【作者】 常文秀；

【导师】 曹书华； 王勇强； 高红梅；

【作者基本信息】 天津医科大学 ， 急诊医学， 2009， 博士

【摘要】 目的观察脓毒症大鼠脾脏T淋巴细胞凋亡与免疫麻痹发生的相关性。观察高糖皮质激素血症及T淋巴细胞过度活化两种促凋亡因素对脾脏T淋巴细胞凋亡、凋亡相关基因mRNA表达及caspase3活性的影响，明确脓毒症时脾脏T淋巴细胞的凋亡机制及调节因素。探讨活血化瘀中药调节T淋巴细胞凋亡，改善脓毒症免疫功能的作用及机制。方法第一部分首先以盲肠结扎穿孔（CLP）法制备脓毒症大鼠模型，假手术组及模型组各10只，检测模型的各项生理及生化指标。然后将96只Wistar大鼠随机分为正常对照组（8只）、假手术组（8只）、脓毒症组（40只）和中药组（40只），中药组动物行CLP术后予活血化瘀中药注射。脓毒症组和中药组再分别分为4 h、8 h、24 h、36 h和48 h五个时间点。分别于术后各个时间点以ELISA法检测血清IL-2、IL-10水平，TUNEL法检测脾细胞凋亡情况，流式细胞仪检测脾脏淋巴细胞中CD₃⁺细胞比例，分光光度法测定脾组织caspase3活性以及免疫组化法检测脾组织Fas、FasL、Bcl-2、Bax蛋白表达水平。第二部分提取BAB／C小鼠脾脏T淋巴细胞并培养，分别以地塞米松和活化诱导细胞凋亡（AICD）方法进行凋亡诱导。于地塞米松诱导6 h、16 h及活化诱导18 h后以流式细胞仪检测T淋巴细胞的凋亡率，并于地塞米松诱导6 h及活化诱导6 h、1 8 h后RT-PCR法检测T淋巴细胞内Fas、FasL、Bcl-2、Bax mRNA表达水平，分光光度法测定细胞内caspase3酶活性。同时观察活血化瘀中药对上述各项指标的影响。第三部分提取BAB／C小鼠脾脏T淋巴细胞并培养，观察活血化瘀中药对静息及活化T细胞增殖活性影响的MTT检测，流式细胞仪检测中药干预后静息T淋巴细胞的自然凋亡率。再分别以地塞米松和活化诱导法进行凋亡诱导，并以活血化瘀中药进行干预，RT-PCR法检测T淋巴细胞内IL-2 mRNA表达水平。结果第一部分实验中脓毒症组大鼠于造模后出现脾组织caspase3活性升高、细胞凋亡数增多，而脾细胞中CD₃⁺细胞比例明显下降。并于造模后36 h、48 h，血清IL-2水平明显下降，IL-10水平显著升高，显示出免疫麻痹的表现。同时脓毒症组脾组织Fas、FasI、Bax表达明显增多，Bcl-2表达下降。活血化瘀中药干预后可提高IL-2水平，降低IL-10水平，减轻脾组织caspase3活化程度，减少脾细胞凋亡，并使CD₃⁺细胞比例恢复正常。同时可明显降低脾组织Fas、FasL、Bax表达，促进Bcl-2表达。第二部分实验中地塞米松诱导6 h后T淋巴细胞凋亡率即明显增加，至16 h细胞较少存活。诱导6 h后T淋巴细胞内BaxmRNA表达升高，Bcl-2 mRNA表达下降，Fas mRNA表达无变化，无FasL mRNA表达。以活化诱导凋亡的T淋巴细胞于诱导18 h后细胞凋亡率明显增加，于诱导6 h时未见FasL、Bax mRNA表达，Fas、Bcl-2 mRNA表达无明显变化；于诱导1 8 h后Fas、FasL、Bax mRNA表达升高，Bcl-2mRNA表达下降，caspase3活性增高。活血化瘀中药可降低两种促凋亡因素诱导的T细胞凋亡，并可分别降低Fas、FasL、Bax mRNA表达，提高Bcl-2 mRNA表达，减轻caspase3酶活性。第三部分实验中活血化瘀中药可以促进静息及活化脾脏T淋巴细胞的增殖活性，降低静息T细胞的自然凋亡率。增加地塞米松诱导6 h后的T淋巴细胞内IL-2 mRNA表达。在活化诱导早期（6 h）促进T淋巴细胞内IL-2 mRNA表达，在晚期（18 h）减少IL-2 mRNA表达。结论脾脏T淋巴细胞的异常凋亡与脓毒症免疫麻痹的发生相关，并受凋亡相关蛋白调节。脓毒症状态下的高糖皮质激素血症及T淋巴细胞过度活化引起的AICD是脾脏T淋巴细胞异常凋亡的诱发因素，地塞米松诱导的凋亡受Bax、Bcl-2 mRNA表达的调节，而AICD的发生与Fas、FasL、Bax、Bcl-2 mRNA表达的改变有关。活血化瘀中药可通过调节IL-2 mRNA表达而减轻两种因素诱导的脾脏T淋巴细胞凋亡，同时可以促进T淋巴细胞的增殖活性，减轻T细胞的自然凋亡率，缓解脓毒症时的免疫麻痹。更多还原

