CytoMPS原位疫苗治疗肝癌的抗瘤作用及其免疫效应

doi:10.3877/cma.j.issn.1674-0793.2010.05.004

中华普通外科学文献(电子版) ›› 2010, Vol. 04 ›› Issue (05) : 415 -419. doi: 10.3877/cma.j.issn.1674-0793.2010.05.004

所属专题：文献；

论著

CytoMPS原位疫苗治疗肝癌的抗瘤作用及其免疫效应

匡铭¹, 刁竞芳², 彭宝岗¹^,^†(

), 沈顺利¹, 王晔¹, 吴浩³, 陈斌¹

^1. 510080　广州，中山大学附属第一医院肝胆外科
^2. 广东省中医院肝胆胰外科
^3. 广州市第十二人民医院普外科

收稿日期:2010-04-30 出版日期:2010-10-01
通信作者: 彭宝岗
基金资助:
国家自然基金资助项目(30672052, 30872486); 广东省自然基金资助项目(8151008901000211)

Cytokines microparticles (CytoMPS)-in situ vaccination for hepatocellular carcinoma: in vivo and in vitro responses

Ming KUANG¹, Jing-fang DIAO², Bao-gang PENG¹^,^†(), Shun-li SHEN¹, Ye WANG¹, Hao WU³, Bin CHEN¹

^1. Department of Hepatobiliary Surgery, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China

Received:2010-04-30 Published:2010-10-01
Corresponding author: Bao-gang PENG
About author:
Corresponding Author: PENG Bao-gang, Email: pengbaogang@tom.com

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目的

制作成细胞因子缓释微粒原位瘤苗（CytoMPS-ISV），观测CytoMPS-ISV抗瘤作用及其免疫效应。

方法

C57BL/6J小鼠皮下移植性肝癌模型瘤内注射CytoMPS 3次制作CytoMPS-ISV，观察其抗肿瘤作用及小鼠生存率；流式细胞仪检测小鼠外周血淋巴细胞变化，免疫组化法检测肿瘤组织的CD4+、CD8+和NK1.1+细胞浸润情况，体外检测小鼠脾CTL和NK细胞的杀瘤活性，检测疫苗诱导脾CTL的抗体阻断作用。

结果

与PBS注射组和对照组相比，瘤内注射CytoMPS后肿瘤生长受到显著抑制（P<0.01）。瘤内注射CytoMPS组血液中及局部浸润的CD4+、CD8+和NK细胞明显高于PBS组和对照组（P<0.05）；淋巴细胞杀伤试验结果显示瘤内注射CytoMPS组小鼠的脾CTL对靶细胞的杀伤率明显高于PBS组（P<0.01）和对照组（P<0.05）；抗体阻断作用试验显示接种疫苗的小鼠脾CTL的杀瘤活性可被抗CD8+、抗MHC-I单克隆抗体所阻断，但不被抗CD4+、抗MHC-II单克隆抗体所阻断。

结论

CytoMPS-ISV可以显著抑制肿瘤生长，能明显增强机体的抗肿瘤免疫作用，其诱导的CTL杀瘤特性是由MHC-I限制的CD8+ T细胞所介导。

关键词: 细胞因子缓释微粒, 肝细胞癌, 疫苗, 免疫治疗

Objective

To prepare the CytoMPS-related in situ vaccine (CytoMPS-ISV) and observe its protective and therapeutic antitumor effects in treatment of mice hepatoma.

Methods

Three times of intratumoral injection of CytoMPS in C57BL/6J mice subcutaneous hepatoma were performed, and the tumor growth and mice survival rates were observed. The proportions of peripheral blood lymphocytes were detected by FACS. The killing efficacy of cytotoxic T lymphocytes(CTLs) derived from spleens of CytoMPS-ISV vaccinated mice was evaluated with and without antibody-blocking preparation. Extent of tumor-infiltrating CD4+、CD8+ and NK1.1+ cells were also detected with immunohistochemical stain(IHC).

Results

Compared with the control group, the tumor growth in CytoMPS-ISV group was significantly inhibited(P<0.01). Moreover, the proportions of CD4+, CD8+ T cell and natural killer cell (NK) in peripheral blood in CytoMPS-ISV group were significantly higher, so were those of tumor-infiltrating lymphocytes in CytoMPS group. Compared with those in the control groups, the cytotoxicities of splenic CTL in CytoMPS-ISV group were significantly enhanced, which could be blocked by anti-mouse MHC Class I and anti-mouse CD8 antibody, but not by anti-mouse MHC Class II and anti-mouse CD4 antibody.

Conclusion

CytoMPS-ISV can significantly inhibit tumor growth and induce anti-tumor immunity. The killing efficacy of CytoMPS-ISV-induced CTLs is related to CD8+ T cells restricted by MHC-I.

Key words: CytoMPS, Hepatocellular carcinoma, Vaccine, Immunotherapy

图1 瘤内注射CytoMPS后肿瘤生长受到显著抑制。A组，瘤内注射CytoMPS组；B组，瘤内注射PBS组；C组，对照组。

图2 瘤内注射CytoMPS后，3组小鼠生存率无显著差异。A组，瘤内注射CytoMPS组；B组，瘤内注射PBS组；C组，对照组。

表1 3组小鼠外周血淋巴细胞组成的比较（%，±s）

组别	n	CD4+	CD8+	NK1.1+
A	3	41.00±1.32^*	34.59±1.71^*	13.08±1.59^*
B	3	23.41±2.74	20.73±1.36	7.62 ±1.06
C	3	24.82±2.22	20.26±0.39	7.39±0.60

表1 3组小鼠外周血淋巴细胞组成的比较（%，±s）

组别	n	CD4+	CD8+	NK1.1+
A	3	41.00±1.32^*	34.59±1.71^*	13.08±1.59^*
B	3	23.41±2.74	20.73±1.36	7.62 ±1.06
C	3	24.82±2.22	20.26±0.39	7.39±0.60

图3 肿瘤组织免疫组化检测CD4+ T细胞3a)瘤内注射CytoMPS组肝癌组织可见中等量CD4 + T细胞浸润（IHC, ×200)；3b PBS对照组肝癌组织未见CD4+ T细胞浸润（IHC, 200×)

图4 肿瘤组织免疫组化检测CD8+ T细胞。4a)瘤内注射CytoMPS组组肝癌组织可见较多CD8+ T细胞浸润(IHC, 200×)。4b）PBS对照组肝癌组织未见CD8+ T细胞浸润(IHC,×200)

图5 肿瘤组织免疫组化检测NK1.1+细胞5a）瘤内注射CytoMPS组肝癌组织可见中等量NK1.1+细胞浸润(IHC,×200)。5b）PBS对照组肝癌组织未见NK1.1+细胞浸润(IHC, ×200)

表2 肝癌组织中浸润的淋巴细胞

组别	切片数量	CD4+, CD8+, NK1.1 +细胞浸润程度
A	9	+ + +^*
B	9	+
C	9	+

表2 肝癌组织中浸润的淋巴细胞

组别	切片数量	CD4+, CD8+, NK1.1 +细胞浸润程度
A	9	+ + +^*
B	9	+
C	9	+

图6 抗体阻断试验：分别用抗- CD8+、抗-MHC-Ⅰ、抗- CD4+、抗-MHC-Ⅱ单克隆抗体封闭后进行LDH释放法检测脾CTLs的杀伤活性。

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