抗Tim3抗体对脓毒症小鼠的治疗作用及其可能机制

发布时间：2018-02-24 13:14

  本文关键词： 毒症 盲肠结扎穿孔 T淋巴细胞 细胞凋亡 抗Tim3抗体　出处：《第二军医大学》2014年硕士论文　论文类型：学位论文

【摘要】：【研究目的】 本课题主要研究脓毒症小鼠经抗Tim3抗体治疗后，其胸腺、脾脏、肺脏和肝脏组织在免疫与病理水平的变化，研究抗Tim3抗体治疗是否改善脓毒症小鼠的生存率，并探讨其可能机制。 【研究方法】 1、建立CLP手术致脓毒症小鼠模型，实验对象为12只C57BL/6小鼠，随机分成脓毒症模型组(CLP，n=6)和假手术组(Sham, n=6)。CLP模型建立24h时，流式细胞术检测各组小鼠脾脏组织CD8+T细胞Tim3分子表达水平。 2、选择C57BL/6小鼠40只，随机分为假手术组(Sham，n=10)、CLP+腹腔注射生理盐水组(CLP，n=10)、CLP+腹腔注射抗Tim3抗体组（anti-Tim3，n=10)和CLP+腹腔注射同型对照抗体组(Isotype，n=10)。各组术后即刻予腹腔注射生理盐水、抗Tim3抗体或同型对照抗体(均每只50μg/150μl)，记录各组小鼠7d的生存时间及生存率。 3、选择C57BL/6小鼠24只，随机分为假手术组(Sham，n=6)、CLP+腹腔注射生理盐水(CLP，n=6)、CLP+腹腔注射抗Tim3抗体组(anti-Tim3，n=6)和CLP+腹腔注射同型对照抗体组(Isotype，n=6)。各组术后即刻予以腹腔注射生理盐水、抗Tim3抗体或同型对照抗体(均每只50μg/150μl)。 ①术后24h时取各组小鼠外周血，检测小鼠外周血细菌清除率； ②术后24h时各组小鼠行腹腔灌洗，取腹腔灌洗液检测细菌清除率。 4、选择C57BL/6小鼠24只，随机分为假手术组(Sham，n=6)、CLP+腹腔注射生理盐水(CLP，n=6)、CLP+腹腔注射抗Tim3抗体组(anti-Tim3，n=6)和CLP+腹腔注射同型对照抗体组(Isotype，n=6)。各组术后即刻给予生理盐水、抗Tim3抗体或同型对照抗体(均每只50μg/150μl)。 ①术后24h时，取各组小鼠肺组织、肝脏组织，进行病理学检验； ②术后24h时，取各组小鼠胸腺组织、脾脏组织，进行TUNEL染色病理检验，同时进行阳性率分析； ③术后24h时，取各组小鼠脾脏组织，流式细胞仪检测T淋巴细胞凋亡； ④术后24h时，取各组小鼠外周血，ELISA法检测细胞因子TNF-α、IL-10、IL-6和IFN-γ水平； ⑤术后24h时，，各组小鼠行腹腔灌洗，流式细胞仪检测腹腔灌洗液内中性粒细胞数量检测。 【结果】 1、建立CLP模型术后24h时，脓毒症模型组小鼠脾脏淋巴细胞特别是CD8+T细胞上Tim3分子表达水平显著高于Sham组（P值均小于0.01）。 2、建立CLP模型术后立刻腹腔给予anti-Tim3组小鼠抗Tim3抗体，CLP组小鼠腹腔注射生理盐水，Isotype组小鼠腹腔注射同型对照抗体，结果表明anti-Tim3组小鼠生存时间及生存率显著高于Isotype组和CLP组（P值小于0.01）。 3、建立CLP模型术后立刻腹腔给予anti-Tim3组小鼠抗Tim3抗体，CLP组小鼠腹腔注射生理盐水，Isotype组小鼠腹腔注射同型对照抗体。 ①24h时anti-Tim3组小鼠外周血检测细菌清除率显著高于CLP组和Isotype组（P值小于0.01）； ②24h时anti-Tim3组小鼠腹腔灌洗液细菌清除率显著高于CLP组和Isotype组（P值小于0.01）。 4、建立CLP模型术后立刻腹腔给予anti-Tim3组小鼠抗Tim3抗体，CLP组小鼠腹腔注射生理盐水，Isotype组小鼠腹腔注射同型对照抗体。 ①24h时anti-Tim3组小鼠肺组织、肝组织病理损伤程度显著轻于Isotype组和CLP组（P值小于0.01）； ②24h时小鼠胸腺、脾脏组织TUNEL染色显示anti-Tim3组细胞凋亡显著少于CLP组和Isotype组，染色阳性的细胞比例也显著降低（P值小于0.01）； ③24h时anti-Tim3组胸腺、脾脏组织细胞凋亡显著少于CLP组和Isotype组（P值小于0.01）； ④24h时anti-Tim3组小鼠外周血细胞因子TNF-α、IL-6和IFN-γ的水平显著低于CLP组和Isotype组。anti-Tim3组小鼠外周血IL-10水平显著高于CLP组和Isotype组（P值小于0.01）； ⑤24h时anti-Tim3组小鼠腹腔灌洗液中性粒细胞数量显著高于CLP组和Isotype组（P值小于0.01）。 【结论】 脓毒症小鼠脾脏CD8+T细胞Tim3分子表达显著增加，抗Tim3抗体可改善脓毒症小鼠7d生存率。抗Tim-3抗体对脓毒症小鼠的保护作用可能与其改善脓毒症小鼠肝脏、肺脏病理损伤程度，抑制脓毒症小鼠胸腺和脾脏细胞凋亡，提高腹腔和血液细菌清除率，以及调节腹腔机体免疫功能中性粒细胞数量相关。
[Abstract]:[purpose]
In this study, we studied the changes of thymus, spleen, lung and liver in immune and pathological level after septic mice treated with anti Tim3 antibody. We studied whether Tim3 antibody therapy could improve the survival rate of septic mice, and explored its possible mechanism.
[research methods]
1, establish a sepsis mouse model induced by CLP operation. 12 C57BL/6 mice were randomly divided into sepsis model group (CLP, n=6) and sham operation group (Sham, n=6).CLP model, 24h was established. Flow cytometry was used to detect the level of CD8+T cell Tim3 molecule in each group of mice.
2, select the 40 C57BL/6 mice were randomly divided into sham operation group (Sham, n=10), CLP+ intraperitoneal injection of saline group (CLP, n=10), CLP+ intraperitoneal injection of anti Tim3 antibody group (anti-Tim3, n=10) and intraperitoneal injection of CLP+ isotype control antibody group (Isotype, n=10). Each group that moment intraperitoneal injection of saline, anti Tim3 antibody or isotype control antibody (each only 50 mu g/150 Mu L), recorded 7d mice survival time and survival rate.
3, select the 24 C57BL/6 mice were randomly divided into sham operation group (Sham, n=6), CLP+ intraperitoneal injection of saline (CLP, n=6), CLP+ intraperitoneal injection of anti Tim3 antibody group (anti-Tim3, n=6) and intraperitoneal injection of CLP+ isotype control antibody group (Isotype, n=6). Each group immediately after the operation to intraperitoneal injection of saline, anti Tim3 antibody or isotype control antibody (each only 50 mu g/150 Mu L).
(1) the peripheral blood of each mouse was taken at 24h after operation to detect the bacterial clearance rate in the peripheral blood of mice.
After 24h, the mice were treated with abdominal lavage, and the peritoneal lavage fluid was taken to detect the bacterial clearance rate.
