【作者】 赵昌林；

【导师】 陈孝银；

【作者基本信息】 暨南大学 ， 中西医结合基础， 2015， 博士

【摘要】 背景:毒邪是癌症发病的中心环节,解毒抗癌法是中医治疗癌症的有效方法,解毒抗癌方对癌症的作用机制仍不清楚。滤泡性辅助性T细胞(Tfh)在B细胞的活化中起主要作用,在前期实验中我们发现HBs Ag和CD57有密切的关系,我们认为在人体中存在CD4+CXCR5+CD57+T细胞亚群,而解毒抗癌方可以调节这一细胞亚群,在肝癌的治疗和预后中起着重要的作用。目的:解毒抗癌法是治疗癌症的有效方法,解毒抗癌方对癌症的作用机制有待阐明,本研究拟通过分析CD4+CXCR5+CD57+T细胞在肝癌中的作用及对预后的影响,探讨解毒抗癌方的抗肿瘤作用和调节CD4+CXCR5+CD57+T细胞的作用机制。方法:临床实验:按照方案筛选实验对象肝癌65例,32例健康志愿者作为对照组。肝癌手术标本行HE染色和免疫组化检测标本中CD57、HBs Ag、CXCR5等蛋白的表达;分离外周血中的淋巴细胞后加入荧光标记的CD57、CXCR5和CD4抗体,孵育后进行流式细胞检测,分析CD4+CXCR5+CD57+T细胞的表达量;血清分离后,使用ELISA方法检测IL-21、IL-6、IFN-γ、CXCL13和CXCR5的浓度;使用生化检测仪分析血清中Ig G、ALT、AST、胆红素的含量;PCR-荧光探针法检测肝癌组织中HBV-DNA表达量;采用荧光定量QPCR分析肝癌组织标本中IL-10、TGFB1、RORC、IL-17A、IL-21、IL-6、CXCR5和CD57等基因的表达量。细胞实验:取健康脐带血或外周血进行淋巴细胞分离,IL-2(300U/ml)、IL-1α(100U/ml)、IL-21(40ng/ml)进行诱导,通过磁珠筛选进行分离,之后加入CD57、CXCR5和CD4荧光标记抗体,检测CD4+CXCR5+CD57+T细胞表达量;取培养液0.5ml加入Hp G2的细胞培养液中,比例为20%,在24-48小时后进行Hp G2的凋亡检测。使用MTT法分析疏肝健脾方、解毒抗癌方、DDP对Hp G2肿瘤细胞生长的抑制作用;分离细胞培养液,使用ELISA检测L-21、IL-6、IFN-γ和CXCR5的含量。动物实验:昆明小鼠和C57BL/6购买自广东省医学实验动物中心,C57BL/6-HBV购买自上海南方模式生物研究中心,C57BL/6-Foxp3GFP购自The Jackson Laboratory。H22肝癌细胞(广东省医学实验动物中心)左前臂腋窝处注射0.1ml,48小时后给与药物干预,干预时间为10d。各组小鼠处死后,取肝、肺和接种的瘤体进行常规HE染色和免疫组化染色分析CD57、CXCL13蛋白表达;无菌分离脾脏研磨分离淋巴细胞后进行CD4+CXCR5+CD57+T细胞的流式细胞检测;ELISA检测外周血IL-21、IL-6、IFN-γ、CXCR5和CXCL13的含量;PCR荧光探针法检测HBV-DNA表达量;荧光定量RT-PCR分析IL-10、TGFB1、RORC、IL-17A、IL-21、IL-6、CXCR5、CD57等基因的表达量。统计方法:使用SPSS(statistics package for social science)19.0软件进行数据分析,定量资料如果符合正态分布,统计描述指标可用均数及标准差,如果不符合正态分布,则指标选用中位数和级差(即:最大值和最小值之差)。对于分组呈正态分布的定量资料,如为两组比较,使用t检验。P<0.05为差异具有统计学意义。结果:临床实验:病理学结果检查表明,HBs Ag阳性与CD57阳性的结果符合率为83%,表明二者之间有密切的相关性;CD4+CXCR5+T细胞的数量在正常人中为5.95%,在I-IIIA期肝癌患者中为5.39%,在IIIB-IV期肝癌患者中为3.21%,正常人群和IIIB-IV期肝癌之间存在着统计学差异(P<0.05);CD57+CXCR5+T细胞在正常人中为20.9%,在I-IIIA期肝癌患者中为13.8%,IIIB-IV期肝癌患者中为8.44%,其表达量在三者之间具有统计学意义;CD4+CD57+T细胞在正常人群为3.5%,在I-IIIA期肝癌患者中为3.18%,在IIIB-IV期肝癌患者中为2.02%,之间具有统计学差异。CD4+CXCR5+CD57+T细胞的阳性表达在正常人群为22.9%,I-IIIA期肝癌患者为8.18%,IIIB-IV期肝癌患者为2.79%,具有统计学意义。ELISA分析发现IL-21的数量则有明显的不同,INF-γ无明显的不同;RT-PCR分结果表明,Hbs Ag阳性标本中的IL-6、LI-21、IL-17A、CD57和CXCR5的基因表达明显下降,具有统计学意义。回归分析发现,CD4+CXCR5+CD57+T细胞与ALT的水平密切相关,当ALT的水平升高时,CD4+CXCR5+CD57+T细胞的数量也随之升高,具有统计学意义,与Ig G的相互关系中发现两者密切相关,而与HBV-DNA的水平没有明显的关系;CD4+CXCR5+CD57+T细胞与生存时间的关系表明,其表达水平越多,肝癌病人的生存时间越长。细胞实验:外周血中的干细胞经IL-2、IL-21和INF-γ诱导可以分化为CD4+CXCR5+CD57+T细胞,并且随着培养时间的增加分化的细胞数量升高,占CD4+细胞数量的22%;诱导的CD4+CXCR5+CD57+T细胞可以分泌IL-6、IL-21、INF-γ,具有时间相关性,而不分泌CXCL13。流式分析发现CD4+CXCR5+CD57+T细胞在24h小时可诱导71.3%的Hp G2细胞凋亡,在48小时诱导89.2%的细胞凋亡,具有统计学意义,分析发现其机制与FAS的表达相关;诱导12d,CD4+的T细胞数量占总数的21.4%,CD4+CXCR5+T细胞的数量为5.9%,而CD4+CXCR5+CD57+T细胞的数量为9.7%,CD57+T细胞的数量为28.2%,随着培养时间的延长,CD4+、CD57+、CD4+CXCR5+和CD4+CXCR5+CD57+T细胞的数量显著增多,具有统计学意义。疏肝健脾方的抑制率为11.1%,解毒抗癌方为72.8%,DDP为74.7%,疏肝健脾方和解毒抗癌方两组之间具有统计学差异;解毒抗癌方可以抑制IL-6的分泌;同时解毒抗癌方抑制IGF-IR的分泌,其作用与DDP相当。