瑞香狼毒药效组分对裸鼠肝癌移植瘤的抑制及其机制

目的:评价瑞香狼毒药效组分抑制人肝癌细胞株BEL-7402裸鼠皮下移植瘤作用的强度,并探讨其机制.方法:建立人肝癌BEL-7402裸小鼠皮下移植瘤模型研究瑞香狼毒药效组分Zp1111抗肿瘤作用与对荷瘤裸鼠体质量与免疫器官的影响,免疫组织化学法检测用药前后BEL-7402皮下移植瘤组织Bcl-2与Bax阳性表达的变化.结果:Zp1111对裸小鼠皮下移植瘤BEL-7402具有较强的抑制作用,187.5mg/kg、250mg/kg(折合成生药量)剂量时对移植瘤的相对肿瘤增殖率T/C小于60%;该组分能显著上调BEL-7402移植瘤组织Bax基因的表达,但对Bcl-2的表达影响较弱.结论:瑞香狼毒药效组分在体内对人肝癌细胞BEL-7402裸鼠移植瘤具有较强的抑制作用并呈现一定的剂量依赖关系,该组分具有一定的诱导凋亡作用,可能与上调Bax有关.     

转染内皮抑素基因联合放射治疗对肝癌移植瘤生长的影响

实体瘤的生长和转移依赖于血管生成。用抗血管生成剂靶向肿瘤血管生成是新的肿瘤治疗方法之一。观察转染内皮抑素基因联合放射治疗对人肝癌BEL7402细胞株裸鼠移植瘤生长的影响，以探索内皮抑素联合放射治疗对肿瘤的治疗作用。     

XAF1基因重组腺病毒载体的构建及其在人肝癌细胞体内外的表达

目的利用携带35型腺病毒纤毛的嵌合型5型腺病毒载体系统Ad5/F35构建XAF1基因重组腺病毒,体内外感染人肝癌细胞SMMC7721并使XAF1基因有效表达。方法将真核表达质粒pcDNA3.1-XAF1和穿梭质粒pDC316用BamHⅠ和EcoRⅠ双酶切、筛选、测序获得重组穿梭质粒pDC316-XAF1。将测序正确的pDC316-XAF1和骨架质粒pBHG-fiber5/F35用Lipofectamine2000共转染HEK293细胞,进行细胞内同源重组,得到重组腺病毒Ad5/F35-XAF1。予终点稀释法测定重组腺病毒的感染滴度。用同样的方法得到携带增强型绿色荧光蛋白(EGFP)的报告病毒Ad5/F35-EGFP。建立人肝癌细胞株SMMC7721裸鼠移植瘤模型,将Ad5/F35-XAF1和Ad5/F35-EGFP重组腺病毒分别感染人肝癌细胞株SMMC7721和瘤内注射;荧光显微镜观察EGFP在细胞和移植瘤冰冻切片中的表达;RT-PCR和Westrenblot法检测XAF1的mRNA和蛋白在细胞和移植瘤组织的表达。结果重组腺病毒Ad5/F35-EGFP感染肝癌细胞和瘤内注射后,予荧光显微镜均可见细胞和冰冻切片中呈现绿色荧光;Ad5/F35-XAF1感染肝癌细胞和瘤内注射后,XAF1mRNA和蛋白表达均显著高于对照组和报告病毒组。结论成功构建重组腺病毒Ad5/F35-XAF1和Ad5/F35-EGFP。该腺病毒载体可携带目的基因在人肝癌细胞株SMMC7721体内和体外进行有效表达。     

双调控双功效溶瘤腺病毒的构建及意义

目的构建表达Survivin基因小发卡RNA和内皮抑素基因的双调控双功效肿瘤特异性溶瘤腺病毒(CNHK500-shSRV-mE,以下简称双调控腺病毒),为肝癌的基因治疗奠定基础。方法以人端粒酶逆转录酶启动子(hTERT)调控腺病毒E1a基因,缺氧调控元件序列(HRE)调控E1b基因,重组双调控双功效的肿瘤特异性溶瘤腺病毒载体;于E1区插入Survivin基因序列设计特异shRNA和全长内皮抑素基因构建双调控腺病毒。将双调控腺病毒分别感染人肝癌细胞株SMMC-7721、BEL-7402,观察病毒的增殖活性。结果双调控腺病毒能在肝癌细胞中特异性高拷贝增殖,而在正常细胞系中几乎不增殖。结论双调控腺病毒成功构建;其为肝癌的基因治疗奠定了基础。     

