摘要
目的探讨基因重组腺相关病毒自杀基因及内皮抑素（ES）联合基因治疗膀胱癌的效果。方法（1）通过重组腺相关病毒（rAAV）-增强绿色荧光蛋白（EGFP）转染膀胱肿瘤T24细胞，来确定rAAV对该细胞的转染情况及转染效率；（2）通过rAAV-胸苷激酶（TK）-核糖体插入位点（IRES）-ES（简称rAAV—TIE）体外转染T24膀胱肿瘤细胞及人脐静脉内皮细胞（HUVEC细胞），应用MTT法、流式细胞仪等方法来检测其对T24细胞及HUVEC细胞凋亡的诱导作用；（3）构建裸鼠膀胱癌模型，分析rAAV—TIE联合基因治疗膀胱癌的体内效果。结果（1）rAAV—EGFP转染T24细胞后可以表达携带的外源基因EGFP；（2）rAAV—TK、rAAV—TIE转染T24细胞72h后，流式细胞仪检测结果显示，rAAV—TK、rAAV—TIE两组凋亡率分别是34．12％和36．91％，明显高于空病毒转染组[rAAV-多克隆位点（MCS）组]（3．08％）和空白对照组（0．84％）；（3）瘤内注射rAAV—ES、rAAV—TK、rAAV—TIE大约9d后，肿瘤生长受到显著抑制，治疗结束后：各组肿瘤的体积分别是：rAAV—ES组（0．75±0．08）cm^3、rAAV-TK组（0．71±0．11）cm^3、rAAV—TIE组（0．52±0．09）cm^3、rAAV—MCS组（1．27±0．13）cm^3和空白对照组（1．24±0．17）cm^3，除rAAV—ES组与rAAV—TK组间比较差异无统计学意义外，其余组间比较差异均有统计学意义（均P〈0．05）。结论体外和体内实验表明rAAV—TIE可有效抑制膀胱癌的血管生成和肿瘤的生长，能够双靶点基因治疗膀胱肿瘤。
Objective To investigate the effect of double targeting gene therapy by using recombinant adeno-associated virus-thymidine kinase （TK）- internal ribosome entry site （IRES）- endostatin （ES） （rAAV-TIE）. Methods Bladder cancer cells of the line T24 were cultured and transfected with rAAV-ES, rAAV-MCS （blank virus）, and rAAV-TIE respectively. 72 hours later the levels of ES in the supernatants were measured by ELISA and annexin V apoptosis test kit was used to examine the apoptosis. Human umbilical vein endothelial cells （HUVECs） were transJP2fected with rAAV-ES, rAAV-TIE, and rAAV-MCS respectively. MTT method and flow cytometry were used to detect the apoptosis of the HUVECs. Balb/c nude rats were inoculated subcutaneously with T24 cells. Twenty rats with tumor were randomly divided into 4 equal groups to be treated by rAAV-MCS, rAAV-TK, rAAV-ES, or rAAV-TIE, and 5 rats were used as control group. Four weeks later, blood samples were collected to detect the ES level by ELISA. The tumors were taken out to undergo microscopy to calculate the mcrovessel density（MVD）. Results 72 h after transfection, ES could be detected in the superuatants of the T24 cells transfected with rAAV-ES, and rAAV-TIE. The apoptotic rates of the T24 cells transfected with rAAV-TK and rAAV-TIE were 34.12% and 36.91% respectively, significantly higher than those of the T24 cells transfected with rAAV-MCS and of the control group （3.08% and 0.84% , all P 〈0.05）. Transfection of rAAV-ES and rAAV-TIE increased the apoptotic rate of the HUVECs time-dependently. Nine days after the transfetion rAAV-ES, rAAV-TK, rAAV-TIE, the tumor volumes of the rAAV-ES, rAAV-TK, and rAAV-TIE groups were （0.75 ± 0.08 ）, （0.71 ± 0.11 ）, and （0.52± 0.09 ）cm^3 respectively, all significantly lower than those of the rAAV-MCS group and control group [ （ 1.27 ±0.13 ） and （ 1.24 ± 0.17 ） cm^3 respectively, all P 〈 0.05 ]. Conclusion rAAV-TIE effectively inhibits the tumorigenesis and angiogenesis in bladder cancer. Double targeting gene therapy against bladder cancer can be achieved by using rAAV.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2008年第38期2700-2704,共5页
National Medical Journal of China
基金
天津市科技发展计划基金资助项目（06YFSZSF03900）
关键词
膀胱肿瘤
基因疗法
基因
转基因
自杀
内皮抑素类
腺相关病毒
Urinary bladder neoplasms
Gene therapy
Genes, transgenic, suicide
Endostatins
Adeno-associated virus