动物实验:解毒抗癌方能抑制肿瘤组织的生长,但是抑制作用小于DDP;C57BL/6-HBV的小鼠肝、肺可见淋巴细胞、浆细胞浸润;C57BL/6-HBV小鼠的H22,Ki67的免疫组化阳性率为80%,中药干预后期阳性率为30%,DDP为10%,IL-21阳性率为10%以下,具有统计学学意义(P<0.05)。ELISA结果显示中药可以提高IL-6、IL-21、INF-γ、CXCL13的水平,具有统计学意义。HBV可以显著的抑制CD4CXCR5的阳性表达,促使IL-6显著升高,IL-21下降,给以中药干预,CD4CXCR5的细胞数量显著的上升,DDP干预后CD4CXCR5的细胞数量减少,进行IL-21的腹腔内注射,可以使CD4CXCR5的数量显著升高。C57BL/6小鼠的CD4+CXCR5+CD57+T细胞的数量为6.2%,而C57BL/6-HBV的数量为2.16%,两组之间有明显的差别。给以C57BL/6-HBV接种H22细胞,在d5,d10和d14进行流式细胞学的检测,结果表明,CD4+CXCR5+CD57+T细胞的数量没有明显的变化,给以解毒抗癌方干预,CD4+CXCR5+CD57+T细胞的数量显著的上升,DDP干预后CD4+CXCR5+CD57+T细胞的数量未见变化,IL-21干预后CD4+CXCR5+CD57+T细胞的显著上升。实时定量RT-PCR分析H22瘤体中Tfh,Th17和Treg相关基因的表达发现,在C57BL/6-HBV接种的H22瘤体IL-6、LI-21、IL-17A、CD57和CXCR5的基因表达明显下降,经解毒抗癌方干预后IL-6、IL-21、CXCR5和CD57的基因表达显著提升(P<0.01),DDP干预后IL-6,IL-21,CXCR5,CD57的基因表达下降;基因表达分析发现,HBV显著的抑制了Tfh,Treg,Th17的相关基因表达,而在DDP和解毒抗癌方的干预方面,则有非常显著的差异,解毒抗癌方则显著的增加CD4+CXCR5+CD57+T细胞的数量。结论:1.CD4+CXCR5+CD57+T细胞是CD4+CXCR5+T细胞的一个亚群,具有抑制肝癌的作用,通过CD95诱导Hp G2细胞凋亡。HBV可以抑制CD4+CXCR5+T细胞和CD4+CXCR5+CD57+T细胞的功能,在肝癌的形成过程中可能是一个重要的因素。2.毒邪是肝癌病因病机中的中心环节,其病变基础是毒邪可以抑制机体的免疫功能,CD4+CXCR5+CD57+T细胞和CD4+CXCR5+T细胞的功能下降使机体表现为正气亏虚。3.解毒抗癌方可以杀死Hp G2细胞,减少IL-6和IGF-IR的分泌。解毒抗癌方可显著的提高CD4+CXCR5+T细胞和CD4+CXCR5+CD57+T细胞的数量和功能,可能是其抗癌的作用机理。更多还原

【Abstract】 Backgrounds:Poison is the central of tumor incidences; detoxification cancer method is an effective method of traditional Chinese medicine for treating cancer, and detoxification cancer prescription is remains unclear of the anticancer mechanism. Follicular helper T cells(Tfh) play a major role in B cell activation; we found HBs Ag and CD57 are closely related in the early experiments, and believe that the presence of CD4+CXCR5+CD57+T cells subsets in the human body, and detoxification cancer prescription can adjust the T cells subsets. CD4+CXCR5+CD57+T cells play an important role in the treatment and prognosis of Hepatic carcinoma.Objectives:Detoxification cancer method is an effective method for therapy cancer,mechanism of detoxification cancer prescription treatment cancer is remains unclear. This study was to analyze the role and prognosis of CD4+CXCR5+CD57+T cells in hepato-carcinogenesis. To discuss anti-tumor effect of detoxification cancer prescription and regulate CD4+CXCR5+CD57+T cells of mechanism for treatment tutor.Methods:Clinical trials:This study screened subjects 65 cases liver cancer patients under the study program and 32 cases of healthy volunteers as a control group. Express ofCD57, HBs Ag, CXCR5 and other proteins by HE staining and immunohistochemical detection of liver cancer specimens by surgical specimens; adding a fluorescent marker CD57, CXCR5 and CD4 antibody after isolated peripheral blood lymphocytes, performed flow cytometry to analysis of CD4+CXCR5+CD57+T cell expression; ELISA method to detect IL-21, IL-6, IFN-γ, CXCL13 and CXCR5 of concentration in serum. ALT,AST, bilirubin, Ig G of concentration