RNA干扰沉默survivin基因对结直肠癌裸鼠移植瘤的影响

目的利用RNA干扰(RNAi)技术抑制survivin基因的体内表达,探讨survivin沉默对结直肠癌裸鼠移植瘤生长的抑制作用。方法将survivin-siRNA重组腺病毒载体pBAsi-survivin导入裸鼠皮下的移植瘤内,观察不同时间点对肿瘤生长的抑制作用以及肿瘤的抑制率。同时用RT-PCR方法检测干预组(成功转染质粒组),对照组(空载体对照组)和空白对照组(生理盐水)中survivin mRNA表达情况。Western印迹检测survivin蛋白的表达。结果与对照组相比,survivin-siRNA重组载体导入裸鼠皮下移植瘤后,肿瘤体积增长缓慢,随着时间的延长,肿瘤生长抑制作用明显,survivin mRNA表达明显降低,survivin蛋白表达下降。结论 survivin-siRNA重组载体能显著抑制结直肠癌裸鼠移植瘤的增殖,Survivin mRNA和蛋白表达明显降低,不同时间点肿瘤抑制率不同,且随时间呈正相关。     

腺病毒携带凋亡素和内皮抑素双基因对小鼠肝癌细胞增殖及凋亡的影响

目的探讨肝癌基因治疗的新方法。方法建立C57BL／6j小鼠皮下种植性肝癌模型,随机分为A、B、C、D组各8只。A组注射腺病毒携带凋亡素和内皮抑素双基因,B组注射腺病毒携带凋亡素单基因,C组注射腺病毒携带绿色荧光蛋白基因,D组注射生理盐水。治疗后连续12d监测各组肿瘤体积,计算抑瘤率,RT-PCR法检测肿瘤组织中凋亡素、内皮抑素mRNA表达;TUNEL染色法检测肿瘤组织的原位凋亡并计算凋亡指数。结果治疗后第7天A组肿瘤体积明显小于其他各组,抑瘤率明显高于其他各组（P均〈0．05）;A组肿瘤组织中有凋亡素和内皮抑素mRNA表达,B组肿瘤组织中有凋亡素mRNA表达;A组凋亡指数明显高于其他各组,P〈0．05。结论腺病毒携带凋亡素和内皮抑素双基因能显著抑制小鼠肝癌细胞生长并能诱导其凋亡;该病毒有望用于肝癌的基因治疗。     

蜂毒素基因重组腺病毒抗肝癌作用的实验研究

目的将编码蜂毒素的基因用于肝癌治疗,为蜂毒素的临床应用提供实验依据.方法采用细菌内同源重组法构建携蜂毒素基因及甲胎蛋白(AFP)启动子的重组腺病毒,X-gal染色检测其转染效率,MTT法测定其对肝癌细胞的抑制作用,并观察其体外转染对肝癌细胞致瘤能力的影响及肿瘤内注射对荷肝癌裸鼠的抑瘤效果.结果蜂毒素基因mRNA在细胞中得到了转录;当感染复数(MOI)为10时,重组腺病毒在体外对BEL-7402人肝癌细胞的转染效率达到100%,体内转染亦有较高的效率.Ad-rAFP-Mel和Ad-rAFP对BEL7402细胞的抑制率分别为66.2%±2.7%和2.9%± 2.3%(t=30.83,P<0.01).Ad-CMV Mel对BEL7402、SMMC7721及L02细胞的抑制率分别为58.9%± 9.6%、65.9%±3.8%和31.7%± 1.2%,而Ad-rAFP-Mel对BEL7402、SMMC7721及L02细胞的抑制率分别为66.2%±2.7%、16.1%±6.6%和7.5%±3.3%,与Ad-CMV-Mel组比较t=1.27,P=0.27,t=11.31、P<0.01和t=12.12、P<0.01.重组腺病毒体外转染后,肝癌细胞致瘤能力减弱,肿瘤内注射,裸鼠皮下移植瘤消退. 结论蜂毒素基因重组腺病毒在体内外对肝癌均有特异性抑制作用,证明动物毒素基因作为抗肿瘤目的基因具有可行性.     

