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1.
利用计算机辅助设计合成针对人VEGF的发卡状核酶(RZ),定向亚克隆于真核表达载体pcDNA3.1^+中,转染肝癌细胞SMMC-7721,RT—PCR鉴定。ELISA法和MTT法检测SMMC-7721细胞对照组、SMMC-7721/pcDNA3.1^+空载体对照组和SMMC-7721/RZ基因转染组细胞VEGF表达和增殖情况,流式细胞术检测各组细胞周期和凋亡情况。结果为SMMC-7721/RZ基因转染组细胞VEGF表达水平和细胞增殖速率显著低于SMMC-7721细胞对照组和SMMC-7721/pcDNA3.1’空载体对照组。MC-7721/RZ基因转染组细胞出现凋亡峰,其凋亡率达11.O%,而细胞对照和空载体细胞对照均末出现。认为SMMC-7721/RZ转基因细胞株的成功构建和生物学特性的初步研究为进-步建立裸鼠人肝癌模型及评价RZ对体内肝癌生长的抑制作用奠定了-定的基础。  相似文献   

2.
目的构建真核表达载体p BI-EGFP-Bach1,并检测其表达及对血红素氧化物(HO-1)表达的影响。方法将Bach1基因与质粒p BIEGFP连接构成重组质粒,RT-PCR法检测重组载体及HO-1在SMMC7721细胞中的表达情况,MTT检测细胞活性。结果 NheⅠ和MluⅠ双酶切以及PCR表明重组质粒p BI-EGFP-Bach1构建成功;RT-PCR显示转染重组Bach1 mRNA表达显著升高,同时HO-1的表达降低;MTT检测未转染组的IC50为0.15μg/ml,转染组的IC50为0.21μg/ml。结论构建了真核表达载体p BI-EGFP-Bach1,转染后Bach1能够在SMMC-7721细胞中表达并抑制了HO-1;MTT验证重组质粒能提高癌细胞对长春新碱(VCR)敏感性。  相似文献   

3.
目的 构建携带人颗粒溶素(GLS)的真核表达质粒pBudCE4.1/GLS并研究其表达后对肝癌细胞SMMC-7721凋亡、线粒体跨膜电位与细胞色素C转位的影响. 方法 将RT PCR扩增出的人GLS编码基因克隆入pBudCE4.1载体中构建真核表达质粒pBudCE4.1/GLS并将其转染肝癌细胞株SMMC-7721,用RT-PCR、免疫细胞化学检测GLS的表达;用Hoechst法及电镜检测细胞的凋亡状况;用荧光染料MitocaptureTM检测线粒体跨膜电位,用Western blot法检测细胞色素C从线粒体的释放. 结果 成功构建了携带GLS的真核表达质粒pBudCE4.1/GLS,在转染的肝癌细胞株SMMC-7721中,从转录和翻译水半都检测到了目标基因GLS的表达产物;重组质粒转染SMMC-7721细胞后,细胞核染色质固缩,旱致密浓染的凋亡状态,线粒体膜跨电位下降并伴随有细胞色素C从线粒体至细胞质的释放. 结论携带人GLS的真核表达质粒在肝癌细胞SMMC-7721中表达后有致细胞凋亡作用,引起线粒体跨膜电位下降与细胞色素C的释放可能是其诱导肿瘤细胞凋亡的途径之一.  相似文献   

4.
目的构建羧基端缺失30个氨基酸的HBx蛋白的真核表达质粒,并在真核细胞内稳定表达。方法提取HBV-DNA阳性血清总DNA,PCR扩增截短变异的HBx基因(HBx),克隆到真核质粒pEGFP-N3中,酶切及测序鉴定。利用脂质体将重组质粒转染HepG2细胞,通过W estern blot方法检测细胞内截短变异的HBx蛋白的表达。结果构建的重组质粒pEGFP-N3/x经酶切及测序鉴定结果正确。质粒pEGFP-N3/x转染细胞后可检测到目的蛋白。结论成功构建在真核细胞内稳定表达的重组质粒pEGFP-N3/x。  相似文献   

5.
目的构建弓形虫致密颗粒蛋白GRA2的真核表达重组质粒。方法设计合成GRA2引物,运用PCR方法扩增其基因片段,经克隆至pMDl8-T载体后,亚克隆至真核表达质粒pcDNA3.1(-)而构建重组表达质粒pcDNA3.1-GRA2。脂质体法将构建的重组质粒转染HFF细胞,RT—PCR法检测转染细胞中GRA2的表达情况。结果PCR扩增GRA2基因序列正确,构建的重组表达质粒pcDNA3.1-GRA2经PCR、EcoRⅠ/HindⅢ双酶切和测序鉴定正确;转染GRA2基因的细胞,RT—PCR可见目的条带。结论成功获得真核表达重组质粒pcDNA3.1-GRA2,为进一步研究弓形虫疫苗的免疫保护性奠定基础。  相似文献   

