重组腺相关病毒介导caveolin-1基因表达抑制肝癌新生血管形成的实验研究

目的 探讨caveolin-1参与肝细胞癌血管生成及影响血管通透性的作用机制.方法 构建肝癌细胞系HepG2裸鼠皮下移植瘤模型,局部注射编码重组可溶性caveolin-1蛋白的重组腺相关病毒载体rAAV-PEG-caveolin-1,观察其对肝癌细胞生长的抑制作用;通过肿瘤微血管密度(microvessel density,MVD)检测肿瘤新生血管的生成;Western blot检测脉管标志蛋白PECAM-1表达改变;FITC标记右旋糖苷测定肿瘤微血管通透性;放射强度测定法检测肝癌组织中eNOS活性,TUNEL法检测caveolin-1对肿瘤细胞的诱导凋亡效应.结果 rAAV-PEG-caveolin-1转染组治疗第14天后,移植瘤的瘤重较对照组和空载体转染组有不同程度的减小(P＜O.05);免疫组化分析及Western blot结果显示rAAV-PEG-caveolin-1转染组较对照组及空载体组MVD及PECAM-1表达显著减少(P＜0.05);FITC-右旋糖苷测定表明rAAV-PEG-caveolin-1可有效降低肿瘤微血管通透性;rAAV-PEG-caveolin-1转染组较对照组及空载体组eNOS活性明显下降(P＜0.05);TUNEL分析显示rAAV-PEG-caveolin-1可诱导肿瘤细胞凋亡.结论 Caveolin-1是血管生成调节的关键分子,能显著抑制肿瘤新生血管的形成,并能有效降低肿瘤微血管的通透性.

Abstract：

Objective To investigate the antitumor effect of the recombined adeno-associated virus encoding caveolin-1 regulated by progression-elevated gene (PEG) promotor on the angiogenesis of implanted human hepatocellular carcinoma(HCC) cell lines in a nude mouse model. Methods HepG2 cells were inoculated subcutaneously into NOD/SCID mice, and animals were treated with rAAV-PEG-caveolin-1 after tumor cell innoculation. The fluorescence ratio of Evans blue to FITC-dextran was used to assess the changes of microvasculature permeability of the tumor. Western blot and immunohistochemical analyses were employed to detect PECAM-1 expression in tumor microvascular endothelium and microvessel density(MVD), respectively; NOS activity was assessed by citrulline generation. Tumor growth was observed and tumor cell apoptosis in tumor tissues were measured by TUNEL. Results Tumor growth was significantly inhibited in mice injected with rAAV-PEG-caveolin-1. The administration of rAAV-PEG-caveolin-1 significantly blocked vascular leakage in the tumor microcirculation. The levels of PECAM-1 protein detected by Western blot were markedly reduced in rAAV-PEG-caveolin-1-treated mice, and there were fewer MVD in tumors from caveolin-1-treated mice, while NOS catalytic activity was much lower in rAAV-PEG-caveolin-1-treated mice compared to the control and empty vector-treated animals. TUNEL demonstrated significant induction of tumor cell specific apoptosis. Conclusions rAAV-PEG-caveolin-1 can reduce tumor progression through blocking microvascular formation by inhibiting eNOS.     

表达端粒酶逆转录酶siRNA的溶瘤腺病毒对裸鼠肾癌移植瘤的治疗作用

Objective To investigate the antitumor effect of oncolytic adenovirus armed with small interference RNA targeting hTERT gene for renal cancer therapy. Methods Nude mice were divid-ed randomly into 4 groups (8 mice/group),and were treated by intratumoral injections of ZD55-hTERT ( an oncolytic adenovirus armed with small interference RNA targeting hTERT gene) ,ZD55-EGFP ( an on-colytic adenovirus) and Ad-hTERT (replication-defective adenovirus armed with small interference RNA targeting hTERT gene) with three consecutive daily at 7 × 108 pfu/day or treated with PBS as a control. The expression of E1A and hTERT, and apoptosis of tumor xenografts were assessed by immunohistochemi-cal technique at the 7th day after injections. The tumor volume was measured at the 50th day after injec-tions. Results The tumor volume in ZD55-hTERT treatment group ( 124.1±27.5) was significantly less than that in ZD-EGFP (499.8±77.1 ) and Ad-hTERT ( 609.0±102.5 ) treatment groups. The E 1A pos-itive expression in ZD55-hTERT treatment group was significantly higher than that in Ad-hTERT treatment group. The hTERT positive expression in ZD55-hTERT treatment group was significantly lower than that in Ad-hTERT treatment group. ZD55-hTERT treatment of tumor xenografts resulted in an increased apoptotie cell death as compared with ZD55-EGFP and Ad-hTERT treatment. Conclusion The antitumor effect of ZD55-hTERT was more potent than oneolytie adenovirus ZD55-EGFP and Ad-hTERT.     

