人肝癌浸润淋巴细胞的分离培养及其CD8阳性亚群特性研究

doi:10.3877/cma.j.issn.1674-0793.2020.03.004

中华普通外科学文献(电子版) ›› 2020, Vol. 14 ›› Issue (03) : 179 -183. doi: 10.3877/cma.j.issn.1674-0793.2020.03.004

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论著

人肝癌浸润淋巴细胞的分离培养及其CD8阳性亚群特性研究

黄镇辉¹, 徐晓艳², 蒋小峰², 薛平², 曾嘉敏³, 魏宜胜¹^,^†(

)

^1. 510260　广州医科大学附属第二医院胃肠外科；510260　广州医科大学附属第二医院外科实验室
^2. 510260　广州医科大学附属第二医院肝胆外科
^3. 510260　广州医科大学附属第二医院病理科

收稿日期:2019-12-28 出版日期:2020-06-01
通信作者: 魏宜胜

In vitro culturing and characteristics of CD8 postive tumor-infiltrating lymphocytes of hepatocellular carcinoma

Zhenhui Huang¹, Xiaoyan Xu², Xiaofeng Jiang², Ping Xue², Jiamin Zeng³, Yisheng Wei¹^,^†()

^1. Department of Gastrointestinal Surgery, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, China; Laboratory of Surgery, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, China
^2. Department of Hepatobiliary Surgery, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, China
^3. Department of Pathology, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, China

Received:2019-12-28 Published:2020-06-01
Corresponding author: Yisheng Wei
About author:
Corresponding author: Wei Yisheng, Email: yswei2004@126.com

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引用本文：

黄镇辉, 徐晓艳, 蒋小峰, 薛平, 曾嘉敏, 魏宜胜. 人肝癌浸润淋巴细胞的分离培养及其CD8阳性亚群特性研究[J]. 中华普通外科学文献(电子版), 2020, 14(03): 179-183.

Zhenhui Huang, Xiaoyan Xu, Xiaofeng Jiang, Ping Xue, Jiamin Zeng, Yisheng Wei. In vitro culturing and characteristics of CD8 postive tumor-infiltrating lymphocytes of hepatocellular carcinoma[J]. Chinese Archives of General Surgery(Electronic Edition), 2020, 14(03): 179-183.

目的

从原发性肝癌（HCC）患者术后的肿瘤组织中提取、培养肿瘤浸润性淋巴细胞（TILs），进一步分选、扩增其CD8阳性亚群（CD8+TILs），并研究其特性。

方法

标本取自38例肝癌根治术后的肿瘤组织，免疫组织化学染色观察癌巢组织和癌旁组织TILs的分布，通过机械剪碎、混合酶消化和不连续密度梯度离心法分离提取TIL前体细胞。采用重组人白介素2进行激活，抗CD3、抗CD28抗体进行共刺激扩增，用磁珠分选出CD8+TILs，CCK8法观察CD8+TILs对肝癌细胞Hep3B、HepG2生长的影响。

结果

TILs富集于癌旁组织，12例成功提取TILs，6例培养并实现大量扩增，扩增17~50倍后细胞数为（1.2~2.5）×10⁸个，CD3+细胞比例为（77.53±16.37）%，CD3+CD4+比例为（27.08±21.56）%，CD3+CD8+比例（44.55±12.73）%，细胞活力为（90.5±3.0）%，CD8+TILs对肿瘤细胞系Hep3B、HepG2有较强的生长抑制作用。

结论

本研究成功建立高效提取、培养CD8+TILs的方法，获得具有高抗肿瘤活性的CD8+TILs，为晚期肝癌等实体瘤的过继免疫治疗提供研究基础。

关键词: 淋巴细胞，肿瘤浸润, 癌，肝细胞, CD8阳性T淋巴细胞, 免疫，细胞

Objective

To investigate the methods of expansion of CD8 positive tumor-infiltrating lymphocytes (CD8+TILs) obtained from human hepatocellular carcinoma (HCC) and to study their immunological characteristics.

Methods

Thirty-eight tumor tissue specimens were obtained from radical liver cancer surgery. Immunohistochemistry staining was performed on cancer nests and side tissues to study the distribution of TILs, and TIL precursor cells were extracted by mechanical shearing, mixed enzyme digestion and discontinuous density gradient centrifugal separation. Activated with high dose of recombinant human interleukin 2 at first, the isolated cells were co-stimulated of anti-CD3 and anti-CD28 antibody. The CD8+TILs were sorted by magnetic beads. The effect of CD8+TILs on the growth of Hep3B and HepG2 cells lines was observed in CCK8 cytotoxicity experiment in vitro.

