NF-κB信号通路调控MMEC凋亡在S.aureus乳腺炎作用研究
本文选题:乳腺上皮细胞 + 金黄色葡萄球菌 ; 参考:《华中农业大学》2015年硕士论文
【摘要】:奶牛乳腺炎是危害我国乃至全世界奶牛业的一种重大疾病,乳腺炎不仅会导致牛奶的质量及产量下降,而且导致额外劳动力以及治疗费用的增加。金黄色葡萄球菌(Staphylococcus aureus,S.aureus)是引起奶牛乳腺炎的一种重要病原菌,由其引起的奶牛乳腺炎通常表现为持续性感染、正常奶牛的早期淘汰。同时,由于抗生素的滥用,大部分乳腺炎源金黄色葡萄球菌表现出多重耐药性,对公共卫生构成严重威胁。近年来,人们通过改善奶牛干奶期管理、调控营养以及制备生物制剂来预防奶牛乳腺炎,但效果均不理想。奶牛金黄色葡萄球菌乳腺炎防治困难,根本原因在于现阶段人们对金黄色葡萄球菌感染损伤乳腺机制还不清楚,严重影响了其防治进程。乳腺上皮细胞是乳腺抵抗病原微生物入侵的第一道防线,病原菌入侵可导致乳腺上皮细胞的凋亡。NF-κB是具有多向性调节作用的蛋白质分子,参与调控多种因子的基因表达,同时,NF-κB是多个信号转导通路的交叉点,在凋亡中起着非常重要的作用。研究NF-κB如何调控S.aurues乳腺炎中乳腺上皮细胞凋亡,探讨S.aureus乳腺感染过程中NF-κB调控乳腺上皮细胞凋亡的机制,有助于更进一步理解奶牛S.aureus乳腺感染及转归机制,为奶牛乳腺炎防治新型策略奠定理论基础。本实验通过建立体内小鼠和体外S.aurues乳腺炎感染模型,通过测定感染后小鼠乳腺上皮细胞(Mouse mammary epithelial cell,MMEC)MMEC的凋亡和NF-κB相关通路蛋白的转录和表达,以期阐明NF-κB调控乳腺上皮细胞凋亡的机制,为阐明奶牛S.aureus感染致乳腺损伤的机理提供理论依据,为寻找干预乳腺炎症的新型药物靶标奠定理论基础。用胰酶消化法分离得到MMEC,将MMEC铺到6孔板中,待细胞长到80%,进行分组实验。体外感染MMEC乳腺炎分为四组,PBS对照组(NC组)、PDTC处理组(PDTC组)、S.aureus感染组(S.aureus组)、PDTC预处理S.aureus感染组(PDTC+S.aureus组)。运用流式细胞术测定感染后乳腺上皮细胞凋亡率;Hoechst33342染色对细胞凋亡形态进行观察;利用荧光定量PCR和Western blot检测了NF-κB P65和P50以及其IκBɑ的m RNA和蛋白水平,酶联免疫法测定炎性细胞因子TNF-ɑ和IL-6的分泌量。主要结果如下:1.1 S.aureus诱导小鼠乳腺上皮细胞凋亡率收集感染3h后的细胞,用Annex V和PI双染法测定MMEC凋亡率,发现S.aureus可以引起小鼠乳腺上皮细胞的凋亡(P0.01),而PDTC+S.aureus组,乳腺上皮的凋亡率比S.aureus感染组显著下降(P0.01);同时,PBS组与PDTC组没有显著差异。1.2小鼠乳腺上皮细胞凋亡形态进行观察细胞发生凋亡后,细胞核皱缩,Hoechst33342染色后,细胞呈亮蓝色。S.aurues感染组呈现亮蓝色的凋亡细胞明显增多,PPDTC+S.aureus组,凋亡的细胞明显减少。1.3 Reeal Time PCR检测NF-κB P65、P50和IκBɑ转录水平表达量对P65、P50、IκBɑ进行转录水平表达量的测定,P65、P50、IκBɑ三个基因的转录水平S.aurues感染组跟PBS对照组相比转录量增高且差异极显著(P0.01),而PDTC+S.aureus组,转录量跟S.aurues感染组相比均下降且差异极显著(P0.01)。1.4 Western blot检测NF-κB P65、IκBɑ、P-P65、P-IκBɑ蛋白水平以β-actin为内参,对P65和IκBɑ蛋白水平和磷酸化水平进行测定,四组中P65蛋白水平没有明显差异,IκBɑ在S.aurues感染组和PDTC+S.aureus组发生降解,P-P65的蛋白水平在S.aurues感染组和PDTC+S.aureus组表达量增加,S.aurues感染组P-IκBɑ相较PBS对照组表达量增加,PDTC+S.aureus组跟S.aurues感染组比较其表达量下降。