表达端粒酶逆转录酶siRNA的溶瘤腺病毒对裸鼠肾癌移植瘤的治疗作用

Objective To investigate the antitumor effect of oncolytic adenovirus armed with small interference RNA targeting hTERT gene for renal cancer therapy. Methods Nude mice were divid-ed randomly into 4 groups (8 mice/group),and were treated by intratumoral injections of ZD55-hTERT ( an oncolytic adenovirus armed with small interference RNA targeting hTERT gene) ,ZD55-EGFP ( an on-colytic adenovirus) and Ad-hTERT (replication-defective adenovirus armed with small interference RNA targeting hTERT gene) with three consecutive daily at 7 × 108 pfu/day or treated with PBS as a control. The expression of E1A and hTERT, and apoptosis of tumor xenografts were assessed by immunohistochemi-cal technique at the 7th day after injections. The tumor volume was measured at the 50th day after injec-tions. Results The tumor volume in ZD55-hTERT treatment group ( 124.1±27.5) was significantly less than that in ZD-EGFP (499.8±77.1 ) and Ad-hTERT ( 609.0±102.5 ) treatment groups. The E 1A pos-itive expression in ZD55-hTERT treatment group was significantly higher than that in Ad-hTERT treatment group. The hTERT positive expression in ZD55-hTERT treatment group was significantly lower than that in Ad-hTERT treatment group. ZD55-hTERT treatment of tumor xenografts resulted in an increased apoptotie cell death as compared with ZD55-EGFP and Ad-hTERT treatment. Conclusion The antitumor effect of ZD55-hTERT was more potent than oneolytie adenovirus ZD55-EGFP and Ad-hTERT.     

表达端粒酶逆转录酶siRNA的溶瘤腺病毒对裸鼠肾癌移植瘤的治疗作用

Objective To investigate the antitumor effect of oncolytic adenovirus armed with small interference RNA targeting hTERT gene for renal cancer therapy. Methods Nude mice were divid-ed randomly into 4 groups (8 mice/group),and were treated by intratumoral injections of ZD55-hTERT ( an oncolytic adenovirus armed with small interference RNA targeting hTERT gene) ,ZD55-EGFP ( an on-colytic adenovirus) and Ad-hTERT (replication-defective adenovirus armed with small interference RNA targeting hTERT gene) with three consecutive daily at 7 × 108 pfu/day or treated with PBS as a control. The expression of E1A and hTERT, and apoptosis of tumor xenografts were assessed by immunohistochemi-cal technique at the 7th day after injections. The tumor volume was measured at the 50th day after injec-tions. Results The tumor volume in ZD55-hTERT treatment group ( 124.1±27.5) was significantly less than that in ZD-EGFP (499.8±77.1 ) and Ad-hTERT ( 609.0±102.5 ) treatment groups. The E 1A pos-itive expression in ZD55-hTERT treatment group was significantly higher than that in Ad-hTERT treatment group. The hTERT positive expression in ZD55-hTERT treatment group was significantly lower than that in Ad-hTERT treatment group. ZD55-hTERT treatment of tumor xenografts resulted in an increased apoptotie cell death as compared with ZD55-EGFP and Ad-hTERT treatment. Conclusion The antitumor effect of ZD55-hTERT was more potent than oneolytie adenovirus ZD55-EGFP and Ad-hTERT.     