in serum with biochemical tests analyzed serum of patients and concentration in serum.HBV-DNA expression of HCC with PCR-fluorescent probe assay; a nalysis IL-10, TGFB1, RORC, IL-17 A, IL-21, the expression of IL-6,CXCR5 and CD57 and other gene of tissue samples by using fluorescence quantitative QPCR in liver cancer specimens.Cell research:Lymphocyte separation from healthy umbilical cord blood or peripheral blood, after IL-2(300U / ml), IL-1α(100U / ml) and IL-21(40ng / ml)induced, isolated by screening the beads, the addition of CD57, CXCR5 and CD4 fluorescently labeled antibodies to detect CD4+CXCR5+CD57+T cell expression with flow cytometry.Lymphocytes Cell culture fluid 0.5ml mixed in Hp G2 Cell culture fluid,the ratio was 20%, after the 24-48 hours to Hp G2 cells apoptosis detection.Analysis of shuganjianpi prescription, detoxification cancer prescription, and DDP inhibition of Hp G2 cells growth by MTT. ELISAdetects L-21, IL-6, IFN-γ and CXCR5 of concentration cell culture fluid.Animal experiment:Kunming mice, C57BL/6(purchased from Guangdong Medical Experimental Animal Center), C57BL/6-HBv(purchased from Shanghai South Model Organism Research Center), and C57BL/6-Foxp3GFP(purchased from the Jackson Laboratory). H22 hepatoma cells(Guangdong Medical Experimental Animal Center) injection 0.1ml in left forearm armpit, 48 hours after giving drug intervention and intervention time is 10 d.After the mice were sacrificed, the liver, lung and tumor tissue HE staining and immunohistochemical analysis expression of CD57, CXCL13protein; after grinding aseptic isolation spleen, separation of lymphocytes,detect CD4+CXCR5+CD57+T cells with flow cytometry. ELISA detect IL-21, IL-6, IFN-γ, CXCR5 and CXCL13 of peripheral blood; PCR fluorescence probe to detect HBV-DNA expression; analysis of IL-10,expression TGFB1, RORC, IL-17 A, IL-21, IL-6, CXCR5, CD57 and other genes of tumor tissue by fluorescence quantitative RT-PCR.Statistical methods:Using SPSS(statistics package for social science) 19.0 software for data analysis, quantitative data if they meet the normal distribution, descriptive statistics available indicators mean and standard deviation, if not normal distribution, the median index selection and differential(ie: difference between maximum and minimum). For normally distributed of quantitativedata, such as the two groups using t test.P<0.05 was considered statistically significant.Result:Clinical trials:Pathological examination results show that HBs Ag positive and CD57-positive results for 83%, indicating that there is a close correlation between the two groups. CD4+CXCR5+CD57+T cells in the normal population is 5.95%, in stage I-IIIA HCC patients is 5.39%, in stage IIIB-IV HCC patients is 3.21%, and it is