胸腺肽α1对原发性肝癌围手术期免疫功能的影响

摘要

目的: 研究胸腺肽α1对原发性肝癌患者围手术期免疫功能的影响.

方法: 103例确诊为原发性肝癌患者分为2组, A组采用胸腺肽α1+手术治疗 (n = 35), B组仅手术治疗 (n = 68). 胸腺肽α1治疗前后及手术前、手术后7, 14, 21 d用流式细胞仪检测CD3⁺, CD4⁺, CD8⁺, CD8⁺CD28⁺, CD16⁺CD56⁺的变化, MTT比色法测定IL-2活性.

结果: A组CD8⁺, CD4⁺/CD8⁺, CD8⁺CD28⁺, CD16⁺CD56⁺及IL-2均明显低于正常对照组 (F = 8.2, P = 0.005); 术前经胸腺肽α1治疗后CD4⁺/CD8⁺, CD16⁺CD56⁺较治疗前增高(F = 8.4, P = 0.004); 术后7 d CD4⁺/CD8⁺, CD8⁺CD28⁺, CD16⁺CD56⁺较术前增高(F = 4.7, P = 0.005或F = 3.5, P = 0.04), 术后14 d IL-2也增高(F = 3.6, P = 0.03). B组术后7 d, CD4⁺, CD8⁺, CD4⁺/CD8⁺, CD8⁺CD28⁺, CD16⁺CD56⁺较术前明显降低(F = 5.4, P = 0.006或F = 3.6, P = 0.02), 术后14 d, CD8⁺, CD8⁺CD28⁺仍然低于术前(F = 5.0, P = 0.007), 其他指标有所恢复; 术后21 d, CD8⁺仍未恢复至术前水平(F = 6.3, P = 0.000); B组术后7 d, CD3⁺, CD8⁺, CD4⁺/CD8⁺, CD8⁺CD28⁺, CD16⁺CD56⁺均明显低于A组(F = 7.2, P = 0.001).

结论: 肝癌患者存在免疫抑制, 术后早期免疫抑制程度加重, 围手术期胸腺肽α1治疗可有效改善肝癌围手术期免疫抑制状况.

关键词: 胸腺肽α1; T淋巴细胞亚群; IL-2; 肝癌; 围手术期

Effects of thymosin α1 on immune function in perioperative patients with primary liver cancer

Qing Xu, Peng Li, Ping Xue, Wei-Ping Zhu, Rui-Xin Chen, Xiu Yu

Qing Xu, Peng Li, Department of General Surgery, the Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu Province, China

Ping Xue, Department of General Surgery, the Affiliated Hai'an Hospital of Nantong University, Nantong Hai'an 226600, Jiangsu Province, China

Wei-Ping Zhu, Department of General Surgery, Nantong Hong Qiao Hospital, Nantong 226000, Jiangsu Province, China

Rui-Xin Chen, Xiu Yu, Research Center of Liver, Gall and Pancreas, the Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu Province, China

Supported by: the Scientific Foundation from Health Office of Jiangsu Province, No. H9327.

Correspondence to: Dr. Qing Xu, Department of General Surgery, the Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong 226001, Jiangsu Province, China. xuching_surg@sina.com

Received: September 21, 2005
Revised: September 25, 2005
Accepted: September 30, 2005
Published online: December 15, 2005

Abstract

AIM: To investigate the immune state of patients with primary hepatic carcinoma (PHC) liver cancer after treatment with thymosin α1 during perioperative period.

METHODS: One hundred and three patients with PHC were divided into two groups. The patients in group A (n = 35) received hepatectomy plus thymosin α1 treatment and the patients in group B (n = 68) underwent hepatectomy only. Twenty normal blood donors were used as controls. Before and 7, 14, and 21 d after treatment, the changes of serum CD3⁺, CD4⁺CD8⁺, CD8⁺CD28⁺, and CD16⁺CD56⁺ were measured by flow cytometry. MTT colometric assay was performed to detect the activity of interleukin-2 (IL-2).