罗格列酮对人胃癌细胞裸鼠移植瘤生长和淋巴管生成的影响

背景:血管内皮生长因子-C(VEGF-C)可激活淋巴管内皮细胞表面的VEGFR-3,诱导淋巴管生成。体外研究显示PPARγ配体罗格列酮(ROS)可通过抑制VEGF-C表达抑制人胃癌细胞的淋巴管生成。目的:明确ROS在体内能否抑制胃癌生长及其淋巴管生成。方法:建立人胃癌细胞株SGC7901裸鼠皮下移植瘤模型,成瘤裸鼠随机分为模型组和3组ROS组,后者分别予50、75、100 mg.kg-1.2 d-1ROS灌胃42 d。处死裸鼠,测定移植瘤体积,D2-40淋巴管内皮免疫组化染色计数微淋巴管密度(LMVD),RT-PCR和蛋白质印迹法检测VEGF-C mRNA和蛋白表达。结果:50、75、100 mg/kg ROS组移植瘤体积、肿瘤组织LMVD以及VEGF-C mRNA和蛋白表达依次降低并均显著低于模型组(P<0.05),作用呈浓度依赖性(P<0.05)。结论:ROS可呈浓度依赖性地抑制人胃癌细胞祼鼠移植瘤生长,并通过下调VEGF-C表达抑制肿瘤淋巴管生成。     

携多药耐药基因1反义RNA重组腺病毒逆转肝癌多药耐药细胞的实验研究

目的探讨携带多药耐药基因1(multidrug resistance gene 1,MDR1)反义RNA重组腺病毒载体在裸鼠体内逆转肝癌多药耐药细胞HepG2/阿霉素(adriamycin,ADM)的疗效及作用机制。方法构建携带AFP和asmdr1的重组腺病毒载体Adeno-asmdr1,ADM分级诱导肝癌细胞HepG2为多药耐药细胞HepG2/ADM,建立HepG2/ADM裸鼠皮下移植瘤模型,Adeno asmdr1局部注射,观察移植瘤的体积、透射电镜检测移植瘤组织细胞凋亡、RT-PCR检测MDR1转录水平,评价Adeno-asmdr1的抗肿瘤活性。结果在Adeno-asmdr1 ADM组,移植瘤体积无增大,而PBS组、ADM组体积明显增大;RT-PCR检测移植瘤细胞1周和4周MDR1 mRNA水平, ADM组无明显变化,Adeno-amdr1 ADM组在4周时MDR1转录水平仅为单纯ADM组的20%,经ADM Adeno-asmdr1处理组,可见凋亡增加,ADM组和PBS处理组的裸鼠移植瘤组织中出现少量或没有凋亡。结论携带MDR1反义RNA重组腺病毒部分逆转HepG2/ADM的多药耐药,阻止肿瘤生长,下调MDR1转录水平,导致肿瘤细胞凋亡。     

腺病毒介导生长抑素2型受体对裸鼠人胰腺癌移植瘤抑制作用的研究

目的构建表达生长抑素2型受体(SSTR2)的重组腺病毒,探讨其对裸鼠人胰腺癌移植瘤生长的影响及机制。方法2004-06-01—2005-12-10,在暨南大学生物工程学系采用位点特异性重组方法构建和包装携带人生长抑素2型受体和报告基因LacZ的重组腺病毒Ad-SSTR2和Ad-LacZ。建立裸鼠人胰腺癌移植瘤模型,分别于瘤内注射生理盐水(阴性对照组)、Ad-LacZ(报告基因对照组)和Ad-SSTR2(实验组),观察肿瘤大小和重量变化。通过逆转录-聚合酶链反应(RT-PCR)和免疫印迹试验(Western blot)鉴定SSTR2在裸鼠肿瘤组织中的表达,Western blot测定信号传导通路蛋白ERK2和ras的表达情况。结果获得滴度分别为6.0×1012pfu/L和6.5×1012pfu/L的重组腺病毒Ad-SSTR2和Ad-LacZ。Ad-SSTR2转染裸鼠胰腺癌移植瘤后SSTR2 mRNA和蛋白都得到有效表达,实验组对移植瘤生长具有明显的抑制作用,抑制率为48.2%。与阴性对照组和报告基因对照组相比,实验组ERK2和ras蛋白的表达量明显减少(P<0.01)。结论腺病毒介导的生长抑素2型受体对裸鼠人胰腺癌移植瘤的生长具有抑制作用,其作用机制可能与信号传导通路蛋白ERK2和ras的表达下调有关,提示其有可能成为胰腺癌基因治疗的一种有效方法。     

