【作者】 李芸茜；

【导师】 冯振卿；

【作者基本信息】 南京医科大学 ， 病理学， 2004， 硕士

【摘要】 系统性炎症反应综合征(Systemic Inflammatory Response Syadrome,SIRS)和多器官功能障碍综合征(Multiple Organ Dysfunction Syndrome,MODS)是严重创伤和感染疾病过程中的疑难问题，死亡率高达40％-70％。SARS CoV引起的非典型性肺炎中的重症病例均属于SIRS-MODS范畴。SIRS的病理基础是免疫应答失去平衡。由于免疫网络的调节系统失控，机体免疫亢进和免疫抑制交替出现或并存，至今仍缺乏有效的治疗手段。而一些具有双向免疫调节作用的中药在临床上显示出良好的效果，如大黄类中药治疗肠道缺血再灌注引发的SIRS-MODS。黄芪是具有免疫调理作用的补益类中药，性味平和，有多种成熟剂型被广泛应用于临床，可用于多种疾病的辅助治疗。在一些SIRS动物模型中也观察到黄芪对某种细胞因子有诱导或抑制的作用。为了验证黄芪对SIRS的整体治疗效果，本研究选用了急性弓形虫感染小鼠作为SIRS模型，观察了黄芪对不同剂量弓形虫速殖子感染小鼠生存情况的影响，并动态观察了黄芪对急性弓形虫小鼠肝、肺虫荷，肝、脑、脾、肺的组织学，血清ALT水平，肝iNOS，血清IFN-γ、IL-18水平，及脾细胞凋亡的影响，探讨黄芪的作用机制及其作为SIRS辅助治疗药物的可行性。 研究方法 1．用10~5(高剂量)，10~3(中剂量)，10~2(低剂量)速殖子腹腔感染小鼠，每个感染剂量组小鼠中设立2个实验组分别给于黄芪0.075g／d／mice，阿齐霉素150mg／d／kg灌胃治疗，1个对照组给于蒸馏水，连续10d。观察10d内每天的小鼠存活率绘制累计生存曲线，并计算平均存活时间。 2．建立10~2速殖子感染小鼠模型，设黄芪治疗组和对照组。2组南京医科大学硕士学位论文小鼠定期分批处死，取血清和肝、肺、脾、脑.用荧光定量PCR检测感染后第4、8天(4、sdpi，day卯st一infeetion)肝、肺的虫荷。用赖氏法测定4、5、6、7dPi血清AIJ水平.取2一sdPi天的肝、脾、肺、膝，中性福尔马林固定后作HE染色切片。取2、4、6、sdPi肝脏做冷冻切片，NADPH一D法进行诱导性一氧化氮合酶(iNOS)酶组化染色.甩ELISA方法测定2、4、6、sdPi血清IFN一Y水平及4、6、sdPi的血清IL一18水平。AN刊旧)JN V-FITC方法经流式细胞仪检测5、7dPi脾细胞洲亡率.研究结果 1.高、中、低剂童速殖子感染时，黄茂组的系计生存曲线均在对膝组右侧.高、中速殖子感染时，黄茂组小鼠平均存活时间为5 .57d和6.23d，与对照组相比差异没有显著性(P>0.05)，阿齐霉素组小鼠平均存活时间为6.96d和8.12d，显著高于对照组(尸<0.05)。1扩个速班子感染时，黄茂组小鼠平均存活时间为8.74d，显著高于对照组(尸切.05)，阿齐霉素组小鼠平均存活时间为7.79d，与对照组相比差弃没有显著性(尸<0.05)。 2.电扣i和8却i肝、肺虫荷两组没有显著差异，感染后黄茂组和冲照纽血清A工T水平持续上升，黄茂组4dPi为87 .20u(卡门氏单位)，质著高于对照组(34.44u)(尸叱0.05)，黄茂组8即i为1 72.5u，显著低争对照厂组(2 25.69u)( P<O.05)。感染后前6d黄茂组和对照组血清IFN-丫水平持续上升，黄茂组4dPi为21 .op到ml，显著高于对照组(5 .62留ml)(产荀.05)。黄茂组叼pi为15.36p岁ml，显著高于对照组(9 .73哪ml)(户刃.05)。黄茂组和对照组血清IL一18水平持续上升，黄茂组名匆i为5舆.75 pg/ml，显著低于对照组(709.00闻ml)(P<o·05)。4dpi起，黄茂组肝脏iNOS染色明显比对照组增强，小叶中央和汇管区均染.对照纽iN0s表达局限于小叶中央，汇管区浅染.黄茂组5、7dPi岭癣细胞料亡率分别为22.5%、28.0%; 36.9%、40.8%，均显著低于南京医科大学硕士学位论文对照组(41.0%、45.4%:53.0%、49.4%)(尸叱0.05).结论 1.黄茂对高、中、低剂量速殖子感染的小鼠均有一定的辅助治疗作用，对低剂童感染小鼠的治疗作用明显。 2.黄茂通过对免疫应答的双向调节对急性弓形虫感染小鼠产生保护作用。更多还原