【Abstract】 Objective To observe the relationship between T lymphocytic apoptosis inspleen and immunoparalysis in sepsis. The effect of glucocorticoid and AICD on theT lymphocyte apoptosis in spleen, apoptotic related gene mRNA levels and theactivity of caspase3 in T lymphocyte were observed. To investigate the mechanism ofthe T lymphocyte apoptosis in sepsis and its influence factors. To investigate themechanism of HuoXueHuaYu injection regulating the apoptosis of T lymphocyte andameliorating the immunoparalysis in sepsis.Methods In the 1st part, the sepsis models were established by cecal ligationand puncture （CLP）,and the parameters of physiology and biochemical in modelswere detected. After that, 96 wistar rats were divided into four groups: control group（n=8）, sham operation group （n=8）, sepsis group （n=40）, and HuoXueHuaYutherapeutic group （n=40）. The sepsis group and HuoXueHuaYu therapeutic groupthen divided into 4h, 8h, 24h, 36h, 48h points. The rats were killed at the point afterthe operation and evaluated the IL-2, IL-10 levels in serum by the methods of ELISA,the apoptotic cells by TUNEL, the CD₃⁺ lymphocyte percent in spleen by flowcytometry, the activity of caspase3 in spleen by spectrophotometric method, and theexpression of Fas, FasL, Bcl-2, Bax proteins in spleen by immunohistochemistry. Inthe 2nd part, the T lymphocytes obtained from the spleens of BAB/C mice werecultured and induced apoptosis by dexamethasone and AICD. Apoptosis was assessedby flow cytometry at 6 h, 16 h after the stimulation of dexamethasone, and 18 hamong the AICD procesure. RT-PCR was used to evaluted the expression of Fas,FasL, Bcl-2, Bax mRNA, and the activity of caspase3 in T lymphocyte was detectedby spectrophotometric method at 6 h after the stimulation of dexamethasone, and 6 h,18 h among the AICD procesure. In the mean time the effect of HuoXueHuaYuinjection on those parameters was observed. In the 3rd part, the T lymphocytes wereobtained from the spleens of BAB/C mice and cultured with HuoXueHuaYu injectionThe MTT was measured to assessed the proliferation activity, and the natureapoptosis of T cells was investigated by flow cytometry. Dexamethasone and AICDwere used to induce apoptosis, and the IL-2 mRNA in T cells were dected by RT-PCR.Results In the 1 st part, the activity of caspase3 increased at 4h after the CLPoperation and there were more apoptosis appeared. Simultaneously the percent ofCD3⁺ lymphocyte in spleen decreased. At 36 h, 48 h after the CLP operation, the IL-2in serum decreased and IL- 10 increased obviously. The immunoparalysis appeared. Inthe mean time, the expression of Fas, FasL, Bax in spleen increased obviously inmodel animals, otherwise the Bcl-2 decreased. After the intervention withHuoXueHuaYu injection, the IL-2 level in serum increased and the IL-10 in serumdecreased. The intervention also lightened the activity of caspase3 and the apoptosis,and made the percent of CD3⁺ lymphocyte returned to normal level. The expressionof Fas, FasL, Bax reduced and Bax raised. In the 2nd part, the apoptosis of Tlymphocyte induced by dexamethasone was increased at 6 h, and there was little alivecells at 16 h. The expressions of Bax mRNA rised and the expression of Bcl-2 mRNAlessened at 6 h. There was no change in the expression of Fas mRNA, and theexpression of FasL mRNA was not detected. After the AICD, T lymphocyte apoptosisraised at 18 h. At 6 h after the AICD, there was no expression of FasL, Bax mRNA,and no change in the expression of Fas and Bcl-2 mRNA. At 18 h after the AICD, theexpressions of Fas, FasL, Bax mRNA rised and the expression of Bcl-2 mRNAlessened. The activity of caspase3 also ascended. HuoueHuaYu injection can cutdown the apoptosis induced by the two pro-apoptotic factors, make the expression ofFas, FasL, Bax mRNA decreased and Bcl-2 mRNA improved. The activity ofcaspase3 also fallen after the HuoueHuaYu injection treated. In the 3rd part,HuoXueHuaYu injection can improve the proliferation activity of T lymphocyte,lessen the nature apoptosis of T lymphocyte. HuoXueHuaYu injection can increasethe expressions of IL-2 mRNA in T lymphocyte induced by dexamethasone, promotethe expressions of IL-2 mRNA at early phase of AICD （6 h） and depress theexpressions at the late period （18 h）.Conclusions There is a close relationship between T lymphocyte apoptosis inspleen and the immunoparalysis in sepsis models. The expression of apoptotic relatedproteins in spleen regulate the T lymphocyte apoptosis. T lymphocyte in spleen canbe induced to apoptotic by dexamethasone and AICD, and be regulated by the expression of apoptotic related gene. The apoptosis induced by dexamethasone wasregulated by the expression of Bax and Bcl-2 mRNA. The AICD of T lymophocytewas regulated by the change of Fas, FasL, Bcl-2, Bax mRNA expression.HuoueHuaYu injection can ameliorate the apoptosis through regulating the expressionof IL-2 mRNA which resulted in the improving the immune function of sepsis. It canimprove the proliferation activity of T lymphocyte, lessen the nature apoptosis of Tlymphocyte.更多还原