RESULTS: Before hepatectomy and thymosin α1 treatment, serum CD8⁺, CD4⁺/CD8⁺, CD8⁺CD28⁺, CD16⁺/CD56⁺ and IL-2 were markedly lower in group A than that in the controls (F = 8.2, P = 0.005), while after treatment, serum CD4⁺/CD8⁺, CD16⁺CD56⁺ were significantly increased (F = 8.4, P = 0.004). Seven days after hepatectomy, serum CD4⁺/CD8⁺, CD8⁺CD28⁺, CD16⁺/CD56⁺ were elevated (F = 4.7, P = 0.005 or F= 3.5, P = 0.04), and 14 d after hepatectomy, the level of serum IL-2 was inceased (F = 3.6, P = 0.03). For group B, serum CD4⁺, CD8⁺, CD4⁺/CD8⁺, CD8⁺/CD28⁺, CD16⁺/CD56⁺ were notably reduced 7 d after hepatectomy (F = 5.4, P = 0.006 or F = 3.6, P = 0.02). The levels of serum CD8⁺ and CD8⁺/CD28⁺ were still lower (F = 5.0, P = 0.007) at 14 d than those before operation, but the levels of CD4⁺, CD4⁺/CD8⁺, and CD16⁺/CD56⁺ restored to some extent. At 21 d, serum CD8⁺ level still didn't restore (F = 6.3, P = 0.000). The levels of serum CD3⁺, CD8⁺, CD4⁺/CD8⁺, CD8⁺/CD28⁺, CD16⁺/CD56⁺ were all lower in group B than those in group A (F = 7.2, P = 0.001).

CONCLUSION: The immunosuppression exists in patients with PHC, and it can be aggravated in early stage after hepatectomy. However, the immunosuppression can be alleviated by thymosin α1 treatment during perioperative period.

Key Words: Thymosin α1; Lymphocyte subsets; Interleukin-2; Hepatic carcinoma; Perioperative period

0 引言

原发性肝癌(简称肝癌)患者存在严重的免疫抑制现象[1]. 我们发现, 肝癌患者围手术期免疫抑制程度加重, 尤其是术后1 wk左右免疫抑制现象尤为严重[2], 免疫调节治疗对预防术后复发及减轻TACE后免疫抑制有良好效果[3], 但围手术期免疫调节治疗一直未引起足够重视. 为此, 我们自1999-01开始, 对35例肝癌患者使用胸腺肽α1进行围手术期免疫治疗, 以探讨胸腺肽α1对肝癌围手术期免疫功能影响.

1 材料和方法

1.1 材料

手术治疗经病理确诊为原发性肝癌, A组胸腺肽α1+手术治疗组, 35例; B组手术治疗组, 68例; 健康义务献血人员20例为正常对照(表1).

表1 原发性肝癌患者临床和病理资料.

项目	A组	B组
男	28	55
女	7	13
平均年龄(岁)	47	44
HBsAg(+)	29	57
肝硬化：
有	34	64
无	1	4
Child分级：
A级	28	56
B级	7	12
AFP(mg/L)：
<20	21	38
≥20	14	30
肿瘤：
单个	33	61
≥2个	2	7
肿瘤大小(mm)：
<50	19	40
≥50	16	28
肿瘤包膜:
完整	11	25
不完整	24	43
门静脉癌栓:
有	8	14
无	27	54
Edmondson分级:
Ⅰ, Ⅱ	7	18
Ⅲ, Ⅳ	28	50

^aP<0.05 vs 对照组;

^cP<0.05 vs 5 wk.

1.2 方法

A组术前4 d每天sc胸腺肽α1(美国赛生公司)1.6 mg, 术后1 wk每2 d sc胸腺肽α1(1.6 mg), 2 wk后每周2次皮下注射胸腺肽α1(1.6 mg)共3 wk. B组病例常规术前准备后进行手术治疗, 手术前后不进行免疫治疗. A组于胸腺肽α1治疗前, 术前、术后7, 14, 21 d, B组于术前、术后7, 14, 21 d分别用流式细胞仪检测外周血CD3⁺, CD4⁺, CD8⁺, CD16⁺CD56⁺, CD8⁺CD28⁺, CD3⁺, CD4⁺, CD8⁺, CD16⁺CD56⁺, CD8⁺CD28⁺ 荧光抗体均购自美国BD公司, MTT比色法检测IL-2. 手术切缘≥2cm为根治性切除, <2cm为姑息性切除, A组根治性切除24例(68.6%), 姑息性切除11例(31.4%), B组根治性切除43例(63.2%), 姑息性切除25例(36.8%). 手术方式包括局部切除57例, 左外叶切除10例、左半肝切除7例、右半肝切除6例, 右三叶切除术3例, 第Ⅷ肝段切除2例. 门静脉癌栓同时行取栓术.