Endostatin gene therapy for liver cancer by a recombinant adenovirus delivery

AIM: To investigate the expression of adenovirus-mediated human endostatin (Ad/hEndo) gene transfer and its effect on the growth of hepatocellular carcinoma (HCC) BEL-7402 xenografted tumors. METHODS: Immunohistochemistry analysis with an anti-endostatin antibody was preformed to detect endostatin protein expression in HCC BEL-7402 cells infected with Ad/hEndo. MTT assay was used to investigate the effects of Ad/hEndo on proliferation of human umbilical vein endothelial cells (HUVEC). Intra-tumoral injections of 1X10(9) pfu Ad/hEndo was given to treat BEL-7402 xenografted tumors in nude mice once weekly for 6 wk. Mice received injections of Ad/LacZ and DMEM were regarded as control groups. After intra-tumoral administration with Ad/hEndo, the endostatin mRNA expression in tumor tissue was analyzed by Northern blotting, and plasma endostatin levels were determined using enzyme-linked immunosorbent assay (ELISA). RESULTS: High level expression of endostatin gene was detected in the infected HCC BEL-7402 cells. Ad/hEndo significantly inhibited HUVEC cell proliferation by 57.2% at a multiplicity of infection (MOI) of 20. After 6-week treatment with Ad/hEndo, the growth of treated tumors was inhibited by 46.50% compared to the Ad/LacZ control group (t=2.729, P<0.05) and by 48.56% compared to the DMEM control group (t=2.485, P<0.05). The ratio of mean tumor volume in treated animals to mean tumor volume in the control animals (T:C ratio) was less than 50% after 24 d of treatment. Endostatin mRNA in tumor tissue was clearly demonstrated as a band of approximately 1.2 kb, which was the expected size of intact and functional endostatin. Plasma endostatin levels peaked at 87.52+/-8.34 ng/mL at d 3 after Ad/hEndo injection, which was significantly higher than the basal level (12.23+/-2.54 ng/mL). By d 7, plasma levels dropped to nearly half the peak level (40.34+/-4.80 ng/mL). CONCLUSION: Adenovirus-mediated human endostatin gene can successfully express endogenous endostatin in vitro and in vivo, and significantly inhibit the growth of BEL-7402 xenografted liver tumors in nude mice.     

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.     

Recurrence or metastasis of HCC:predictors, early detection and experimental antiangiogenic therapy

AIM To investigate the predictors for recurrence or metastasis of HCC, and to evaluate the effect of antiangiogenic therapy on the growth of transplantable human HCC in nude mice. METHODS RT-PCR was used to measure the expression of matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) in 56 pairs of nontumorous liver and tumor samples. Sixty blood samples from human HCC were examined by nested RT-PCR to find out AFP mRNA. Recombinant human endostatin and polyclonal antibody against VEGF were administered to treat human HCC transplanted in nude mice. RESULTS Thirty of 56 HCC samples showed stronger expression of MMP-9 in tumorous tissues than in nontumorous tissues. Fifteen of the 26 patients with relative expression level of MMP-9 more than 0.34 developed tumor recurrence or metastasis, whereas only 7 of 30 patients with relative expression level less than 0.34 developed tumor recurrence (P＜0.05).There was no significant difference in the relative expression level of VEGF between patients with postoperative recurrence or metastasis and those without recurrence. AFP mRNA was detectable in 53.3% of patients with HCC. The sensitivity and specificity of AFP mRNA as a marker to detect hematogenous dissemination of HCC cells was 81.8% and 84.4%, respectively. Recombinant human endostatin and polyclonal antibody against VEGF inhibited the growth of transplantable HCC in nude mice by 52.2% and 45.7%, respectively.CONCLUSION MMP-9 expression in HCC correlates with the postoperative recurrence or metastasis of HCC. Patients with high level of MMP-9 expression in HCC are susceptible to metastasis. AFP mRNA could serve as an indicator of hematogenous spreading of HCC cells in circulation and a predictor of recurrence or metastasis of HCC. Antiangiogenesis may be an adjuvant therapy for HCC.     