6.
目的探讨人剪切修复基因着色性干皮病基因D(XPD)对人脐静脉内皮细胞(HUVEC)的促凋亡作用。方法用脂质体转染法瞬时转染HUVEC,转染重组质粒pEGFP-N2/XPD和空载质粒pEGFP-N2,并用未转染的与重组质粒pEGFP-N2/XPD和空载质粒pEGFP-N2具有相同遗传背景和代数的HUVEC作为空白对照。实验分为3组:正常对照组、pEGFP-N2组和pEGFP-N2/XPD组。用荧光显微镜观察绿色荧光蛋白报告基因表达情况,用流式细胞仪检测细胞凋亡情况,用RT-PCR和Western Blot检测XPD、Bcl-2、Bax和wt-p53表达量的变化,用MTT法观察细胞增殖活力。结果在荧光显微镜下,可在转染了重组质粒pEGFP-N2/XPD或空载质粒pEGFP-N2的细胞中观察到绿色荧光,即转染成功;流式细胞仪结果显示,重组质粒pEGFP-N2/XPD的转染引起细胞凋亡增加(P<0.05或P<0.01);RT-PCR和Western Blot检测发现,重组质粒pEGFP-N2/XPD的转染使得XPD表达增高(P<0.05),同时使得Bcl-2表达降低,Bax和wt-p53表达增高(P<0.05或P<0.01);MTT结果显示,重组质粒pEGFP-N2/XPD的转染抑制细胞增殖活力(P<0.05)。结论 XPD能促进HUVEC凋亡,下调XPD的表达,有望成为治疗动脉粥样硬化的一个新靶点。  相似文献   

7.
目的:观察人剪切修复基因人类着色性干皮病D组基因(xeroderma pigmentosum group D,XPD)转染至人肝癌细胞株SMMC-7721细胞后XPD、DNp73和GADD45β基因的表达变化以及对肝癌细胞生长的影响.方法:实验分4组:重组质粒SMMC-7721-pEGFP-N2-XPD(XPD组)、空载质粒SMMC-7721-pEGFP-N2组(N2组),脂质体组和SMMC-7721细胞空白对照组.应用Lipofectamine2000脂质体瞬时转染,逆转录聚合酶链反应(RT-PCR)和蛋白印迹(Western blot)法检测转XPD基因后,人肝癌细胞株SMMC-7721细胞中DNp73以及GADD45β的mRNA和蛋白质的表达量变化,并用四甲基偶氮唑盐(MTT)法检测细胞增殖的活力,流式细胞仪检测细胞凋亡的变化.结果:荧光显微镜下,XPD组和N2组细胞中观察到绿色荧光蛋白表达,说明转染成功;RT-PCR检测显示:XPD组中DNp73 mRNA相对表达量较其他3组显著下调,XPD和GADD45βmRNA相对表达量较其他3组明显上调(均P<0.01);Western blot检测显示:XPD、DNp73以及GADD45β蛋白相对表达量在各组间的差异与其mRNA各组间差异一致;MTT检测示:SMMC-7721细胞空白对照组、脂质体组、N2组、XPD组的吸光度(A)值分别为0.633±0.012,0.623±0.009,0.628±0.016,0.384±0.011,XPD组低于其他3组,差异均有统计学意义(均P<0.01),表明转染XPD后SMMC-7721细胞的增殖能力减弱.流式细胞仪检测SMMC-7721肝癌细胞凋亡:转染XPD的SMMC-7721细胞凋亡显著,凋亡率达56.53%,而其他3组均未见明显凋亡.结论:XPD基因在肝癌的发生发展中起抑制作用,癌基因DNp73的表达随XPD表达增加而降低,抑癌基因GADD45β则随XPD表达增加而增加,提示两者可能在XPD抑制肝癌细胞的生长机制中起重要作用.  相似文献   

8.
目的探讨血清糖皮质激素调节蛋白激酶3(SGK3)基因过表达对乳腺癌细胞凋亡的影响。方法构建pEGFP-N1-SGK3重组质粒,用载体质粒pEGFP-N1和重组质粒pEGFP-N1-SGK3转染乳腺癌MCF7细胞;MTT法观察转染细胞的增殖情况,流式细胞术分析细胞凋亡情况,RT-PCR检测凋亡相关基因表达。结果利用pEGFP-N1-SGK3质粒转染乳腺癌MCF7细胞,建立表达SGK3蛋白的细胞系;通过细胞增殖实验发现,SGK3的过表达可促进MCF7细胞增殖;流式细胞术分析显示,外源性SGK3可抑制MCF7细胞凋亡的发生,并影响凋亡相关基因bad、bcl-xl的表达。结论 SGK3的过表达可抑制乳腺癌MCF7细胞凋亡。  相似文献   

