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.     

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．     

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.     

Genistein对肝癌细胞株SMMC-7721的抑制作用及对其癌基因表达的影响

原发性肝癌为高度恶性肿瘤,其病死率在我国各种恶性肿瘤中仅次于胃癌和食管癌,居第三位。虽然目前临床治疗方法较多,但疗效欠佳,预后不良。4′,5,7-三羟基异黄酮(Genistein)是近年来受到广泛关注的非营养素成分。本实验采     

Inhibitory effect of all-trans retinoic acid on human hepatocellular carcinoma cell proliferation

AIM: To study the inhibitory effect of all-trans retinoic acid on human hepatocellular carcinoma cell line SMMC-7721 and to explore the mechanism of its effect. METHODS: SMMC-7721 cells were divided into two groups, one treated with all-trans retinoic acid (ATRA) for 5 days and the other as a control group. Light microscope and electron microscope were used to observe the morphological changes. Telomerase activity was analyzed with silver-stained telomere repeated assay protocal (TRAP). Expression of Caspase-3 was demonstrated with western blot. RESULTS: ATRA-treated cells showed differentiation features including small and pyknotic nuclei, densely stained chromatin and fewer microvilli. Besides, ATRA could inhibit the activity of telomerase, promote the expression of Caspase-3 and its activation. CONCLUSION: Telomerase activity and Caspase-3 expression are changed in human hepatocellular carcinoma cell line SMMC-7721 treated with all-trans retinioc acid. The inhibition of telomerase activity and the activation of Caspase-3 may be the key steps through which ATRA inhibits the proliferation of SMMC-7721 cell line.     

Up-regulation expression of MLC1 in human liver cancer tissue and enhanced SMMC7721 cell tumorigenesis in vivo and vitro

BACKGROUND/AIMS: We screened a novel gene MLC1 in human liver cancer tissue by differential display, and its cDNA full-length is 1600bp. The purpose of this study is to find expression of MLC1 gene in human liver cancer tissue and the affect to SMMC7721 cell tumorigenesis in vivo and vitro. METHODOLOGY: 250 cases of primary HCC tissue samples were studied for MLC1 mRNA and protein expression using RT-PCR, western blot, immunohistochemistry, MLC1 stable transfection into SMMC771, and SMMC7721 cells growth curve was analyzed by MTT method and SMMC7721 cells tumorigenesis in vivo. RESULTS: RT-PCR results showed that 98.1% (245/250) was MLC1 up-regulation expression, 1.9% (5/250) was MLC1 down-regulation (p<0.01). Immunohistochemistry showed that 97.2% (243/250) was MLC1 up-regulation expression, 2.8% (7/250) was down-regulation (p<0.01) in cancer tissue compared with paracancerous tissue (p<0.01). Western blotting results showed that 98.9% (247/250) was MLC1 up-regulation expression, 1.1% (3/250) was MLC1 down-regulation. Overexpression of MLC1 enhanced the growth of SMMC7721 cells compared with the control cells. pcDNA3.1-MLC1 cells accelerated tumor formation compared with pcDNA3.1. CONCLUSIONS: MLC1 gene showed up-regulation expression at both the mRNA and protein levels in HCC tissues and that MLC1 plays an important role in the growth of hepatoma cell SMMC7721 in vitro and vivo.     

健脾理气药诱导人肝癌细胞SMMC7721凋亡的研究

目的观察健脾理气药的诱导凋亡效应,为其临床应用进一步提供依据.方法采用血清药理学方法研究中药的体外效应.应用四甲基偶氮唑蓝(MTT)比色法检测含中药兔血清对肝癌细胞的抑制效应,以Annexin V标记法、DNA含量测定、电子显微镜方法检测含中药血清诱导凋亡及细胞周期阻滞效应.免疫组化法观察含中药血清对P53,P21WAF1/CIP1蛋白的影响,RT-PCR法观察含中药血清对P21WAF1/CIP1 mRNA表达水平的影响.结果含中药血清有一定的抑制肝癌细胞作用,其作用3 d的抑制率为6.6%,作用6 d的抑制率为36.2%.含中药血清作用2 d诱导9.8%±4.0%的肝癌细胞凋亡,使细胞周期阻滞于S期,并上调P53蛋白、P21WAF1/CIP1 mRNA及蛋白的表达.结论健脾理气药具一定的诱导凋亡及抑制肝癌细胞效应,上调p53,p21WAF1/CIP1基因的表达为分子机制.     