Effect of arsenic trioxide on human hepatocarcinoma in nude mice

AIM: To study the effect of arsenic trioxide (As(2)0(3)) on human hepatoma cell line BEL-7402 in vivo. METHODS: Human hepatoma cell line BEL-7402 cultured in vitro was inoculated into nude mice and arsenic trioxide, 5-Fu and saline were injected into abdominal cavity of the nude mice respectively. The volumes of tumor and general conditions of the nude mice and structural changes of the liver and kidney were observed. Morphologic changes were studied under electron microscope. Expression of AFP was investigated by immunohistochemical method. RESULTS: As(2)O(3) could inhibit the growth of tumor. The tumor growth inhibitory rate in mice treated with 2.5 mg/kg As(2)O(3) was 53.42% on the tenth day. The tumor growth inhibitory rate in mice treated with 5 mg/kg As(2)O(3) was 79.28% on the fifth day and 96.58% on the tenth day respectively. As(2)O(3) did not damage the liver and kidney of nude mice, or affect the blood system. Typical apoptotic morphological changes were found under electron microscope, and the change of mitochondria was obvious. The expression rate of AFP declined after treatment. CONCLUSION: Arsenic trioxide can induce apoptosis of human hepatoma cells, and inhibit proliferation of tumor with no obvious side effects on liver and kidney.     

Genistein inhibits invasive potential of human hepatocellular carcinoma by altering cell cycle, apoptosis, and angiogenesis

AIM: To study the in vitro and in vivo inhibitory effects of genistein on invasive potential of Bel 7402 hepatocellular carcinoma (HCC) cells and to explore the underlying mechanism. METHODS: Bel 7402 HCC cells were exposed to genistein. The invasive activity of tumor cells was assayed in transwell cell culture chamber. p125FAK expression and cell cycle were evaluated by a functional assay. Cell apoptosis analysis was performed with TUNEL method. In addition, bilateral subrenal capsule xenograft transplantation of HCC was performed in 10 nude mice. Genistein was injected and the invasion of HCC into the renal parenchyma was observed. Microvessels with immunohistochemical staining were detected. RESULTS: Genistein significantly inhibited the growth of Bel 7402 cells, the inhibitory rate of tumor cells was 26 -42%. The invasive potential of Bel 7402 cells in vitro was significantly inhibited, the inhibitory rate was 11-28%. Genistein caused G2/M cell cycle arrest, S phase decreased significantly. The occurrence of apoptosis in genistein group increased significantly. The expression of p125FAK in 5 μg/mL genistein group (15.26±0.16%) and 10 μg/mL genistein group (12.89±0.36%) was significantly lower than that in the control group (19.75±1.12%,P<0.05). Tumor growth in genistein-treated nude mice was significantly retarded in comparison to control mice, the inhibitory rate of tumor growth was about 20%. Genistein also significantly inhibited the invasion of Bel 7402 cells into the renal parenchyma of nude mice with xenograft transplant. The positive unit value of microvessels in genistein-treated group (10.422±0.807) was significantly lower than that in control group (22.330±5.696, P< 0.01). CONCLUSION: Genistein can effectively inhibit the invasive potential of Bel 7402 HCC cells by altering cell cycle, apoptosis and angiogenesis, inhibition of focal adhesion kinase may play a significant role in this process.     

Heat shock protein 70 chaperoned alpha-fetoprotein in human hepatocellular carcinoma cell line BEL-7402

AIM: To investigate the interaction between heat shock protein 70 (HSP70) and a-fetoprotein (AFP) in human hepatocellular carcinoma (HCC) cell line BEL-7402. METHODS: The expression and localization of HSP70 and AFP in human HCC cell line BEL-7402 were determined by immunocytochemistry and indirect immunofluorescence cytochemical staining. The interaction between HSP70 and AFP in HCC cells was analyzed by immunoprecipitation and Western blot. RESULTS: Immunocytochemical staining detection showed that HCC cell BEL-7402 expressed a high level of HSP70 and AFP synchronously. Both were stained in cell plasma. AFP existed in the immunoprecipitate of anti-HSP70 mAb, while there was HSP70 in the immunoprecipitate of anti-AFP mAb. CONCLUSION: HSP70 chaperones AFP in human HCC cell BEL-7402. The interaction between HSP70 and AFP in human HCC cell can be a new route to study the pathogenesis and immunotherapy of HCC.