9.
目的 研究shRNA干扰GRP78对肝癌细胞株SMMC-7721增殖和凋亡的影响。方法 将GRP78特异性shRNA质粒载体Pla-anti-GRP78转染SMMC-7721,采用流式细胞术(FCM)检测转染效率、分析细胞周期分布和凋亡,从蛋白和mRNA水平检测干扰GRP78效果,MTT 法检测干扰GRP78对SMMC-7721增殖的影响。结果Pla-anti-GRP78转染SMMC-7721细胞48h后,GRP78表达下降,空白对照组GRP78 mRNA的表达量为1,无关shRNA组为0.95,而Pla-anti-GRP78组为0.25(P<0.05);转染Pla-anti-GRP78的SMMC-7721细胞G2期阻滞(55.2%,P<0.05);空白对照组凋亡率为6.6%,无关shRNA组为8.1%,而Pla-anti-GRP78组高达58.2%(P<0.05)。 结论 shRNA干扰GRP78表达抑制SMMC-7721的增殖,并诱导其凋亡。  相似文献   

10.
P16真核表达载体的构建及其对肝癌细胞生长的抑制作用   总被引:10,自引:8,他引:2  
目的构建pcDNA3/p16真核表达质粒并了解其对肝癌细胞BEL-7404生长的抑制作用.方法将p16 cDNA亚克隆至pcDNA3真核表达载体上,并经脂质体介导转染至BEL-7404细胞中.应用MTT法和流式细胞仪分析转染细胞生长情况和细胞周期.结果重组pcDNA3/p16表达质粒构建成功.经pcDNA3/p16转染的BEL-7404细胞生长速度受到明显抑制,且细胞多停滞于G0/G1期.结论重组pcDNA3/p16质粒能在BEL-7404细胞内表达,且能抑制BEL-7404细胞的生长.  相似文献   

11.
AIM: To investigate the effects of the WWOX gene on the human hepatic carcinoma cell line SMMC-7721.METHODS: Full-length WWOX cDNA was amplified from normal human liver tissues. Full-length cDNA was subcloned into pEGFP-N1, a eukaryotic expression vector. After introduction of the WWOX gene into cancer cells using liposomes, the WWOX protein level in the cells was detected through Western blotting. Cell growth rates were assessed by methyl thiazolyl tetrazolium (MTT) and colony formation assays. Cell cycle progression and cell apoptosis were measured by flow cytometry. The phosphorylated protein kinase B (AKT) and activated fragments of caspase-9 and caspase-3 were examined by Western blotting analysis.RESULTS: WWOX significantly inhibited cell proliferation, as evaluated by the MTT and colony formation assays. Cells transfected with WWOX showed significantly higher apoptosis ratios when compared with cells transfected with a mock plasmid, and overexpression of WWOX delayed cell cycle progression from G1 to S phase, as measured by flow cytometry. An increase in apoptosis was also indicated by a remarkable activation of caspase-9 and caspase-3 and a dephosphorylation of AKT (Thr308 and Ser473) measured with Western blotting analysis.CONCLUSION: Overexpression of WWOX induces apoptosis and inhibits proliferation of the human hepatic carcinoma cell line SMMC-7721.  相似文献   

12.
13.
AIM:To investigate the growth-inhibiting and apoptosis-inducing effects of the gene MOB2 on human hepatic carcinoma cell line SMMC-7721.METHODS:The full-length cDNA of the MOB2 gene was amplified from human umbilical vein endothelial cells.The correct full-length MOB2 cDNA was subcloned into the eukaryotic expression vector pEGFP-C1.After lipofection of the MOB2 gene into cancer cells,the levels of MOB2 protein in the cancer cells were detected by immunoblotting.To transfect the recombined plasmid vector pEGFP-CI-MOB2 into SMMC-7721 cells,the cells were cultured in Dulbecco's Modified Eagle'sMedium with 10% fetal calf serum and glutamine,and then mixed with liposomes,Lipofectamine 2000 and the plasmid vector pEGFP-CI-MOB2.RESULTS:We observed the growth and proliferation of SMMC-7721 cells containing pEGFP-CI-MOB2 and analyzed their apoptosis and growth cycle phases by flow cytometry.We successfully transfected the recombined plasmid vector pEGFP-CI-MOB2 into SMMC-7721 cells and screened for a single clone cell containing MOB2.After transfection,MOB2 enhanced growth suppression,induced apoptosis,increased the ratio of G0/G1,significantly inhibited the advance of cell cycle phase,and arrested cells in G0/G1 phase.CONCLUSION:MOB2 overexpression induces apoptosis and inhibits the growth of human hepatic cancer cells,which may be useful in gene therapy for hepatic carcinoma.  相似文献   

