活化态肝星状细胞促进肝癌血管新生的机制

doi:10.3877/cma.j.issn.2095-3232.2016.05.013

中华肝脏外科手术学电子杂志 ›› 2016, Vol. 05 ›› Issue (05) : 323 -327. doi: 10.3877/cma.j.issn.2095-3232.2016.05.013

所属专题：文献；

基础研究

活化态肝星状细胞促进肝癌血管新生的机制

唐亚军¹, 潘楚芝¹, 卢逸¹, 汤照峰¹, 林楠¹^,^†(

)

^1. 510630　广州，中山大学附属第三医院肝胆外科

收稿日期:2016-05-14 出版日期:2016-10-10
通信作者: 林楠
基金资助:
广东省自然科学基金(2014A030313067，2014A030313144); 农村科技领域项目(2016A050502014)

Mechanism of activated hepatic stellate cells promote angiogenesis of hepatocellular carcinoma

Yajun Tang¹, Chuzhi Pan¹, Yi Lu¹, Zhaofeng Tang¹, Nan Lin¹^,^†()

^1. Department of Hepatobiliary Surgery, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China

Received:2016-05-14 Published:2016-10-10
Corresponding author: Nan Lin
About author:
Corresponding author: Lin Nan, Email: drlinnan@vip.163.com

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目的

探讨活化态肝星状细胞（aHSC）促进肝癌血管新生的机制。

方法

aHSC分别连续培养1、3、5、7 d后收集细胞，实时荧光定量PCR检测aHSC血管生成素-1（Ang-1）mRNA相对表达量。免疫荧光染色法观察aHSC的Ang-1蛋白表达情况。Transwell小室观察aHSC对肝血管内皮细胞（HVEC）增殖的影响。小管形成实验观察aHSC对HVEC成管作用的影响。实验数据比较采用单因素方差分析和LSD-t检验。

结果

aHSC培养1、3、5、7 d，其Ang-1 mRNA的相对表达量分别为1.000±0.024、1.920±0.080、6.230±0.320、7.820±0.380，随培养时间的延长逐渐增加（LSD-t=7.32，13.68，8.34；P<0.05）。免疫荧光染色结果显示，Ang-1与平滑肌肌动蛋白抗体（aSMA）共同表达于aHSC。Transwell小室间接共培养结果显示，aHSC促进HVEC的增殖，加入Ang-1抗体后，aHSC促进HVEC增殖的能力减弱。小管形成实验结果显示，在aHSC条件培养基的作用下，HVEC可以聚合并逐渐形成管状结构，而加入Ang-1抗体后，微血管形成明显减少。

结论

aHSC可能通过分泌Ang-1促进肝癌中HVEC的增殖及血管生成。

关键词: 癌，肝细胞, 活化态肝星状细胞, 血管生成素-1, 血管生成

Objective

To investigate the mechanism of activated hepatic stellate cells (aHSC) promote the angiogenesis of hepatocellular carcinoma (HCC).

Methods

aHSCs were collected after being cultured for 1, 3, 5, 7 d respectively . The relative expression of aHSC angiopoietin-1 (Ang-1) messenger ribonucleic acid (mRNA) was detected by real-time fluorescence quantitative polymerase chain reaction (PCR). The expression of aHSC Ang-1 protein was observed by immunofluorescence staining. The impact of aHSC on the proliferation of hepatic vascular endothelial cells (HVEC) was observed by Transwell chamber. The impact of aHSC on HVEC tube formation was observed by tube formation assay. The experimental data were compared using one-way analysis of variance and LSD-t test.

Results

After aHSC were cultured for 1, 3, 5, 7 d, the relative expression of aHSC Ang-1 mRNA was respectively 1.000±0.024, 1.920±0.080, 6.230±0.320 and 7.820±0.380, which gradually increased as the culture time went on (LSD-t=7.32, 13.68, 8.34; P<0.05). The immunofluorescence staining showed that Ang-1 and smooth muscle actin antibody (aSMA) were co-expressed in the aHSC. Transwell chamber indirect co-culture showed that aHSC could promote the proliferation of HVEC, and the effect weakened after Ang-1 antibody was added. Tube formation assay showed that HVEC could polymerize and gradually developed tubular structure in the aHSC conditioned medium, and the effect significantly weakened after Ang-1 antibody was added.

Conclusion

aHSC may promote the proliferation and angiogenesis of HVEC in HCC by secreting Ang-1.

Key words: Carcinoma, hepatocellular, Activited hepatic stellate cell, Angiopoietin-1, Angiogenesis

图1 aHSC连续培养1、5 d时Ang-1和SMA的表达情况（荧光显微镜 ×100）

图2 aHSC和HCC对HVEC增殖能力的影响

图3 aHSC促进HVEC成管实验（显微镜 ×100）

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