摘要
目的:探讨桑黄多糖对脑胶质瘤细胞免疫监视的增强作用及其对主要组织相容性复合物Ⅰ链相关基因B(MICB)/自然杀伤细胞活化性受体2D(NKG2D)信号通路的调控机制。方法:取6~8周龄C57BL/6小鼠60只和GL261细胞株培养传代,随机取其中的50只小鼠建立脑胶质瘤模型,其余10只小鼠记为对照组,另将建模成功小鼠采用随机数字表分组,榄香烯组予以100 mg/kg榄香烯与1 ml/100 g生理盐水混匀灌胃,桑黄多糖各剂量组分别予以50、100、200 mg/kg桑黄多糖溶于1 ml/100 g生理盐水中灌胃,对照组和模型组均予以等量生理盐水灌胃,均1次/d,共4周。干预后处死小鼠,比较各组瘤重和抑瘤率;苏木素-伊红(HE)染色观察瘤组织病理变化;以CD57抗体标记检测瘤组织中NK细胞浸润;实时-逆转录聚合酶链反应(RT-qPCR)检测瘤组织主要组织相容性复合物Ⅰ链相关基因A(MICA)、MICB、UL16结合蛋白1(ULBP1)、UL16结合蛋白2(ULBP2)、UL16结合蛋白3(ULBP3)mRNA表达,Western blot检测瘤组织MICA、MICB、ULBP1、ULBP2、ULBP3蛋白表达;采用磁珠亲和细胞分选法分离各组组织中NK细胞,采用流式细胞仪检测其表面NKG2D活性。结果:与模型组比较,榄香烯组和桑黄多糖各剂量组瘤重下降,抑瘤率升高,瘤组织细胞变性、坏死增多,NK细胞浸润增多,MICA、ULBP2、ULBP3 mRNA和蛋白表达下降,MICB、ULBP1 mRNA和蛋白表达升高,且NK细胞表面NKG2D活性增强,上述差异均有统计学意义(P<0.05);桑黄多糖的作用呈剂量依赖性,桑黄多糖低剂量组和榄香烯组所有指标比较差异均无统计学意义(P>0.05)。结论:桑黄多糖可抑制脑胶质瘤增长,促进瘤细胞变性、坏死,增加NK细胞浸润,推测d与调控MICB/NKG2D信号通路,增加MICB、ULBP1表达和NK细胞表面NKG2D活性,下调MICA、ULBP2、ULBP3表达,增强对脑胶质瘤细胞的免疫监视有关。
Objective:To investigate the inhibitory effect of polysaccharide from Phellinus Linteus on immune surveillance of glioma cells and its regulatory mechanism on the major histocompatibility complexⅠchain related gene B(MICB)/natural killer cell activated receptor 2D(NKG2D)signaling pathway.Methods:Total 60 C57BL/6 mice aged 6~8 weeks and GL261 cell line were selected.Fifty of them were randomly selected to establish brain glioma models,and the remaining 10 mice were recorded as control group.In addition,the model mice were randomly divided into groups.The elemene group was given 100 mg/kg elemene and 1 ml/100 g normal saline by gavage,and the different doses of polysaccharide from Phellinus Linteus groups were given 50,100 and 200 mg/kg of polysaccharide from Phellinus Linteus dissolved in 1 ml/100 g normal saline by gavage,while the control group and model group were given the same amount of normal saline,all once a day for 4 weeks.After intervention,the mice were killed,and the tumor weight and tumor inhibition rates were compared among groups.Hematoxylin eosin(HE)staining was used to observe the pathological changes.NK cell infiltration was detected by CD57 antibody.Real time reverse transcription polymerase chain reaction(RT-qPCR)was used to detect the mRNA expression of major histocompatibility complexⅠchain related genes A(MICA),MICB,UL16 binding protein 1(ULBP1),UL16 binding protein 2(ULBP2),UL16 binding protein 3(ULBP3).The expressions of MICA,MICB,ULBP1,ULBP2 and ULBP3 were detected by Western blot.NK cells were separated by magnetic bead affinity cell sorting,and the activities of NKG2D on the surface of NK cells were detected by flow cytometry in the tissues of mice.Results:Compared with model group,the tumor weight of elemene group and the different doses of polysaccharide from Phellinus Linteus groups were decreased,the tumor inhibition rates were increased,the degeneration and necrosis of tumor cells were increased,the infiltration of NK cells was increased,the mRNA and protein expressions of MICA,ULBP2 and ULBP3 were decreased,the mRNA and protein expressions of MICB and ULBP1 were increased,and the NKG2D activities on the surface of NK cells were increased in elemene group and the three doses of polysaccharide from Phellinus Linteus groups,with statistically significant differences(P<0.05).There was no significant difference in all of the indexes between low-dose polysaccharide from Phellinus Linteus group and elemene group(P>0.05).Conclusion:Polysaccharide from Phellinus Linteus can inhibit the growth of glioma,promote the degeneration and necrosis of tumor cells,increase NK cell infiltration,and it is suggested that it is related to regulating the signaling pathway of MICB/NKG2D,increasing the expression of MICB,ULBP1 and NKG2D activity on NK cells,decreasing the expressions of MICA,ULBP2 and ULBP3,and enhance immunosurveillance of glioma cells.
作者
张正平
李坤正
巨虎
刘熹
ZHANG Zhengping;LI Kunzheng;JU Hu;LIU Xi(Department of Neurosurgery,Affiliated Hospital of Qinghai University,Xining 810000,China)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2023年第3期578-585,共8页
Chinese Journal of Immunology
关键词
桑黄多糖
主要组织相容性复合物Ⅰ链相关基因B
自然杀伤细胞活化性受体2D
脑胶质瘤
Polysaccharide from Phellinus Linteus
Major histocompatibility complexⅠchain related gene B
Natural killer cell activated receptor 2D
Brain glioma
