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

AIM: To constnuct a stable transfectant of human livercarcinoma cell line SMMC7721 that could secret humanencicstatin and to explore the effect of human encostatinexpressed by the transfectant on enciotheliai cell proliferation.METHODS: Recombinant retroviral plasmid pLncx-Endocontaining the eDNA for human endoslsin gene togetherwith mt albumin signal peptide was engineered andtransferred into SMMC7721 cell by lipofectamine. Afterselection with G418, endcotatin-transfected SMMC7721 ceiiswere chosen and expanded. Immunohistochemical stainingand Western blot were used to detect the expression ofhuman endosatin in transfected SMMC7721 cells and itsmedium. The conditioned medium of endostatin-transfectedand control SMMC7721 cells were collected to cultivate withhuman umbilical vein endothelial cells for 72 hours. Theinhibitory effect of endoststin, expressed by transfectedSMMC7721 cells, on endothelial proliferation in vitro wasobserved by using Mn assay.RESULTS: A 550 bp specific fragment of endostatin gene wasdetected from the PCR product of endostatin-transfeclsdSMMC7721 cells. Immunohistochemistry and Western blotanalysis confirmed the expression and secretion of foreighhuman endostatin protein by endoslstin-transfeclsdSMMC7721 cells. In vitro endothelial proliferation assayshowed that 72 hours after cultivation with human umbilicalvein endothelial cells, the optical density (OD) in groupusing the medium from endostatin-transfected SMMC7721cells was 0.51 ±0.06, lower than that from RPMI 1640 group(0.98 ± 0.09) or that from control plasmid pLncx-transfeotedSMMC7721 cells (0. 88 ± 0. 11). The inhibitory rate formedium from endostatin-transfeclsd SMMC7721 cells was 48%, significantly higher than that from empty plasmid plncx-transfected SMMC7721 cells (10.2 %, P＜ 0.01).CONCLUSION: Human endoslstin can he stably expressedby SMMC7721 cell tran sferred with human endoslsin geneand its product can significantly inhibit the proliferation ofhuman umbilical vein endothelial cell in vitro.     

Retrovirus—mediated gene transfer of human endostatin inhibits growth of human liver carcinoma cells SMMC7721 in nude mice

AIM: To study the effect of human endostatin mediated by retroviral gene transfer on the growth of human hepatocarcinoma cell line SMMC7721 in nude mice. METHODS: Human endostatin gene together with rat serum albumin signal peptide was transferred into human liver carcinoma SMMC7721 cells by retroviral vector pLncx to build a stable transfectant (SMMC-endo). PCR and Western blot analysis were used to verify the transfection and secretion of human endostatin gene in SMMC7721 cells. The endothelial cell proliferation assay in vitro was conducted to test the biological activity of the expressed human endostatin. The inhibitory effect of endostatin expressed by transfected SMMC7721 on the growth rates of tumor cells in vivo was observed. The mean microvessel density in the specimen was also counted. RESULTS: PCR amplification proved that the genome of SMMC-endo cells contained a 550bp specific fragment of endostatin gene. Western blot analysis confirmed the secretion of human endostatin gene in the conditioned medium of transfected SMMC-endo cells. The endothelial proliferation assay showed that the conditioned medium of SMMC-endo cells significantly inhibited the proliferation of human umbilical vein endothelial cells by 48 %, significantly higher than that of SMMC-pLncx (10.2 %, P<0.01). In vitro experiments revealed that only in 3 out of 5 mice tumors were formed and the mean size of flank tumors from SMMC-endo cells was 94.5 % smaller than that from the control SMMC-pLncx cells 22 days after tumor inoculation (P<0.001). The mean microvessel density in tumor samples from SMMC-endo cells was only 8.6+/-1.1, much fewer than that of 22.6+/-4.5 from SMMC-pLncx cells (P<0.01). CONCLUSION: Human endostatin mediated by retroviral gene transfer can inhibit human liver carcinoma cell SMMC7721 growth in nude mice.     

