Effect of interleukin-12 gene modified bone marrow mesenchymal stem cells on the growth of ovarian cancer cells

doi:10.3969/j.issn.2095-4344.2017.09.007

Abstract

Abstract:

BACKGROUND: Bone marrow mesenchymal stem cells are characterized by wide sources and low immunogenicity. Especially, these cells are easy to import and express foreign genes, and thus have obvious superiorities as anti-tumor gene therapy vectors.

OBJECTIVE: To investigate the effects of interleukin-12 gene modified bone marrow mesenchymal stem cells on the proliferation, cell cycle and apoptosis of ovarian cancer cells.

METHODS: Interleukin-12 recombinant adenovirus vector was used to transfect bone marrow mesenchymal stem cells. Then, the expression of interleukin-12 mRNA and protien in transfected bone marrow mesenchymal stem cells was detected by RT-PCR and western blot assay, respectively. The level of interleukin-12 in cell supernatant was determined by ELISA. The SKOV3 cells were co-cultured with the supernatant of bone marrow mesenchymal stem cells transfected with (transfection group) or without (control group) interleukin-12 recombinant adenovirus vector. The proliferation of SKOV3 cells was determined by MTT assay. Flow cytometry was used to detect the cell cycle and apoptosis of SKOV3 cells.

RESULTS AND CONCLUSION: RT-PCR and western blot results showed that interleukin-12 mRNA and protein were expressed in transfected bone marrow mesenchymal stem cells, but not found in empty vector group and blank control group. ELISA results showed that the content of interleukin-12 in the supernatant of bone marrow mesenchymal stem cells was (68.78±12.35) μg/L in the interleukin-12 transfection group after 48 hours culture, and no interleukin-12 expression was detected in the empty vector group and the blank control group. Interleukin-12-transfected bone marrow mesenchymal stem cells significantly inhibited the proliferation of SKOV3 cells, and the proliferation inhibition rate was increased with time (P < 0.05). The proportion of G₁-phased SKOV3 cells was higher in the transfection group than in the control group (P < 0.05), and the percentage of G₂-phased SKOV3 cells was lower in the transfection group than in the control group (P < 0.05). The apoptosis rate of SKOV3 cells in the transfection group was higher than that in the control group (P < 0.05). Our experimental findings indicate that bone marrow mesenchymal stem cells transfected with interleukin-12 recombinant adenovirus vector can express interleukin-12, inhibit the proliferation and induce apoptosis of ovarian cancer cells.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Ovarian Neoplasms, Bone Marrow, Mesenchymal Stem Cells, Interleukin-12, Tissue Engineering

CLC Number:

R394.2

Zhang Xia, Du Zhi-xiang, Wang Na, Meng Ya-li. Effect of interleukin-12 gene modified bone marrow mesenchymal stem cells on the growth of ovarian cancer cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(9): 1346-1351.

Figures/Tables 2

2.1 重组腺病毒的获得重组质粒转染293细胞后48 h可见细胞内有绿色荧光，荧光随时间延长而增多，光镜下可见细胞变圆、聚集、脱落，出现细胞病变，经过多轮感染后，重组病毒的效价达1012 pfu/L，见图1。 2.2 各组骨髓间充质干细胞中白细胞介素12 mRNA的表达 RT-PCR结果显示：白细胞介素12组骨髓间充质干细胞在632 bp处出现阳性条带，符合白细胞介素12扩增片段长度，说明转染后骨髓间充质干细胞内白细胞介素12基因在mRNA水平获成功表达，而空病毒载体组和空白对照组骨髓间充质干细胞中均未检测到白细胞介素12 mRNA表达，见图2。 2.3 各组骨髓间充质干细胞中白细胞介素12蛋白的表达 Western Blotting结果表明：白细胞介素12组骨髓间充质干细胞有清晰条带出现，说明转染后骨髓间充质干细胞中白细胞介素12基因在蛋白水平获成功表达，而空病毒载体组和空白对照组骨髓间充质干细胞均未检测到白细胞介素12蛋白表达，见图3和表1。 2.4 各组骨髓间充质干细胞上清液中白细胞介素12水平 ELISA结果显示：培养48 h后，白细胞介素12组骨髓间充质干细胞上清液中白细胞介素12质量浓度为(68.78± 12.35) μg/L，空病毒载体组和空白对照组骨髓间充质干细胞上清液中未检测到白细胞介素12表达。 2.5 白细胞介素12转染组骨髓间充质干细胞上清液对SKOV3细胞增殖的影响白细胞介素12转染组骨髓间充质干细胞上清液对SKOV3细胞增殖有抑制作用，细胞增殖抑制率随时间的延长而增加，培养第2天和第3天SKOV3细胞增殖抑制率高于培养第1天(P < 0.05)，培养第3天细胞增殖抑制率高于培养第2天(P < 0.05)，见表2。 2.6 白细胞介素12转染组骨髓间充质干细胞上清液对SKOV3细胞周期的影响转染组SKOV3细胞G1期细胞比例高于对照组(P < 0.05)，G2期细胞比例低于对照组(P < 0.05)，两组S期细胞比例比较差异无显著性意义(P > 0.05)，见表3。 2.7 白细胞介素12转染组骨髓间充质干细胞上清液对SKOV3细胞凋亡的影响转染组SKOV3细胞凋亡率高于对照组[(8.98±1.21)%，(2.32±0.64)%，P < 0.05]。"

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