Human umbilical cord mesenchymal stem cells promote the proliferation of HepG-2 cells through interleukin-6/STAT3 signaling pathway

doi:10.3969/j.issn.2095-4344.2016.50.002

Abstract

Abstract:

BACKGROUND: Human umbilical cord mesenchymal stem cells (hUC-MSCs) can secrete a variety of factors involved in the regulation of tumor proliferation, metastasis and angiogenesis. Probably, interleukin-6 (IL-6) is one of the most important inflammatory factors.
OBJECTIVE: To explore the effect of hUC-MSCs on the proliferation and migration of HepG-2 hepatocyte carcinoma cells via the IL-6/STAT3 signaling pathway.
METHODS: IL-6 expression levels in hUC-MSCs and HepG-2 cells were determined by ELISA. STAT3 and p-STAT3 expression levels were determined by western blot assay. Transcription levels of PCNA, CyclinD1 and STAT3 genes were measured by RT-PCR. HepG-2 cell proliferation was analyzed by flow cytometry and cell counting kit-8 assays. The migration capacity of HepG-2 cells was evaluated through a scratch test and Transwell assays.
RESULTS AND CONCLUSION: The IL-6 level in the hUC-MSCs was significantly higher than that in the HepG-2 cells (P < 0.05). Both the hUC-MSC conditioned culture medium and IL-6 could be used for STAT3 activation. The addition of an IL-6 neutralizing antibody significantly weakened the activation of STAT3 in HepG-2 cells by the hUC-MSCs-conditioned culture medium. In the presence of the IL-6 neutralizing antibody or the STAT3 inhibitor, AG490, the mRNA expression levels of HepG-2 proliferation-related genes (PCNA, CyclinD1 and Survivin) were significantly reduced. The proliferation and migration capacity of HepG-2 cells were also significantly decreased by this treatment. Taken together, hUC-MSCs can secrete IL-6 to activate the STAT3 signaling pathway, thereby promoting the proliferation and migration of HepG-2 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: Umbilical Cord, Mesenchymal Stem Cells, Liver Neoplasms, Interleukin-6, STAT3 Transcription Factor, Tissue Engineering

CLC Number:

R394.2

Zheng Sheng, Yang Juan, Chen Wen-qin, Liu Jing, Zhang Fan, Wang Yu-bo. Human umbilical cord mesenchymal stem cells promote the proliferation of HepG-2 cells through interleukin-6/STAT3 signaling pathway[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(50): 7460-7468.

Figures/Tables 2

2.1 人脐带间充质干细胞的生物学特性脐带组织原代培养3 d后，可见少许细胞贴壁生长，细胞多呈短梭形；培养7-10 d后，细胞聚集融合成单层，细胞形态大多为长梭形或多角形；传代至第3、4代，细胞形态均一，胞质透光性良好，细胞形态类似成纤维细胞，呈漩涡状生长(图1)。流式细胞仪检测第5代细胞高表达CD29、CD90、CD105(> 95%)，极低表达CD19(0.1%)，符合间充质干细胞鉴定标准。 2.2 白细胞介素6和人脐带间充质干细胞条件培养基对HepG-2细胞STAT3和p-STAT3蛋白表达的影响 ELISA结果提示人脐带间充质干细胞条件培养基中白细胞介素6水平为(1 856.2±96.6) ng/L，HepG-2细胞白细胞介素6表达水平仅为(180.8±35.5) ng/L，两组相比较，差异有显著性意义(P < 0.05，图2)。 20 μg/L重人白细胞介素6及40%人脐带间充质干细胞条件培养基分别作用于HepG-2细胞30 min后，p-STAT3表达明显升高。白细胞介素6中和抗体加入条件培养基后STAT3的活性被抑制，同时p-STAT3的表达明显下降。JAK2特异性抑制剂AG490显著抑制了HepG-2细胞STAT3的活化并降低了条件培养基对STAT3的激活作用，组间比较差异有显著性意义(P < 0.05)。各组细胞STAT3蛋白表达差异无显著性意义(P > 0.05，图3)。 2.3 白细胞介素6和人脐带间充质干细胞条件培养基对HepG-2细胞增殖相关基因表达的影响 RT-PCR检测结果显示：条件培养基组增殖相关基因PCNA、CyclinD1、Survivin表达水平均明显增高，白细胞介素6中和抗体可明显减弱条件培养基的促增殖基因表达作用，与对照组相比差异均有显著性意义(P < 0.05)。AG490加入条件培养基后，HepG-2细胞的PCNA、CyclinD1、Survivin mRNA表达明显下调，与对照组相比差异均有显著性意义(P < 0.05)。重组白细胞介素6对各增殖相关基因均具有显著的上调作用，差异均有显著性意义(P < 0.05)。STAT3基因的转录水平在各处理因素作用24 h后，未见明显变化(图4)。 2.4 白细胞介素6和人脐带间充质干细胞条件培养基对HepG-2细胞增殖的影响细胞计数实验及CCK-8实验结果显示外源性白细胞介素6及人脐带间充质干细胞条件培养基均能促进HepG-2细胞增殖，与对照组相比差异均有显著性意义(P < 0.05)。AG490可明显延缓了HepG-2细胞的增殖，加入条件培养基后HepG-2细胞增殖与其他组比较差异仍有显著性意义(P < 0.05，图5)。 2.5 白细胞介素6和人脐带间充质干细胞条件培养基对HepG-2细胞迁移的影响 20 μg/L重组白细胞介素6和40%人脐带间充质干细胞条件培养基对HepG-2细胞分别预处理24 h，细胞迁移能力明显增强，与对照组相比差异均有显著性意义(P < 0.05)；白细胞介素6中和抗体加入条件培养基后，HepG-2细胞迁移能力下降，与对照组相比差异有显著性意义(P < 0.05)；条件培养基+ AG490组穿过聚碳酸酯膜的细胞数量明显少于对照组及条件培养基组，组间比较差异显著性意义(P < 0.05，图6，图7)。划痕实验结果显示，白细胞介素6及人脐带间充质干细胞条件培养基均能提高HepG-2细胞的损伤愈合能力，而条件培养基中的白细胞介素6被中和后，细胞的愈合能力明显下降，与Transwell实验结果一致。使用AG490抑制HepG-2细胞JAK2/STAT3信号通路后，无论人脐带间充质干细胞条件培养基是否存在，细胞的愈合能力均严重受损(图8)。"

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