孕酮对大肠杆菌感染山羊子宫内膜的影响机制
发布时间：2018-07-16 20:16
【摘要】：奶牛子宫内膜炎是奶牛产后常发的生殖疾病,常造成奶牛不孕率增加,产奶量下降,给奶牛业造成巨大的损失。孕酮作为类固醇类激素,能够引起子宫腔中细胞因子的积聚,抑制体外淋巴细胞的功能和体内抗体的形成,对子宫免疫产生一定的影响。为了探究孕酮对大肠杆菌感染山羊子宫内膜的影响机制,本试验以成年杂交母山羊为试验动物,通过对试验羊肌肉注射孕酮,观察山羊子宫感染大肠杆菌后生理指标、血液和子宫分泌物的WBC数目变化、子宫内膜组织结构变化、子宫内膜组织中TLR4、IL-1β、IL-6、IL-8和TNF-α的mRNA表达水平变化以及子宫内膜组织中TGF-β、VEGF和EGFR的mRNA表达水平变化。试验方法:12只体重30～40kg杂交母山羊随机分为4组:对照组、大肠杆菌组、孕酮组和孕酮+大肠杆菌组。实验第1天开始,每天上午9点对孕酮组和孕酮+大肠杆菌组实施肌内注射黄体酮注射液(1mg/kg),实验第3天通过宫腔镜向大肠杆菌组和孕酮+大肠杆菌组山羊子宫内注入大肠杆菌液5mL(109cfu/mL),同时向对照组和孕酮组山羊子宫内注入生理盐水5mL;动物试验连续进行10天;分别在注菌后0h、12h、24h、96h和168h测定试验山羊的体温(T)、脉搏(P)和呼吸(R),同时颈静脉无菌采集外周血检测WBC数目变化;分别在注菌后第24h、96h和168h用灭菌棉拭子采集子宫分泌物涂布于载玻片上,Diff Quick染色后镜检WBC数目变化;于注菌后第1天、第4天、第7天通过宫腔镜无菌采集各组试验羊子宫内膜组织,部分组织置于10%中性福尔马林溶液固定,用于病理组织学观察,部分组织用PBS液洗涤后放于无RNA酶的冻存管中并立即放入液氮保存,用于基因表达检测。结果如下:(1)在接菌后第12h和第24h,与对照组相比,大肠杆菌组和孕酮+大肠杆菌组试验羊体温明显上升,且差异极显著(p0.01);在接菌后第168h,与大肠杆菌组相比,孕酮+大肠杆菌组试验羊体温下降,且差异显著(p0.05);在接菌后第12h、24h,与对照组相比,大肠杆菌组试验羊外周血WBC数目明显升高,且差异极显著(p0.01),与大肠杆菌组相比,孕酮+大肠杆菌组试验羊外周血WBC数目明显降低,且差异显著(p0.05);接菌后第1天,与对照组相比,大肠杆菌组与孕酮+大肠杆菌组试验羊子宫分泌物中WBC数目明显增多;与大肠杆菌组相比,孕酮+大肠杆菌组试验羊子宫分泌物中WBC数目明显减少。(2)在接菌后第1天,大肠杆菌组山羊子宫组织中可见大量白细胞浸润,子宫黏膜损伤,毛细血管充血;孕酮+大肠杆菌组山羊子宫组织切片可见少量白细胞浸润。在接菌后第4天,大肠杆菌组山羊子宫组织中依然可见大量白细胞浸润,其他各组子宫组织未见明显变化。(3)在接菌后第1天,与对照组相比,大肠杆菌组试验羊子宫内膜组织TLR4、IL-6、TNF-α、IL-1β和IL-8的mRNA相对表达量明显升高,且差异显著(p0.05);与大肠杆菌组相比,孕酮+大肠杆菌组的试验羊子宫内膜组织TLR4、IL-6、IL-8的mRNA相对表达量明显降低,且差异显著(p0.05)。在接菌后第4天,与大肠杆菌组相比,孕酮+大肠杆菌组试验羊子宫内膜组织TLR4、TNF-α的mRNA相对表达量明显下降,且差异显著(p0.05)。(4)在接菌后第1天,与对照组相比,大肠杆菌组与孕酮组试验羊子宫内膜组织TGF-βmRNA相对表达量升高,但差异不显著(p0.05),大肠杆菌组、孕酮组和孕酮+大肠杆菌组试验羊子宫内膜组织VEGF和EGFR的mRNA相对表达量明显降低,仅大肠杆菌组差异显著(p0.05)。与大肠杆菌组相比,孕酮+大肠杆菌组试验羊子宫内膜组织TGF-βmRNA相对表达量降低,但差异不显著(p0.05),孕酮+大肠杆菌组试验羊子宫内膜组织VEGFmRNA相对表达量升高,但差异不显著(p0.05)。结论:山羊子宫接注大肠杆菌后,山羊的T、P和R以及外周血与子宫分泌物中WBC数目增多,山羊子宫发生急性感染。孕酮能够调节子宫接注大肠杆菌后山羊子宫内膜中TLR4、IL-6、TNF-α、IL-1β 和 IL-8 的基因表达,同时能够调节 TGF-β、VEGF 和 EGFR的基因表达,提示孕酮通过对炎性细胞因子以及细胞增殖因子表达的调节对子宫局部免疫起促进作用,对子宫内膜修复无明显作用。
[Abstract]:Cow endometritis is a common reproductive disease after dairy cows. It is often made to increase the infertility rate and decrease the milk production and cause huge loss to dairy cattle. Progesterone as a steroid hormone can cause the accumulation of cytokines in the uterine cavity, inhibit the function of the lymphocytes in vitro and the formation of antibodies in the body, and produce an immune system to the uterus. In order to explore the effect mechanism of progesterone on the endometrium of Escherichia coli infected goat endometrium, this experiment took the adult female goat as experimental animal. By injecting progesterone into the sheep muscle, the physiological indexes of the infected goat uterus after infection of Escherichia coli, the change of the number of WBC in the blood and uterus secretion and the change of the endometrium tissue structure were observed. Changes in the level of mRNA expression of TLR4, IL-1 beta, IL-6, IL-8 and TNF- alpha in endometrium and the change of TGF- beta, VEGF and EGFR mRNA expressions in endometrium. Experimental methods: 12 groups of 30 to 40kg hybrid female goats were randomly divided into 4 groups: control group, coliform group, progesterone group and progesterone + Escherichia coli group. The experiment began on day first days. Intramuscular injection of Progesterone Injection (1mg/kg) was performed on progesterone group and progesterone + Escherichia coli group at 9 a.m., and 5mL (109cfu/mL) was injected into the uterus of the Escherichia coli group and the progesterone + Escherichia coli group by hysteroscopy for the third day, and the normal saline 5mL was injected into the uterus of the control group and the progesterone group. The animal test was continuous. After 10 days, the body