【作者】 谭明娟；

【导师】 王勇；

【作者基本信息】 南京医科大学 ， 病原生物学， 2008， 硕士

【摘要】 血吸虫病（schistosomiasis）是一种世界分布的人兽共患寄生虫病,是所有寄生虫病中分布最广、危害最严重的疾病之一。人群流行病学和动物模型研究均提示,随着日本血吸虫感染的慢性化进程,宿主出现免疫下调现象。除了血吸虫病外,很多慢性感染性疾病,如利什曼病,结核病等,也存在免疫下调现象。近几年,一些研究者将这种持续慢性感染状态与CD4⁺CD25⁺T细胞诱导的免疫抑制作用联系起来。对曼氏血吸虫慢性感染的研究已经开始涉及CD4⁺CD25⁺T细胞的免疫调节作用,但迄今为止,在日本血吸虫感染的研究中却少有报道。基于日本血吸虫病慢性期的免疫下调现象,本课题组前期实验结果提示,CD4⁺CD25⁺T细胞与日本血吸虫病慢性期的免疫下调现象有关,结合其它慢性感染性疾病的研究资料,设计本研究,进一步探讨日本血吸虫慢性感染小鼠在解除抗原持续刺激后,体内CD4⁺CD25⁺调节性T细胞（CD4⁺CD25⁺Tregs）功能和数量的变化以及对机体免疫应答状态的影响,并初步探讨其变化机制,为进一步研究CD4⁺CD25⁺Tregs在日本血吸虫感染慢性化过程中的免疫下调作用提供实验资料。我们首先建立日本血吸虫慢性感染小鼠模型,在感染后13wk,给予吡喹酮化疗。在化疗后的不同时间点上,为了观察CD4⁺CD25⁺Tregs数量变化以及影响其变化因素,采用免疫组织化学方法检测肝脏肉芽肿内Foxp3⁺细胞的分布;以三色流式细胞术检测小鼠脾脏组织中CD4⁺CD25⁺Foxp3⁺T细胞相对比例的变化;运用实时荧光定量PCR方法检测Foxp3 mRNA在脾脏CD4⁺T细胞中的表达水平。为进一步观察吡喹酮化疗后CD4⁺CD25⁺Tregs抑制功能变化以及机体对非相关抗原OVA的免疫应答反应,运用免疫磁珠分离纯化脾脏CD4⁺CD25⁺T细胞,在有丝分裂原ConA（刀豆蛋白A）刺激下,采用同位素³H-TdR掺入细胞增殖实验分析CD4⁺CD25⁺T细胞对脾脏CD4⁺CD25^-T细胞体外增殖的抑制作用;以OVA免疫吡喹酮化疗后14d的慢性感染期小鼠,用ELISA检测血清中抗OVA（卵清蛋白）抗体水平。本研究获得如下主要结果:1.吡喹酮化疗对日本血吸虫慢性感染小鼠肝脏虫卵肉芽肿内Foxp3⁺细胞分布的影响免疫组织化学染色显示,正常小鼠肝脏中几乎没有Foxp3⁺细胞分布,而日本血吸虫感染慢性期,肝脏虫卵肉芽肿内分布大量的Foxp3⁺细胞。给予吡喹酮化疗后3d,虫卵肉芽肿内Foxp3⁺细胞无明显变化,到化疗后7d时,Foxp3⁺细胞明显变少,到14d时几乎见不到Foxp3⁺细胞。Foxp3蛋白是CD4⁺CD25⁺Tregs的主要标志分子。本观察结果提示,日本血吸虫慢性感染鼠经吡喹酮有效治疗后,肝脏虫卵肉芽肿内CD4⁺CD25⁺Tregs数量减少到正常水平。2.吡喹酮化疗对日本血吸虫慢性感染小鼠脾脏CD4⁺CD25⁺Foxp3⁺T细胞数量的影响流式细胞仪检测结果表明,脾脏CD4⁺CD25⁺Foxp3⁺T细胞占CD4⁺T细胞比例在慢性感染期鼠为（15.37±0.38）%,在正常小鼠脾脏中其比例为（11.37±0.51）%,二者之间差异有显著性意义（P＜0.01）。给予吡喹酮化疗后,感染鼠脾脏CD4⁺CD25⁺Foxp3⁺T细胞占CD4⁺T细胞比例逐渐下降,其比例在化疗后3d为（14.37±0.64）%,7d时为（12.62±1.05）%,与慢性期比较差异有统计学意义（P＜0.05）;到化疗后14d时为（11.47±0.46）%,已下降到正常水平。结果提示,日本血吸虫慢性感染鼠在有效病原治疗后,脾脏CD4⁺CD25⁺Tregs数量也逐渐降至正常水平。3.吡喹酮化疗对小鼠脾脏CD4⁺T细胞Foxp3 mRNA表达水平的影响实时荧光定量PCR结果显示,在慢性感染期小鼠脾脏CD4⁺T细胞Foxp3 mRNA的相对表达水平为1.01±0.15,在正常小鼠中其相对表达水平为0.75±0.16,二者差异有统计学意义（P＜0.05）。给予吡喹酮化疗后,小鼠脾脏CD4⁺T细胞Foxp3 mRNA相对表达水平在第3d为0.70±0.18,与慢性期比较差异有统计学意义（P＜0.05）,到化疗后7d和14d时,Foxp3 mRNA相对表达水平仍然保持在化疗后3d的水平上。结果提示,日本血吸虫慢性感染鼠在给予吡喹酮化疗后,CD4⁺CD25⁺Tregs的标志分子Foxp3在基因转录水平上的变化与CD4⁺CD25⁺Tregs数量上的变化相一致,并且在时相上先于数量变化。4.影响脾脏CD4⁺CD25⁺Foxp3⁺T细胞数量变化相关因素的研究上述实验结果显示,日本血吸虫慢性感染期小鼠给予吡喹酮化疗后,脾脏CD4⁺CD25⁺Foxp3⁺T细胞数量及Foxp3基因表达下降。为排除吡喹酮对实验结果的影响,给予正常小鼠吡喹酮处理,分别在3d、7d和14d,流式细胞仪检测脾脏CD4⁺CD25⁺Foxp3⁺T占CD4⁺T的比例。结果显示,各组之间的比例变化差异无显著性意义（P＞0.05）。为探讨CD4⁺CD25⁺Tregs的数量变化是否与病原治疗后血吸虫成虫被杀死并在短时间内释放大量抗原有关,将成虫可溶性抗原经腹腔注射到慢性感染小鼠体内,7d后流式细胞仪检测脾脏CD4⁺CD25⁺Foxp3⁺T占CD4⁺T的比例。结果显示,未处理和注射PBS的慢性感染小鼠脾脏CD4⁺CD25⁺Foxp3⁺T占CD4⁺T的比例分别为15.2%和15.1%,注射成虫可溶性抗原能使日本血吸虫慢性感染小鼠脾脏CD4⁺CD25⁺Foxp3⁺T细胞占CD4⁺T的比例（12.5%）明显降低。结果提示,成虫可溶性抗原刺激可能是导致日本血吸虫慢性感染鼠脾脏CD4⁺CD25⁺Tregs数量下降的因素。5.吡喹酮化疗对CD4⁺CD25⁺T细胞免疫抑制功能的影响³H-TdR掺入法检测细胞增殖实验结果显示,与正常小鼠来源的CD4⁺CD25⁺T细胞比较（cpm值为4092.00±122.76）,慢性期来源的CD4⁺CD25⁺T细胞对CD4⁺CD25T细胞增殖（cpm值为2952.25±88.03）的抑制作用较强（P＜0.01）;给予吡喹酮化疗后,抑制作用逐渐减弱,化疗后3d时（cpm值为2954.00±295.57）,抑制作用无明显变化,到化疗后7d时（cpm值为3620.20±237.87）,抑制作用明显下降（P＜0.05）,而到化疗后14d时,其抑制作用已下降到正常水平（cpm值为4283.60±517.89）。