Results

TILs were enriched in paracancer tissues, 12 cases were successfully extracted, 6 cases were achieved a large amplification among them. The number of amplificated cells was (1.2-2.5)×10⁸ after 17-50 times expansion fold, and the proportion of CD3+ cells in the paracancer tissues was (77.53±16.37)%, CD3+CD4+ ratio was (27.08±21.56)%, CD3+CD8+ ratio was (44.55±12.73)%, and the cell viability was (90.5±3.0)%. Moreover, CD8+TILs showed strong growth inhibition effect on Hep3B and HepG2 cell lines.

Conclusions

This study successfully establishes a method for efficient extraction and cultivation of CD8 + TILs, and obtains CD8 +TILs with high antitumor activity, which provides a research basis for adoptive immunotherapy of solid tumors such as advanced liver cancer.

Key words: Lymphocytes, tumor-infiltrating, Carcinoma, hepatocellular, CD8-positive T-lymphocytes, Immunity, cellular

图1 肝癌标本的苏木精-伊红染色（HE）和细胞表位抗原CD8免疫组织化学（IHC）结果

图2 分离和激活的TILs图　A为刚分离出来的TILs（100×）；B、C、D分别为100、200、400倍镜下激活的TILs

表1 癌旁组织和癌巢组织提取肿瘤浸润淋巴的细胞表型及占比

组织来源	n	CD3+	CD3+CD4+	CD3+CD8+
癌旁	6	77.53±16.37	27.08±21.56	44.55±12.73
癌巢	6	57.50±20.41	37.57±16.12	22.39±20.69
t值	?	3.077	-0.953	4.193
P值	?	0.028	0.365	0.009

表1 癌旁组织和癌巢组织提取肿瘤浸润淋巴的细胞表型及占比

组织来源	n	CD3+	CD3+CD4+	CD3+CD8+
癌旁	6	77.53±16.37	27.08±21.56	44.55±12.73
癌巢	6	57.50±20.41	37.57±16.12	22.39±20.69
t值	?	3.077	-0.953	4.193
P值	?	0.028	0.365	0.009

图3 TILs的吖啶橙（AO）染色（100×） A为培养第2天；B为培养第6天；C为培养第14天

表2 CD8+TILs和非CD8+TILs对肝癌细胞系HepG2、Hep3B生长的抑制率（%，±s）

组别	HepG2细胞			Hep3B细胞
组别	ET=11	ET=51	ET=101	ET=11	ET=51	ET=101
CD8+TILs	1.60±0.23	85.16±3.66	87.74±5.81	3.35±2.59	81.34±2.32	91.03±2.41
非CD8+TILs	0.53±2.21	0.30±1.67	75.00±4.90	1.29±2.83	1.94±2.04	43.54±1.98
t值	0.834	36.535	2.903	0.930	44.516	26.372
P值	0.490	<0.001	0.045	0.405	<0.001	<0.001

表2 CD8+TILs和非CD8+TILs对肝癌细胞系HepG2、Hep3B生长的抑制率（%，±s）

组别	HepG2细胞			Hep3B细胞
组别	ET=11	ET=51	ET=101	ET=11	ET=51	ET=101
CD8+TILs	1.60±0.23	85.16±3.66	87.74±5.81	3.35±2.59	81.34±2.32	91.03±2.41
非CD8+TILs	0.53±2.21	0.30±1.67	75.00±4.90	1.29±2.83	1.94±2.04	43.54±1.98
t值	0.834	36.535	2.903	0.930	44.516	26.372
P值	0.490	<0.001	0.045	0.405	<0.001	<0.001

表3 CD8+TILs对肝癌细胞系HepG2、Hep3B最适效靶比分析

肿瘤细胞	组别（1）	组别（2）	P值
HepG2	ET=11	ET=51	<0.001
	ET=11	ET=101	<0.001
	ET=51	ET=11	<0.001
	ET=51	ET=101	1.000
Hep3B	ET=11	ET=51	<0.001
	ET=11	ET=101	<0.001
	ET=51	ET=11	<0.001
	ET=51	ET=101	0.011

表3 CD8+TILs对肝癌细胞系HepG2、Hep3B最适效靶比分析

肿瘤细胞	组别（1）	组别（2）	P值
HepG2	ET=11	ET=51	<0.001
	ET=11	ET=101	<0.001
	ET=51	ET=11	<0.001
	ET=51	ET=101	1.000
Hep3B	ET=11	ET=51	<0.001
	ET=11	ET=101	<0.001
	ET=51	ET=11	<0.001
	ET=51	ET=101	0.011

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