反义局部粘着斑激酶脱氧寡核苷酸对肝癌细胞侵袭性生长的抑制作用

目的 观察反义局部粘着斑激酶脱氧寡核苷酸(FAK ODN)转染对肝癌细胞侵袭性生长的影响,并探讨其作用机制。 方法 以LipofecTAMINE介导的反义FAK ODN转染Bel 7402肝癌细胞株,测定Bel 7402肝癌细胞株体外生长曲线、细胞活力,测定不同时间点该细胞体外黏附能力变化,以Transwell小室测定细胞的体外侵袭能力,同时行FAK表达与细胞DNA含量的双参数流式细胞仪检测及细胞凋亡的流式细胞仪检测。 结果 p125FAK表达在反义转染组(6.49％±0.10％)显著低于正义转染组(14.33％±1.88％)与对照组(16.68％±1.62％),F=7.66,P<0.01;反义FAK ODN转染显著抑制Bel 7402肝癌细胞株的生长,其细胞活力显著下降,肿瘤细胞抑制率在30％-60％之间;细胞体外黏附能力受到显著抑制,黏附抑制率在25％-55％间;细胞的体外侵袭能力显著下降,侵袭抑制率在15％-25％之间;细胞凋亡显著增加;细胞周期分析显示S期细胞比率显著降低,细胞生长主要阻滞在G2/M期。 结论 FAK在Bel 7402肝癌细胞的黏附与迁移运动中发挥重要作用,其表达阻断显著抑制肝癌细胞的体外黏附与侵袭活性。FAK表达阻断显著抑制肝癌细胞的体外增殖,促进细胞凋亡。     

Survivin反义寡核苷酸诱导肝癌细胞凋亡的作用

目的 用反义寡核苷酸封闭肝癌细胞中survivin基因的表达,研究其诱导细胞凋亡的作用及其机制。方法 用脂质体介导survivin反义寡核苷酸转染人肝癌细胞株SMMC-7721细胞,流式细胞仪检测细胞周期变化及细胞凋亡比率,激光共聚焦显微镜观察细胞骨架微丝系统变化,激酶活性检测方法测定细胞内caspase-3活性变化,免疫沉淀法测定细胞内丝裂原活化蛋白激酶p38(p38MAPK)活性的变化。结果 脂质体介导survivin反义寡核苷酸转染肝癌细胞后Ⅰ～Ⅵ组(空白对照、正义对照、400、600、800、1000ng/ml反义转染组)细胞内p38MAPK活性分别为7.03、7.07、13.47、16.37、43.97及47.87;caspase-3活性分别为0.015±0.010、0.014±0.002、0.026±0.003、0.042±0.001、0.093±0.001及0.100±0.001;Ⅳ～Ⅵ组细胞内p38MAPK及caspase-3活性较对照组明显升高。转染后细胞发生G_2/M期阻滞,Ⅰ～Ⅵ组细胞凋亡率分别为0.70％、0.76％、2.43％、7.82％、23.11％及31.35％,各实验组细胞凋亡较对照组明显增加。细胞内微丝形态结构破坏,Ⅰ～Ⅵ组细胞肌动蛋白平均荧光强度分别为189.69±6.68、184.23±8.76、173.14±8.15、99.48±6.57、76.69±10.05及63.80±6.79,Ⅳ～Ⅵ组细胞肌动蛋白平均荧光强度较对照组明显降低。结论 脂质体介导转染survivi     

环氧合酶-2反义核酸抑制胃癌细胞的恶性表型

目的　通过环氧合酶 2 (COX 2 )反义核酸基因转染 ,逆转COX 2高表达的人胃癌细胞系中其表达水平 ,观察细胞生物学行为的变化情况 ,以初步探讨COX 2表达在胃癌发生中的某些具体机制。方法　使用脂质体介导的方法 ,用反义重组质粒及空载体分别转染COX 2异常高表达的人胃癌细胞系SGC 790 1(转染细胞分别命名为 790 1 AS及 790 1 P细胞 )。通过免疫细胞化学及RNA斑点杂交试验检测反义核酸转染的细胞中COX 2的蛋白及mRNA表达水平。四唑盐 (MTT)比色试验检测转染细胞的体外增殖速度。应用裸鼠成瘤试验比较转染前后细胞体内成瘤能力的差别。结果　免疫细胞化学及RNA斑点杂交试验证实 :在反义核酸转染的 790 1 AS细胞中COX 2的蛋白及mRNA水平均显著下调。MTT比色试验显示 790 1 AS细胞的增殖速度低于亲本SGC 790 1细胞。裸鼠成瘤试验表明 ,反义核酸转染细胞成瘤潜伏期延长 ,成瘤体积减小。 3组细胞接种裸鼠 30d后 ,瘤体的平均重量( x±s)分别为 (82 6 6 7± 77 6 7)mg(SGC 790 1细胞 ) ,(776 6 7± 30 0 0 6 )mg(790 1 P细胞 )和 (486 6 7±15 2 8)mg (790 1 AS细胞 )。转染反义核酸的细胞成瘤性显著低于未转染细胞及转染载体对照细胞(P <0 0 1)。结论　胃癌细胞中过表达的COX 2与细胞的恶性表型相关。用     