Inhibitory effect of adeno-associated virus-mediated gene transfer of human endostatin on hepatocellular carcinoma

AIM: To investigate the effect of adeno-associated virus-mediated gene transfer of human endostatin on the growth of hepatocellular carcinoma (HCC). METHODS: HCC cell line Hep3B was infected with recombinant adeno-associated virus containing human endostatin gene (rAAV2-hEndo). The results of transfection were detected by RT-PCR and SDS-PAGE assay. MTT assay was used to observe the effects of supernatant of transfected cells on ECV304 cell proliferation. An animal model of HCC was established by injecting Hep3B cells subcutaneously into the back of nude mice. Intratumoral injection of rAAV2-hEndo, empty virus and phosphate-buffered saline were given sequentially. Serum endostatin was determined by ELISA, the inhibitory effect of endostatin on the growth of xenograft was assessed in 3 wk. RESULTS: The results of RT-PCR and SDS-PAGE assay confirmed that rAAV2-hEndo successfully transfected Hep3B cells, and endostatin was secreted from Hep3B cells to medium. The supernatant of transfected cells markedly inhibited the proliferation of ECV304 cells (P<0.01). Intratumoral injection of rAAV2-hEndo (2×1010 v.g.) led to a sustained serum endostatin level of approximately (86.71±5.19) ng/mL. The tumor volume and microvessel density were less in rAAV2-hEndo group than in control groups(P<0.01). CONCLUSION: Human endostatin can be stably expressed by adeno-associated virus-mediated gene transfer and effectively inhibit the growth of HCC.     

蛋白酶体β亚基4型对人肝癌SMMC7721细胞增殖存活的影响

目的旨在探讨蛋白酶体β亚基4型(PSMB4)对人肝癌SMMC7721细胞增殖存活的影响及其可能的机制。方法利用特异性短发夹RNA(shRNA)技术构建干扰PSMB4表达的SMMC7721实验组细胞;运用MTT、克隆形成实验检测细胞增殖的影响,流式细胞术检测细胞凋亡率的变化,Western Blot检测相关蛋白的表达变化。计量资料两组间比较采用独立样本t检验。结果成功构建了干扰PSMB4表达的SMMC7721实验组细胞(shRNA1:t=22.67,P<0.0001;shRNA2;t=30.88,P<0.0001;shRNA3:t=67.82,P<0.0001)。MTT实验显示第4天(0.4770±0.0135 vs 0.3237±0.0127,t=8.286,P=0.0012)、第5天(0.5893±0.0088 vs 0.3847±0.0090,t=16.220,P<0.0001)实验组细胞OD490值显著低于对照组。克隆形成实验显示细胞集落数显著减少。流式细胞术显示实验组细胞的早期凋亡率[(5.5570±0.2589)%vs(3.8870±0.3324)%,t=3.964,P=0.0166]、晚期凋亡率[(12.6300±0.4198)%vs(5.3100±0.3062)%,t=14.080,P=0.0001]及总凋亡率[(18.1800±0.6785)%vs(9.1970±0.6313)%,t=9.967,P=0.0006]均明显高于对照组,且干扰PSMB4表达的实验组细胞中核因子-κB亚基p65蛋白表达降低(0.8015±0.0120 vs 0.2841±0.0110,t=31.830,P<0.0001),核因子-κB抑制蛋白α表达增加(0.4816±0.0112 vs 0.6583±0.0142,t=9.774,P=0.0006)。结论干扰PSMB4表达可能通过抑制NF-κB信号通路,从而导致肝癌SMMC7721细胞的增殖存活能力下降。     

载体肝干细胞分泌表达的Endostatin对血管内皮细胞增殖和凋亡的影响

目的观察携带内皮抑素基因的载体肝干细胞分泌表达的内皮抑素蛋白（Endostatin,ES）对血管内皮细胞体外增殖和凋亡的影响。方法体外扩增培养本室构建的携带内皮抑素基因的载体肝干细胞WB-ES,收集合有分泌型Endostatin的上清液。将人脐静脉内皮细胞ECV304体外增殖培养,加入不同浓度的含倍比稀释Endostatin的上清液,在不同的作用时间（24、48、72h）,通过四甲基偶氮唑蓝比色法（MTT）检测细胞生长抑制率。采用流式细胞仪检测细胞周期和凋亡率,分析载体肝干细胞WB—ES分泌表达的Endostatin对ECV304的增殖和凋亡的影响。结果载体肝干细胞胞外表达的Endostatin对人脐静脉内皮细胞ECV304生长有显著抑制作用,48h作用达到高峰,随浓度增加抑制作用增强;Endostatin作用的实验组,G1期细胞比例增加,s期细胞数下降,抑制细胞生长的机制可能主要是通过对细胞增殖的影响（P〈0．05）;实验组细胞存在细胞凋亡增加现象,但与对照组比较凋亡率差异无统计学意义。结论携带内皮抑素基因的载体肝干细胞WB-ES胞外表达的Endostatin在体外可有效抑制血管内皮细胞增殖,载体肝干细胞WB-ES有望成为靶向抗肿瘤血管生成的肝癌基因治疗载体细胞。     

