【作者】 王伟；

【导师】 王勇；

【作者基本信息】 南京医科大学 ， 病原生物学， 2015， 硕士

【摘要】 血吸虫病(schistosomiasis)是血吸虫寄生人体引起的寄生虫病,血吸虫病危害严重,是全球重要的公共卫生问题之一。我国主要流行的是日本血吸虫病,至2012年底,大约还有20万的血吸虫病患者。日本血吸虫病造成的病理损伤主要是由于虫卵沉积在肝脏形成肉芽肿,导致门静脉阻塞,继而发生肝脏纤维化和脾脏肿大并伴脾脏功能亢进的临床现象。目前针对血吸虫病患者脾脏肿大及脾脏功能亢进,并无有效的内科治疗方法,通常选择脾脏全切或部分切除的姑息治疗方式。血吸虫病造成的门静脉持续高压致脾脏淤血和脾脏自身的免疫细胞功能改变是导致脾脏肿大的两个关键因素。日本血吸虫感染至慢性期,可见小鼠脾脏异常肿大。吡喹酮作为一种传统的抗血吸虫药物,过去人们主要关注其杀虫作用。本实验室前期研究中发现,慢性血吸虫病小鼠经吡喹酮治疗后,门静脉高压得到明显缓解,且脾脏及巨噬细胞系的炎性因子基因表达和细胞因子表达均受到明显抑制,但巨噬细胞作为介导脾脏炎症和脾功能亢进病理过程的重要细胞亚群,吡喹酮是否能影响其数量和功能尚未见详细研究报道。本研究从脾脏巨噬细胞入手,研究感染及吡喹酮治疗前后脾脏巨噬细胞数量及功能变化情况,进一步揭示吡喹酮治疗血吸虫感染后脾脏肿大及脾功能亢进的机制。已知巨噬细胞受不同诱导因素作用分别可通过经典途径和替代途径活化并分化为M1和M2两型细胞。相关文献报道,M1和M2型分别与机体Th1型和Th2型免疫应答相关,且在不同临床疾病中,M1和M2型巨噬细胞分化趋势不同。M1型巨噬细胞活化呈现出炎性免疫应答,常与糖尿病,关节炎,动脉硬化等疾病发生相关。M2型巨噬细胞活化呈现抑制炎性应答,常参与纤维化发生,伤口愈合,肿瘤转移等病理生理现象。在血吸虫感染中,已知肝脏及腹腔巨噬细胞存在极性转化的现象,证明其在感染早期以表达M1型巨噬细胞为主,而感染慢性期主要表达M2型巨噬细胞。但在日本血吸虫感染慢性期脾脏组织中,巨噬细胞的极化方向尚不明确。为此,我们通过观察感染慢性期脾脏巨噬细胞极化情况,分析巨噬细胞不同亚群在脾脏肿大中可能发挥的调控作用,及吡喹酮对脾脏巨噬细胞极化的影响。本课题主要研究吡喹酮对日本血吸虫感染慢性期脾脏巨噬细胞数量和功能的影响。首先,我们建立日本血吸虫感染12周模型及吡喹酮化疗模型,观察吡喹酮对脾脏肿大的治疗效果;接着,我们研究吡喹酮化疗后脾脏巨噬细胞的数量及功能变化情况;最后,进一步研究吡喹酮化疗后,脾脏巨噬细胞的极化情况。本研究获得如下主要结果:1.日本血吸虫感染鼠经吡喹酮治疗后脾脏病理状况明显改善我们采用日本血吸虫感染12周小鼠模型,用300mg/kg剂量吡喹酮100μl体积给药治疗。结果显示,杀虫3天后小鼠脾脏大小,脾脏重量和脾重指数有一定恢复,治疗4周后小鼠脾脏大小基本恢复正常,脾脏重量和体重指数均恢复正常水平,与同期感染血吸虫16周组小鼠相比有明显差异。脾脏组织病理切片H.E染色结果显示,感染血吸虫16周小鼠脾脏组织结构遭到明显破坏,红髓、白髓分布不明显,可见部分脾窦充血,脾细胞排列不规则,且有坏死的组织。经吡喹酮3天杀虫后,脾脏组织结构有一定程度恢复,但尚没形成清楚红、白髓结构。经吡喹酮4周治疗后对脾脏实质内结构有修复作用,形成清晰白髓、红髓结构,脾细胞分布较规则。以上结果提示:吡喹酮持续4周化疗对感染血吸虫16周小鼠脾脏肿大效果明显且对病理损伤的脾脏组织结构有显著改善作用。2.日本血吸虫感染鼠经吡喹酮治疗后脾脏巨噬细胞数量比例明显减少,分泌炎性细胞因子功能有下降趋势为了观察吡喹酮对脾脏中巨噬细胞的影响,对脾脏组织进行免疫组化分析,我们采用标记巨噬细胞表面分子CD68为一抗,HRP标记山羊抗鼠兔通用二抗,DAB显色来区分巨噬细胞,阳性巨噬细胞显棕黄色。通过照片观察及数据统计分析,结果显示:感染16周后脾脏巨噬细胞显色面积和数量比例明显上调,经吡喹酮3天杀虫后巨噬细胞显色密度和数量有一定降低。经吡喹酮4周治疗后巨噬细胞显色密度和数量比例显著下降,趋于正常对照组。进一步地,我们采用流式分析脾脏中巨噬细胞的比例变化,标记F4/80~+CD11b~+为阳性巨噬细胞,结果显示:感染16周鼠脾脏中巨噬细胞比例明显上升,而经过吡喹酮治疗后脾脏巨噬细胞比例明显下降。且持续吡喹酮治疗4周,巨噬细胞比例进一步下降,较吡喹酮3天杀虫有明显差异。同时取脾脏单个核细胞培养,贴壁分离巨噬细胞并培养24小时,取上清检测细胞因子,显示吡喹酮4周治疗后炎性因子IL-6的水平有所下降。这些结果提示:吡喹酮持续4周治疗对感染血吸虫16周鼠脾脏的巨噬细胞数量比例有显著下调作用,对其分泌炎性因子有一定抑制作用。3.日本血吸虫感染鼠经吡喹酮治疗后脾脏巨噬细胞极化方向发生变化,M1型巨噬细胞数量比例下调为了进一步评价吡喹酮对巨噬细胞两群细胞的极化的影响,我们用流式分析两群细胞的变化,标记F4/80~+CD11b~+CD16/32~+为阳性M1型巨噬细胞,标记F4/80~+CD11b~+CD206~+为M2型巨噬细胞,结果显示:感染血吸虫16周后脾脏巨噬细胞以M1型巨噬细胞为主,经吡喹酮治疗后M1型巨噬细胞比例下调,吡喹酮4周治疗后较吡喹酮3天杀虫后M1型巨噬细胞比例下降更明显,而M2型巨噬细胞变化均不明显。这些结果提示:吡喹酮持续4周治疗使感染血吸虫16周鼠脾脏的M1型巨噬细胞比例下调。4.日本血吸虫感染鼠经甲氟喹治疗后脾脏病理状况,及巨噬细胞数量比例和M1型巨噬细胞比例变化为了进一步确认吡喹酮对巨噬细胞数量功能及极化方向产生的影响,我们采用另一种杀虫效果显著的药物甲氟喹,观察其对血吸虫感染12周小鼠脾脏肿大治疗及对脾脏巨噬细胞的作用。结果显示:甲氟喹治疗后,脾脏依然呈现肿大状态且脾脏组织病理损伤没有得到改善,脾脏巨噬细胞所占比例与感染组比例相近,且仍表达与感染组相似的M1型巨噬细胞为主的分型极化趋势。这些结果提示:从另一角度证明,吡喹酮不仅是通过杀虫缓解门静脉持续高压,从而使肿大的脾脏恢复正常,而且吡喹酮对脾脏肿大作用中关键的巨噬细胞群可能发挥作用,对巨噬细胞数量比例、功能和极化方向有调节作用。更多还原

【Abstract】 Schistosomiasis is a parasitic disease that caused by Schistosoma japonicum.It has led to a serious public health problem in China and worldwide.To the end of2012,there are still about 240,000 schistosomiasis patients in China.The pathological damage caused by schistosomiasis is mainly due to the deposition of eggs in the liver which results in granuloma formation,portal vein obstruction,liver fibrosis,and spleen abnormal enlargement and hypersplenism.Until now,reports about the treatment of spleen pathological damage caused by S.japonicum are rare.For