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。     

Liposome transfected to plasmid-encoding endostatin gene combined with radiotherapy inhibits liver cancer growth in nude mice

AIM: To evaluate whether intratumoral injection of liposome-endostatin complexes could enhance the antitumor efficacy of radiation therapy in human liver carcinoma (BEL7402) model. METHODS: Recombinant plasmid pcDNA3.End was transfected into human liver carcinoma cell line (BEL7402) with lipofectamine to produce conditioned medium. Then BEL7402 cells and human umbilical vein endothelial cells (HUVECs) were treated with the conditioned medium. Cell cycle and apoptosis were analyzed by flow cytometer and endothelial cell proliferation rates were determined by MTT assay. The antitumor efficacy of endostatin gene combined with ionizing radiation in mouse xenograft liver tumor was observed. RESULTS: Endostatin significantly suppressed the S phase fraction and increased the apoptotic index in HUVECs. In contrast, endostatin treatment had no effect on BEL7402 cell apoptosis(2.1±0.3% vs 8.9±1.3%, t= 8.83, P=0.009<0.01) or cell cycle distribution (17.2±2.3% vs 9.8±1.2%, t=4.94, P= 0.016<0.05). The MTT assay showed that endostatin significantly inhibited the proliferation of HUVECs by 46.4%. The combination of local endostatin gene therapy with radiation therapy significantly inhibited the growth of human liver carcinoma BEL7402 xenografts, the inhibition rate of tumor size was 69.8% on d 28 compared to the untreated group. The tumor volume in the pcDNA3.End combined with radiation therapy group (249±83 mm3) was significantly different from that in the untreated group (823±148 mm3, t=5.86, P=0.009<0.01) or in the pcDNA3 group (717±94 mm3, t=6.46, P= 0.003<0.01). Endostatin or the radiation alone also inhibited the growth of liver tumor in vivo, but their inhibition effects were weaker than those of endostatin combined with radiation, the inhibition rates on d 28 were 44.7% and 40.1%, respectively. CONCLUSION: Endostatin not only significantly suppresses tumor growth but also enhances the antitumor efficacy of radiation therapy in human carcinoma xenograft.     

人端粒酶催化亚单位锤头状核酶诱导肝癌细胞凋亡的作用

目的 构建带有U6启动子的人端粒酶催化亚单位锤头状核酶真核表达质粒及其突变体,转染入肝癌细胞株SMMC7721,观察端粒酶活性、细胞增殖和凋亡的情况。 方法 用分子克隆技术构建由U6作为启动子、绿色荧光蛋白基因作为报告基因的核酶真核表达质粒pGTRz-U6及其突变体pGTmRz-U6,并以空质粒pEGFP-C1作为对照。Lipofectamine2000转染人肝癌细胞株SMMC7721,G418筛选阳性克隆。RT-PCR检测核酶及hTERT基因的表达,四甲基偶氮唑盐(MTT)作细胞生长曲线观察其生长情况,TRAP-银染法检测端粒酶活性变化,流式细胞计数(FCM)法检测细胞的凋亡水平。 结果 核酶、突变核酶在SMMC7721中持续表达;凝胶成像系统分析SMMC7721-pEGFP-C1、SMMC7721-mRz、SMMC7721-Rz hTERT基因表达,用SPSS10.0软件对3种细胞进行分析,发现三者hTERT基因表达水平不同(F=47.987,P<0.01);t检验分析得出SMMC7721-Rz hTERT基因表达明显低于SMMC7721-mRz和SMMC7721- pEGFP-C1(t值分别为-7.640和-11.602,P值均<0.01)。SMMC7721-pEGFP-C1和SMMC7721-mRz hTERT表达没有区别(t=-0.178,P>0.05)。TRAP-银染及FCM结果分别显示,随着细胞的分裂,SMMC7721-Rz和SMMC7721-mRz细胞端粒酶活性逐渐降低,凋亡水平逐渐增加,7PDS细胞凋亡率分别是29.86％和9.87％,而对照组SMMC     

MAGE-1修饰的树突状细胞体外诱导杀伤人肝癌细胞

目的通过观察肿瘤相关抗原基因MAGE-1转导的树突状细胞(dendritic     

Growth-inhibitory effects of MOB2 on human hepatic carcinoma cell line SMMC-7721

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.     