statistically significant differencebetween the normal population and IIIB-IV liver cancer(P <0.05).CD57+CXCR5+T cells in the normal population is 20.9%, it is 13.8% in stage I-IIIA HCC patients, it was 8.44% IIIB-IV patients with hepatocellular carcinoma, and it is statistical significance among the three groups;CD4+CD57+T cells in the normal population is 3.5% and 3.18% in stage I-IIIA HCC patients and 2.02% in stage IIIB-IV HCC patients, it is a significant difference between the three groups.CD4+CXCR5+CD57+T cells expression of in the normal population is22.9%, and I-IIIA patients with liver cancer are 8.18%, and IIIB-IV is 2.79%of liver cancer patients, with statistical significance. ELISA analysis found that the number of IL-21 is significantly different, and INF-γ was no significant difference; RT-PCR results showed that Hbs Ag positive samples of IL-6, LI-21, IL-17 A, CD57 and CXCR5 of gene expression wassignificantly decreased, with statistical significance.Regression analysis showed that CD4+CXCR5+CD57+T cells is closely related to the level of ALT, and CD4+CXCR5+CD57+T cells will be increased when elevated levels of ALT, with statistical significance.CD4+CXCR5+CD57+T cells levels are closely correlated with the level of Ig G; there is no significant relationship with HBV-DNA.CD4+CXCR5+CD57+T cells increase survival time and the longer the survival time of patients.Cell Research:Peripheral blood stem cells differentiate into CD4+CXCR5+CD57+T cells by IL-2, IL-21 and INF-γ induced, and increasing the number of cells increases with culture time, accounting for CD4+cells count of 22%; induced the CD4+CXCR5+CD57+T cells secrete IL-6, IL-21, INF-γ, with time correlation, and no secreted CXCL13.Flow analysis showed CD4+CXCR5+CD57+T cells induced 71.3% of Hp G2 apoptosis in 24 h hours, 89.2% in the 48 hours with statistical significance, the analysis found that the expression of the mechanisms associated with FAS. CD4+T-cell count 21.4 percent of the total,CD4+CXCR5+T cells was 5.9%, while the CD4+CXCR5+CD57+T cells was9.7%, CD57+T cells was 28.2 % when induce 12 d. With the incubation time,CD4+, CD57+, CD4+CXCR5+and CD4+CXCR5+CD57+T T cells was significantly increased, with statistical significance.Shuganjianpi prescription inhibition rate of 11.1%, detoxification cancer prescription is 72.8%, DDP 74.7%, with significant differences between the detoxification cancer prescription and Shuganjianpi prescription groups.Detoxification cancer prescription can be suppressed secretion of IL-6; and inhibiting the secretion to IGF-IR, the effect is quite DDP.Animal Research:Detoxification cancer prescription can inhibit the growth of tumor tissue, but its role is lower than DDP; liver and lung of C57BL/6-HBV appear lymphocytes and