4, select the 24 C57BL/6 mice were randomly divided into sham operation group (Sham, n=6), CLP+ intraperitoneal injection of saline (CLP, n=6), CLP+ intraperitoneal injection of anti Tim3 antibody group (anti-Tim3, n=6) and intraperitoneal injection of CLP+ isotype control antibody group (Isotype, n=6). Each group immediately after the operation to saline, anti Tim3 antibody or isotype control antibody (each only 50 mu g/150 Mu L).
(1) after 24h, the lung tissues and liver tissues of all the mice were examined by pathological examination.
(2) after 24h, the thymus and spleen tissues of each group were examined by TUNEL staining, and the positive rate was analyzed at the same time.
(3) after 24h, the spleen tissues of all the mice were taken and the apoptosis of T lymphocytes was detected by flow cytometry.
(4) after 24h, the peripheral blood of all mice was taken, and the levels of cytokines TNF- a, IL-10, IL-6 and IFN- gamma were detected by ELISA.
At 24h after operation, the mice in each group were treated with abdominal lavage, and the flow cytometry was used to detect the number of neutrophils in the peritoneal lavage fluid.
[results]
1, when the CLP model was established at 24h, the expression level of Tim3 in splenic lymphocytes, especially CD8+T cells in sepsis model group was significantly higher than that in Sham group (P value was less than 0.01).
2, the establishment of CLP model operation immediately after intraperitoneal administration of anti-Tim3 mice anti Tim3 antibody, CLP group of mice by intraperitoneal injection of saline, Isotype group of mice by intraperitoneal injection of isotype control antibody, the results showed that anti-Tim3 mice survival time and survival rate was significantly higher than that of Isotype group and CLP group (P value less than 0.01).
3, establish the CLP model. Immediately after the operation, anti-Tim3 group mice were given anti Tim3 antibody immediately, CLP group was intraperitoneally injected with normal saline, and Isotype group was intraperitoneally injected with the same type of control antibody.
(1) the bacterial clearance rate of peripheral blood test in group anti-Tim3 mice was significantly higher than that of group CLP and Isotype group at 24h (P value was less than 0.01).
(2) the bacterial clearance rate of peritoneal lavage fluid in group anti-Tim3 mice was significantly higher than that of group CLP and Isotype group (P value was less than 0.01) at 24h.
4, establish the CLP model. Immediately after the operation, anti-Tim3 group mice were given anti Tim3 antibody immediately, CLP group was intraperitoneally injected with normal saline, and Isotype group was intraperitoneally injected with the same type of control antibody.
(1) the pathological damage of lung tissue in group anti-Tim3 mice was significantly lighter than that of group Isotype and CLP group (P value was less than 0.01) at 24h.
At 24h, TUNEL staining in thymus and spleen showed that apoptosis in anti-Tim3 group was significantly less than that in group CLP and Isotype, and the proportion of staining positive cells was also significantly decreased (P value was less than 0.01).
(3) the apoptosis of the thymus in group anti-Tim3 was significantly less than that of group CLP and Isotype (P value was less than 0.01) at 24h.
At 24h, the levels of cytokines TNF-, IL-6 and IFN- in peripheral blood of anti-Tim3 group were significantly lower than those of CLP group and Isotype group, and IL-10 level of.Anti-Tim3 group was significantly higher than that of CLP group and Isotype group (the value of IL-6 was less than 0.01).
5. At 24h, the number of neutrophils in the peritoneal lavage fluid of group anti-Tim3 mice was significantly higher than that of group CLP and Isotype group (P value was less than 0.01).
[Conclusion]
The expression of sepsis mice spleen CD8+T cell Tim3 molecule increased significantly, the anti Tim3 antibody can improve sepsis mice survival rate of 7D. The protective effect of anti Tim-3 antibody in sepsis mice may be related to the improvement of sepsis in mice liver, lung injury, inhibition of sepsis mice thymus and spleen cell apoptosis, increase peritoneal the blood and the bacterial clearance rate, and regulate the immune function of peritoneal neutrophil number.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R459.7