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.     

B7转染人肝癌细胞株的建立及生物学特性的实验研究

研究共刺激信号在肿瘤免疫中的作用。脂质体介导DNA转移法将入B7基因转入人肝癌细胞株Smmc7721,建立新的细胞株B7^ Smmc7721,PCR及逆转录PCR（RT-PCR）法鉴定。四唑盐（MTT）比色试验体外检测白细胞介素-2（IL-2）激活的LAK细胞（LAK-C）对两种肝癌细胞的杀伤活性。PCR,RT-PCR法证实B7^ Smmc7721细胞稳定表达B7基因,LAK细胞对B7^ Smmc7721细胞的杀伤活性明显高于Smmc7721,结果有统计学意义。B7基因可以体外转染人肝癌细胞,并表达有活性的B7分子,并可明显增强IL-2激活的LAK细胞的肿瘤杀伤作用,为进一步开展临床应用奠定基础。     

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.     

Potent inhibition of angiogenesis and liver tumor growth by administration of an aerosol containing a transferrin-liposome-endostatin complex

AIM: To obtain an efficient delivery system for transporting endostatin gene to mouse liver tumor xenografts by administration of aerosol.METHODS: Recombinant plasmid pcDNA3.0/endostatin containing human endostatin gene together with signal peptide from alkaline phosphatase were transferred into human umbilical vein endothelial cell (HUVEC) by transferrin(TF)-Iiposome-endostatin complex. Western blot was used to detect the expression of human endostatin in transfected HUVEC cells and its medium. After the tumor-bearing mice were administrated with TF-liposome-endostatin complex,the lung tissue was analyzed by immunohistochemical method for expression of endostatin and the tumors were treated with CD-31 antibody to detect the density of microvesseles in tumor tissues. The inhibition of tumor growth was estimated by the weight of tumors from groups treated with different doses of TF-liposome-endostatin complex. DNA fragmentation assay was used to detect the apoptosis of the cells from primary liver tumor.RESULTS: Western blot analysis and immunohistochemical method confirmed the expression of endostatin protein in vitro and in vivo. After the tumor sections were treated with CD-31 antibody, the positive reaction cells appeared brown while the negative cells were colorless. The positively stained area of the TF-liposome-endostatin treated group was significantly smaller (P&lt;0.01, 645.8+55.2 μm^2) than that of the control group (1325.4&#177;198.5 μm^2). The data showed a significant inhibition of angiogenesis. After administration of TF-liposome-endostatin, comparing with the control group administrated with TF-liposome-pcDNA3.0, liver tumor growth in the mice treated with 50, 250 and 500 mg DNA/kg was inhibited by 36.6 %, 40.8%, and 72.8%, respectively(P&lt;0.01). And a typical DNA fragmentation of apoptosis was found in the cells from tumor tissues of the mice treated with TF-liposome-endostatin but none in the control group.CONCLUSION: Endostatin gene could be efficiently transported into the mice with TF-liposome-DNA delivery system by administration of aerosol. TF-liposome-mediated endostatin gene therapy strongly inhibited angiogenesis and the growth of mouse xenograft liver tumors. It also could promote the development of apoptosis of tumors without direct influence on tumor cells.     

