本文關(guān)鍵詞： 毒癥 盲腸結(jié)扎穿孔 T淋巴細(xì)胞 細(xì)胞凋亡 抗Tim3抗體　出處：《第二軍醫(yī)大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：【研究目的】 本課題主要研究膿毒癥小鼠經(jīng)抗Tim3抗體治療后，其胸腺、脾臟、肺臟和肝臟組織在免疫與病理水平的變化，研究抗Tim3抗體治療是否改善膿毒癥小鼠的生存率，并探討其可能機(jī)制。 【研究方法】 1、建立CLP手術(shù)致膿毒癥小鼠模型，實(shí)驗(yàn)對(duì)象為12只C57BL/6小鼠，隨機(jī)分成膿毒癥模型組(CLP，n=6)和假手術(shù)組(Sham, n=6)。CLP模型建立24h時(shí)，流式細(xì)胞術(shù)檢測各組小鼠脾臟組織CD8+T細(xì)胞Tim3分子表達(dá)水平。 2、選擇C57BL/6小鼠40只，隨機(jī)分為假手術(shù)組(Sham，n=10)、CLP+腹腔注射生理鹽水組(CLP，n=10)、CLP+腹腔注射抗Tim3抗體組（anti-Tim3，n=10)和CLP+腹腔注射同型對(duì)照抗體組(Isotype，n=10)。各組術(shù)后即刻予腹腔注射生理鹽水、抗Tim3抗體或同型對(duì)照抗體(均每只50μg/150μl)，記錄各組小鼠7d的生存時(shí)間及生存率。 3、選擇C57BL/6小鼠24只，隨機(jī)分為假手術(shù)組(Sham，n=6)、CLP+腹腔注射生理鹽水(CLP，n=6)、CLP+腹腔注射抗Tim3抗體組(anti-Tim3，n=6)和CLP+腹腔注射同型對(duì)照抗體組(Isotype，n=6)。各組術(shù)后即刻予以腹腔注射生理鹽水、抗Tim3抗體或同型對(duì)照抗體(均每只50μg/150μl)。 ①術(shù)后24h時(shí)取各組小鼠外周血，檢測小鼠外周血細(xì)菌清除率； ②術(shù)后24h時(shí)各組小鼠行腹腔灌洗，取腹腔灌洗液檢測細(xì)菌清除率。 4、選擇C57BL/6小鼠24只，隨機(jī)分為假手術(shù)組(Sham，n=6)、CLP+腹腔注射生理鹽水(CLP，n=6)、CLP+腹腔注射抗Tim3抗體組(anti-Tim3，n=6)和CLP+腹腔注射同型對(duì)照抗體組(Isotype，n=6)。各組術(shù)后即刻給予生理鹽水、抗Tim3抗體或同型對(duì)照抗體(均每只50μg/150μl)。 ①術(shù)后24h時(shí)，取各組小鼠肺組織、肝臟組織，進(jìn)行病理學(xué)檢驗(yàn)； ②術(shù)后24h時(shí)，取各組小鼠胸腺組織、脾臟組織，進(jìn)行TUNEL染色病理檢驗(yàn)，同時(shí)進(jìn)行陽性率分析； ③術(shù)后24h時(shí)，取各組小鼠脾臟組織，流式細(xì)胞儀檢測T淋巴細(xì)胞凋亡； ④術(shù)后24h時(shí)，取各組小鼠外周血，ELISA法檢測細(xì)胞因子TNF-α、IL-10、IL-6和IFN-γ水平； ⑤術(shù)后24h時(shí)，，各組小鼠行腹腔灌洗，流式細(xì)胞儀檢測腹腔灌洗液內(nèi)中性粒細(xì)胞數(shù)量檢測。 【結(jié)果】 1、建立CLP模型術(shù)后24h時(shí)，膿毒癥模型組小鼠脾臟淋巴細(xì)胞特別是CD8+T細(xì)胞上Tim3分子表達(dá)水平顯著高于Sham組（P值均小于0.01）。 2、建立CLP模型術(shù)后立刻腹腔給予anti-Tim3組小鼠抗Tim3抗體，CLP組小鼠腹腔注射生理鹽水，Isotype組小鼠腹腔注射同型對(duì)照抗體，結(jié)果表明anti-Tim3組小鼠生存時(shí)間及生存率顯著高于Isotype組和CLP組（P值小于0.01）。 3、建立CLP模型術(shù)后立刻腹腔給予anti-Tim3組小鼠抗Tim3抗體，CLP組小鼠腹腔注射生理鹽水，Isotype組小鼠腹腔注射同型對(duì)照抗體。 ①24h時(shí)anti-Tim3組小鼠外周血檢測細(xì)菌清除率顯著高于CLP組和Isotype組（P值小于0.01）； ②24h時(shí)anti-Tim3組小鼠腹腔灌洗液細(xì)菌清除率顯著高于CLP組和Isotype組（P值小于0.01）。 4、建立CLP模型術(shù)后立刻腹腔給予anti-Tim3組小鼠抗Tim3抗體，CLP組小鼠腹腔注射生理鹽水，Isotype組小鼠腹腔注射同型對(duì)照抗體。 ①24h時(shí)anti-Tim3組小鼠肺組織、肝組織病理損傷程度顯著輕于Isotype組和CLP組（P值小于0.01）； ②24h時(shí)小鼠胸腺、脾臟組織TUNEL染色顯示anti-Tim3組細(xì)胞凋亡顯著少于CLP組和Isotype組，染色陽性的細(xì)胞比例也顯著降低（P值小于0.01）； ③24h時(shí)anti-Tim3組胸腺、脾臟組織細(xì)胞凋亡顯著少于CLP組和Isotype組（P值小于0.01）； ④24h時(shí)anti-Tim3組小鼠外周血細(xì)胞因子TNF-α、IL-6和IFN-γ的水平顯著低于CLP組和Isotype組。anti-Tim3組小鼠外周血IL-10水平顯著高于CLP組和Isotype組（P值小于0.01）； ⑤24h時(shí)anti-Tim3組小鼠腹腔灌洗液中性粒細(xì)胞數(shù)量顯著高于CLP組和Isotype組（P值小于0.01）。 【結(jié)論】 膿毒癥小鼠脾臟CD8+T細(xì)胞Tim3分子表達(dá)顯著增加，抗Tim3抗體可改善膿毒癥小鼠7d生存率�？筎im-3抗體對(duì)膿毒癥小鼠的保護(hù)作用可能與其改善膿毒癥小鼠肝臟、肺臟病理損傷程度，抑制膿毒癥小鼠胸腺和脾臟細(xì)胞凋亡，提高腹腔和血液細(xì)菌清除率，以及調(diào)節(jié)腹腔機(jī)體免疫功能中性粒細(xì)胞數(shù)量相關(guān)。
[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.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R459.7

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