盲肠结扎穿孔模型小鼠脾NK细胞计数及其亚群改变
发布时间：2018-09-18 20:08
【摘要】：脓毒症是针对感染的失调的宿主反应引起的危及生命的器官功能障碍,是重症监护室(ICU)的常见疾病,其发生率和死亡率一直居高不下。近十年来研究发现,脓毒症后期的免疫抑制是脓毒症死亡的主要原因,且越来越多的研究显示,自然杀伤细胞(natural killer cells,NK cells)是参与脓毒症免疫反应的一类重要的免疫细胞。作为一类同时参与固有免疫反应和适应性免疫反应的大颗粒淋巴细胞,NK细胞在脓毒症的免疫中发挥着至关重要的作用,已有临床研究证实,人类NK细胞及NK细胞亚群CD56brightNK细胞和CD56dimNK细胞百分比在脓毒症后期明显下降。然而,CD56却并不存在于小鼠的NK细胞上,这在一定程度上限制了NK细胞在脓毒症中的研究。近年来,有研究发现,根据NK细胞表面标记CD27和CD11b可将人类和鼠科动物NK细胞分为四个亚群CD27-CD11b+,CD27+CD11b+,CD27+CD11b-,CD27-CD11b-,也可根据功能不同将NK细胞分为三个功能亚群,NKcytotoxic,NKregulatory,NKtolerant,其中NKcytotoxic主要包括CD27-CD11b+NK细胞,NKregulatory主要包括CD27+CD11b+和CD27+CD11b-NK细胞,NKtolerant主要包括CD27-CD11b-NK细胞。本研究分析了小鼠脾NK细胞、CD27和CD11b标记的NK细胞亚群、以及相应的功能亚群在脓毒症免疫抑制期的变化,为NK细胞在脓毒症中的基础研究和临床研究提供了桥梁,同时希望为脓毒症机制和治疗的进一步研究提供理论依据。第一部分:脓毒症小鼠生存率变化、脓毒症小鼠肺组织和小肠组织的病理变化及脓毒症小鼠血清炎症因子浓度变化目的:通过盲肠结扎和穿孔术(CLP)制备脓毒症模型,(1)观察记录脓毒症小鼠生存率的变化。(2)通过HE染色检测脓毒症小鼠肺组织和小肠组织不同时间点的病理变化。(3)通过ELISA法检测不同时间点的血清炎症因子TNF-α和IL-10的变化。方法:(1)选择体重20~30g,8~10周龄雄性SPF级C57bl/6小鼠60只,采用随机数字表法随机分为2组:假手术组(Sham组)和脓毒症组(CLP组),每组30只小鼠,制备盲肠结扎穿孔(CLP)模型和假手术模型,分别记录8 d内脓毒症小鼠的生存率。(2)选择体重20~30 g,8~10周龄雄性SPF级C57bl/6小鼠,制备盲肠结扎穿孔(CLP)模型,分别于术后24h、48h和72 h点断颈处死小鼠,取小鼠小肠组织,肺组织(每组样本量为3)。根据HE染色结果比较sham组小鼠、脓毒症组24h、48 h和72 h小鼠小肠组织和肺组织的病理改变。(3)选择体重20~30 g,8~10周龄雄性SPF级C57bl/6小鼠,制备盲肠结扎穿孔(CLP)模型,于术后2h、4h、6h、24h、48h和72 h时将小鼠断颈处死,摘取小鼠眼球取血,制备血清后将血清放置于-80℃冰箱冻存(每组样本量为6只)。采用ELISA试剂盒分别检测脓毒症2h、4h、6h、24h、48h和72 h时与Sham组比较血清TNF-α和IL-10的浓度变化。结果:(1)造模后8d内,Sham组的生存率为100%,脓毒症组则在不同时间点的生存率都较Sham组降低。(2)HE染色结果显示,Sham组小肠黏膜结构正常,可见清晰完整的小肠绒毛,而CLP各组小鼠小肠组织排列紊乱,充血明显,可见明显的炎细胞浸润,肠绒毛大片断裂、脱落。于CLP24h小肠组织病理改变较明显。Sham组肺组织结构正常,肺泡结构完整,没有毛细血管扩张、肺泡壁增宽及炎细胞浸润。而CLP各组肺组织表现为肺泡壁增厚、充血、水肿,肺泡结构紊乱,肺泡内有大量炎细胞浸润、毛细血管扩张等改变。