E-selectin is required for the antiangiogenic activity of endostatin

Endostatin, a 20-kDa fragment of collagen XVIII, is a potent angiogenesis inhibitor. E-selectin, an inducible leukocyte adhesion molecule specifically expressed by endothelial cells, has also been implicated in angiogenesis. By using in vivo, ex vivo, and in vitro angiogenic assays, we investigated the functional relationship between endostatin and E-selectin. In corneal micropocket assays, recombinant endostatin administered i.p. by osmotic pump inhibited basic fibroblast growth factor-induced angiogenesis in WT, but not E-selectin-deficient, mice. Similarly, endostatin inhibited vascular endothelial growth factor-stimulated endothelial sprout formation from aortic rings dissected from WT but not from E-selectin-deficient mice. To further explore this apparent requirement for E-selectin in endostatin action, we manipulated E-selectin expression in cultured human endothelial cells. When E-selectin was induced by IL-1beta, or lipopolysaccharide, human umbilical vein endothelial cells and human dermal microvascular endothelial cells each became markedly more sensitive to inhibition by endostatin in a vascular endothelial growth factor-induced cell migration assay. To dissociate E-selectin expression from other consequences of endothelial activation, human umbilical vein endothelial cells were transduced with an adenoviral human E-selectin expression construct; these cells also showed increased sensitivity to endostatin, and this effect required the E-selectin cytoplasmic domain. Taken together, these results indicate that E-selectin is required for the antiangiogenic activity of endostatin in vivo and ex vivo and confers endostatin sensitivity to nonresponsive human endothelial cells in vitro. E-selectin may be a useful predictor and modulator of endostatin efficacy in antiangiogenic therapy.     

缺氧对人肝癌细胞株SMMC7721中HCCR表达的影响及其调控机制

背景：缺氧是实体瘤微环境的基本特征之一。人宫颈癌基因（HCCR）是新近发现的一种癌基因．在多种人类肿瘤中过表达。然而,尚缺乏HCCR在肿瘤缺氧环境中的表达及其调控机制的研究。目的：探讨缺氧对人肝癌细胞株SMMC7721中HCCR表达的影响及其调控机制。方法：SMMC7721细胞分别接受缺氧培养以及LY294002（P13K特异性抑制剂）和PD98059（MEK1特异性抑制剂）预处理＋缺氧培养,以蛋白质印迹法检测HCCR表达：将含HCCR启动子片段的荧光素酶报告基因质粒转染SMMC7721细胞,检测缺氧对HCCR启动子转录活性的影响。结果：缺氧环境下SMMC7721细胞中HCCR蛋白表达和转录活性均较常氧环境中升高,LY294002和PD98059可抑制缺氧环境下HCCR蛋白表达。结论：缺氧状态下人肝癌细胞株SMMC7721中HCCR蛋白表达和启动子转录活性增高。可能是肿瘤细胞在缺氧实体瘤组织中存活和快速增殖的重要机制之一。缺氧状态下HCCR蛋白表达受P13K／Akt和MAPK这两条信号通路的调节。     

RNA interference-mediated silencing of Stat5 induces apoptosis and growth suppression of hepatocellular carcinoma cells

It has been reported that Stat5 is overexpressed in a variety of human cancer cell lines and primary tumors. Inhibition of Stat5 in tumor cell lines has been associated with growth suppression and induction of apoptosis. However, no one of published studies have investigated the expression and role of Stat5 in hepatocellular carcinoma. In this study, we used human hepatocellular carcinoma cell line SMMC7721 as a model to demonstrate that Stat5 was highly expressed in these cells. Next we showed that RNAi mediated Stat5 knockdown could inhibit the proliferation and induce the apoptosis of SMMC7721 cells in vitro. Furthermore, we demonstrated that Stat5 knockdown inhibited the growth and induced the apoptosis of SMMC7721 cells in xenografts in nude mice. Taken together, our in vitro and in vivo data suggest that Stat5 plays an important role in human hepatocellular carcinoma. Inhibition of Stat5 by RNAi holds promise to be a novel gene therapy vector for hepatocellular carcinoma.     

人内皮抑素小环载体在真核细胞中的生物活性

目的通过对人内皮抑素小环载体在真核细胞中的表达和生物学效应的研究,探讨小环载体作为生物治疗载体的可行性。方法将mc-hES、pcDNA-hES分别转染人肝癌细胞HepG2后,通过ELISA、RT-PCR和Westernblot观察hES表达。MTT法检测其对人脐静脉内皮细胞（HUVEC）的增殖抑制作用。结果 mc-hES和pcDNA-hES均能表达hES,mc-hES能明显抑制HUVEC的生长,而对HepG2无明显作用（P〈0.05）。在相同条件下,小环较常规质粒在体外能快速介导转基因的表达,且较传统质粒高6～8.3倍。结论 mc-hES较普通质粒pcDNA-hES在肝癌细胞中能高效表达转基因,为小环载体进一步研究提供了很好的实验基础。     