temperature (T), pulse (P) and respiration (R) of the goats were measured after 0h, 12h, 24h, 96h and 168h respectively. Meanwhile, the number of WBC changes was detected in the peripheral blood of the jugular vein. After first days, fourth days, seventh days after the injection of bacteria, the endometrium of sheep was tested by hysteroscopy. Some tissues were fixed in 10% neutral formalin solution and used for histopathological observation. Some tissues were washed with PBS liquid in the cryopreservation tube without RNA enzyme and stored in liquid nitrogen immediately. The results were as a result, for example. (1) (1) compared with the control group, the body temperature of the Escherichia coli group and the progesterone + Escherichia coli group increased significantly, and the difference was very significant (P0.01) compared with the control group. Compared with the Escherichia coli group, the progesterone + Escherichia coli group was compared with the Escherichia coli group, and the difference was significant (P0.05), and 12h, 24h, and the control group after receiving the bacteria. Compared with the coliform group, the number of WBC in the peripheral blood of the sheep was significantly increased and the difference was very significant (P0.01). Compared with the Escherichia coli group, the number of WBC in the peripheral blood of the progesterone + Escherichia coli group was significantly reduced and the difference was significant (P0.05). Compared with the control group, the Escherichia coli group and the progesterone + Escherichia coli group tested the W of the uterine secretion in the sheep first days after the inoculation. The number of BC increased significantly. Compared with the Escherichia coli group, the number of WBC in the uterus secretions of the progesterone + Escherichia coli group decreased significantly. (2) a large number of leukocyte infiltration, uterine mucosa injury, capillaries were found in the uterus tissue of the Escherichia coli group, and the uterus tissue section of the progesterone + Escherichia coli group goat was less visible. A large amount of leukocyte infiltration still visible in the uterus tissue of the Escherichia coli group at fourth days after the inoculation, and there was no obvious change in the other groups of uterus. (3) the relative expression of TLR4, IL-6, TNF- a, IL-1 beta and IL-8 in the endometrium of the sheep endometrium was significantly higher than that of the control group after first days after the inoculation. Compared with the Escherichia coli group, the mRNA relative expression of TLR4, IL-6, IL-8 in the endometrium of progesterone + Escherichia coli decreased significantly (P0.05), and was significantly different (P0.05). Compared with the Escherichia coli group, the progesterone + Enterobacteriaceae group tested the TLR4 of the endometrium and the mRNA relative table of TNF- a in the sheep endometrium fourth days after the inoculation of the Escherichia coli group. The difference was significant (P0.05). (4) the relative expression of TGF- beta mRNA in the endometrium of the coliform group and progesterone group increased, but the difference was not significant (P0.05) compared with the control group after first days after the inoculation, and the mRNA relative to the mRNA of the endometrium of sheep endometrium in the sheep endometrium of the coliform group and progesterone group and the progesterone + Escherichia coli group were relative to the mRNA in the endometrium of the sheep. Compared with the Escherichia coli group, the relative expression of TGF- beta mRNA in the endometrium tissue of the progesterone + Escherichia coli group decreased, but the difference was not significant (P0.05), but the relative expression of VEGFmRNA in the endometrium endometrium of progesterone + Escherichia coli group increased, but the difference was not significant (P0.05). Conclusion: the number of T, P and R in goat's uterus increased and the number of WBC in the peripheral blood and uterine secretions increased and the goat uterus had an acute infection. Progesterone could regulate the gene expression of TLR4, IL-6, TNF- a, IL-1 beta and IL-8 in the endometrium of the uterus and could regulate TGF- beta, VEGF and EGFR. Gene expression suggests that progesterone promotes the local immunization of the uterus by regulating the expression of inflammatory cytokines and cell proliferators, and has no significant effect on endometrial repair.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.27
本文编号：2127557
[Abstract]:Cow endometritis is a common reproductive disease after dairy cows. It is often made to increase the infertility rate and decrease the milk production and cause huge loss to dairy cattle. Progesterone as a steroid hormone can cause the accumulation of cytokines in the uterine cavity, inhibit the function of the lymphocytes in vitro and the formation of antibodies in the body, and produce an immune system to the uterus. In order to explore the effect mechanism of progesterone on the endometrium of Escherichia coli infected goat endometrium, this experiment took the adult female goat as experimental animal. By injecting progesterone into the sheep muscle, the physiological indexes of the infected goat uterus after infection of Escherichia coli, the change of the number of WBC in the blood and uterus secretion and the change of the endometrium tissue structure were observed. Changes in the level of mRNA expression of TLR4, IL-1 beta, IL-6, IL-8 and TNF- alpha in endometrium and the change of TGF- beta, VEGF and EGFR mRNA expressions in endometrium. Experimental methods: 12 groups of 30 to 40kg hybrid female goats were randomly divided into 4 groups: control group, coliform group, progesterone group and progesterone + Escherichia coli group. The experiment began on day first days. Intramuscular injection of Progesterone Injection (1mg/kg) was performed on progesterone group and progesterone + Escherichia coli group at 9 a.m., and 5mL (109cfu/mL) was injected into the uterus of the Escherichia coli group and the progesterone + Escherichia coli group by hysteroscopy for the third day, and the normal saline 5mL was injected into the uterus of the control group and the progesterone group. The animal test was continuous. After 10 days, the body temperature (T), pulse (P) and respiration (R) of the goats were measured after 0h, 12h, 24h, 96h and 168h respectively. Meanwhile, the number of WBC changes was detected in the peripheral blood of the jugular vein. After first days, fourth days, seventh days after the injection of bacteria, the endometrium of sheep was tested by hysteroscopy. Some tissues were fixed in 10% neutral formalin solution and used for histopathological observation. Some tissues were washed with PBS liquid in the cryopreservation tube without RNA enzyme and stored in liquid nitrogen immediately. The results were as a result, for example. (1) (1) compared with the control group, the body temperature of the Escherichia coli group and the progesterone + Escherichia coli group increased significantly, and the difference was very significant (P0.01) compared with the control group. Compared with the Escherichia coli group, the progesterone + Escherichia coli group