盲腸結(jié)扎穿孔模型小鼠脾NK細胞計數(shù)及其亞群改變
發(fā)布時間：2018-09-18 20:08
【摘要】：膿毒癥是針對感染的失調(diào)的宿主反應引起的危及生命的器官功能障礙,是重癥監(jiān)護室(ICU)的常見疾病,其發(fā)生率和死亡率一直居高不下。近十年來研究發(fā)現(xiàn),膿毒癥后期的免疫抑制是膿毒癥死亡的主要原因,且越來越多的研究顯示,自然殺傷細胞(natural killer cells,NK cells)是參與膿毒癥免疫反應的一類重要的免疫細胞。作為一類同時參與固有免疫反應和適應性免疫反應的大顆粒淋巴細胞,NK細胞在膿毒癥的免疫中發(fā)揮著至關重要的作用,已有臨床研究證實,人類NK細胞及NK細胞亞群CD56brightNK細胞和CD56dimNK細胞百分比在膿毒癥后期明顯下降。然而,CD56卻并不存在于小鼠的NK細胞上,這在一定程度上限制了NK細胞在膿毒癥中的研究。近年來,有研究發(fā)現(xiàn),根據(jù)NK細胞表面標記CD27和CD11b可將人類和鼠科動物NK細胞分為四個亞群CD27-CD11b+,CD27+CD11b+,CD27+CD11b-,CD27-CD11b-,也可根據(jù)功能不同將NK細胞分為三個功能亞群,NKcytotoxic,NKregulatory,NKtolerant,其中NKcytotoxic主要包括CD27-CD11b+NK細胞,NKregulatory主要包括CD27+CD11b+和CD27+CD11b-NK細胞,NKtolerant主要包括CD27-CD11b-NK細胞。本研究分析了小鼠脾NK細胞、CD27和CD11b標記的NK細胞亞群、以及相應的功能亞群在膿毒癥免疫抑制期的變化,為NK細胞在膿毒癥中的基礎研究和臨床研究提供了橋梁,同時希望為膿毒癥機制和治療的進一步研究提供理論依據(jù)。第一部分:膿毒癥小鼠生存率變化、膿毒癥小鼠肺組織和小腸組織的病理變化及膿毒癥小鼠血清炎癥因子濃度變化目的:通過盲腸結(jié)扎和穿孔術(shù)(CLP)制備膿毒癥模型,(1)觀察記錄膿毒癥小鼠生存率的變化。(2)通過HE染色檢測膿毒癥小鼠肺組織和小腸組織不同時間點的病理變化。(3)通過ELISA法檢測不同時間點的血清炎癥因子TNF-α和IL-10的變化。方法:(1)選擇體重20~30g,8~10周齡雄性SPF級C57bl/6小鼠60只,采用隨機數(shù)字表法隨機分為2組:假手術(shù)組(Sham組)和膿毒癥組(CLP組),每組30只小鼠,制備盲腸結(jié)扎穿孔(CLP)模型和假手術(shù)模型,分別記錄8 d內(nèi)膿毒癥小鼠的生存率。(2)選擇體重20~30 g,8~10周齡雄性SPF級C57bl/6小鼠,制備盲腸結(jié)扎穿孔(CLP)模型,分別于術(shù)后24h、48h和72 h點斷頸處死小鼠,取小鼠小腸組織,肺組織(每組樣本量為3)。根據(jù)HE染色結(jié)果比較sham組小鼠、膿毒癥組24h、48 h和72 h小鼠小腸組織和肺組織的病理改變。(3)選擇體重20~30 g,8~10周齡雄性SPF級C57bl/6小鼠,制備盲腸結(jié)扎穿孔(CLP)模型,于術(shù)后2h、4h、6h、24h、48h和72 h時將小鼠斷頸處死,摘取小鼠眼球取血,制備血清后將血清放置于-80℃冰箱凍存(每組樣本量為6只)。采用ELISA試劑盒分別檢測膿毒癥2h、4h、6h、24h、48h和72 h時與Sham組比較血清TNF-α和IL-10的濃度變化。結(jié)果:(1)造模后8d內(nèi),Sham組的生存率為100%,膿毒癥組則在不同時間點的生存率都較Sham組降低。(2)HE染色結(jié)果顯示,Sham組小腸黏膜結(jié)構(gòu)正常,可見清晰完整的小腸絨毛,而CLP各組小鼠小腸組織排列紊亂,充血明顯,可見明顯的炎細胞浸潤,腸絨毛大片斷裂、脫落。于CLP24h小腸組織病理改變較明顯。Sham組肺組織結(jié)構(gòu)正常,肺泡結(jié)構(gòu)完整,沒有毛細血管擴張、肺泡壁增寬及炎細胞浸潤。而CLP各組肺組織表現(xiàn)為肺泡壁增厚、充血、水腫,肺泡結(jié)構(gòu)紊亂,肺泡內(nèi)有大量炎細胞浸潤、毛細血管擴張等改變。尤其于CLP24h其病理改變最明顯。(3)運用ELISA試劑盒檢測血清促炎因子TNF-α和血清抗炎因子IL-10濃度在膿毒癥不同時間點與Sham組的變化。結(jié)果顯示,在CLP2h血清促炎因子TNF-α升高(P0.05),于CLP6h其升高程度達到峰值(P0.05)。CLP24h、CLP48hTNF-α與Sham組比較,結(jié)果無統(tǒng)計學差異。與CLP6h比較明顯下降。血清抗炎因子IL-10則在CLP6h,CLP24h,CLP 72h也較Sham組明顯升高(P0.05),在48h無明顯變化。結(jié)論:盲腸結(jié)扎穿孔模型小鼠的生存率較低顯示,其模型建立有效。膿毒癥小鼠炎癥最早侵犯部位小腸及最早轉(zhuǎn)移部位肺均發(fā)生炎癥性病理改變。膿毒癥早期表現(xiàn)為以釋放大量的促炎因子TNF-α為主,膿毒癥后期則表現(xiàn)為以釋放大量抗炎因子IL-10為主。這一結(jié)論為NK細胞在膿毒癥免疫抑制期的研究提供了時間依據(jù)。第二部分:膿毒癥小鼠脾NK細胞及NK細胞亞群在膿毒癥后期的變化目的:通過盲腸結(jié)扎和穿孔術(shù)(CLP)制備膿毒癥模型,探討膿毒癥小鼠脾NK細胞及其亞群的變化,為膿毒癥機制和治療的進一步研究提供參考。方法:體重20~30g,8~10周齡雄性SPF級C57bl/6小鼠,制備盲腸結(jié)扎穿孔(CLP)模型。運用流式細胞儀技術(shù)分別檢測膿毒癥24h,48h,72h與Sham組比較,NK細胞數(shù)目及其各亞群(CD27-CD11b+,CD27+CD11b+,CD27+CD11b-,CD27-CD11b-)的百分比變化,同時分析各功能亞群的變化(每組樣本量為6只)。結(jié)果:小鼠脾NK細胞數(shù)目在CLP24h,48h,72h都較對照組明顯降低(P0.05);NK細胞的各個細胞亞群與Sham組相比,NKcytotoxic(CD27-CD11b+)在CLP48h顯著降低(P0.05);NKregulatory(CD27+CD11b+和CD27+CD11b-)在CLP48h顯著升高(P0.05);NKtolerant(CD27-CD11b-)在CLP24h,48h降低(P0.05)在CLP72h卻表現(xiàn)為升高(P0.05)。結(jié)論:NK細胞及其亞群參與膿毒癥免疫抑制期的形成,并且在其中發(fā)揮著重要的作用。
[Abstract]:Sepsis is a life-threatening organ dysfunction caused by an imbalanced host response to infection. It is a common disease in intensive care unit (ICU). The incidence and mortality rate of sepsis remain high. In the past decade, studies have found that immunosuppression in the late stage of sepsis is the main cause of death in sepsis. More and more studies have shown that natural causes of death are natural. Natural killer cells (NK cells) are a class of important immune cells involved in the immune response to sepsis. As a kind of large granular lymphocytes involved in both innate and adaptive immune responses, NK cells play an important role in the immune response to sepsis. The percentage of CD56 