Inhibitory effect of antisense vascular endothelial growth factor RNA on the profile of hepatocellular carcinoma cell line in vitro and in vivo

AIM:To evaluate the effect of antisense vascularendothelial growth factor(VEGF)RNA(PCMV-FGEV)transfection on the profile of hepatocellular carcinoma(HCC)SMMC-7721 cells in vitro and in vivo.METHODS:SMMC-7721 cells were transfectedwith PCMV-FGEV antisense,PCMV-VEGF sense andempty vector plasmid encapsulated by lipofectamineas antisense group,sense group and control grouprespectively.The positive cell clones were selectedwith G418.The stable transfection and expressionof VEGF in the cells were determined by RT-PCR andimmunohistochemistry.Cell proliferation was observedby MTT assay.FACS analysis was used to determine theeffect of PCMV-FGEV transfection on cell apoptosis.Thegrowth of transfected cells in Wvo was also observed innude mice.RESULTS:VEGF expression was reduced in SMMC-7721transfected with PCMV-FGEV,which was confirmed byRT-PCR and immunohistochemistry.No effect of PCMV-FGEV transfection was found on cell proliferation andcell apoptosis of SMMC-7721 in vitro.The growth of cellstransfected with PCMV-FGEV was slow in nude miceand accompanied with obvious apoptosis.The latenttime of tumors in the antisense group was 25.0±1.8d,which was longer than that in sense and controlgroups(F=19.455,P<0.01).The average tumor weightin antisense group(0.96 g±0.28 g)was the smallestamong the three groups(F=21.501,P<0.01).CONCLUSION:The expression of VEGF can be inhibitedby antisense PCMV-FGEV.Antisense PCMV-FGEV has no effect on cell proliferation and apoptosis of SMMC-7721in vitro but can inhibit tumor growth and induce cellapoptosis in vivo.     

血管内皮细胞生长因子反义核糖核酸对肝癌细胞生长的抑制作用

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。     

腺病毒介导XAF1基因诱导人肝癌细胞株SMMC7721凋亡的实验研究

背景：X连锁凋亡抑制蛋白（XIAP）相关因子1（XAF1）是新近鉴定的肿瘤抑制基因,在许多人类恶性肿瘤中低表达甚至不表达。目的：研究Ad5/F35腺病毒介导XAF1基因诱导人肝癌细胞株SMMC7721凋亡的作用及其可能机制。方法：构建重组腺病毒Ad5/F35-XAF1、对照病毒Ad5/F35-Null和报告病毒Ad5/F35-增强型绿色荧光蛋白（EGFP）,分别按不同感染复数（MOI）在同一作用时间点感染人肝癌细胞株SMMC7721。以荧光显微镜和流式细胞仪检测Ad5/F35-EGFP的感染效率;分别以逆转录聚合酶链反应（RT-PCR）和蛋白质印迹法检测XAF1mRNA和蛋白表达;以甲基噻唑基四唑（MTT）法检测细胞增殖率;以Annexin V-FITC/PI双染法和原位末端标记TUNEL法检测细胞凋亡率;以蛋白质印迹法检测凋亡相关蛋白caspase-3、caspase-8和caspase-9的表达。结果：Ad5/F35-EGFP感染48h,MOI为2.0时,92%以上的SMMC7721细胞表达绿色荧光蛋白。Ad5/F35-XAF1感染48h后,SMMC7721细胞中XAF1 mRNA和蛋白表达增高,细胞增殖受到抑制,细胞凋亡剂量依赖性地增多,并伴随凋亡相关蛋白caspase-3、caspase-8和caspase-9的裂解。结论：重组腺病毒Ad5/F35-XAF1在人肝癌细胞株SMMC7721中具有很强的感染效率,可促进XAF1基因表达,且能明显抑制人肝癌细胞增殖和诱导凋亡,此作用可能与XAF1激活了内、外源性凋亡通路有关。