表达端粒酶逆转录酶siRNA的溶瘤腺病毒对裸鼠肾癌移植瘤的治疗作用

Objective To investigate the antitumor effect of oncolytic adenovirus armed with small interference RNA targeting hTERT gene for renal cancer therapy. Methods Nude mice were divid-ed randomly into 4 groups (8 mice/group),and were treated by intratumoral injections of ZD55-hTERT ( an oncolytic adenovirus armed with small interference RNA targeting hTERT gene) ,ZD55-EGFP ( an on-colytic adenovirus) and Ad-hTERT (replication-defective adenovirus armed with small interference RNA targeting hTERT gene) with three consecutive daily at 7 × 108 pfu/day or treated with PBS as a control. The expression of E1A and hTERT, and apoptosis of tumor xenografts were assessed by immunohistochemi-cal technique at the 7th day after injections. The tumor volume was measured at the 50th day after injec-tions. Results The tumor volume in ZD55-hTERT treatment group ( 124.1±27.5) was significantly less than that in ZD-EGFP (499.8±77.1 ) and Ad-hTERT ( 609.0±102.5 ) treatment groups. The E 1A pos-itive expression in ZD55-hTERT treatment group was significantly higher than that in Ad-hTERT treatment group. The hTERT positive expression in ZD55-hTERT treatment group was significantly lower than that in Ad-hTERT treatment group. ZD55-hTERT treatment of tumor xenografts resulted in an increased apoptotie cell death as compared with ZD55-EGFP and Ad-hTERT treatment. Conclusion The antitumor effect of ZD55-hTERT was more potent than oneolytie adenovirus ZD55-EGFP and Ad-hTERT.     

表达端粒酶逆转录酶siRNA的溶瘤腺病毒对裸鼠肾癌移植瘤的治疗作用

Objective To investigate the antitumor effect of oncolytic adenovirus armed with small interference RNA targeting hTERT gene for renal cancer therapy. Methods Nude mice were divid-ed randomly into 4 groups (8 mice/group),and were treated by intratumoral injections of ZD55-hTERT ( an oncolytic adenovirus armed with small interference RNA targeting hTERT gene) ,ZD55-EGFP ( an on-colytic adenovirus) and Ad-hTERT (replication-defective adenovirus armed with small interference RNA targeting hTERT gene) with three consecutive daily at 7 × 108 pfu/day or treated with PBS as a control. The expression of E1A and hTERT, and apoptosis of tumor xenografts were assessed by immunohistochemi-cal technique at the 7th day after injections. The tumor volume was measured at the 50th day after injec-tions. Results The tumor volume in ZD55-hTERT treatment group ( 124.1±27.5) was significantly less than that in ZD-EGFP (499.8±77.1 ) and Ad-hTERT ( 609.0±102.5 ) treatment groups. The E 1A pos-itive expression in ZD55-hTERT treatment group was significantly higher than that in Ad-hTERT treatment group. The hTERT positive expression in ZD55-hTERT treatment group was significantly lower than that in Ad-hTERT treatment group. ZD55-hTERT treatment of tumor xenografts resulted in an increased apoptotie cell death as compared with ZD55-EGFP and Ad-hTERT treatment. Conclusion The antitumor effect of ZD55-hTERT was more potent than oneolytie adenovirus ZD55-EGFP and Ad-hTERT.     

表达端粒酶逆转录酶siRNA的溶瘤腺病毒对裸鼠肾癌移植瘤的治疗作用

Objective To investigate the antitumor effect of oncolytic adenovirus armed with small interference RNA targeting hTERT gene for renal cancer therapy. Methods Nude mice were divid-ed randomly into 4 groups (8 mice/group),and were treated by intratumoral injections of ZD55-hTERT ( an oncolytic adenovirus armed with small interference RNA targeting hTERT gene) ,ZD55-EGFP ( an on-colytic adenovirus) and Ad-hTERT (replication-defective adenovirus armed with small interference RNA targeting hTERT gene) with three consecutive daily at 7 × 108 pfu/day or treated with PBS as a control. The expression of E1A and hTERT, and apoptosis of tumor xenografts were assessed by immunohistochemi-cal technique at the 7th day after injections. The tumor volume was measured at the 50th day after injec-tions. Results The tumor volume in ZD55-hTERT treatment group ( 124.1±27.5) was significantly less than that in ZD-EGFP (499.8±77.1 ) and Ad-hTERT ( 609.0±102.5 ) treatment groups. The E 1A pos-itive expression in ZD55-hTERT treatment group was significantly higher than that in Ad-hTERT treatment group. The hTERT positive expression in ZD55-hTERT treatment group was significantly lower than that in Ad-hTERT treatment group. ZD55-hTERT treatment of tumor xenografts resulted in an increased apoptotie cell death as compared with ZD55-EGFP and Ad-hTERT treatment. Conclusion The antitumor effect of ZD55-hTERT was more potent than oneolytie adenovirus ZD55-EGFP and Ad-hTERT.     