结果提示,日本血吸虫慢性感染鼠给予吡喹酮化疗后,脾脏CD4⁺CD25⁺Tregs受感染因素诱导增强的抑制功能可在较短时间恢复到正常水平。6.吡喹酮化疗对日本血吸虫慢性感染小鼠接受其它抗原刺激免疫应答的影响吡喹酮化疗后14d,用日本血吸虫非相关抗原OVA免疫小鼠,4wk后ELISA法检测血清特异性抗体水平。结果显示,日本血吸虫慢性感染小鼠在给予吡喹酮化疗前后血清中抗OVA抗体IgG A₄₅₀值分别是0.68±0.14和1.04±0.22;IgG1抗体亚类A₄₅₀值分别是1.16±0.13和1.35±0.06;IgG2a抗体亚类A₄₅₀值分别是0.68±0.12和1.12±0.17;IgG2a与IgG1比值分别是0.59和0.86。结果表明,在解除抗原持续刺激后,宿主对非特异性抗原的免疫应答也恢复到正常水平,尤其是在感染慢性期被明显抑制的Th1应答上升明显。本试验采用日本血吸虫慢性感染鼠模型,研究吡喹酮化疗前后机体CD4⁺CD25⁺Tregs以及免疫应答状态的变化情况,发现慢性感染期鼠体内CD4⁺CD25⁺Foxp3⁺T细胞的数量增加,抑制功能增强,而化疗后其数量下降,功能也恢复到正常水平,并且宿主对非相关抗原免疫应答由低应答状态恢复到正常水平。进一步研究发现,化疗后CD4⁺CD25⁺Foxp3⁺T细胞的数量下降可能与成虫释放的抗原刺激有关。研究结果提示,CD4⁺CD25⁺Tregs可能是日本血吸虫慢性感染期维持免疫抑制状态的重要因素,并且,经吡喹酮有效病原治疗后,血吸虫感染机体的免疫抑制状态,能在较短时间内得到改善。更多还原

【Abstract】 Schistosomiasis,a parasitic disease,is one of the most important challenges to the population living in the epidemic areas.At present,the main problem is the long-term persistence of pathogen in the host and no strong protective immunity available.Although adult worms of schistosome can be effectively killed by praziquantel,reinfection is difficult to be interrupted.Accordingly,vaccine strategies on control of this disease have been expected.For several decades of efforts,however, there are still few effective vaccines against schistosomiasis.Studies on human populations and experimental models showed that the specific host immunity to schistosome is gradually down-regulated along with the progress of schistosomiasis,which is similar to other chronic infections, including tuberculosis and leishmaniasis.Recently,growing evidence has been presented to support the association between the chronic infections and CD4~+CD25~+ regulatory T cells,but to date,most data focused on the role of CD4~+CD25~+ regulatory T cells in some diseases while the functions of these subset T cells in schistosomiasis japonica are unclear. Our previous study demonstrated that there were some relationships between the down-regulated immunity of the chronic infections and CD4~+CD25~+ regulatory T cells in murine schistosomiasis.This study focused on the changes of CD4~+CD25~+ regulatory T cells and immunosuppression of mice chronically infected with Schistosoma japonicum after treatment with praziquantel.In this study,murine models infected with Schistosoma japonicum were established.Thirteen weeks after the infection,the mice were treated with praziquantel.At different time points,foxp3~+ cells distribution in the hepatic granulomas were examined by immunohistochemistry method.Then the numbers of CD4~+CD25~+ regulatory T cells were detected by flow cytometry and foxp3 mRNA