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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 ›› 2015, Vol. 04 ›› Issue (05) : 318 -322. doi: 10.3877/cma.j.issn.2095-3232.2015.05.015

所属专题： 文献；

基础研究

杀伤细胞免疫球蛋白样受体基因在肝癌细胞免疫杀伤中的作用
何免1, 邱小惠1, 张文峰1, 沈晗1, 王辉1, 薄华本1, 黄树林1, 曾宪成2, 邵红伟1,†()   
  1. 1. 510006　广州，广东药学院生命科学与生物制药学院　广东省生物技术候选药物研究重点实验室
    2. 513000　广州市增城区人民医院普通外科
  • 收稿日期:2015-06-04 出版日期:2015-10-10
  • 通信作者: 邵红伟
  • 基金资助:
    国家自然科学基金基金(31100664，31300737，81303292); 广东省自然科学基金(10151022401000024，2014A030313586); 广东药学院师资队伍建设专项经费(20080927); 广州市卫生局项目(20141A011117，20151A011112); 广州市科技计划项目(2013J4100081)

Effect of killer cell immunoglobulin-like receptor gene in immune killing of hepatoma cells

Mian He1, Xiaohui Qiu1, Wenfeng Zhang1, Han Shen1, Hui Wang1, Huaben Bo1, Shulin Huang1, Xiancheng Zeng2, Hongwei Shao1,†()   

  1. 1. School of Life Science and Biopharmaceutics, Guangdong Pharmaceutical University, Guangdong Key Laboratory for Biotechnology Drug Candidate, Guangzhou 510006, China
    2. Department of General Surgery, Zengcheng District People's Hospital of Guangzhou, Guangdong 513000, China
  • Received:2015-06-04 Published:2015-10-10
  • Corresponding author: Hongwei Shao
  • About author:
    Corresponding author:Shao Hongwei, Email:
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引用本文：

何免, 邱小惠, 张文峰, 沈晗, 王辉, 薄华本, 黄树林, 曾宪成, 邵红伟. 杀伤细胞免疫球蛋白样受体基因在肝癌细胞免疫杀伤中的作用[J]. 中华肝脏外科手术学电子杂志, 2015, 04(05): 318-322.

Mian He, Xiaohui Qiu, Wenfeng Zhang, Han Shen, Hui Wang, Huaben Bo, Shulin Huang, Xiancheng Zeng, Hongwei Shao. Effect of killer cell immunoglobulin-like receptor gene in immune killing of hepatoma cells[J]. Chinese Journal of Hepatic Surgery(Electronic Edition), 2015, 04(05): 318-322.

目的

探讨杀伤细胞免疫球蛋白样受体（KIR）基因在肝癌细胞免疫杀伤中的作用。

方法

外周血单个核细胞（PBMC）与肝癌细胞HepG2按不同效靶比共孵育，观察不同效靶比共孵育下PBMC中KIR基因家族的表达、γ干扰素（IFN-γ）分泌、肝癌细胞形态学改变及PBMC对肝癌细胞的杀伤情况。杀伤率比较采用单因素方差分析和LSD-t检验。

结果

与肝癌细胞共孵育12 h后PBMC活化性KIR基因表达开始增加，24 h后下降。抑制性KIR基因共孵育12 h后开始下降。辅助活化基因DAP12一直保持高表达水平。PBMC中IFN-γ的含量随着效靶比的升高而降低，12 h达到高峰。共孵育后不同效靶比组的肝癌细胞均表现为染色质浓缩，细胞核呈半球状或半月形状且边缘化的细胞比例增加，细胞停止旺盛分裂。效靶比1∶1组的相对杀伤率为（8±3）%，10∶1组为（14±4）%，50∶1组为（32±6）%，50∶1组明显高于1∶1组和10∶1组（LSD-t=5.97，4.61；P<0.05）。

结论

活化性KIR基因在肝癌细胞免疫杀伤中发挥重要作用。

关键词: 受体，KIR, 白细胞，单核, 受体，自然杀伤细胞, 癌，肝细胞, 干扰素γ
Objective

To investigate the effect of killer cell immunoglobulin-like receptor (KIR) gene in immune killing of hepatoma cells.