表达端粒酶逆转录酶siRNA的溶瘤腺病毒对裸鼠肾癌移植瘤的治疗作用

Objective To investigate the antitumor effect of oncolytic adenovirus armed with small interference RNA targeting hTERT gene for renal cancer therapy. Methods Nude mice were divid-ed randomly into 4 groups (8 mice/group),and were treated by intratumoral injections of ZD55-hTERT ( an oncolytic adenovirus armed with small interference RNA targeting hTERT gene) ,ZD55-EGFP ( an on-colytic adenovirus) and Ad-hTERT (replication-defective adenovirus armed with small interference RNA targeting hTERT gene) with three consecutive daily at 7 × 108 pfu/day or treated with PBS as a control. The expression of E1A and hTERT, and apoptosis of tumor xenografts were assessed by immunohistochemi-cal technique at the 7th day after injections. The tumor volume was measured at the 50th day after injec-tions. Results The tumor volume in ZD55-hTERT treatment group ( 124.1±27.5) was significantly less than that in ZD-EGFP (499.8±77.1 ) and Ad-hTERT ( 609.0±102.5 ) treatment groups. The E 1A pos-itive expression in ZD55-hTERT treatment group was significantly higher than that in Ad-hTERT treatment group. The hTERT positive expression in ZD55-hTERT treatment group was significantly lower than that in Ad-hTERT treatment group. ZD55-hTERT treatment of tumor xenografts resulted in an increased apoptotie cell death as compared with ZD55-EGFP and Ad-hTERT treatment. Conclusion The antitumor effect of ZD55-hTERT was more potent than oneolytie adenovirus ZD55-EGFP and Ad-hTERT.     

表达端粒酶逆转录酶siRNA的溶瘤腺病毒对裸鼠肾癌移植瘤的治疗作用

Objective To investigate the antitumor effect of oncolytic adenovirus armed with small interference RNA targeting hTERT gene for renal cancer therapy. Methods Nude mice were divid-ed randomly into 4 groups (8 mice/group),and were treated by intratumoral injections of ZD55-hTERT ( an oncolytic adenovirus armed with small interference RNA targeting hTERT gene) ,ZD55-EGFP ( an on-colytic adenovirus) and Ad-hTERT (replication-defective adenovirus armed with small interference RNA targeting hTERT gene) with three consecutive daily at 7 × 108 pfu/day or treated with PBS as a control. The expression of E1A and hTERT, and apoptosis of tumor xenografts were assessed by immunohistochemi-cal technique at the 7th day after injections. The tumor volume was measured at the 50th day after injec-tions. Results The tumor volume in ZD55-hTERT treatment group ( 124.1±27.5) was significantly less than that in ZD-EGFP (499.8±77.1 ) and Ad-hTERT ( 609.0±102.5 ) treatment groups. The E 1A pos-itive expression in ZD55-hTERT treatment group was significantly higher than that in Ad-hTERT treatment group. The hTERT positive expression in ZD55-hTERT treatment group was significantly lower than that in Ad-hTERT treatment group. ZD55-hTERT treatment of tumor xenografts resulted in an increased apoptotie cell death as compared with ZD55-EGFP and Ad-hTERT treatment. Conclusion The antitumor effect of ZD55-hTERT was more potent than oneolytie adenovirus ZD55-EGFP and Ad-hTERT.     

表达端粒酶逆转录酶siRNA的溶瘤腺病毒对裸鼠肾癌移植瘤的治疗作用

Objective To investigate the antitumor effect of oncolytic adenovirus armed with small interference RNA targeting hTERT gene for renal cancer therapy. Methods Nude mice were divid-ed randomly into 4 groups (8 mice/group),and were treated by intratumoral injections of ZD55-hTERT ( an oncolytic adenovirus armed with small interference RNA targeting hTERT gene) ,ZD55-EGFP ( an on-colytic adenovirus) and Ad-hTERT (replication-defective adenovirus armed with small interference RNA targeting hTERT gene) with three consecutive daily at 7 × 108 pfu/day or treated with PBS as a control. The expression of E1A and hTERT, and apoptosis of tumor xenografts were assessed by immunohistochemi-cal technique at the 7th day after injections. The tumor volume was measured at the 50th day after injec-tions. Results The tumor volume in ZD55-hTERT treatment group ( 124.1±27.5) was significantly less than that in ZD-EGFP (499.8±77.1 ) and Ad-hTERT ( 609.0±102.5 ) treatment groups. The E 1A pos-itive expression in ZD55-hTERT treatment group was significantly higher than that in Ad-hTERT treatment group. The hTERT positive expression in ZD55-hTERT treatment group was significantly lower than that in Ad-hTERT treatment group. ZD55-hTERT treatment of tumor xenografts resulted in an increased apoptotie cell death as compared with ZD55-EGFP and Ad-hTERT treatment. Conclusion The antitumor effect of ZD55-hTERT was more potent than oneolytie adenovirus ZD55-EGFP and Ad-hTERT.     

