMC-7721的形态及端粒酶活性的影响，应用光镜、电镜及端粒序列重复扩增银染法。结果发现该药能引起SMMC-7721细胞核浓缩、线粒体空泡化、微绒毛减少。该药没能抑制端粒酶活性，但能诱导分化抑制肝癌细胞的增殖。