【Abstract】 The innate immune system plays a crucial role in protecting the host gainst infectious microorganisms. An inappropriate control of this system may have profound consequences such as SIRS (systemic inflammatory response syndrome) and MODS (multiple organ dysfunction syndrome) because of the maintained production of specific proinflammatory molecules. Monoclonal antibody or binding protein of some cytokines failed to imorove the survival or may place the host at increased risk of infectious complications. There are not yet any effective therapy in clinical practice. Therefore in China, many herbs are universaly adnministered on patients to sustain the balance of immune responses. Astragalus has a long history of medicinal use within the traditional Chinese system. Astragalus has demonstrated a wide range of immunopotentiating effects. In this study, a SIRS model of acute murine toxoplasmosis were established to access the efficacy of Astraglus on sustaining the balance of immune responses. Previous researches have suggested that Astraglus had the function of modulating some cytokine during the development of SIRS, but the resultes failed to describe the immune status of hosts. In our model of acute murine toxoplasmosis, the efficacy of Astraglus can be quantified by the survival of mice which is a consequence of delebrate regulation of immune response. In our study, mice were intraperitoneally infected with different doses of tachyzoites, and the effecacy of Astraglus were observed. Serum levels of ALT, IFN-r, IL-18, iNOS prodece in liver, apoptosis of spleen were assayed at intervals to study the mechanism.-4-MethodsICR Mice respectedly infected with 10 10 10 tachyzoites of RH strain, Toxoplasma Gondii were orally treated with Astraglus memberane and Azithromycin from 1st day post infection.Control groups were infected with same dose of tachyzoites and given water. The dose of infection with which Astragli demonstrated best effectiveness was decided as the one to establish the target model. Mice treated with Astraglus and water were killed at intervals to collect sera and tissues. Parasites loads were deterimined with quantitative fluorescence Poly merase Chain Reaction (PCR). Serum enzyme levels of alanine aminotransferase (ALT) were quantified using a commercial kit. Serum levels of IFN-r, IL-18 were assayed using two-site ELISA sets. Paraffin sections of tissues were stained with H&E. Frozen sections were stained by the method of nicotinamide adenosine dinudeotide phosphate diaphorase to quantitate iNOS. Splenocytes were stained by annexin V before being analyzed on a FACScan to evaluate the apoptosis rates.ResultsNo mice but 20% of those who were infected with low dose of tachyzoites (10) and treated with Astragli survived 10 days or above.As to the average survival time post infection, mice receiving Astragli demonstrated no benefit comparing control groups when infected with middle (10) or high (10) dose of tachyzoites. But when infected with low dose of tachyzoites (10), mice receiving Astragli survived 8.74d that was significant longer than the control(7.63d) (p<0.05) .The following results were obtained from the murine model infected with low dose of tachyzoites (10). Parasite loads in live and lung detected at 4dpi and 8dpi were common between Astrgli group and control group.The ALT levels in-5-sera of both groups kept elevating. ALT level was observed increasing to 87.20u at 4dpi in Astrgali group,while it remained low(34.44u) in control group. But at 7dpi, ALT level in Astragli group(172.5u) were significant lower than that in control group(225.69u)(P<0.05). Serum levels of IL-18 increased until death, whereas IFN- Y levels peaked and then decreased IFN- y levels of Astragli group were higher than that of control group at 4dpi and 8dpi (P0.05) . IL-18 levels of Astragli group were lower than that of control group at 8dpi (P<0.05) .Control groups demonstrated low levels of iNOS expression until death,while Astragli induced higher levels of iNOS and more extensive expression from 4 dpi. Apl更多还原