蝎毒多肽提取物调控肝脏移植瘤中自然杀伤细胞浸润的机制研究  

Regulatory mechanism of PESV on tumor-infiltrating natural killer cells in liver orthotopic transplantation tumor

作　　者：韩琛[1,2] 王朝霞[1,2] 贾青[1,2] 王兆朋[1,2] 张月英[1,2] 张钰[1,2] 王恒孝[1,2] 

机构地区：[1]山东省医学科学院基础医学研究所 [2]济南大学山东省医学科学院医学与生命科学学院,济南250062

出　　处：《中国免疫学杂志》2016年第3期390-395,400,共7页Chinese Journal of Immunology

基　　金：国家自然科学基金青年基金项目(81303077);山东省医药卫生科技发展计划项目(2013WS0369)

摘　　要：目的:观察蝎毒多肽提取物(PESV)对H22细胞小鼠肝脏原位移植瘤模型中肿瘤浸润自然杀伤细胞(Natural killer cell,NK cell)的浸润分布及活性的影响。方法:C57BL/6小鼠肝脏接种H22肝癌细胞株悬液构建原位移植瘤模型,设立正常对照组、荷瘤对照组、PESV小剂量组和PESV大剂量组,分别给予生理盐水及PESV灌胃干预14 d,观察比较各组小鼠给药后的肿瘤体积、肿瘤质量及生存期变化,通过HE染色比较各组肿瘤组织形态学变化,流式细胞术和免疫组化检测肝脏及癌组织中浸润NK1.1^+细胞的比例及分布特点,real-time PCR法检测癌组织中穿孔素、颗粒酶B的表达。结果:PESV大、小剂量组肝脏移植瘤的生长受到抑制,肿瘤体积和瘤质量均低于荷瘤对照组,肿瘤抑制率分别为30.77%和15.38%,生存期观察显示PESV大剂量组能显著延长荷瘤小鼠生存时间,生命延长率为34.06%,P<0.05;荷瘤对照组肿瘤细胞排列紧密,异型性显著,呈浸润性生长,PESV大、小剂量组出现明显坏死区,细胞异型性减轻;PESV大剂量组小鼠肝脏NK细胞占肝脏总淋巴细胞的比例是(5.91±0.49)%,显著高于荷瘤对照组(3.69±0.50)%(P<0.05),且大量浸润分布在肿瘤组织及癌旁组织中;PESV大剂量组瘤组织中穿孔素和颗粒酶的mRNA表达量分别是荷瘤对照组的3.62倍和5.82倍(P<0.05)。结论:PESV可通过提高H22细胞肝脏原位移植瘤中浸润NK细胞的比例,促进NK细胞向肿瘤组织迁移,诱导穿孔素和颗粒酶B的产生,有助于NK细胞杀伤活性的恢复,从而增强对肿瘤细胞的清除,抑制肿瘤的浸润生长。Objective： To investigate the regulatory mechanism of PESV on tumor-infiltrating natural killer（ NK） cells in a mice model with H22 orthotopic transplantation tumor. Methods： Suspensions of H22 cells were injected into the lobe of liver on C57 BL /6mice for establishing liver orthotopic transplantation tumor model,then the mice were randomly divided into four groups： normal group,control group,PESV low dose group（ PESV-L） and PESV high dose group（ PESV-H）. Mice were either sacrificed for mechanistic studies or survival followed 14 days of therapy. The volume and weight of the tumor were measured. The proportion of infiltrating NK cells was measured by flow cytometry and the expression of NK1. 1（ NK） cells was investigated by immunohistochemistry method. The expression of perforin and granzyme B were further investigated by real-time PCR. Results： In contrast to control group,the tumor inhibition rate was 15. 38% and 30. 77% in PESV-L group and PESV-H group respectively. The survival showed that PESV-H could significantly prolong the survival time of mice,and life extension rate was 34. 06%,（ P〈0. 05）. Histological analysis revealed significant pleomorphism of the neoplastic cells and invasive extendion in control group,while there were more necrosis and less degree of atypia in PESV-L and PESV-H. The level of tumor-infiltrating NK cell was significantly higher in PESV-H than in tumor-bearing control group[（ 5. 91±0. 49） % vs.（ 3. 69±0. 50） %,P〈0. 05],and NK cells were infiltrating in peritumoral lesions. The mRNA of perforin and granzyme B in PESV-H were respectively 3. 62 and 5. 82 times than that of control group（ P〈0. 05）. Conclusion： These findings suggest that the treatment of PESV might increase the infiltration of natural killer cells in the orthotopic transplantation tumor and contribute to NK cells migration to the tumor,which induct and maintain the activities of natural killer cells against tumor cells by expressing perforin and granzyme B in vivo.

关 键 词：蝎毒 肝肿瘤 自然杀伤细胞 穿孔素 颗粒酶B 

分 类 号：R731-36[医药卫生—肿瘤]