Antiangiogenic effect of ZD1839 against murine renal cell carcinoma (RENCA) in an orthotopic mouse model

INTRODUCTION: ZD1839 (Iressa) is a selective epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI). We evaluated the antitumor and antiangiogenesis activities of ZD1839 in a murine renal cell carcinoma (RENCA) model. MATERIALS AND METHODS: The effect of ZD1839 on the cellular proliferation of RENCA cells in vitro was measured by colorimetric assay. For the in vivo studies, RENCA cells were adsorbed in Gelfoam and implanted into BALB/cJ mouse parenchyma with an agarose bar. Mice were treated with ZD1839 (40 mg/kg/day s.c.), genistein or saline for 14 days. Western blot analysis was performed to observe EGFR expression in RENCA cells and tumor tissues. Microvessel density (MVD) was quantified by immunostaining for factor VIII-related antigens and VEGF level was assayed by ELISA. RESULTS: ZD1839 showed a dose-dependent inhibition of RENCA cellular proliferation. ZD1839 treatment resulted in a marked decrease in tumor growth compared with saline treatment. The MVD and VEGF in the RENCA tumors were decreased significantly by ZD1839 (p<0.01 and p>0.05, respectively). Genistein also suppressed tumor growth and decreased MVD and VEGF level, but the efficacies were less than with ZD1839. CONCLUSION: The suppressive activity of ZD1839 on RENCA tumor growth was accompanied by decreases in the MVD and VEGF production. These results suggest that the antitumor effect of ZD1839 in a RENCA model is mediated partially by the inhibition of tumor angiogenesis.     

基因重组腺相关病毒内皮抑素基因治疗膀胱癌的实验研究

目的 探讨基因重组腺相关病毒(rAAV)内皮抑素(ES)基因治疗膀胱癌的效果.方法 通过rAAV增强绿色荧光蛋白(EGFP)转染膀胱肿瘤T24细胞株确定rAAV对细胞的转染效率;rAAV-ES体外转染T24细胞及人脐静脉血管内皮细胞(HUVEC),偶氮唑盐法、流式细胞仪检测其对T24细胞及HUVEC细胞凋亡的诱导作用;构建裸鼠膀胱癌模型,观察肿瘤生长情况及测量微血管密度(MVD),分析rAAV-ES基因治疗膀胱癌的体内效果.结果 rAAV-EGFP转染T24细胞后可以稳定表达EGFP;rAAV-ES转染T24细胞72 h后.细胞凋亡率37.02%,明显高于空病毒转染组(7.90%)和空白对照组(0.30%).差异均有统计学意义(均P＜0.05).荷瘤裸鼠瘤内注射rAAV-ES9 d后,肿瘤生长受到明显抑制;治疗4周结束后rAAV-ES组、rAAV-MCS组和空白对照组肿瘤体积分别为(0.75±0.08)、(1.27±0.13)、(1.24±0.17)cm~3,差异有统计学意义(P＜0.05).结论 体内外实验表明rAAV-ES可有效抑制膀胱癌的血管生成和肿瘤生长.     