expression on CD4~+ T cells in splenocytes was analyzed by real-time PCR.The inhibitory function of CD4~+CD25~+ regulatory T cells was assessed by the[~3H]-thymidine incorporation method.Finally,the serous specific antibody responses after OVA inoculation were assessed by ELISA.The main results are as follows:1.Foxp3~+ cells in the hepatic egg granulomas decreased when mice chronically infected with Schistosoma japonicum were treated with praziquantel In the hepatic sections of mice with the chronic infection,foxp3~+ cells were aggregated in the egg granulomas.On the day 3 after treatment,there were few changes on the number of foxp3~+ cells.Howerer,on the day 7 after treatment,the foxp3~+ cells were significantly decreased.On day 14 after treatment,almost no foxp3~+ cells could be found in the granulomas.The results demonstrated that praziquantel chemotherapy played roles on reduction of local foxp3~+ cells in the egg granulomas of mice infected with Schistosoma japonicum.2.The relative ratio of CD4~+CD25~+Foxp3~+ T cells from splenocytes was significantly decreased in the chronically infected mice after treatment with praziquantel Flow cytometry analysis showed that the ratio of CD4~+CD25~+Foxp3~+T cells in CD4~+T cells was higher in the chronic infection group than that in normal control.After praziquantel treatment,the ratio was decreased slightly on day 3 and significantly on day 7.Till day 14,the ratio of CD4~+CD25~+ Foxp3~+T cells fell to the level of normal control.The results indicated that praziquantel intervention also caused markedly reduction of splenic CD4~+CD25~+ Foxp3~+T cells.3.The expressions of foxp3 mRAN in splenic CD4~+T cells were down-regulated after mice with chronic infection were treated with praziquantel Real-time PCR analysis showed that in the infection group,the expression of foxp3 mRNA was significantly down-regulated on day 3 after treatment,compared with that in the normal control and sustained the same levels on day 7 and day 14. This indicated that the changes of foxp3 mRNA expressions were prior to the reduction of the CD4~+CD25~+ Foxp3~+T cell numbers.4.SWAP injection may cause reduction of splenic CD4~+CD25~+ Foxp3~+T cell numbers Flow cytometry analysis showed that praziquantel per se didn’t cause any significant changes of splenic CD4~+CD25~+Foxp3~+ T cell numbers.However,the splenic CD4~+ CD25~+Foxp3~+T cell numbers decreased obviously when mice were injected i.p with SWAP.We presumed that the antigen injection may mimic the circumstance which the adult worms were killed by praziquantel,and consequently released lots of antigens in a short period of time.The mechanism