Methods

Peripheral blood mononuclear cell (PBMC) and hepatoma cells were co-cultured with different effector-target ratios. The expression of KIR gene family in PBMC, the content to interferon-γ (IFN-γ), the morphological change of hepatoma cell and the cytotoxicity to hepatoma cell by PBMC were observed after the co-incubation with different effector-target ratios. The comparison on cytotoxicity rates was conducted using one-way analysis of variance and LSD-t test.

Results

The expression of activating KIR gene increased after 12 h of co-culture, but decreased after 24 h of co-culture. The expression of inhibitory KIR gene decreased after 12 h of co-culture. DAP12 maintained high expression all the time. The content of IFN-γ in PBMC decreased with the increase of effector-target ratio and reached the peak at 12 h of co-culture. Hepatoma cells co-cultured with different effector-target ratios were observed with increased chromatin condensation, rising proportion of cells with hemispherical or half moon shape and marginalized nucleus, and stagnant of active cell division. The cytotoxicity rate of effector-target ratio 1∶1, 10∶1 and 50∶1 was (8±3) %, (14±4) % and (32±6) %, respectively, with 50∶1 group significantly higher than 11∶1 and 10∶1 group (LSD-t=5.97, 4.61; P<0.05).

Conclusion

The activating KIR gene plays an important role in immune killing of hepatoma cells.

Key words: Receptors, KIR, Leukocytes, mononuclear, Receptors, natural killer cell, Carcinoma, hepatocellular, Interferon-gamma
表1 RT-PCR检测的KIR基因引物
KIR基因 正向引物（5'-3'） 反向引物（5'-3'） 产物长度（bp）
2DL1 TCTCCATCAGTCGCATGAC CAGAATGTGCAGGTGTCG 488
2DL2 GCAATGTTGGTCAGATGTCAG GCCCTGCAGAGAACCTACA 383
2DL3 CCACTGAACCCAGCTCCG CAGGAGACAACTTTGATCA 352
2DL4 AACATCTTCACGCTGTACAAGA GCCTGGAATGTTCCATTG AT 368
2DL5 TGGTCACAGGTCTATTTGG GGTTCAGTGGGTGAAGATG 340
2DS1 TCTCCATCAGTCGCATGAA AGGGCCCAGAGGAAAGTT 314
2DS2 TGCACAGAGAGGGGAAGTA CACGCTCTCTCCTGCCAA 257
2DS3 TCACTCCCCCTATCAGTTT GCATCTGTAGGTTCCTCCT 280
2DS4 GGTTCAGGCAGGAGAGAAT TTGACCACTCGTAGGGAGC 218/196a
2DS5 AGAGAGGGGACGTTTAACC TCCGTGGGTGGCAGGGT 396
3DL1 ACATCGTGGTCACGGGTCC ACAACTTTGGATCTGGGCTT 663
3DL2 CGGTCCCTTGATGCCTGT GACCACACGCAGGGCAG 369
3DL3 CACGATGCGGGTTCCC AGAAGAGGAGGATAGCAAAGG 508
3DS1 GGCACCCAGCAACCCCA AAGGGCACGCATCATGGA 247
DAP10 ATGATCCATCTGGGTCACATC CTGCCTGGCATGTTGATG 274
DAP12 CAGTTGCTCTACGGTGAGC TGTGTGTTGAGGTCGCTG 224
GAPDH AGGTCGGAGTCAACGGATTTG GTGATGGCATGGACTGTGGT ?
图1 PBMC与肝癌细胞共孵育时KIR基因的表达
图2 ELISA检测共孵育后PBMC的IFN-γ分泌情况
图3 激光共聚焦扫描显微镜下肝癌细胞形态图（DAPI染色　×100）
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