plasma cells infiltratied. H22 of C57BL/6-HBV mice that Ki67 immunohistochemistry the positive rate was 80%, TCM intervention later positive rate was 30%, DDP was 10%, IL-21-positive rate of 10% or less, with statistically significance(P<0.05). ELISA results show that TCM can improve the IL-6, IL-21, INF-γ, CXCL13 levels, with statistical significance. HBV can significantly inhibit the expression CD4CXCR5, prompting IL-6 was significantly increased, IL-21 decreased,TCM intervention after, was significant increased in CD4CXCR5 of cell number. It reduced the number of cells CD4CXCR5 after DDP intervention.It can significantly increase the number of CD4CXCR5, with IL-21 intraperitoneal injection.CD4+CXCR5+CD57+T cells of C57BL/6 mice were was 6.2%, while C57BL/6-HBV was 2.16%, there are significant differences between the two groups. Infect H22 cells to C57BL/6-HBV, d5, d10 and d14 to detect flowcytometry, and the results show, CD4+CXCR5+CD57+regulatory T cells did not change significantly, give detoxification cancer prescription interventied,CD4+CXCR5+CD57+T significant increase in the number of T cells,CD4+CXCR5+CD57+T cells showed no change after DDP interventied,CD4+CXCR5+CD57+T cells increased significantly after IL-21 interventied.Real-time quantitative PCR analysis of the expression of H22 tumor in Tfh, Th17 and Treg-related genes found that IL-6, LI-21, IL-17 A, CD57 and gene expression of CXCR5 decreased significantly in the C57BL/6-HBV;IL-6 and IL-21 significantly improve, CXCR5 and gene expression of CD57(P <0.01) after detoxification cancer prescription interventions; after DDP intervention, IL-6, IL-21, CXCR5, CD57 gene decreased expression. Gene expression analysis that HBV significantly inhibited Tfh, Treg, Th17-related genes, but in DDP and detoxification cancer prescription interventions after,there are very significant differences, the significant increase CD4+CXCR5+CD57+T cells in detoxification cancer rescription.Conclusion:1. CD4+CXCR5+CD57+T cells are a subpopulation of CD4+CXCR5+T cells;inhibit liver cancer effects and induced Hp G2 cells apoptosis through CD95.HBV can inhibit CD4+CXCR5+T cells and CD4+CXCR5+CD57+T cells function, which may be an important factor in the formation of liver cancer.2. The toxin is central place of liver cancer pathogenesis, and based on the toxin can inhibit the body’s immune function, CD4+CXCR5+CD57+T cellsand CD4+CXCR5+T cell function decreased so that the body showed righteousness deficiency.3. Detoxification cancer prescription can kill Hp G2 cells; reduce the secretion of IL-6 and IGF-IR. Detoxification cancer prescription significant increased number and function of CD4+CXCR5+T cells and CD4+CXCR5+CD57+T cells. It may be the mechanism of action against cancer.更多还原