bright NK cells and CD56 dim NK cells in NK cell subsets decreased significantly in the late stage of sepsis. However, CD56 did not exist in NK cells of mice, which limited the study of NK cells in sepsis to a certain extent. In recent years, it has been found that NK cells from human and rodents can be labeled with CD27 and CD11b on the surface of NK cells. Cells can be divided into four subgroups: CD27-CD11b+, CD27+CD11b+, CD27+CD11b-, CD27-CD11b-, and CD27-CD11b-. NK cells can also be divided into three functional subgroups according to their functions, NK cytotoxicity, NK regulatory, NK tolerant. NK cytotoxicity mainly includes CD27-CD11b+NK cells, NK regulatory mainly includes CD27+CD11b+ and CD27+CD11b-NK cells, NK tolerant mainly includes CD27+CD11b-NK cells. 27-CD11b-NK cells. This study analyzed the changes of splenic NK cells, CD27 and CD11b-labeled NK cell subsets, and the corresponding functional subsets in the immunosuppressive phase of sepsis. It provided a bridge for basic and clinical research of NK cells in sepsis, and hoped to provide theoretical basis for further research on the mechanism and treatment of sepsis. Objective: To establish sepsis model by cecum ligation and perforation (CLP). (1) To observe and record the changes of survival rate in septic mice. (2) To detect small sepsis by HE staining. Methods: (1) Sixty male SPF C57bl/6 mice weighing 20-30 g and aged 8-10 weeks were randomly divided into two groups: sham operation group (Sham group) and sepsis group (CLP group). Cecal ligation and perforation (CLP) model and sham operation model were made in 30 mice in each group, and the survival rate of septic mice in 8 days was recorded respectively. (2) Male SPF C57bl/6 mice weighing 20-30 g and 8-10 weeks were selected to establish cecal ligation and perforation (CLP) model. The mice were executed at 24, 48 and 72 hours after operation, and the small intestine tissues and lung tissues (every time) were taken out. According to the HE staining results, the pathological changes of intestinal and lung tissues of sham mice, sepsis mice at 24, 48 and 72 hours were compared. Serum samples were frozen at - 80 C (6 in each group). Serum TNF - alpha and IL - 10 levels were measured by ELISA kit at 2, 4, 6, 24, 48, and 72 hours after sepsis compared with Sham group. Results: (1) Survival rate was 100% in Sham group and 100% in sepsis group within 8 days after modeling. The results of HE staining showed that the intestinal mucosa in Sham group was normal and the intestinal villi were clear and intact, while the intestinal tissues in CLP groups were disordered and hyperemia was obvious, inflammatory cell infiltration, large intestinal villi fragmentation and shedding were observed. The pathological changes of small intestinal tissues in Sham group were obvious at 24 hours after CLP24. Often, the alveolar structure is intact, without capillary dilatation, alveolar wall widening and inflammatory cell infiltration. The lung tissue of CLP groups showed thickening of alveolar wall, congestion, edema, alveolar structure disorder, a large number of inflammatory cells infiltration in alveoli, telangiectasia and other changes. Especially in CLP24h, the pathological changes were most obvious. (3) ELISA kit was used to detect serum. The results showed that the levels of proinflammatory factor TNF-alpha and serum anti-inflammatory factor IL-10 increased at CLP2h (P 0.05) and reached the peak at 6 h (P 0.05). There was no significant difference between CLP24h, 48 h TNF-alpha and Sham group. Factor IL-10 was also significantly