PUMA基因转染对胰腺癌细胞生长的影响

目的探讨PUMA基因转染抑制胰腺肿瘤生长的体内外效果。方法利用脂质体转染法将表达PUMA的质粒转染导入胰腺癌细胞株PC-3中,G418筛选出阳性克隆,Western和RT-PCR法检测PUMA转染后PC-3PUMA的表达,流式细胞仪检测转染后细胞凋亡率;分别将转染PUMA的PC-3细胞(实验组)和未转染的PC-3细胞移植到裸鼠体内,比较裸鼠移植肿瘤的大小和重量以及PUMA表达。结果PUMA表达质粒转染的PC-3细胞(PC-3/PUMA)稳定表达PUMA,其细胞凋亡率为(5.50±0.90)%,明显高于未转染组的(1.073±0.248)%和空载体转染组的(1.08±0.35)%(P<0.05);裸鼠接种4周后PC-3/PUMA细胞成瘤率为70%,PC-3细胞和空载体PC-3细胞成瘤率为100%(P>0.05),PC-3/PUMA细胞形成的肿瘤体积比PC-3细胞和空载体PC-3细胞明显减小(P<0.05),并且形成的肿瘤组织中PUMA高表达。结论胰腺癌细胞中缺失PUMA基因表达,PUMA转染胰腺癌细胞后表达PUMA,并能促进细胞凋亡。     

VEGF165正反义表达载体的构建及其对人胃癌细胞VEGF表达的调节

目的构建VEGF165(vascular endothelial growth factor,血管内皮生长因子)正、反义表达载体,将其转染人胃癌细胞,观察其对胃癌细胞VEGF在mRNA和蛋白质水平表达的调节作用.方法用限制性内切酶将VEGF165 cDNA从pGEM-hVEGF165克隆载体上切下来,以正、反方向插入质粒pCDNA3中,构建VEGF165正、反义真核表达载体;用限制性内切酶和Sanger双脱氧终止DNA测序法证明VEGF165正、反义表达载体目的基因的序列和方向的正确性;用脂质体将VEGF165正、反义表达载体转染人胃癌细胞,用RNA斑点杂交及免疫荧光流式细胞仪检测VEGF mRNA和蛋白质的表达水平.结果酶谱分析及DNA测序表明VFGF165正、反义真核表达载体目的基因的序列及方向正确;转染正义表达载体后VEGF mRNA和蛋白质表达水平增加(蛋白免疫强度为75.4%;P＜0.05vs对照组,31.6%),转染反义表达载体后VEGF mRNA和蛋白表达水平降低(蛋白免疫强度为8.9%;P＜0.05vs对照组).结论成功地构建了VEGF165正、反义真核表达载体;构建的载体能在胃癌细胞内有效表达,调节血管内皮生长因子的水平.     

血管内皮生长因子反义核酸治疗裸鼠皮下种植胰腺癌

目的:探讨抗血管生成基因转染治疗胰腺癌的可行性. 方法:构建反向插入VEGF165cDNA的复制缺陷型腺病毒载体,体外转染SW1990细胞,MTT法检测重组腺病毒转染对细胞生长的影响.Northern blot和ELISA检测转染前后SW1990细胞VEGF的mRNA水平和蛋白表达.裸鼠皮下种植瘤瘤体内注射重组腺病毒,CD31染色观察反义VEGF重组腺病毒转染对裸鼠种植瘤微血管密度和肿瘤生长速度的影响. 结果:重组腺病毒体外转染并不影响SW1990细胞的生长速度.Northern blot和ELISA检测在mRNA水平和蛋白水平证实反义VEGF重组腺病毒转染对体外培养的SW1990细胞内源性VEGF表达有明显的下调作用.体内实验表明反义VEGF重组腺病毒转染可减少肿瘤内微血管数量,肿瘤生长受到抑制. 结论:反义VEGF165重组腺病毒可以抑制胰腺癌的血管生成和肿瘤的生长,为抗血管生成的基因治疗奠定了基础.     

