溶瘤腺病毒修饰间充质干细胞裂解上清对小鼠乳腺癌细胞4T1生物学特性的影响

Biological effect of lysates of oncolytic adenoviral transduced mesenchymal stem cells on mouse breast cancer 4T1 cells

目的评价溶瘤腺病毒(rAd)修饰间充质干细胞(MSC)裂解上清对小鼠乳腺癌4T1细胞生物学特性的影响。方法采用携带核心蛋白聚糖(DCN)基因的溶瘤腺病毒rAd.DCN和对照病毒rAd.Null感染MSC,命名为MSC.DCN和MSC.Null。感染后48 h,收集MSC.DCN,MSC.Null和MSC的裂解上清,将其与小鼠乳腺癌细胞4T1共培养,同时设4T1细胞对照组,用实时定量PCR法和Western印迹法检测小鼠乳腺癌4T1细胞DCN mRNA和蛋白表达;通过Dye670标记,用流式细胞术检测4T1细胞增殖;用FITCAnnexinⅤ/PI凋亡试剂盒检测4T1细胞凋亡;通过细胞划痕实验检测4T1细胞的迁移能力。结果 MSC.DCN裂解上清处理后,4T1细胞DCN mRNA和蛋白表达均高于其余2组(P<0.01)。共培养3 d后,与细胞对照组相比,MSC组4T1细胞增殖指数下降(P<0.01);共培养3和5 d后,与MSC组相比,MSC.Null组和MSC.DCN组4T1细胞增殖指数下降(P<0.05),凋亡比例上升(P<0.05);MSC.Null与MSC.DCN组间差异无统计学意义。MSC,MSC.Null和MSC.DCN裂解上清与4T1细胞共培养12 h后,与细胞对照组相比,MSC组迁移率升高(P<0.01);与MSC组和MSC.Null组相比,MSC.DCN组迁移率下降(P<0.05)。结论MSC.DCN裂解上清可抑制小鼠乳腺癌细胞4T1细胞增殖和MSC裂解上清所诱导的4T1细胞迁移,同时促进4T1细胞凋亡。

OBJECTIVE To evaluate the biological effect of lysates from oncolytic adenoviral transfected mesenchymal stem cells(MSCs)on mouse breast cancer 4T1 cells.METHODS MSCs were infected with oncolytic adenovirus carrying decorin(DCN)gene(rAd.DCN)and control virus(rAd.Null),respectively,and named MSC.DCN and MSC.Null.48 h after infection,the lysates of MSC,MSC.DCN and MSC.Null were collected and incubated with mouse breast cancer cells 4T1.Real-time quantitative PCR and Western blotting were used to detect the DCN mRNA and protein expression of 4T1 cells after incubation with the lysates.After Dye670 labeling,the proliferation of 4T1 cells was detected by flow cytometry after incubation.The apoptosis of 4T1 cells was detected by FITC-AnnexinⅤ/PI apoptosis kit.The migration of 4T1 cells was detected by wound healing assay after treatment.RESULTS After the incubation with the lysate of MSCs.DCN,the mRNA and protein expression of DCN in 4T1 cells was higher than that in the other two groups(P<0.01).After 3 d of treatment with lysates,compared with 4T1 cell control group,the proliferation index of 4T1 cells in MSCs group decreased(P<0.01).And after 3 and 5 d of treatment with lysates,compared with MSC group,the proliferation index of 4T1 cells decreased(P<0.05)and the proportion of apoptosis increased(P<0.05)in MSC.Null group and MSC.DCN group,but there was no significant difference between MSC.Null group and MSC.DCN group.After 12 h of treatment with lysates,the 4T1 cell mobility of MSC group was higher than that of cell control group(P<0.01),while that of MSC.DCN group was lower than that of MSCs group and MSC.Null group(P<0.05).CONCLUSION The lysate of MSC.DCN could inhibit the proliferation of mouse breast cancer cells 4T1 and the migration of 4T1 cells induced by the supernatant of MSCs′lysate,and promote the apoptosis of 4T1 cells.