siRNA沉默P2Y12受体基因对肝细胞癌血管生成能力的影响

doi:10.3877/cma.j.issn.2095-3232.2015.01.015

中华肝脏外科手术学电子杂志 ›› 2015, Vol. 04 ›› Issue (01) : 53 -56. doi: 10.3877/cma.j.issn.2095-3232.2015.01.015

所属专题：文献；

基础研究

siRNA沉默P2Y12受体基因对肝细胞癌血管生成能力的影响

邹德志¹, 郭明明¹, 刘江辉¹, 蔡锐彬¹, 徐丹苹²^,^†(

)

^1. 510080　广州，中山大学附属第一医院急诊科
^2. 510140　广州，广东省中医院心血管科

收稿日期:2014-10-19 出版日期:2015-02-10
通信作者: 徐丹苹
基金资助:
国家自然科学基金(81403341)

Impact of purinergic receptor P2Y12 gene inhibited by siRNA on the angiogenesis of hepatocellular carcinoma

Dezhi Zou¹, Mingming Guo¹, Jianghui Liu¹, Ruibin Cai¹, Danping Xu²^,^†()

^1. Department of Emergency, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China
^2. Department of Cardiovascular, Guangdong Provincial Chinese Medicine Hospital, Guangzhou 510140, China

Received:2014-10-19 Published:2015-02-10
Corresponding author: Danping Xu
About author:
Corresponding author: Xu Danping, Email: xudanping@hotmail.com

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目的

研究siRNA沉默P2Y12嘌呤受体基因对肝细胞癌（肝癌）血管生成能力的影响。

方法

P2Y12-siRNA慢病毒表达载体及空载体对照质粒感染人肝癌细胞株MHCC97H，建立siRNA组和control组。采用Western blot检测P2Y12蛋白表达，体外血管生成实验检测肝癌细胞促血管生成能力，观察裸鼠皮下肝癌成瘤微血管密度(MVD)。两组实验数据比较采用t检验。

结果

siRNA组和control组人肝癌细胞MHCC97H的P2Y12蛋白平均相对表达量分别为0.07±0.01、0.26±0.02，siRNA组明显少于control组（t=-35.64，P<0.05）。经siRNA组细胞上清刺激的人脐静脉血管内皮细胞（HUVEC）未形成连续的封闭小管状结构，经control组细胞上清刺激的HUVEC细胞形成大量封闭小管。siRNA组裸鼠皮下成瘤的微血管较少，MVD为5±1，明显低于control组的23±6 （t=-17.01，P<0.05）。

结论

siRNA沉默P2Y12受体基因可抑制肝癌血管生成。

关键词: 受体，嘌呤能P2Y12, 肝肿瘤，实验性, 新生血管化，病理性

Objective

To investigate the impact of purinergic receptors P2Y12 gene inhibited by small interference RNA (siRNA) on the angiogenesis of hepatocellular carcinoma (HCC).

Methods

Human HCC cell line MHCC97H was infected using P2Y12-siRNA lentiviral expression vector and empty negative control vector to establish siRNA group and control group. The expression of P2Y12 protein in two groups was detected by Western blot assay. In vitro angiogenesis assay was used to define the angiogenesis potential of HCC cell. The microvessel density (MVD) of nude mice subcutaneous tumors was observed. The experimental data between two groups were compared using t test.

Results

The average relative expression of P2Y12 protein were 0.07±0.01 and 0.26±0.02 respectively in siRNA group and control group. The expression of P2Y12 protein in siRNA group decreased significantly compared with that in control group (t=-35.64, P<0.05). No continuous closed small tubular structure was observed in the human umbilical vein endothelial cell (HUVEC) stimulated by the supernatant of siRNA group, while large number of closed tubules was observed in the HUVEC stimulated by the supernatant of control group. A few microvessels of nude mice subcutaneous tumors were observed in siRNA group. The MVD was 5±1, which was significantly lower than that in control group (23±6) (t=-17.01, P<0.05).

Conclusion

Purinergic receptors P2Y12 gene inhibited by siRNA can suppress the angiogenesis of HCC.

Key words: Receptors, purinergic P2Y12, Liver neoplasms, experimental, Neovascularization, pathologic

图1 两组细胞体外血管生成实验（光镜×400）

图2 两组裸鼠皮下肿瘤CD34表达（免疫组化法×400）

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