尤其于CLP24h其病理改变最明显。(3)运用ELISA试剂盒检测血清促炎因子TNF-α和血清抗炎因子IL-10浓度在脓毒症不同时间点与Sham组的变化。结果显示,在CLP2h血清促炎因子TNF-α升高(P0.05),于CLP6h其升高程度达到峰值(P0.05)。CLP24h、CLP48hTNF-α与Sham组比较,结果无统计学差异。与CLP6h比较明显下降。血清抗炎因子IL-10则在CLP6h,CLP24h,CLP 72h也较Sham组明显升高(P0.05),在48h无明显变化。结论:盲肠结扎穿孔模型小鼠的生存率较低显示,其模型建立有效。脓毒症小鼠炎症最早侵犯部位小肠及最早转移部位肺均发生炎症性病理改变。脓毒症早期表现为以释放大量的促炎因子TNF-α为主,脓毒症后期则表现为以释放大量抗炎因子IL-10为主。这一结论为NK细胞在脓毒症免疫抑制期的研究提供了时间依据。第二部分:脓毒症小鼠脾NK细胞及NK细胞亚群在脓毒症后期的变化目的:通过盲肠结扎和穿孔术(CLP)制备脓毒症模型,探讨脓毒症小鼠脾NK细胞及其亚群的变化,为脓毒症机制和治疗的进一步研究提供参考。方法:体重20~30g,8~10周龄雄性SPF级C57bl/6小鼠,制备盲肠结扎穿孔(CLP)模型。运用流式细胞仪技术分别检测脓毒症24h,48h,72h与Sham组比较,NK细胞数目及其各亚群(CD27-CD11b+,CD27+CD11b+,CD27+CD11b-,CD27-CD11b-)的百分比变化,同时分析各功能亚群的变化(每组样本量为6只)。结果:小鼠脾NK细胞数目在CLP24h,48h,72h都较对照组明显降低(P0.05);NK细胞的各个细胞亚群与Sham组相比,NKcytotoxic(CD27-CD11b+)在CLP48h显著降低(P0.05);NKregulatory(CD27+CD11b+和CD27+CD11b-)在CLP48h显著升高(P0.05);NKtolerant(CD27-CD11b-)在CLP24h,48h降低(P0.05)在CLP72h却表现为升高(P0.05)。结论:NK细胞及其亚群参与脓毒症免疫抑制期的形成,并且在其中发挥着重要的作用。
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R-332;R459.7
本文编号：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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R-332;R459.7
【参考文献】
相关期刊论文 前6条
1 郑丹阳;吴惠玲;陈晓明;李涛;刘良明;;血管内皮细胞微粒在脓毒症大鼠血管渗漏中的作用[J];第三军医大学学报;2016年08期
2 ;中国严重脓毒症/脓毒性休克治疗指南(2014)[J];全科医学临床与教育;2015年04期
3 罗勇;刘强;田先雨;刘纯义;郑晓芬;;严重脓毒症患儿外周血自然杀伤细胞比例、亚群的变化[J];社区医学杂志;2014年14期
4 朱晓娜;王邦松;李庆兴;陈迎晓;张慧芳;;大鼠鲍氏不动杆菌性脓毒症血清中肿瘤坏死因子α与白细胞介素-18的变化[J];中华医院感染学杂志;2009年18期
5 姚咏明,盛志勇;内毒素与革兰阳性菌致病因子的协同效应与意义[J];中国危重病急救医学;2005年04期
6 李文雄,陈惠德,王小文,邢晓燕,赵松,万力,庞宝森,黄儦庭;脓毒症患者血清TNFα、IL-10水平、IL-10/TNFα比值的变化及其临床意义[J];中华普通外科杂志;2003年04期
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