抗人VEGF发卡状核酶基因的构建及其对肝癌细胞VEGF蛋白表达的影响

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。     

重组人内皮抑素腺病毒抑制肝癌裸鼠移植瘤生长

目的 观察重组人内皮抑素腺病毒(Ad/hEndo)对人肝癌裸鼠移植瘤生长的影响。方法 人脐静脉内皮细胞ECV-304经Ad/hEndo感染后,western印迹检测人内皮抑素的表达。人肝癌BEL-7402细胞移植到裸鼠背脊部后,检测Ad/hEndo对肝癌移植瘤生长的抑制作用。逆转录聚合酶链反应(RT-PCR)检测肿瘤组织中内皮抑素mRNA的表达。分析人内皮抑素在裸鼠体内的表达分布。结果 Western印迹检测到人内皮抑素基因在ECV-304细胞内高效表达。Ad/hEndo明显抑制人肝癌BEL-7402裸鼠移植瘤生长(F=4.061,P<0.05)。Ad/hEndo组血管密度计数为6.88±1.08,DMEM组为13.60±1.71(t=9.216,P<0.01)。瘤内注射Ad/hEndo后3d,RT-PCR在肿瘤组织检测到内皮抑素mRNA的表达,7d后表达不明显。人内皮抑素蛋白主要分布在肿瘤组织。结论 腺病毒介导的人内皮抑素基因在体内、体外获得高效表达,并明显抑制肝癌裸鼠移植瘤的生长与血管生成。     

多药耐药相关蛋白-3表达与肝癌耐药的相关性

目的 探讨多药耐药相关蛋白-3(MRP-3)的表达与肝癌耐药的相关性.方法 应用RT-PCR方法检测正常肝细胞L-02、肝癌细胞BEL及肝癌耐药细胞HepG2/ADM中MRP-3 mRNA的差异表达,再将MRP-3反义及正义寡核苷酸片段分别用脂质体转染进肝癌耐药细胞HepG2/ADM中,用MTT法、流式细胞仪及RT-PCR检测转染后细胞内阿霉素(ADM)的荧光强度、耐药指数及MRP-3 mRNA的表达情况.结果 肝癌耐药细胞HepG2/ADM中MRP-3 mRNA的表达明显高于正常肝细胞L-02和肝癌细胞BEL(P<0.05).MRP-3反义转染进耐药细胞后,可明显降低细胞内ADM的耐药指数,提高细胞内ADM浓度(P<0.05),细胞内MRP-3 mRNA的表达较未转染细胞下降了62.5%(P<0.05).结论 MRP-3的高表达可能是导致肝癌多药耐药的机制之一.     

甲胎蛋白启动子介导反义乙型肝炎病毒X基因在肝癌细胞中的表达及其作用

目的 构建含甲胎蛋白(AFP)启动子和增强子的反义乙型肝炎病毒X基因(HBX)真核表达载体，研究其特异性和有效性，为开发肝癌细胞特异性HBX反义RNA基因治疗乙型肝炎病毒(HBV)奠定基础。方法 聚合酶链反应(PCR)扩增HBX(1370—1872nt)基因，克隆至EB病毒表达载体，双轮PCR筛选、鉴定基因插入方向。脂质体转染肝癌细胞和ECV304细胞，Northernblot检测HBX mRNA的表达，酶联免疫试验(ELISA)检测HBV抗原，荧光定量PCR检测HBV DNA。结果 成功构建正、反义RNA表达载体pEBAF—s—HBX、pEBAF—as—HBX。Northernblot证实反义RNA仅在AFP阳性的肝癌细胞中表达。pEBAF—as—HBX转染3d后，可显著抑制2．2．15细胞HBV复制和抗原表达，其HBsAg、HBeAg抗原表达较正义对照分别下降37．9％和36．8％，HBV DNA降低25％。结论 反义RNA表达载体pEBAF—as—HBX仅在肝癌细胞中特异表达、并可有效抑制HBV，有良好的开发应用前景。     

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.     