增殖型腺病毒介导的人内皮抑素基因治疗胰腺癌

目的 观察携带人内皮抑素基因的双重调控增殖型腺病毒(AdTPHre-hEndo)对胰腺癌的治疗作用.方法 通过病毒重组技术将人内皮抑素基因克隆入双重调控增殖型腺病毒基因组中,获得腺病毒滴度为3.25×1010pfu/ml;通过建立SW-1990胰腺癌细胞Balb/c裸鼠皮下移植瘤模型,分析基因转导后胰腺癌组织中内皮抑素的表达情况及对肿瘤血管的抑制作用.结果 构建了AdTPHre-hEndo;AdTPHre-hEndo组荷瘤裸鼠肿瘤体积显著低于携带人内皮抑素基因的重组腺病毒(Ad-hEndo)组(P＜0.01)和选择性增殖型腺病毒(ONYX-015)组(P＜0.05);AdTPHre-hEndo组内皮抑素表达量明显高于Ad-hEndo组和对照组(P＜0.01).AdTPHre-hEndo组肿瘤微血管密度(MVD)为6.8±2.5,Ad-hEndo组和对照组MVD分别为16.0±4.6(P＜0.01)、47.2±10.0(P＜0.01).结论 所构建的AdTPHre-hEndo可有效表达具有生物学活性的内皮抑素,使肿瘤内微血管生成减少,肿瘤细胞增殖减慢,具备应用于胰腺癌临床治疗的潜力.     

缺氧诱导因子-1α小干扰RNA对人膀胱癌小鼠移植瘤生长的实验研究

目的 观察缺氧诱导因子-1α(HIF-1α)siRNA对人膀胱癌小鼠膀胱原位移植瘤生长的抑制作用,探讨HIF-1α作为膀胱癌基因治疗靶点的有效性.方法 把已稳定转染pGC-siHIF-1α的人膀胱癌细胞株T24接种至小鼠膀胱.通过CT观察肿瘤生长,应用免疫组化(SABC)检测肿瘤细胞HIF-1α和肿瘤间质CD34的水平,根据CD34表达情况评价肿瘤微血管密度(MVD).末端脱氧核苷酸转移酶标注法(TUNEL)测定肿瘤细胞凋亡指数;二苯基溴化四氮唑蓝(MTT)法测定肿瘤细胞增值率;流式细胞仪(FCM)测定肿瘤细胞周期分布.结果 实验组与对照组相比较,实验组成瘤率低,肿瘤生长速度缓慢(p＜0.05);HIF-1α水平下降(P＜0.01);肿瘤微血管密度减少(P＜0.01);凋亡指数升高(P＜0.01);增殖能力减弱(P＜0.01);G0/GI期细胞增多,G2/M期和S期细胞均减少(P＜0.05);与对照组相比较,组间差异均具有统计学意义.结论 以HIF-1α为靶点的siRNA有抑制H1F-1α表达,遏制膀胱癌T24细胞在体内生长的作用,HIF-1 0有可能成为临床膀胱癌基因治疗的新的有效靶点.     

全反式维甲酸对人结肠癌生长、肝转移的抑制作用

目的 观察全反式维甲酸(ATRA)对人结肠癌细胞生长及肝转移的抑制作用.方法 0、2、4、8μmol／LATRA分别作用于SW480/M5细胞48 h,免疫组织化学检测SW480/M5细胞血管内皮生长因子-A(VEGF-A)的表达水平.以SW480/M5细胞建立裸鼠原位肿瘤种植模型,随机分为5组,分别以生理盐水、溶剂对照液及5、15、45 mg/kg ATRA对裸鼠隔日灌胃,6周后观察裸鼠原位种植瘤及肝转移瘤的生长,实时荧光定量聚合酶链反应(RT-PCR)检测肿瘤组织VEGF-A mRNA表达水平,免疫组织化学检测裸鼠肿瘤组织微血管密度(MVD).结果 SW480/M5细胞VEGF-A阳性率随着ATRA作用浓度的增加逐渐下降,差异有统计学意义(x2=630.96,P＜0.01).各ATRA作用组原位肿瘤的抑瘤率为4.1％、31.6％及49.7％,肝转移发生率为63.6％、36.4％及20.0％,差异均有统计学意义(分别为P＜0.01及P＜0.05).与对照组比较,中剂量及高剂量组裸鼠原位肿瘤显著缩小(P＜0.05)、肿瘤肝转移程度明显降低(P＜0.05),肿瘤组织的VEGF-A mRNA水平及MVD值亦显著下降(P＜0.05).结论 ATRA能下调人结肠癌原位种植肿瘤和肝转移瘤组织VEGF-A的表达,减少肿瘤新生血管的形成,对结肠癌生长和肝转移具有抑制作用.     