those patients,splenectomy or partial splenectomy have to be used to alleviate hypersplenism.During chronic stage of schistosomiasis,spleen abnormal enlargement is obvious.However,splenomegaly is significantly relieved after praziquantel treatment.The change of immune cell function in the spleen and the portal hypertension are considered to be two key factors in causing splenomegaly.Praziquantel,a traditional anti-S.japonicum drug,is well known its insecticidal effect.Previous studies found that praziquantel could relieve portal hypertension,and inhibit the expression of both inflammatory factor and cytokine gene in spleen macrophage of mice infected with S.japonicum.But whether praziquantel has effect on macrophages,the key cells of splenomegaly,has not yet been reported.Our study focus on splenic macrophages,especially the functional and number changes of macrophages after infection and praziquantel treatment,in order to further revealed the mechanism of praziquantel treatment on the splenomegaly and hypersplenism caused by schistosomiasis.It is acknowledged that macrophages may differentiate into M1 and M2 types of cells when induced by different factors through the classical pathway and the alternative pathway.It is reported that M1 and M2 type of cells are associated with Th1-type and Th2-type immune responses,respectively.In various clinical conditions,M1 macrophages mediated activation of inflammatory immune response,often associated with diabetes,arthritis,atherosclerosis and other diseases,while M2type macrophages usually represent immune modulation,often involved in fibrosis,wound healing,cancer metastasis and other physiological phenomena.In schistosome infection,liver and peritoneal macrophages expressed M1 phenotype in early stage,and transfor to M2 phenogype in chronic stage.However,it is not clear that how it occur for splenic macrophages of mice with chronic schistosomiasis.For this reason,we observed changes on the population and the polarization of splenic macrophages of mice with chronic schistosomiasis with or without praziquantel treatment.At first,we established mice models of chronic schistosomiasis.The infected mice received continuous four weeks praziquantel treatment.Then,we observed the changes of splenic macrophages.Finally,we analysed the polarization of splenic macrophages after praziquantel treatment.The main findings are as follows:1.Praziquantel treatment significantly ameliorated spleen pathological condition of mice infected with S.japonicum.The infected mice were given praziquantel treatment in 300mg/kg dosage for continuous.The results showed that after treatment 3 days,the spleens of mice had a reduction.The spleen weight and body mass index had a reduction.After treatment4 weeks,the spleens of mice returned to the normal size.The spleen weight and body mass index were also restored to the normal level,in comparison with that of infected group of mice.In addition,H.E staining showed that the histological structure of spleen in mice infected with S.japonicum was disorganized,with chaotic red pulp and white pulp distribution and the visible part of splenic