棘霉素诱导肝癌SMMC7721细胞凋亡的研究

目的研究棘霉素诱导肝癌SMMC7721细胞凋亡的作用,探讨其抗肿瘤的作用机制。方法通过细胞计数获得不同浓度棘霉素作用SMMC7721细胞的生长抑制率,观察棘酶素对肝癌细胞生长的影响;通过流式细胞仪分析棘霉素对细胞凋亡和细胞周期的影响。结果棘霉素浓度〉10 ng/ml时可抑制SMMC7721细胞的生长。结论棘霉素在体外可诱导肝癌细胞凋亡,具有很强的抗肿瘤作用。     

奥曲肽体外抑制人肝癌细胞株SMMC-7721的增殖及其机制研究

目的研究生长抑素类似物奥曲肽(octreotide)在体外对人肝癌细胞株SMMC-7721的抗增殖作用,并从细胞周期调控的角度探讨其作用机制.方法采用MTT比色法观察octreotidedui对细胞增殖的抑制作用,流式细胞仪分析细胞周期,免疫细胞化学检测cyclin A、增殖细胞核抗原(PCNA)蛋白的表达.结果octreotide能明显地抑制SMMC-7721细胞的增殖,呈剂量依赖性;与对照组比较,octreotide使实验组G0/G1、G2/M期细胞数减少,S期细胞数增加.经octreotide作用72 h后,cyclinA、PCNA蛋白表达水平明显下降(对照组2.833±0.074,4.851±0.059;octreotide组1.674±0.043,3.257±0.036).结论octreotide能影响细胞周期的进程,通过下调S期细胞周期素A、PCNA蛋白的表达来干扰SMMC-7721细胞增殖环节,可能是其发挥抑制肝癌细胞增殖的机制之一.     

牛蒡子苷元对肝癌SMMC-7721细胞增殖、凋亡的影响及机制探讨

目的观察牛蒡子苷元（ARG）对肝癌SMMC-7721细胞增殖、凋亡的影响,并探讨其机制。方法将不同浓度的ARG作用于SMMC-7721细胞,并设不加ARG对照组。MTT法测算细胞增殖抑制率、流式细胞术检测细胞周期及凋亡率,RT-PCR法检测细胞中的Bcl-2 mRNA。结果与对照组比较,随ARG浓度增加,SMMC-7721细胞增殖率逐渐降低（P均〈0.05）,G0/G1期细胞比例显著增加（P均〈0.05）,G2、M、S期细胞比例减少（P均〈0.05）;随ARG浓度增加、作用时间延长,SMMC-7721细胞凋亡率显著增加（P均〈0.05）,Bcl-2 mRNA表达量减少（P均〈0.05）。结论 ARG可明显抑制SMMC-7721细胞增殖并诱导其凋亡;可能与ARG下调细胞中Bcl-2基因的表达有关。     

KPNA2基因沉默对人肝癌细胞株SMMC7721和Bel7404增殖和侵袭能力的影响

目的观察核转运蛋白基因α2(Karyopherinα-2,KPNA2)基因沉默对人肝癌细胞株SMMC7721和Bel7404增殖以及侵袭能力的影响。方法将KPNA2 siRNA干扰质粒用LipofectaminTM 2000方法瞬时转染人肝癌细胞株SMMC7721和Bel7404,转染后48 h应用蛋白质印迹法检测转染细胞中KPNA2蛋白表达。采用MTT法检测基因沉默细胞增殖能力,采用Transwell法检测基因沉默细胞侵袭能力。结果 SMMC7721细胞株对照组mRNA为1.02±0.13,高于siRNA转染组的0.37±0.07(t=10.78,P0.01);肝癌Bel7404细胞株对照组mRNA为1.05±0.17,高于siRNA转染组的0.36±0.06(t=9.38,P0.01)。肝癌SMMC7721细胞株对照组蛋白定量值为0.96±0.10,高于转染组的0.42±0.05(t=11.83,P0.01);肝癌Bel7404细胞株对照组蛋白定量值为0.93±0.09,高于转染组的0.48±0.06(t=10.19,P0.01)。SMMC7721和Bel7404细胞株在24、48和72 h时对照组增殖能力高于转染组,且差异有统计学意义(P0.05)。SMMC7721细胞株对照组侵袭能力值为126.20±21.61,高于siRNA转染组的51.13±10.2(t=7.68,P0.01);肝癌Bel7404细胞株对照组侵袭能力值为125.124±8.04,高于siRNA转染组的55.20±18.54(t=8.48,P0.01)。结论KPNA2基因沉默可以调节人肝癌细胞株SMMC7721和Bel7404的增殖能力和侵袭能力。     

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...