人脐带间充质干细胞对人卵巢癌SKOV3细胞凋亡的影响及可能机制

Effect of human umbilical cord-derived mesenchymal stem cells on the apoptosis of SKOV3 cells and its possible mechanism

目的探讨人脐带间充质干细胞(human umbilical cord-derived mesenchymal stem cells,hUC-MSCs)对人卵巢癌SKOV3细胞凋亡的影响及可能作用机制。方法取传代培养至第5代的hUC-MSCs细胞,应用显微镜下观察细胞形态,采用流式细胞术检测细胞表面标志物。取细胞数分别为0个/mL(对照组)、1×10^6个/mL(低剂量组)、2×10^6个/mL(中剂量组)、4×10^6个/mL(高剂量组)第5代hUC-MSCs细胞悬液0.5 mL,接种于Transwell小室膜上,应用Transwell小室与对数生长期SKOV3细胞共培养60 h,采用CCK-8法检测SKOV3细胞增殖相对抑制率,采用流式细胞术检测SKOV3细胞凋亡率,采用实时荧光定量PCR法检测SKOV3细胞PI3K mRNA、Akt mRNA、B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)mRNA、caspase-3 mRNA相对表达量。结果显微镜下第5代hUC-MSCs呈贴壁长梭状细胞,单核,大小较均一,呈漩涡状、放射状或平行排列,仍可保持原代hUC-MSCs细胞典型形态;流式细胞仪检测结果显示,第5代hUC-MSCs细胞表面CD44、CD29表达率分别为(98.4067±0.1332)%、(98.3933±0.3669)%,CD34、CD45表达率分别为(0.0133±0.0058)%、(0.0067±0.0058)%。第5代hUC-MSCs细胞与SKOV3细胞共培养60 h,低剂量组、中剂量组、高剂量组SKOV3细胞凋亡率[(40.63±0.74)%、(45.53±0.67)%、(56.53±0.57)%]、细胞增殖相对抑制率[(23.96±1.55)%、(40.35±1.34)%、(46.25±2.41)%]和caspase-3 mRNA相对表达量(1.46±0.02、1.91±0.04、2.37±0.01)依次增高(P均<0.05),且均高于对照组[(31.60±0.53)%、0、1](P<0.05)。低剂量组、中剂量组、高剂量组PI3K mRNA(0.60±0.02、0.46±0.02、0.34±0.04)、Akt mRNA(0.85±0.04、0.66±0.03、0.46±0.05)、Bcl-2 mRNA(0.85±0.04、0.69±0.04、0.39±0.03)相对表达量依次降低(P均<0.05),且均低于对照组(1、1、1)(P<0.05)。结论hUC-MSCs细胞与SKOV3细胞共培养可促进SKOV3细胞凋亡,且hUC-MSCs细胞数越多促凋亡作用越强,其机制可能与下调Bcl-2、上调caspase-3表达,抑制PI3K/Akt信号通路有关。

Objective To investigate the effect of human umbilical cord-derived mesenchymal stem cells(hUC-MSCs)on the apoptosis of human ovarian cancer SKOV3 cells and its possible mechanism.Methods The morphology of the fifth generation of hUC-MSCs cells was observed on microscope,and the hUC-MSCs surface markers were detected by flow cytometry.The hUC-MSCs cell suspension(0.5 mL)at the inoculation densities of 0 cells/mL(control group),1×10^6 cells/mL(low-density group),2×10^6 cells/mL(medium-density group),and 4×10^6 cells/mL(high-density group)was inoculated on Transwell membrane,respectively.After culture with SKOV3 cells in logarithmic growth phase in Transwell chamber for 60 h,the relative inhibitory rate of SKOV3 cells was detected by CCK-8,the apoptotic rate was detected by flow cytometry,and the relative expressions of PI3 KmRNA,Akt mRNA,B-cell lymphoma-2(Bcl-2)mRNA and caspase-3 mRNA of SKOV3 cells were detected by real-time fluorescence quantitative PCR technique.Results The fifth generation of hUC-MSCs still maintained the typical morphology of primary hUC-MSCs cells on microscope,in adherent long spindle shape,single nucleus,relatively uniform size,swirling,radial or parallel arrangement.Flow cytometry results revealed that the expression rates of CD44,CD29,CD34 and CD45 of the fifth generation of hUC-MSCs were(98.4067±0.1332)%,(98.3933±0.3669)%,(0.0133±0.0058)%and(0.0067±0.0058)%.After 60 h culture,the apoptotic rate of SKOV3 cells((31.60±0.53)%,(40.63±0.74)%,(45.53±0.67)%,(56.53±0.57)%)and the relative proliferation inhibitory rate(0,(23.96±1.55)%,(40.35±1.34)%,(46.25±2.41)%)increased,the relative expressions of PI3 K mRNA(1,0.60±0.02,0.46±0.02,0.34±0.04),Akt mRNA(1,0.85±0.04,0.66±0.03,0.46±0.05)and Bcl-2 mRNA(1,0.85±0.04,0.69±0.04,0.39±0.03)decreased,and the relative expression of caspase-3 mRNA(1,1.46±0.02,1.91±0.04,2.37±0.01)increased gradually in turn in control group,low-density group,medium-density group and high-density group(P<0.05).Conclusion The co-culture of hUC-MSCs and SKOV3 can significantly promote the apoptosis of SKOV3 cells,and the more the hUC-MSCs cells,the stronger the effect,probably by down-regulating Bcl-2,up-regulating caspase-3 and suppressing the PI3 K/Akt signaling pathway.