in detail of how SWAP causes decrease of CD4~+CD25~+Foxp3~+T cell remains unclear.5.Suppressive potential of CD4~+CD25~+ T cells on the proliferation of CD4~+CD25~- T cells was attenuated in the chronically infected mice after treatment with praziquantel ~3H-TdR incorporation method was used to evaluate the Suppressive ability of CD4~+CD25~+ T cells.The experiments showed that the CD4~+CD25~+ T cells isolated from mice with chronic infection presented higher suppressive potential on the proliferation of CD4~+CD25~- T cells from the same mice under ConA stimulation,compared with the CD4~+CD25~+T cells isolated from normal mice.However,these suppressive capabilities began to subside gradually after CD4~+CD25~+ T cell donated mice were treated with praziquantel.It became obvious on day 7 after the treatment.On the day 14,the suppressive capabilities of CD4~+CD25~+ T cells decreased to the level approximately resembling that from normal mice.The results suggests that praziquantel chemotherapy could not only reduce both the numbers and foxp3 mRNA expression of CD4~+CD25~+ T cells,but also degrade the suppressive functions of this cell subset.6.Specific anti-OVA antibody was increased in chronically infected mice after treatment with praziquantel ELISA detection showed that the level of specific anti-OVA antibody IgG and its isotype IgG1 as well as IgG2a were higher in treated group than those in chronic infection group.Meanwhile,the ratio of IgG2a to IgG1 was rose in treated group compared to that in chronic infection group.The results demonstrated that schistosome infection could induce immunosuppression of the hosts and the immune responses of the infected mice were rebounded and skewed to Th1 type shortly after praziquantel chemotherapy.In conclusion,our study demonstrated that the numbers and the inhibitory activities of CD4~+CD25~+ regulatory T cells of mice chronically infected with Schistosoma japonicum were down-regulated after praziquantel chemotherapy.The same changes in the numbers of CD4~+CD25~+ regulatory T cells could also be induced by SWAP injection.Furthermore,the immunosuppressive status of mice caused by the infection could be reversed by the chemotherapy.The results suggested that CD4~+CD25~+ regulatory T cells may play important roles in sustaining the immunosuppressive status of mice chronically infected with Schistosoma japonicum.This also revealed that praziquantel,as a drug of the first choice for anti-schistosomiasis,could terminate the persistent infection by effectively killing the adult worms and simultaneously restore the immunologic balance that once was broken by the infection.更多还原