【参考文献】

相关期刊论文 前6条

1 张志英;栾斌;冯晓霞;;Galectin-9及其受体Tim-3在哮喘小鼠肺组织中的表达[J];中国当代儿科杂志;2011年05期

2 姚强;;基质金属蛋白酶及其与恶性肿瘤的关系[J];中外医疗;2010年06期

3 薛克营;熊盛道;熊维宁;倪望;陈仕新;李丹;;丹参注射液对哮喘大鼠气道炎症及CD4~+CD25~+调节性T细胞的影响[J];华中科技大学学报(医学版);2008年01期

4 张艳芬;许重洁;杨保胜;张光谋;;环磷酰胺对小鼠腹腔巨噬细胞功能的影响[J];新乡医学院学报;2008年04期

5 顾危页;庞智;郑家驹;沈必武;周春立;皇甫照;;CD_4~+CD_(25)~(high)Tr和CTLA-4与炎症性肠病的关系研究[J];实用临床医药杂志;2010年15期

6 杨子彬;李善华;张纪军;刘莹;冯国鹏;;强直性脊柱炎与Tim分子[J];中国煤炭工业医学杂志;2010年10期

相关博士学位论文 前1条

1 娄景盛;抗LAG-3抗体对脓毒症小鼠保护作用及免疫功能影响的研究[D];第二军医大学;2012年

本文编号：1530359