was compared with the Escherichia coli group, and the difference was significant (P0.05), and 12h, 24h, and the control group after receiving the bacteria. Compared with the coliform group, the number of WBC in the peripheral blood of the sheep was significantly increased and the difference was very significant (P0.01). Compared with the Escherichia coli group, the number of WBC in the peripheral blood of the progesterone + Escherichia coli group was significantly reduced and the difference was significant (P0.05). Compared with the control group, the Escherichia coli group and the progesterone + Escherichia coli group tested the W of the uterine secretion in the sheep first days after the inoculation. The number of BC increased significantly. Compared with the Escherichia coli group, the number of WBC in the uterus secretions of the progesterone + Escherichia coli group decreased significantly. (2) a large number of leukocyte infiltration, uterine mucosa injury, capillaries were found in the uterus tissue of the Escherichia coli group, and the uterus tissue section of the progesterone + Escherichia coli group goat was less visible. A large amount of leukocyte infiltration still visible in the uterus tissue of the Escherichia coli group at fourth days after the inoculation, and there was no obvious change in the other groups of uterus. (3) the relative expression of TLR4, IL-6, TNF- a, IL-1 beta and IL-8 in the endometrium of the sheep endometrium was significantly higher than that of the control group after first days after the inoculation. Compared with the Escherichia coli group, the mRNA relative expression of TLR4, IL-6, IL-8 in the endometrium of progesterone + Escherichia coli decreased significantly (P0.05), and was significantly different (P0.05). Compared with the Escherichia coli group, the progesterone + Enterobacteriaceae group tested the TLR4 of the endometrium and the mRNA relative table of TNF- a in the sheep endometrium fourth days after the inoculation of the Escherichia coli group. The difference was significant (P0.05). (4) the relative expression of TGF- beta mRNA in the endometrium of the coliform group and progesterone group increased, but the difference was not significant (P0.05) compared with the control group after first days after the inoculation, and the mRNA relative to the mRNA of the endometrium of sheep endometrium in the sheep endometrium of the coliform group and progesterone group and the progesterone + Escherichia coli group were relative to the mRNA in the endometrium of the sheep. Compared with the Escherichia coli group, the relative expression of TGF- beta mRNA in the endometrium tissue of the progesterone + Escherichia coli group decreased, but the difference was not significant (P0.05), but the relative expression of VEGFmRNA in the endometrium endometrium of progesterone + Escherichia coli group increased, but the difference was not significant (P0.05). Conclusion: the number of T, P and R in goat's uterus increased and the number of WBC in the peripheral blood and uterine secretions increased and the goat uterus had an acute infection. Progesterone could regulate the gene expression of TLR4, IL-6, TNF- a, IL-1 beta and IL-8 in the endometrium of the uterus and could regulate TGF- beta, VEGF and EGFR. Gene expression suggests that progesterone promotes the local immunization of the uterus by regulating the expression of inflammatory cytokines and cell proliferators, and has no significant effect on endometrial repair.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.27
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