Effects of insulin-like growth factors-IR and -IIR antisense gene transfection on the biological behaviors of SMMC-7721 human hepatoma cells

BACKGROUND AND AIMS: Insulin-like growth factors (IGFs) are closely related to hepatocellular carcinoma growth. The study aim was to investigate the effects of IGF-IR and IGF-IIR antisense gene transfection on the biological behaviors of SMMC-7721 human hepatoma cells. METHODS: 7721-IGF-IR-AS cells (human hepatoma SMMC-7721 cells transfected with IGF-IR antisense gene in our previous study) were transfected with a plasmid vector expressing IGF-IIR cDNA in the antisense orientation by DOTAP liposome.7721-IGF-R-AS cells were obtained by selection with G418 and hygromycin. Morphological changes of the cells were observed with optic and electron microscopes. In vitro growth of the 7721-IGF-R-AS cells was observed with a soft agar test, MTT test and with naked mice inoculation test in vivo. RESULTS: The following changes were found in the SMMC-7721 cells after being transfected with the IGF-IR and IGF-IIR antisense genes: (i) the degree of malignancy of the tumor cells as revealed by cell morphology was ameliorated; (ii) the growth capability of the tumor cells in soft agar and their tumorigenicity in naked mice were significantly depressed. However, in the control groups, the SMMC-7721 cells transfected both with IGF-IR and IGF-IIR sense cDNA and SMMC-7721 cells transfected without any external genes, had no such changes. However, the cell growth curves had no significant differences among these three groups. CONCLUSION: IGF-IR and IGF-IIR antisense genes could significantly restrain the malignant behavior of human hepatoma cells and might be useful in investigating a potential route for hepatocellular carcinoma gene therapy.     

Reduction of tumorigenicity of SMMC-7721 hepatoma cells by vascular endothelial growth factor antisense gene therapy

AIM To test the hypothesis to block VEGFexpression of SMMC-7721 hepatoma cells mayinhibit tumor growth using the rat hepatomamodel.METHODS Amplifiy the 200 VEGF cDNAfragment and insert it into human U6 genecassette in the reverse orientation transcribingsmall antisense RNA which could specificallyinteract with VEGF165, and VEGF121 mRNA.Construct the retroviral vector containing thisantisense VEGF U6 cassette and package thereplication-deficient recombinant retrovirus.SMMC-7721 cells were transduced with thesevirus and positive clones were selected withG418. PCR and Southern blot analysis wereperformed to determine if U6 cassette integratedinto the genomic DNA of positive clone,Transfected tumor cells were evaluated for RNAexpression by ribonuclease protection assays.The VEGF protein in the supernatant of parentaltumor cells and genetically modified tumor cellswas determined with ELISA. In vitro and in vivogrowth properties of antisense VEGF cell clonein nude mice were analyzed.RESULTS Restriction enzyme digestion andPCR sequencing verified that the antisense VEGFRNA retroviral vector was successfullyconstructed. After G418 selection, resistantSMMC-7721 cell clone was picked up, PCR andSouthern blot analysis suggested that U6cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transducedwith U6 antisense RNA cassette could express200bp small antisense VEGF RNA and secretereduced levels of VEGF in culture condition.Production of VEGF by antisense transgene-expressing cells was 65±10 ng/L per 10~6 cells,420±45 ng/L per 10~6 cells in sense group and 495±30 ng/L per 10~6 cells in the negative controlgroup, (P<0.05), The antisense-VEGF cellclone appeared phenotypically indistinguishablefrom SMMC-7721 cells and SMMC-7721 cellstransfected sense VEGF. The growth rate of theantisense-VEGF cell clone was the same as thecontrol cells. When S. C. was implanted intonude mice, growth of antisense-VEGF cell lineswas greatly inhibited compared with controlcells.CONCLUSION Expression of antisense VEGFRNA in SMMC-7721 cells could decrease thetumorigenicity, and antisense-VEGF genetherapy may be an adjuvant treatment forhepatoma.     

