中文摘要：

目的构建人前列腺特异性抗原（hPSA）启动子调控的趋化因子受体4（CXCR4）的RNA干扰逆转录病毒载体，并探讨其对激素依赖性前列腺癌LNCaP细胞CXCR4基因表达的靶向抑制作用。方法PCR扩增CXCR4特异性小干扰RNA（siRNA），转入带有增强型绿色荧光蛋白（EGFP）和启动子u6的pGensil-1质粒，以hPSA启动子代替U6启动子，再将重组基因片段导入到逆转录病毒真核表达载体pLXSN中，进行限制性酶切鉴定。转染PA317包装细胞，收获病毒上清，分别转染前列腺癌细胞PC-3m、LNCaP和人乳腺癌细胞MCF7。采用RT-PCR、Westernblot检测CXCR4基因表达。Transwell小室侵袭实验检测前列腺癌细胞体外侵袭能力的变化。结果通过限制性酶切鉴定该重组质粒，成功构建了CXCR4的RNA干扰质粒pLXSN／EGFP—hPSA-siCXCR4。与空载体组比较，CXCR4-siRNA对LNCaP细胞CXCR4mRNA和蛋白表达抑制显著，48h抑制率分别为（81．53±10．22）％和（90．52±9．31）％；对PC-3m、MCF-7细胞CXCR4mRNA和蛋白表达无抑制作用。Transwell侵袭实验结果显示，LNCaP细胞干扰组微膜下孔细胞数为（139．9±14．2）个，空载体组为（348．4±36．4）个，两组差异有统计学意义（P〈0．05）；PC-3m、MCF-7细胞干扰组微膜下孔细胞数与空载体组比较，差异无统计学意义（P〉0．05）。结论该逆转录病毒系统中，hPSA启动子调控的下游RNA干扰序列特异性地抑制激素依赖前列腺肿瘤细胞的CXCR4基因表达，具有明显的靶向性。hPSA启动子调控的靶向CXCR4基因的RNA干扰技术在激素依赖性前列腺癌的基因治疗中具有一定价值。

英文摘要：

Objective To construct RNA interfering retrovirus vector targeting CXCR4 gene driven by human prostate-specific antigen promoter and investigate its targeted inhibition effects in androgen- responsive prostate cancer cells LNCaP. Methods To clone the CXCR4 targeting siRNA gene by PCR. The PCR products were inserted into the pGensil-Ⅰ plasmid containing U6 promoter and EGFP. U6 promoter was replaced by hPSA promoter. Then, the recombinant EGFP-hPSA-siCXCR4 fragment was sub-cloned into pLXSN, which was evaluated by restriction enzyme. The pLXSN-EGFP-hPSA-siCXCR4 was transfected into PA317 cells with LipofeetamineTM 2000. The virus obtained from transfected PA317 cells was transfected into PC-3m, LNCaP and MCF-7 cells, respectively. The expression of CXCR4 mRNA and protein was detected by RT-PCR and Western blot. The invasion ability of prostate carcinoma cells was detected by Transwell experiment. Results The recombinant pLXSN-hPSA-siCXCR4 was successfully constructed. The expression of CXCR4 mRNA and protein in LNCaP cells was blocked by pLXSN-hPSA-siCXCR4. The expression inhibition rate was （81.53± 10.22）% at mRNA level detected by semi-quantitive RT-PCR and （90.52 ± 9.31 ）% at protein level detected by Western blot, respectively, in LNCaP cells at 48 h. The expression of CXCR4 mRNA and protein was effectively inhibited by sequence-specific hPSA-siCXCR4 in LNCaP cells, but not in PC-3m and MCF-7 cells. The results of Transwell experiment showed that the number of cells in down-pore of micro-membrane was 139.9 ± 14.2 in the treated group, significantly less in comparison with 348.4 ±36.4 in the controlled group （P 〈0.05）. However, the number of PC-3m and MCF-7 cells in down-pore of micro-membrane was not significantly different among the control and treated groups （ P 〉 0.05）. Conclusion The downstream siRNA controlled by hPSA promoter in retrovirus system can be expressed selectively in androgen-responsive prostate carcinoma cells, showing an apparent targeting character. RNAi targeted t