统计学处理 数据均用SPSS(10.0)软件处理, 结果采用mean±SD表示, 用方差分析进行统计学处理, P<0.05有统计学意义.

2 结果

2.1 胸腺肽α1对术前肝癌患者免疫功能的影响

术前A组经胸腺肽α1治疗后CD4⁺/CD8⁺, CD16⁺ CD56⁺较治疗前增高, 肝癌患者在治疗前CD8⁺, CD4⁺/CD8⁺, CD8⁺CD28⁺, CD16⁺ CD56⁺, IL-2均明显低于正常对照组(表2).

表2 胸腺肽α1对术前肝癌患者免疫功能的影响(mean±SD).

免疫指标	A组治疗前	A组治疗后	正常对照
CD3+(%)	60.5±7.5	64.2±9.0	69.1±7.6
CD4+(%)	30.5±8.1	35.5±7.5	47.0±6.9
CD8+(%)	42.2±7.5	33.8±8.2	26.4±5.8b
CD4+/CD8+	0.8±0.3	1.4±0.3b	1.7±0.2b
CD8+CD28+(%)	8.5±2.2	10.2±2.0	18.0±2.2b
CD16+CD56+(%)	14.8±3.1	21.0±2.5b	27.5±3.0b
IL-2(kU/L)	40.2±4.3	49.8±3.5	81.5±4.7b

^bP<0.01 vs A组治疗前.

2.2 AB组患者手术前后免疫指标的变化

A组经胸腺肽α1治疗后, 术后7 d CD4⁺/CD8⁺, CD8⁺CD28⁺, CD16⁺ CD56⁺较术前增高, 术后14 d IL-2也增高(表3). B组术后7 d CD4⁺, CD8⁺, CD4⁺/CD8⁺, CD8⁺CD28⁺, CD16⁺ CD56⁺较术前明显降低, 术后14 d CD8⁺, CD8⁺CD28⁺仍然低于术前, 其他指标有所恢复, 术后21 d CD8⁺仍未恢复至术前水平. B组术后7 d CD3⁺, CD8+, CD4⁺/CD8⁺, CD8⁺CD28⁺, CD16⁺ CD56⁺均明显低于A组(P<0.01)．

表3 AB组患者手术前后免疫指标的变化 (mean±SD).

	A组				B组
指标	术前	术后7 d	术后14 d	术后21 d	术前	术后7 d	术后14 d	术后21 d
CD3+(%)	60.5±7.5	64.1±8.0	63.6±8.2	79.3±8.1	54.3±8.4	30.6±8.4	39.4±9.6	44.2±8.8
CD4+(%)	30.5±8.1	48.0±7.9	40.5±6.3	44.6±7.1	28.8±7.6	16.1±7.6a	29.1±8.4	36.0±7.4
CD8+(%)	42.2±7.5	34.5±8.5	33.7±7.7	32.4±7.0	44.1±6.6	21.4±5.0b	24.0±5.1b	23.5±5.7b
CD4+/CD8+	0.8±0.3	1.4±0.3a	1.6±0.2b	1.6±0.3b	0.8±0.3	0.4±0.3a	1.3±0.2a	1.2±0.2a
CD8+CD28+(%)	8.5±2.2	16.7±2.1b	19.0±2.3b	16.7±2.3b	8.6±2.2	3.94±2.0b	4.8±2.3b	9.8±2.1
CD16+CD56+(%)	14.8±3.1	23.5±3.2b	24.7±3.4b	29.1±3.0b	11.3±2.1	6.1±2.1b	16.6±2.6	19.6±3.0
IL-2(kU/L)	40.2±4.3	45.7±4.0	70.2±4.3a	87.5±4.1b	38.6±4.2	30.5±3.9	31.8±4.2	59.3±4.2

^aP<0.05,

^bP<0.01 vs 术前.

