孕酮對(duì)大腸桿菌感染山羊子宮內(nèi)膜的影響機(jī)制
發(fā)布時(shí)間：2018-07-16 20:16
【摘要】：奶牛子宮內(nèi)膜炎是奶牛產(chǎn)后常發(fā)的生殖疾病,常造成奶牛不孕率增加,產(chǎn)奶量下降,給奶牛業(yè)造成巨大的損失。孕酮作為類固醇類激素,能夠引起子宮腔中細(xì)胞因子的積聚,抑制體外淋巴細(xì)胞的功能和體內(nèi)抗體的形成,對(duì)子宮免疫產(chǎn)生一定的影響。為了探究孕酮對(duì)大腸桿菌感染山羊子宮內(nèi)膜的影響機(jī)制,本試驗(yàn)以成年雜交母山羊?yàn)樵囼?yàn)動(dòng)物,通過(guò)對(duì)試驗(yàn)羊肌肉注射孕酮,觀察山羊子宮感染大腸桿菌后生理指標(biāo)、血液和子宮分泌物的WBC數(shù)目變化、子宮內(nèi)膜組織結(jié)構(gòu)變化、子宮內(nèi)膜組織中TLR4、IL-1β、IL-6、IL-8和TNF-α的mRNA表達(dá)水平變化以及子宮內(nèi)膜組織中TGF-β、VEGF和EGFR的mRNA表達(dá)水平變化。試驗(yàn)方法:12只體重30～40kg雜交母山羊隨機(jī)分為4組:對(duì)照組、大腸桿菌組、孕酮組和孕酮+大腸桿菌組。實(shí)驗(yàn)第1天開(kāi)始,每天上午9點(diǎn)對(duì)孕酮組和孕酮+大腸桿菌組實(shí)施肌內(nèi)注射黃體酮注射液(1mg/kg),實(shí)驗(yàn)第3天通過(guò)宮腔鏡向大腸桿菌組和孕酮+大腸桿菌組山羊子宮內(nèi)注入大腸桿菌液5mL(109cfu/mL),同時(shí)向?qū)φ战M和孕酮組山羊子宮內(nèi)注入生理鹽水5mL;動(dòng)物試驗(yàn)連續(xù)進(jìn)行10天;分別在注菌后0h、12h、24h、96h和168h測(cè)定試驗(yàn)山羊的體溫(T)、脈搏(P)和呼吸(R),同時(shí)頸靜脈無(wú)菌采集外周血檢測(cè)WBC數(shù)目變化;分別在注菌后第24h、96h和168h用滅菌棉拭子采集子宮分泌物涂布于載玻片上,Diff Quick染色后鏡檢WBC數(shù)目變化;于注菌后第1天、第4天、第7天通過(guò)宮腔鏡無(wú)菌采集各組試驗(yàn)羊子宮內(nèi)膜組織,部分組織置于10%中性福爾馬林溶液固定,用于病理組織學(xué)觀察,部分組織用PBS液洗滌后放于無(wú)RNA酶的凍存管中并立即放入液氮保存,用于基因表達(dá)檢測(cè)。結(jié)果如下:(1)在接菌后第12h和第24h,與對(duì)照組相比,大腸桿菌組和孕酮+大腸桿菌組試驗(yàn)羊體溫明顯上升,且差異極顯著(p0.01);在接菌后第168h,與大腸桿菌組相比,孕酮+大腸桿菌組試驗(yàn)羊體溫下降,且差異顯著(p0.05);在接菌后第12h、24h,與對(duì)照組相比,大腸桿菌組試驗(yàn)羊外周血WBC數(shù)目明顯升高,且差異極顯著(p0.01),與大腸桿菌組相比,孕酮+大腸桿菌組試驗(yàn)羊外周血WBC數(shù)目明顯降低,且差異顯著(p0.05);接菌后第1天,與對(duì)照組相比,大腸桿菌組與孕酮+大腸桿菌組試驗(yàn)羊子宮分泌物中WBC數(shù)目明顯增多;與大腸桿菌組相比,孕酮+大腸桿菌組試驗(yàn)羊子宮分泌物中WBC數(shù)目明顯減少。(2)在接菌后第1天,大腸桿菌組山羊子宮組織中可見(jiàn)大量白細(xì)胞浸潤(rùn),子宮黏膜損傷,毛細(xì)血管充血;孕酮+大腸桿菌組山羊子宮組織切片可見(jiàn)少量白細(xì)胞浸潤(rùn)。在接菌后第4天,大腸桿菌組山羊子宮組織中依然可見(jiàn)大量白細(xì)胞浸潤(rùn),其他各組子宮組織未見(jiàn)明顯變化。(3)在接菌后第1天,與對(duì)照組相比,大腸桿菌組試驗(yàn)羊子宮內(nèi)膜組織TLR4、IL-6、TNF-α、IL-1β和IL-8的mRNA相對(duì)表達(dá)量明顯升高,且差異顯著(p0.05);與大腸桿菌組相比,孕酮+大腸桿菌組的試驗(yàn)羊子宮內(nèi)膜組織TLR4、IL-6、IL-8的mRNA相對(duì)表達(dá)量明顯降低,且差異顯著(p0.05)。在接菌后第4天,與大腸桿菌組相比,孕酮+大腸桿菌組試驗(yàn)羊子宮內(nèi)膜組織TLR4、TNF-α的mRNA相對(duì)表達(dá)量明顯下降,且差異顯著(p0.05)。(4)在接菌后第1天,與對(duì)照組相比,大腸桿菌組與孕酮組試驗(yàn)羊子宮內(nèi)膜組織TGF-βmRNA相對(duì)表達(dá)量升高,但差異不顯著(p0.05),大腸桿菌組、孕酮組和孕酮+大腸桿菌組試驗(yàn)羊子宮內(nèi)膜組織VEGF和EGFR的mRNA相對(duì)表達(dá)量明顯降低,僅大腸桿菌組差異顯著(p0.05)。與大腸桿菌組相比,孕酮+大腸桿菌組試驗(yàn)羊子宮內(nèi)膜組織TGF-βmRNA相對(duì)表達(dá)量降低,但差異不顯著(p0.05),孕酮+大腸桿菌組試驗(yàn)羊子宮內(nèi)膜組織VEGFmRNA相對(duì)表達(dá)量升高,但差異不顯著(p0.05)。結(jié)論:山羊子宮接注大腸桿菌后,山羊的T、P和R以及外周血與子宮分泌物中WBC數(shù)目增多,山羊子宮發(fā)生急性感染。孕酮能夠調(diào)節(jié)子宮接注大腸桿菌后山羊子宮內(nèi)膜中TLR4、IL-6、TNF-α、IL-1β 和 IL-8 的基因表達(dá),同時(shí)能夠調(diào)節(jié) TGF-β、VEGF 和 EGFR的基因表達(dá),提示孕酮通過(guò)對(duì)炎性細(xì)胞因子以及細(xì)胞增殖因子表達(dá)的調(diào)節(jié)對(duì)子宮局部免疫起促進(jìn)作用,對(duì)子宮內(nèi)膜修復(fù)無(wú)明顯作用。
[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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S858.27
本文編號(hào)：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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S858.27
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