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    丁猛, 毛立军, 于海元, 潘俊. 荷载SPAG9基因shRNA的溶瘤腺病毒对前列腺癌细胞增殖及侵袭作用的影响[J]. 徐州医科大学学报, 2018, 38(6): 357-362.
    引用本文: 丁猛, 毛立军, 于海元, 潘俊. 荷载SPAG9基因shRNA的溶瘤腺病毒对前列腺癌细胞增殖及侵袭作用的影响[J]. 徐州医科大学学报, 2018, 38(6): 357-362.
    shu
    Effects of Oncolytic adenovirus carrying SPAG9 gene on the proliferation and invasion of prostate cancer cells[J]. Journal of Xuzhou Medical University, 2018, 38(6): 357-362.
    Citation: Effects of Oncolytic adenovirus carrying SPAG9 gene on the proliferation and invasion of prostate cancer cells[J]. Journal of Xuzhou Medical University, 2018, 38(6): 357-362.
    shu

    荷载SPAG9基因shRNA的溶瘤腺病毒对前列腺癌细胞增殖及侵袭作用的影响

    Effects of Oncolytic adenovirus carrying SPAG9 gene on the proliferation and invasion of prostate cancer cells

      摘要
    • 摘要: 目的perm associated antigen9 ，SPAG9）基因的方法SPAG9基因的SPAG9，PCR法鉴定病毒、HEK-293细胞扩增病毒、病毒纯化试剂盒纯化病毒及TCID50法测定病毒滴度。实验分为3组：ZD55-SPAG9组（10MOI）、ZD55-EGFP组（10MOI）、PBS组。荧光显微镜观察病毒转染效率。CCK-8法检测前列腺癌细胞的增殖抑制作用。结晶紫法观察前列腺癌细胞的毒性作用。Transwell检测前列腺癌细胞的侵袭作用。Western Blot检测前列腺癌细胞中SPAG9上皮标志蛋白E-钙黏蛋白（E-cadherin）、间质标志蛋白波形蛋白（Vimentin）基质金属蛋表达结果 PCR验证SPAG9。ZD55-SPAG9及ZD55-EGFP大量扩增、纯化后的滴度分别为ZD55-SPAG9：9.5×109pfu/ml；ZD55-EGFP：9.0×109pfu/ml。48h时，PC-3和DU145细胞中荧光蛋白的表达量最高，72h后由于溶瘤腺病毒对细胞的杀伤作用，细胞存活率降低，荧光蛋白表达量随之降低。在同一时间，与对照病毒ZD55-EGFP组相比，ZD55-SPAG9组的绿色荧光蛋白表达更高。48h时，ZD55-SPAG9组PC-3细胞生存率为（54.83±5.04）%，与ZD55-EGFP组（70.91±2.35）%相比，生存率显著降低，差异具有统计学意义（P＜0.0）；DU145细胞中，ZD55-SPAG9组生存率为（60.02±2.87）%，与ZD55-EGFP组（75.45±4.32）%相比，生存率显著降低，差异具有统计学意义（P＜0.0）SPAG9对前列腺癌细胞的毒性作用明显强于ZD55-EGFP。Transwell实验结果显示，PC-3细胞ZD55-SPAG9组侵袭细胞目为58.40±3.20， ZD55-EGFP组为74.80±7.60、PBS组为121.60±12.20，ZD55-SPAG9组分别与ZD55-EGFP组和PBS组比较，侵袭细胞明显减少，差异具有统计学有意义(P＜0.05)。DU145细胞ZD55-SPAG9组侵袭细胞目为37.00±3.20，ZD55-EGFP组为84.20±6.60、PBS组为164.66±10.45，ZD55-SPAG9组分别与ZD55-EGFP组和PBS组比较，侵袭细胞明显减少，差异具有统计学有意义(P＜0.05)。Western Blot 实验SPAG9作用于前列腺癌细胞PC-3和DU145后，可上调E-cadherin的表达同时下调MMP2和imentin的表达SPAG9基因的溶瘤腺病毒SPAG9，实验表明ZD55-SPAG9能够显著抑制增殖，SPAG9抑制前列腺癌可能与EMTepithelial-mesenchymal transition）过程有关。

       

      Abstract: Objective To observe the effect of oncolytic adenovirus carrying SPAG9 gene on the proliferation and invasion of prostate cancer PC-3 and DU145 cells. Methods 1.Construction of oncolytic adenovirus carrying SPAG9 gene (ZD55-SPAG9) and identification of adenovirus was tested by PCR. ZD55-SPAG9 were amplify in low passage HEK293 cells and purified by AAV Purification Miniprep Kit. The viral titer was determined by TCID50 method. 2. The transfection efficiency of adenovirus was detected with fluorescence microscopy compared with ZD55-EGFP. 3.The viability of PC-3 and DU145 cells was assessed by cell counting kit-8(CCK-8) assay.4.The cytotoxic effects of adenoviruses ZD55-SPAG9 to PC-3 and DU145 cells were detected by Crystal Violet Staining. 5. Cell invasion capacities of PC-3 and DU145 cells were detected by using transwell assay, respectively. 6.The expression of SPAG9 and invasion associated protein MMP-2, Vimentin and E-cadherin in PC-3 and DU145 cells were detected by Western Blot. Results The results of PCR assay showed that adenovirus ZD55-SPAG9 was constructed successfully. 2. After amplification and purification of adenovirus,the titer of ZD55-SPAG9 and ZD55-EGFP were 9.5×109pfu/ml and 9.0×109pfu/ml, respectively.The transfection efficiency ZD55-SPAG9 and ZD55-EGFP were highest at 48h. The cell viability decreased due to the killing effect of adenovirus with fluorescent protein expression decline after 72h. Moreover, the transfection efficiency of ZD55-SPAG9 was higher than ZD55-EGFP at the same time.CCK-8 assay showed that compared with the viability of PC-3 cells of ZD55-EGFP group (70.91±2.35)%, the viability of ZD55-SPAG9 group was (54.83±5.04)%, the difference with statistical significance. The viability of DU145 cells of ZD55-EGFP group and ZD55-SPAG9 group were respectively (75.45±4.32)% and (60.02±2.87)%, the difference with statistical significance. Crystal Violet Staining showed that ZD55-SPAG9 had the strongest cytotoxicity in PC-3 and DU145 cells compared with ZD55-EGFP. Transwell assay showed that the number of transmembrane PC-3 cells of ZD55-SPAG9 group was (58.40±3.20), compared with ZD55-EGFP group (74.80±7.60) and PBS group(121.60±12.20), the difference was statistically significant (P<0.05). The number of transmembrane DU145 cells of ZD55-SPAG9 group was (37.00±3.20), compared with ZD55-EGFP group (84.20±6.60) and PBS group(164.66±10.45), the difference was statistically significant (P<0.05). Western Blot showed that the SPAG9 expression of ZD55-SPAG9 group was lower than PBS and ZD55-EGFP group.Western Blot analysis also confirmed that ZD55-SPAG9 could increase the expression level of E-cadherin, concomitant with down-regulating the proteins expression of MMP-2 and Vimentin, their?difference?was significant(P<0.05). Conclusion Oncolytic adenovirus carrying SPAG9 gene could inhibit the growth and invasion effect in PC-3 and DU145 cells. We preliminarily explore the mechanism of ZD55-SPAG9 on the effect of cell invasion and the regulatory effect may related to EMT(epithelial-mesenchymal transition)

       

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