人脐带间充质干细胞条件培养基对肝癌细胞增殖、迁移和侵袭的影响

Effects of mesenchymal stem cell derived conditioned medium on proliferation,migration and invasion of hepatocellular carcinoma cells and its mechanism

目的探讨人脐带间充质干细胞条件培养基(UCMSC-CM)对肝癌细胞增殖、凋亡、迁移及侵袭的影响及其机制。方法体外培养肝癌细胞系HepG2和BEL-7402,常规培养肝癌细胞系作为对照组,UCMSC-CM处理的肝癌细胞系作为UCMSC-CM组。采用细胞增殖与毒性检测(CCK-8)法检测细胞增殖;流式细胞术检测细胞凋亡;划痕试验和Transwell侵袭试验分别检测细胞迁移和侵袭能力;Western blot检测细胞中糖原合成酶激酶-3β(GSK-3β)、p-GSK-3β、Bak、Bax、兔抗人大分子B淋巴细胞瘤蛋白(BCL-XL)、髓细胞白血病-1蛋白(MCL-1)、存活素蛋白表达;实时荧光定量PCR(RT-qPCR)检测细胞中上皮间质转化(EMT)相关标志物mRNA的表达水平。结果CCK-8法检测结果显示,UCMSC-CM处理HepG2细胞和BEL-7402肝癌细胞24、48、72 h后的光密度值与对照组比较,差异无统计学意义(P>0.05);流式细胞术检测结果显示,UCMSC-CM组与对照组处理HepG2、BEL-7402细胞早期(Q4)、晚期(Q2)凋亡率比较,差异均无统计学意义(P>0.05)。UCMSC-CM处理组处理HepG2细胞24、48 h后划痕愈合面积百分率[(53.00±2.45)%、(87.33±1.25)%]均高于对照组[(21.00±2.45)%、(43.67±4.64)%],差异有统计学意义(P<0.05);UCMSC-CM处理组处理BEL-7402细胞48 h后划痕愈合面积百分率[(65.33±11.26)%]高于对照组[(36.67±8.81)%],差异有统计学意义(P<0.05)。侵袭试验结果显示,UCMSC-CM处理HepG2细胞24 h后,UCMSC-CM组侵袭细胞数[(630.00±62.33)个]高于对照组[(386.00±42.53)个],差异有统计学意义(P<0.05);UCMSC-CM处理BEL-7402细胞24 h后,UCMSC-CM组侵袭细胞数[(228.00±17.91)个]低于对照组[(491.00±10.34)个],差异有统计学意义(P<0.05)。Western blot检测结果显示,对照组与UCMSC-CM组肝癌细胞凋亡相关信号分子GSK-3β、p-GSK-3β、Bak、Bax、BCL-XL、MCL-1及存活素蛋白表达比较,差异均无统计学意义(P>0.05)。RT-qPCR结果显示,UCMSC-CM处理的HepG2、BEL-7402细胞E-钙黏蛋白的mRNA表达水平[(0.18±0.06)、(0.48±0.25)]较对照组[(1.33±0.06)、(1.01±0.12)]均明显下调(P<0.05)。BEL-7402细胞中snail的mRNA表达水平(3.37±0.41)较对照组(1.01±0.18)明显上调(P<0.05),而HepG2细胞中N-钙黏蛋白、snail和波形蛋白的mRNA表达水平均无明显变化(P>0.05)。结论UCMSC-CM可能通过EMT过程促进肝癌细胞的迁移和侵袭能力。

Objective To investigate the effects of human umbilical cord mesenchymal stem cells-derived conditioned medium(UCMSC-CM)on proliferation,apoptosis,migration and invasion of hepatocellular carcinoma(HCC)cells and its mechanism.Methods HCC lines HepG2 and BEL-7402 were cultured in vitro,conventional cultured HCC lines were used as control group,and UCMSC-CM treated hepatocellular carcinoma cell lines were used as UCMSC-CM group.Cell proliferation was detected by cell counting kit-8(CCK-8)method.Cell apoptosis was detected by flow cytometry.The ability of cell migration and invasion were detected by scratch assay and Transwell invasion assay,respectively.The expression levels of GSK-3β,p-GSK-3β,Bak,Bax,BCL-XL,MCL-1 and Survivin were detected by Western blot.The mRNA expression levels of epithelial-mesenchymal transition(EMT)markers in cells were detected by Quantitative real-time PCR(RT-qPCR).Results Compared with the control group,CCK-8 method showed that there was no significant difference in optical density of HepG2 cells and Bel-7402 liver cancer cells after UCMSC-CM treatment for 24,48,72 h(P>0.05).Flow cytometry results showed that there was no significant difference in early(Q4)and late(Q2)apoptosis rates of HepG2 and BEL-7402 cells in both groups(P>0.05).The percentage of scratch healing area[(53.00±2.45)%and(87.33±1.25)%]in UCMSC-CM treatment group was higher than that in control group[(21.00±2.45)%and(43.67±4.64)%]after 24 and 48 h treatment,and the difference was statistically significant(P<0.05).The percentage of scratch healing area of BEL-7402 cells treated with UCMSC-CM for 48 h[(65.33±11.26)%]was higher than that of the control group[(36.67±8.81)%],the differences were statistically significant(P<0.05).The results of invasion assay showed that after HepG2 cells were treated with UCMSC-CM for 24 h,the number of invasion cells in UCMSC-CM group(630.00±62.33)was higher than that in the control group(386.00±42.53),the differences were statistically significant(P<0.05).After UCMSC-CM treated BEL-7402 cells for 24 h,the number of invasive cells in UCMSC-CM group(228.00±17.91)was lower than that in control group(491.00±10.34),and the difference was statistically significant(P<0.05).Western blot analysis showed that there was no significant difference in the expression of GSK-3β,p-GSK-3β,Bak,Bax,BCl-XL,MCL-1 and Survivin proteins between both groups(P>0.05).RT-qPCR results showed that,the mRNA expression of E-Cadherin in HepG2 and BEL-7402 cells treated with UCMSC-CM[(0.18±0.06),(0.48±0.25)]was significantly down-regulated compared with control group[(1.33±0.06)and(1.01±0.12)],the differences were statistically significant(P<0.05).Snail mRNA expression in BEL-7402 cells(3.37±0.41)was significantly up-regulated compared with control group(1.01±0.18),the difference was statistically significant(P<0.05),but there were no significant changes in mRNA expression levels of N-Cadherin,snail and vimentin in HepG2 cells(P>0.05).Conclusions UCMSC-CM may promote the migration and invasion of HCC cells through the EMT process.