C-Met信号阻断对肝癌细胞生长和运动能力的影响

目的 用合成的c-Met反义寡核苷酸链,构建的c—Met反义质粒及U1SnRNA/核酶/反义c-Met复合体来封闭c-Met信号传导,探讨c-Met信号传导阻滞对肝癌生长和转移的影响。方法 以脂质体法将c-Met反义质粒、U 1SnRNA/核酶/反义c—Met复合体转染肝癌SF7721细胞,分别用四甲基偶氮唑蓝、生长曲线、穿膜试验观察转染前后细胞的变化。将转染前后细胞分别接种裸鼠,观察移植瘤生长及转移情况。结果c-Met反义寡核苷酸链能抑制肝癌SF7721细胞的增殖,t=3.58,P< 0.05,c-Met反义核酸质粒及U1SnRNA/核酶/反义c-Met复合体质粒转染后细胞表达受体c—Met的量均减少,且转染后细胞增殖明显减慢,为对照组的50％,F=4.87,P<0.05,侵袭运动能力减弱。初步动物实验显示转染后细胞生长较慢,潜伏期延长,35 d后反义(6.37 g)与核酶(6.14 g)实验组肿瘤生长明显低于对照组(11.01g),F=5.17,P<0.05。结论 c-Met受体基因表达在肝癌生长转移中起重要作用,阻断c-Met—SF的信号传导降低肿瘤生长甚至转移的能力。     

Antihepatoma effect of alpha-fetoprotein antisense phosphorothioate oligodeoxyribonucleotides in vitro and in mice

AIM To evaluate antihepatoma effect ofantisense phosphorothioate oligodeo-xyribonucleotides(S-ODNs)targeted to alpha-fetoprotein(AFP)genes in vitro and in nudemice.METHODS AFP gene expression was examinedby immunocytochemical method or enzyme-linked immunosorbent assay.Effect of S-ODNson SMMC-7721 human hepatoma cell growth invitro was determined using microculturetetrazolium assay.In vivo antitumor activitiesof S-ODNs were monitored by measuring tumorweight differences in treated and control micebearing SMMC-7721 xenografts.Induction of cellapoptosis was evaluated by fluorescence-activated cell sorter(FACS)analysis.RESULTS Antisense S-ODN treatment led toreduced AFP gene expression.Specificantisense S-ODNs,but not control S-ODNs,inhibited the growth of heaptoma cells in vitro.In vivo,only antisense S-ODNs exhibitedobvious antitumor activities.FACS analysisrevealed that the growth inhibition by antisenseS-ODNs was associated with their cell apoptosisinduction.CONCLUSION Antisense S-ODNs targeted toAFP genes inhibit the growth of human hepatomacells and solid hepatoma,which is related totheir cell apoptosis induction.     

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.     

脂质体法转染人肿瘤坏死因子-α基因对裸鼠人肝癌移植瘤的抑制效应及其机制

背景：外源性肿瘤坏死因子（TNF）-α联合化疗药物对肿瘤的疗效较单独应用更佳,为肿瘤治疗提供了新的方向。目的：对裸鼠人肝癌移植瘤模型行脂质体介导的TNF-α基因瘤内转染,研究肝癌移植瘤的生长抑制情况及其机制。方法：经脂质体介导,以真核表达质粒pSVK3-TNF-α分别转染人肝癌细胞株SMMC-7721和裸鼠皮下SMMC-7721细胞移植瘤。测定SMMC-7721细胞的TNF-α浓度,甲基噻唑基四唑（MTT）法测定细胞杀伤率,流式细胞仪和原位末端标记（TUNEL）法检测细胞周期和凋亡情况。结果：TNF-α转基因治疗裸鼠人肝癌移植瘤结束第5d,移植瘤体积为（75.28±35.35）mm^3,显著低于对照组的（326.45±103.64）mm^3（P〈0.05）。TNF-α基因体外转染SMMC-7721细胞24、48、72h后,基因转染组每106个细胞的TNF-α表达量分别为（1680±187）pg、（1702±205）pg和（1650±164）pg,细胞杀伤率分别为（37.1±2.4）%、（79.4±4.3）%和（84.2±4.6）%。基因转染72h后,SMMC-7721细胞增殖指数为（30.5±3.2）%,显著低于对照组的（46.1±3.9）%（P〈0.05）;凋亡指数为（10.0±2.1）%,显著高于对照组的（2.7±0.4）%（P〈0.01）。结论：脂质体介导的TNF-α基因转染裸鼠人肝癌移植瘤可明显抑制肿瘤生长,其机制可能为影响肿瘤细胞生长周期以及诱导肿瘤细胞凋亡。     

Inhibitory effect of antisense vascular endothelial growth factor 165 eukaryotic expression vector on proliferation of hepatocellular carcinoma cells

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.