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中华肝脏外科手术学电子杂志

中华肝脏外科手术学电子杂志 ›› 2016, Vol. 05 ›› Issue (06) : 404 -408. doi: 10.3877/cma.j.issn.2095-3232.2016.06.014

所属专题： 文献；

基础研究

肝癌来源间充质干细胞对肝癌细胞侵袭能力的影响
姚志成1, 邬杰忠2, 熊志勇2, 徐见亮2, 邓美海2, 方和平2,†()   
  1. 1. 510530　广州，中山大学附属第三医院岭南医院普通外科
    2. 510630　广州，中山大学附属第三医院肝胆外科
  • 收稿日期:2016-08-25 出版日期:2016-12-10
  • 通信作者: 方和平
  • 基金资助:
    国家自然科学基金(81572726); 广东省医学科研基金(A2016312); 广东省科技计划资助项目(2010B060900035，2013B010404014，2014A020212122); 广州市科技计划项目(1563000226)

Effect of liver cancer derived mesenchymal stem cell on invasion of liver cancer cells

Zhicheng Yao1, Jiezhong Wu2, Zhiyong Xiong2, Jianliang Xu2, Meihai Deng2, Heping Fang2,†()   

  1. 1. Department of General Surgery, Lingnan Hospital, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510530, China
    2. Department of Hepatobiliary Surgery, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China
  • Received:2016-08-25 Published:2016-12-10
  • Corresponding author: Heping Fang
  • About author:
    Corresponding author: Fang Heping, Email:
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引用本文：

姚志成, 邬杰忠, 熊志勇, 徐见亮, 邓美海, 方和平. 肝癌来源间充质干细胞对肝癌细胞侵袭能力的影响[J]. 中华肝脏外科手术学电子杂志, 2016, 05(06): 404-408.

Zhicheng Yao, Jiezhong Wu, Zhiyong Xiong, Jianliang Xu, Meihai Deng, Heping Fang. Effect of liver cancer derived mesenchymal stem cell on invasion of liver cancer cells[J]. Chinese Journal of Hepatic Surgery(Electronic Edition), 2016, 05(06): 404-408.

目的

探讨肝癌来源间充质干细胞（LCMSC）对肝癌细胞侵袭能力的影响及其机制。

方法

采用RT-PCR检测骨髓来源间充质干细胞（BMSC）和LCMSC中IL-6、IL-8以及趋化因子CXCL1、CXCL5、CXCL12的mRNA表达差异，ELISA和Western blot检测LCMSC上清液中CXCL12蛋白水平。采用细胞共培养法设为HepG2+BMSC、HepG2+LCMSC、HepG2+LCMSC+siRNA-CXCL12组，Transwell细胞侵袭实验检测CXCL12对肝癌细胞HepG2侵袭能力的影响。实验数据比较采用单因素方差分析和LSD-t检验或t检验。

结果

LCMSC中CXCL12的mRNA表达量为60.3±2.4，明显高于BMSC中的13.8±1.8（t=15.68，P<0.05）。LCMSC上清液中CXCL12蛋白含量为（31.5±1.7）ng/L，明显高于BMSC中的（14.3±1.5）ng/L（t=7.60，P<0.05），且CXCL12蛋白表达增强。Transwell细胞侵袭实验显示，HepG2+LCMSC组穿膜细胞数为（110±12）个，明显多于HepG2+BMSC组的（65±9）个和HepG2+LCMSC+siRNA-CXCL12组的（76±7）个（LSD-t=5.25，4.86；P<0.05）。

结论

LCMSC高表达CXCL12，LCMSC可能通过上调CXCL12表达增强HepG2细胞的侵袭能力，采用siRNA沉默CXCL12基因可有效降低肝癌细胞的侵袭能力。

关键词: 间充质干细胞, 癌，肝细胞, 肿瘤侵润, 趋化因子CXCL12
Objective

To investigate the effect and mechanism of liver cancer derived mesenchymal stem cell (LCMSC) on the invasion of liver cancer cells.

Methods

The expressions of interleukin (IL)-6, IL-8 and chemotactic factors CXCL1, CXCL5 and CXCL12 mRNA in the bone marrow derived mesenchymal stem cell (BMSC) and LCMSC were detected by reverse transcription-polymerase chain reaction (RT-PCR). The expression of protein CXCL12 in the supernate of LCMSC was detected by enzyme-linked immunosorbent assy (ELISA) and Western blot. Different cells were co-cultured and divided into the HepG2+BMSC, HepG2+LCMSC and HepG2+LCMSC+siRNA-CXCL12 groups. The effect of CXCL12 on the invasion of liver cancer HepG2 cells were detected by Transwell migration assay. The experiment data were compared using one way analysis of variance and LSD-t test or t test.

Results

The expression of CXCL12 mRNA in LCMSC was 60.3±2.4, significantly higher than 13.8±1.8 in BMSC (t=15.68, P<0.05). The expression of protein CXCL12 in the supernate of LCMSC was (31.5±1.7) ng/L, significantly higher than (14.3±1.5) ng/L in BMSC (t=7.60, P<0.05). And the expression of protein CXCL12 was up-regulated. Transwell migration assay indicated that the quantity of membrane-invasion cells in the HepG2+LCMSC group was 110±12, significantly higher than 65±9 in the HepG2+BMSC group and 76±7 in the HepG2+LCMSC+siRNA-CXCL12 group (LSD-t=5.25, 4.86; P<0.05).

Conclusion

CXCL12 is highly expressed in LCMSC. LCMSC may enhance the invasion of HepG2 cells through up-regulating the expression of CXCL12. The invasion of liver cancer cells can be effectively weakend by silencing the CXCL12 gene with siRNA.

Key words: Mesenchymal stem cells, Carcinoma, hepatocellular, Neoplasm invasiveness, Chemokine CXCL12
表1 RT-PCR引物序列
基因 引物序列
IL-6 F:5'-CCAGTACCCCCAGGAGAAGAT-3'
R:5'-TTGCCTTTTTCTGCAGGAAC-3'
IL-8 F:5'-TTGGCAGCCTTCCTGATTT-3'
R:5'-TCAAAAACTTCTCCACAACCC-3'
CXCL1 F:5'-CACCCAAACCGAAGTCATAG-3'
R:5'-AAGCCAGCGTTCACCAGA-3'
CXCL5 F:5'-CGCTGTCATCCTCATTGCTA-3'
R:5'-GAGCACTTGCCACTGGTGTA-3'
CXCL12 F:5'-AGTCAGGTGGTGGCTTAACAG-3'
R:5'-AGAGGAGGTGAAGGCAGTGG-3'
GAPDH F:5'-ACCACAGTCCATGCCATCAC-3'
R:5'-TCCACCACCCTGTTGCTGTA-3'
图1 BMSC和LCMSC中的细胞因子mRNA表达
图2 CXCL12蛋白在BMSC和LCMSC的表达水平
图3 Western blot检测CXCL12蛋白表达
图4 Transwell细胞侵袭实验检测穿膜细胞数（Giemsa染色 ×50）
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