14.
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.  相似文献   

15.
AIM: To study the function of α-fetoprotein (AFP) in SMMC-7721 hepatoma cells. METHODS: A hairpin siRNA expressing plasmid pSilencer3.0-H1-afp was constructed and transfected into SMMC-7721 cells with Lipofectamine 2000. The expression of AFP was monitored by real-time RT-PCR and immunoassays, its effect on SMMC-7721 cell proliferation and cell death was detected by MTT and fluorescenceactivated cell sorter (FACS). RESULTS: The AFP-siRNA expressing plasmid downregulated the expression of AFP obviously (about 34%), and inhibited SMMC-7721 cell proliferation, but did not induce apoptosis. CONCLUSION: Downregulation of AFP siRNA inhibits proliferation of SMMC-7721 cells, but cannot cause apoptosis.  相似文献   

16.
目的 探讨血管内皮细胞生长因子(VEGF)反义RNA转染人肝癌细胞后对细胞体内外生物学性状的影响。方法 将含正义、反义VEGFcDNA序列的质粒PCMV—VEGF、PCMV—FGEV及空载体质粒pcDNA3.1,在脂质体介导下导入SMMC—7721肝癌细胞,分别称为正义、反义及对照组,并通过G418筛选获得阳性克隆。细胞原位杂交和免疫组织化学方法检测转染后VEGF在肝癌细胞内的表达情况;MTT法和FCM检测转染后细胞在体外的增殖和凋亡情况;并制备裸鼠动物模型,观察转染后细胞的体内生长情况。结果 转染PCMV—FGEV后肝癌细胞内VEGF的转录及其蛋白的表达水平显著下降,但转染后体外细胞的增殖与凋亡情况均无明显变化。转染PCMV—FGEV后细胞在裸鼠体内的生长缓慢,反义组成瘤时间为(25.0±1.8)d,明显长于正义组(15.7±2.5)d和对照组(18.5±2.1)d,F=19.455,P<0.01;而平均瘤重以反义组最轻,为(0.96±0.28)g,F=21.501,P<0.01;同时反义组裸鼠肿瘤细胞发生明显的凋亡。结论 VEGF反义RNA转染人肝癌细胞可抑制肿瘤细胞VEGF的表达,在体外对细胞增殖和凋亡无影响,而体内可显著诱导细胞凋亡并抑制肿瘤生长。  相似文献   

17.
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   总被引:1,自引:0,他引:1  
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  相似文献   

18.
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.  相似文献   

19.
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。  相似文献   

20.
AIM: To construct antisense VEGF(165) eukaryotic expression vector PCDNA(3)-as-VEGF(165) and to study its expression and effect on the proliferation of hepatocarcinoma SMMC-7721 cells. METHODS: VEGF(165) cDNA was inserted into polylinker sites of eukaryotic expression vector PCDNA(3) to construct PCDNA(3)-as-VEGF(165). Then the vector was transferred into human hepatocarcinoma cell strain SMMC-7721 with cation lipofectamine 2000 mediated methods to evaluate the expression of VEGF protein and the inhibitory effect on the proliferation of hepatocarcinoma SMMC-7721 cells. RESULTS: The detection indicated the presence of VEGF cDNA in normally cultured SMMC-7721 cells by PCR. VEGF mRNA expression was notably decreased in SMMC-7721 cells by RT-PCR after PCDNA(3)-as-VEGF(165) transfection. The expression of VEGF protein was dramatically inhibited (142.01+/-7.95 vs 1 625.52+/-64.46 pg/ml(-1), P<0.01) 2 days after transfection, which correlated with the dose of PCDNA(3)-as-VEGF(165)5 gene. VEGF protein was most expressed in PCDNA(3) transferred SMMC-7721 cells but few in PCDNA(3)-as-VEGF(165) transferred cells by immunohistochemical staining. The apoptotic rate of hepatocarcinoma SMMC-7721 cells was significantly promoted (17.98+/-0.86% vs 4.86+/-0.27%, P<0.01) and the survival rate was notably decreased (80.99+/-3.20% vs 93.52+/-3.93%, P<0.05) due to antisense VEGF(165) by flow cytometry (FCM). The transfection of antisense VEGF(165) gene resulted in the inhibitory effect on the proliferation of hepatocarcinoma cells by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and the death of all hepatocarcinoma cells on day 6 after transfection. CONCLUSION: It is confirmed that antisense VEGF(165) can inhibit the expression of VEGF protein, interfere with the proliferation and induce the apoptosis of hepatocarcinoma cells in our study. Antisense VEGF(165) gene therapy may play an important role in the treatment of human hepatocarcinoma.  相似文献   

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