3 讨论

肝癌术后复发是患者死亡主要原因, 复发多在术后1-2年内[4], 术后化疗结合免疫治疗预防肝癌复发为大部分学者所肯定[5]. 周伟平et al[6]报道TACE+LAK+白细胞介素-2联合治疗有效降低肝癌术后复发率, 化疗结合起来α-干扰素治疗能预防复发或延迟复发时间, 但对肝癌围手术期免疫治疗长期以来未引起足够重视. 我们发现肝癌患者存在严重免疫抑制, 手术治疗、麻醉、输血等因素均可进一步损害患者免疫功能, 表现为术后7 d CD4⁺, CD8⁺, CD4⁺/CD8⁺, CD8⁺CD28⁺, CD16⁺CD56⁺较术前下降, 出现较术前更为严重的免疫抑制期, 在以往的研究中我们称之为免疫极度抑制期[2], 此后由于肿瘤切除, 免疫抑制虽有不同程度恢复但仍不满意. 临床研究发现, 肝癌患者术后免疫功能愈低, 恢复愈慢, 术后复发愈早, 复发率愈高, 提高或加快免疫功能的恢复有利于降低复发率[6]. 围手术期成为一个综合治疗空白区, 此期间循环中残存的肿瘤或肝内残留肿瘤细胞极易逃逸肿瘤免疫监视成为转移和复发的主要因素. 许多研究表明, 恶性肿瘤患者外周血中存在微转移癌细胞, 骆成玉et al[7]在研究大肠癌微转移癌细胞中发现许多大肠癌患者本身在术前就存在循环中微转移癌细胞, 术中解剖对组织的牵拉或挤压等可导致癌细胞种植与转移. Capurro et al[8]在研究肝癌外周血GPC-3mRNA表达时也发现高表达GPC-3mRNA与肝癌微转移的发生有密切关系, 并指出肝癌患者外周血中存在微转移癌细胞. 在肝癌免疫中起重要作用的效应细胞是T细胞, CD8⁺按CD28⁺表达与否分为CD8⁺CD28⁺细胞毒T细胞和CD8⁺CD28^-抑制性T细胞, 细胞毒T细胞在抗肿瘤效应中起关键作用, 该细胞与肿瘤直接接触, 通过分泌穿孔素和释放颗粒酶和淋巴毒素直接杀伤肿瘤细胞或通过高表达Fasl与靶细胞表面Fas结合而诱导肿瘤细胞凋亡[9]. 该效应在荷瘤早期、肿瘤缓解期或清除手术后残余肿瘤细胞中发挥重要作用. 本组资料显示, 未经免疫治疗患者术前CD8⁺CD28⁺与CD8⁺T细胞已处于低水平, 术后CD8⁺CD28⁺与CD8⁺T细胞同时下降, 此时机体免疫功能受到抑制, 残余肿瘤细胞或微转移癌细胞易于逃逸免疫攻击, 出现扩散与转移, 因此, 围手术期综合治疗及免疫治疗应该是预防复发的重要内容之一.

Tα1是氨基酸乙酰化的28个氨基酸组成的多肽, 除作为免疫增强剂外, 还具有直接抗瘤毒和抗肿瘤作用[10], Tα1通过对CD3⁺, CD4⁺, CD16⁺CD56⁺ NK细胞刺激, 增强NK细胞的杀伤活性, 促使致敏淋巴细胞分泌IL-2, α及γ-干扰素, 增强致敏淋巴细胞IL-2R的表达[11], 还可促进T细胞表面抗原Thy1、2, Ly1、2、3表达, 增强T细胞介导同种或自身淋巴细胞反应能力, 增细机体免疫应答功能[12]. 因此, 具有免疫调节和抑制肿瘤细胞增殖作用. 本研究中我们发现Tα1可使术前肝癌患者CD4⁺/CD8⁺比例恢复, CD16⁺CD56⁺ (NK细胞)增多. 术后继续使用Tα1可有效消除术后7 d左右的免疫极度抑制期, 表现为A组经胸腺肽α1治疗后, 术后7 d CD4⁺/CD8⁺ , CD8⁺CD28⁺, CD16⁺ CD56⁺较术前增高, 术后14 d IL-2也增高, A组术后7 d CD3⁺, CD8⁺, CD4⁺/CD8⁺ , CD8⁺CD28⁺, CD16⁺ CD56⁺均明显高于B组. 在此后的使用中治疗组(A组)肝癌患者免疫指标恢复较未治疗组(B组)满意, 这不仅有利于减少术后感染等并发症, 也为术后TACE及其他免疫治疗提供了良好的条件.

志谢

南通大学公共卫生学院统计学教研室沈毅帮助数据处理及统计.

备注

电编:张勇 编辑:潘伯荣 审读:张海宁

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