higher in CLP6h, CLP24h, and CLP 72h than in Sham group (P 0.05). There was no significant change at 48h. Conclusion: The survival rate of cecum ligation and perforation model mice was low, and the establishment of the model was effective. This conclusion provides a time basis for the study of NK cells in the immunosuppressive phase of sepsis. Part II: Changes of splenic NK cells and NK cell subsets in septic mice in the late stage of sepsis: through cecum node Methods: Male SPF C57bl/6 mice weighing 20-30 g and aged 8-10 weeks were used to establish the model of cecum ligation and perforation (CLP). Flow cytometry was used to detect the changes of splenic NK cells and their subsets in septic mice for 24 hours and 4 weeks respectively. Compared with Sham group, the number of NK cells and their subsets (CD27-CD11b+, CD27+CD11b+, CD27+CD11b-, CD27-CD11b-) were significantly decreased at 8h, 72h, and the number of splenic NK cells in mice was significantly lower at CLP24h, 48h and 72h (P 0.05). NK cytotoxic (CD27-CD11b+) was significantly lower at 48 hours of CLP (P 0.05), NK regulatory (CD27+CD11b+ and CD27+CD11b-) was significantly higher at 48 hours of CLP (P 0.05), NK tolerant (CD27-CD11b-) was significantly lower at 24 hours of CLP 24, but decreased at 48 hours of CLP 72 hours (P 0.05). Play an important role.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R-332;R459.7
本文編號：2248985
[Abstract]:Sepsis is a life-threatening organ dysfunction caused by an imbalanced host response to infection. It is a common disease in intensive care unit (ICU). The incidence and mortality rate of sepsis remain high. In the past decade, studies have found that immunosuppression in the late stage of sepsis is the main cause of death in sepsis. More and more studies have shown that natural causes of death are natural. Natural killer cells (NK cells) are a class of important immune cells involved in the immune response to sepsis. As a kind of large granular lymphocytes involved in both innate and adaptive immune responses, NK cells play an important role in the immune response to sepsis. The percentage of CD56 bright NK cells and CD56 dim NK cells in NK cell subsets decreased significantly in the late stage of sepsis. However, CD56 did not exist in NK cells of mice, which limited the study of NK cells in sepsis to a certain extent. In recent years, it has been found that NK cells from human and rodents can be labeled with CD27 and CD11b on the surface of NK cells. Cells can be divided into four subgroups: CD27-CD11b+, CD27+CD11b+, CD27+CD11b-, CD27-CD11b-, and CD27-CD11b-. NK cells can also be divided into three functional subgroups according to their functions, NK cytotoxicity, NK regulatory, NK tolerant. NK cytotoxicity mainly includes CD27-CD11b+NK cells, NK regulatory mainly includes CD27+CD11b+ and CD27+CD11b-NK cells, NK tolerant mainly includes CD27+CD11b-NK cells. 27-CD11b-NK cells. This study analyzed the changes of splenic NK cells, CD27 and CD11b-labeled NK cell subsets, and the corresponding functional subsets in the immunosuppressive phase of sepsis. It provided a bridge for basic and clinical research of NK cells in sepsis, and hoped to provide theoretical basis for further research on the mechanism and treatment of sepsis. Objective: To establish sepsis model by cecum ligation and perforation (CLP). (1) To observe and record the changes of survival rate in septic mice. (2) To detect small sepsis by HE staining. Methods: (1) Sixty male SPF C57bl/6 mice weighing 20-30 g and aged 8-10 weeks were randomly divided into two groups: sham operation group (Sham group) and sepsis group (CLP group). Cecal ligation and perforation (CLP) model and sham operation model were made in 30 mice in each group, and the survival rate of septic mice in 8 days was recorded respectively. (2) Male SPF C57bl/6 mice weighing 20-30 g and 8-10 weeks were selected to establish cecal ligation and perforation (CLP) model. The mice were executed at 24, 48 and 72 hours after operation, and the small intestine tissues and lung tissues (every time) were taken out. According to the HE staining results, the pathological changes of intestinal and lung tissues of sham mice, sepsis mice at 24, 48 and 72 hours were compared. Serum samples were frozen at - 80 C (6 in each group). Serum TNF - alpha and IL - 10 levels were measured by ELISA kit at 2, 4, 6, 24, 48, and 72 hours after sepsis compared with Sham group. Results: (1) Survival rate was 100% in Sham group and 100% in sepsis group within 8 days after modeling. The results of HE staining showed that the intestinal mucosa in Sham group was normal and the intestinal villi were clear and intact, while the intestinal tissues in CLP groups were disordered and hyperemia was obvious, inflammatory cell infiltration, large intestinal villi fragmentation and shedding were observed. The pathological changes of small intestinal tissues in Sham group were obvious at 24 hours after CLP24. Often, the alveolar structure is intact, without capillary dilatation, alveolar wall widening and inflammatory cell infiltration. The lung tissue of CLP groups showed thickening of alveolar wall, congestion, edema, alveolar structure disorder, a large number of inflammatory cells infiltration in alveoli, telangiectasia and other changes. Especially in CLP24h, the pathological changes were most obvious. (3) ELISA kit was used to detect serum. The results showed that the levels of proinflammatory factor TNF-alpha and serum anti-inflammatory factor IL-10 increased at CLP2h (P 0.05) and reached the peak at 6 h (P 0.05). There was no significant difference between CLP24h, 48 h TNF-alpha and Sham group. Factor IL-10 was also significantly higher in CLP6h, CLP24h, and CLP 72h than in Sham group (P 0.05). There was no significant change at 48h. Conclusion: The survival rate of cecum ligation and perforation model mice was low, and the establishment of the model was effective. This conclusion provides a time basis for the study of NK cells in the immunosuppressive phase of sepsis. Part II: Changes of splenic NK cells and NK cell subsets in septic mice in the late stage of sepsis: through cecum node Methods: Male SPF C57bl/6 mice weighing 20-30 g and aged 8-10 weeks were used to establish the model of cecum ligation and perforation (CLP). Flow cytometry was used to detect the changes of splenic NK cells and their subsets in septic mice for 24 hours and 4 weeks respectively. Compared with Sham group, the number of NK cells and their subsets (CD27-CD11b+, CD27+CD11b+, CD27+CD11b-, CD27-CD11b-) were significantly decreased at 8h, 72h, and the number of splenic NK cells in mice was significantly lower at CLP24h, 48h and 72h (P 0.05). NK cytotoxic (CD27-CD11b+) was significantly lower at 48 hours of CLP (P 0.05), NK regulatory (CD27+CD11b+ and CD27+CD11b-) was significantly higher at 48 hours of CLP (P 0.05), NK tolerant (CD27-CD11b-) was significantly lower at 24 hours of CLP 24, but decreased at 48 hours of CLP 72 hours (P 0.05). Play an important role.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R-332;R459.7
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