慢病毒介导的microRNA-21-siRNA对肝癌细胞系HepG2生物学行为的影响

目的 通过构建慢病毒介导( lentivirus,LV)的microRNA-21-siRNA,探讨下调microRNA-21对肝癌细胞系HepG2生物学行为的影响.方法 针对目标序列合成特异性siRNA干扰片段并克隆入慢病毒载体pGCSIL-GFP,将重组microRNA-21-RNAi-LV转染肝细胞癌HepG2.体外设为干扰组(microRNA-21 -siRNA,SI组)、阴性对照组(negative control,NC组)和正常组(normal,N组);分别采用聚合酶链反应方法检测microRNA-21目的基因的表达的情况;用噻唑蓝比色法检测增殖情况、transwell小室迁移实验检测迁移情况、Hochest33258凋亡实验检测凋亡情况.体内实验设为SI组和NC组,观察反义microRNA-21对裸鼠移植瘤生长的影响.结果 (1)micmRNA-21 -RNAi-LV可显著降低microRNA-21的表达.(2) MTT实验96h后SI组增殖能力显著低于NC组和N组(P=0.0031,P＜0.05).(3)迁移能力减弱(P =0.0004,P＜0.05).(4)SI组细胞凋亡明显增加,促凋亡基因caspase3基因表达上调(P =0.0002,P＜0.05).(5)各组裸鼠皮下肿瘤生长比较差异无统计学意义(P=0.0624,P＞0.05).结论 microRNA-21 -siRNA通过抑制microRNA-21的表达抑制细胞增殖,促进细胞凋亡,降低其迁移能力.     

围介入手术期联合应用IFN-α及ATRA治疗肝癌的机制探讨

目的 在围介入治疗间期,探讨一种疗效好、副作用少的辅助治疗方法 .方法 建立肝癌细胞CBRH7919的大鼠动物模型,选22只接种瘤块组织的Wistar大鼠,3周瘤块形成后全部给予肝动脉结扎.分为2组:(1)对照组(12只):给予腹腔注射等量生理盐水,(2)干扰素-α(IFN-α)和全反式维甲酸(ATRA)联合高剂量组(10只):腹腔给予IFN-α(1000 000 U·kg~(-1)·qod~(-1))隔天1次,腹腔给予ATRA(10 mg·kg~(-1)·d~(-1))1次/d,于肝动脉结扎后第1天开始用药,治疗10 d后处死动物,测肿瘤组织中转移相关基因表达水平、蛋白表达水平、肿瘤微血管密度及肿瘤细胞凋亡.结果 RT-PCR法检测肿瘤组织中转移相关基因VEGF、bFGF的mRNA表达,两组相比,IFN-α联合ATRA处理组的肿瘤组织中bFGF和VEGF表达显著下降(P＜0.05).免疫组化法检测肿瘤组织中VEGF、bFGF的蛋白表达水平与RT-PCR结果 一致,IFN-α和ATRA联合处理组的肿瘤组织中bFGF和VEGF下降明显,bFGF表达很弱,而VEGF几乎不表达.ELISA法检测血清中VEGF水平,对照组血清VEGF浓度为(92.5±13.9)pg/ml,IFN-α和ATRA联合处理组血清VEGF浓度为(69.85±20.4)pg/ml,两者相比有统计学差异(P＜0.05).免疫组化法检测肿瘤微血管密度(MVD),对照组的肿瘤组织中可见到较多的新生血管,而在IFN-α和ATRA联合处理组的肿瘤组织中新生血管明显减少.MVD在对照组为(114±18)/HP,IFN-α和ATRA联合处理组为(66±5)/HP(P＜0.01).肿瘤细胞凋亡的检测,两组相比,IFN-α和ATRA联合处理组,肿瘤组织出现较大面积的坏死,流式细胞仪检测发现坏死细胞(Annexin V~-PI~+)显著增多,而早期凋亡细胞比例并明显增高.TUNEL法镜下观察到凋亡细胞的细胞核被染成蓝紫色,染色质分布不均.对照组和治疗组的凋亡指数分别为(3.74±1.57)%、(12.34±4.78)%,差异具有显著性(P＜0.05).结论 联合应用IFN-α及ATRA可以抑制肿瘤新生血管形成,进而抑制肝癌生长和转移,提示两种作为抗肿瘤血管形成的药物在介入治疗间期的早期联合应用,在临床防治肝癌术后的复发转移中有一定的作用.     