sinus congestion,irregular cells arranged and tissue necrosis.After praziquantel treatment 4 weeks,the splenic parenchymal structure repaired,with the re-formation of clear white pulp and red pulp and the regular cell distribution.The above results indicated that praziquantel treatment 4 weeks has a obvious effect on splenomegaly and have significantly improved in histological structure of spleen in mice infected with S.japonicum.2.After praziquantel treatment,the proportion of splenic macrophages and its secretion of inflammatory cytokine of mice infected with S.japonicum decreased significantly.In order to observe the effect of praziquantel on splenic macrophages,we used immunohistochemistry method to label surface molecule CD68 on splenic macrophages The proportion of the number of macrophages was increased significantly after the infection,after praziquantel treatment 3 days,had a reduction but declined significantly after praziquantel treatment 4 weeks,in comparison with the control group.Flow cytometric analysis further confirmed that the proportion of splenic macrophages obviously increased in infected mice,and decreased significantly after praziquantel treatment.Detection on the cultured supernatant of isolated macrophages revealed that the levels of inflammatory factor IL-6 also decreased after praziquantel treatment.These results suggested that praziquantel treatment 4 weeks may significantly decreased the number and the secretion of inflammatory factor of infected mice with splenomegaly.3.After praziquantel treatment,the polarization of splenic macrophages changed in mice infected with S.japonicum and the ratio of M1 macrophages decreased.In order to further evaluate the effect of praziquantel on macrophage polarization,we used flow cytometry to analyse the changes of M1/M2phenotype population.M1 positive macrophages were labeled CD16/32~+and M2macrophages labeled CD206~+.The results showed that M1 type of cells were predominant in splenic macrophages of mice after infection,and decreased after praziquantel treatment,and praziquantel treatment 4 weeks was more obvious than praziquantel treatment 3 days.However,M2 type cells did not change significantly either with praziquantel treatment of not.These results suggested that praziquantel 4weeks down-regulated M1 macrophages of mice with splenomegaly.4.After mefloquine treatment,spleen pathological condition,the number of macrophages and the ratio of M1 macrophage has not been changed in mice infected with S.japonicum.In order to discriminate the effects of praziquantel on macrophages and splenomegaly were not just the consequent event of helminthicide of this drug,we used mefloquine,the another strong helminthicide drug,to treat the chronically infected mice.The results showed that splenomegaly and pathological injury had not been changed after treated with mefloquine.Furthmore,the ratio of splenic macrophage were similar with the infected group,and still expressed M1 dominant cell population.These results even confirmed that praziquantel not only had the helminthicide effect,but also had a regulatory effect on splenic macrophages.更多还原