慢病毒介导shRNA沉默STAT3表达治疗结直肠癌的体内外研究

目的 探讨慢病毒介导shRNA沉默STAT3表达对结直肠癌生长的影响.方法 分别用慢病毒干扰质粒pRNAT-shSTAT3、慢病毒空质粒pRNAT-GFP和慢病毒包装质粒混合物共转染293T细胞,获得慢病毒分别感染结直肠癌HT-29细胞,筛选培养后获得HT-29-shSTAT3、HT-29-GFP细胞株.将3种细胞株用于建立裸鼠皮下移植瘤模型,分为3组:第1组为HT-29组,第2组为HT-29-GFP组,第3组为HT-29-shSTAT3组,每组5只.MTT法检测细胞生长情况,流式细胞仪检测细胞周期变化,取瘤组织切片行CD34免疫组织化学观察肿瘤微血管密度(MVD)的变化.应用单因素方差分析检测结果.结果 结直肠癌HT-29、HT-29-GFP细胞株生长能力明显强于HT-29-shSTAT3细胞株,3者比较,差异有统计学意义(F=632.50,P<0.05).HT-29-shSTAT3细胞株生长明显减慢,G0/G1期细胞占68.7%±2.9%,HT-29-GFP细胞株为38.5%±1.6%,HT-29细胞株为38.7%±2.3%;3者比较,差异有统计学意义(F=166.53,P<0.05).HT-29组、HT-29-GFP组和HT-29-sh STAT3组MVD分别为29±5、28±4、10±3,3组比较,差异有统计学意义(F=31.60,P<0.05).结论 慢病毒介导的shRNA沉默STAT3表达能明显抑制结直肠癌的生长.     

胃肠道平滑肌肿瘤微血管密度的测定及其临床意义

目的　探讨微血管密度(MVD)与胃肠道平滑肌肿瘤(GISMT)良恶性、恶性程度、转移及预后的关系。　方法　采用免疫组织化学S-P法检测86例GISMT中MVD。　结果　MVD按平滑肌瘤、低度恶性平滑肌肉瘤、高度恶性平滑肌肉瘤的顺序依次明显增高,差异有非常显著性意义(P＜0.01)。同时,MVD与肿瘤良恶性生长方式、肿瘤大小、肿瘤中心有无坏死亦有明显关系,差异有非常显著性意义(P＜0.01)。MVD低表达组的5年生存率明显高于高表达组,差异有非常显著性意义(P＜0.01)。　结论　MVD是反映GISMT生物学特性的良好标志物,其测定可以作为判断GISMT良恶性、恶性程度、转移及预后的客观指标。     

血管内皮生长因子受体启动子双自杀基因治疗乳腺癌

目的 探讨腺病毒介导的KDRP-CD/TK融合基因对乳腺癌裸鼠体内抑瘤作用的特点.方法 以MCF-7细胞株建立裸小鼠乳腺癌动物模型,随机分为Ⅰ组[注射重组腺病毒AdKDRP-CDglyTK与前药5-氟胞嘧啶(5-FC)与丙氧鸟苷(GCV)]、Ⅱ组(仅注射前药5-FC与GCV)、Ⅲ组(仅注射重组腺病毒AdKDRP-CDglyTK)及Ⅳ组(空白对照,不施加任何处理).治疗结束后,计算抑瘤率、常规病理检查、逆转录-聚合酶链反应(RT-PCR)检测瘤体CD/TK融合基因的表达以及微血管密度(MVD)变化的检测,同时观察该治疗体系有无系统毒性.结果 各组瘤重如下:Ⅰ组(实验组)(25.04±3.09)mg、Ⅱ组(498.07±4.47)mg、Ⅲ组(501.94±4.50)mg、Ⅳ组(503.79±6.27)mg.可见Ⅰ组肿瘤逐渐缩小,余各组肿瘤逐渐增大(F=12 727.420,P＜0.01);第Ⅱ、Ⅲ、Ⅳ组肿瘤生长差异无统计学意义(P＞0.05);Ⅰ组有CD/TK基因表达且MVD(1.05±0.04)较对照组(4.15±0.10)变小(t=64.126,P＜0.01);常规病理检查发现Ⅰ组组织片状坏死,且该治疗体系对重要脏器无毒性作用.结论 KDR启动子驱动双自杀基因体系对裸鼠皮下移植瘤有明显的生长抑制作用,其机制涉及对肿瘤及其血管内皮细胞的双重杀伤.