1.5 ELISA检测促炎因子TNF-ɑ和IL-6的分泌量S.aurues感染组相较PBS组可显著提高TNF-ɑ和IL-6的分泌量(P0.05),PDTC+S.aureus组的TNF-ɑ分泌量与S.aurues感染组比较下降显著(P0.05),IL-6的分泌量与S.aurues感染组比较下降但没有显著差异。用分娩1周后的昆明小鼠进行乳腺炎模型的建立,信号通路抑制剂PDTC使用方式为腹腔注射,实验分为四组:PBS灌注乳腺组(NC组);PDTC预处理组(PDTC组);S.aurues感染组;PDTC预处理后S.aurues感染组(PDTC+S.aureus组)。对不同处理进行全血细胞计数;乳腺组织细菌分离、回收鉴定、计数;评估不同处理组的乳腺临床症状变化及病理变化;Tunel检测乳腺上皮细胞凋亡评价NF-κB信号通路对乳腺炎的影响,结果如下:2.1不同处理组全血细胞计数对不同处理组取抗凝血进行全血细胞计数,血小板及红细胞、血红蛋白、红细胞压积、平均红细胞体积、平均红细胞血红蛋白含量、平均红细胞血红蛋白浓度指标各处理组之间没有差异,S.aurues感染组与PDTC+S.aureus组白细胞差异显著(P㩳0.05),S.aureus感染组与PDTC+S.aureus组中性粒细胞差异显著,S.aurues感染组与PDTC+S.aureus组淋巴细胞差异显著。2.2不同处理组分菌和临床症状变化对不同处理组S.aureus分菌结果以LOG10表示,PBS组与PDTC组没有分离到细菌,S.aureus感染组分离到的CFU为8.4±0.05,PDTC+S.aureus组CFU的为6.27±0.048,两组差异极显著(P0.01)。PBS和PDTC组小鼠乳腺组织健康呈乳白色,S.aureus感染组和PDTC+S.aureus组均有出血现象,临床症状没有差异性。病理组织切片评分,S.aureus感染组分数为3.4±0.54,PDTC+S.aureus组为2.4±0.54,两者差异显著(P0.05)。2.3 Tunel原位凋亡检测乳腺组织乳腺上皮细胞凋亡对乳腺上皮细胞进行原位凋亡实验,结果显示PBS组与PDTC组可见清晰清乳腺腺泡结构,凋亡细胞数目少,S.aureus感染组乳腺腺泡结构被破坏,有大量的MMEC凋亡,PDTC+S.aureus组凋亡细胞数减少。在体内动物实验表现了跟体外实验相似的规律性。结果:1乳腺上皮细胞的凋亡是由金黄色葡萄球菌引起的,信号通路抑制剂PDTC不能引起乳腺上皮细胞凋亡作用。NF-κB信号通路参与了对乳腺上皮细胞凋亡的调节。2金黄色葡萄球菌引起乳腺上皮细胞的凋亡是通过使NF-κB信号通路及其抑制蛋白IκBɑ的转录水平升高和P-P65和P-IκBɑ表达量升高发挥作用。3 TNF-ɑ和IL-6的分泌量受NF-κB信号通路的调控,而IL-6的分泌还可能受其他信号通路的调控。4动物实验HE染色,S.aurueus可引起小鼠乳腺炎,PDTC抑制信号通路后可减轻乳腺炎炎症发应,同时也可以减少凋亡细胞数目,与体外实验结果一致。
[Abstract]:Cow mastitis is a major disease that endangers the dairy industry in China and in the world. Mastitis not only leads to the decline in the quality and production of milk, but also leads to the increase of extra labor and treatment costs. Staphylococcus aureus (Staphylococcus aureus, S.aureus) is an important pathogen of cow mastitis. Dairy cow mastitis is usually characterized by persistent infection, early elimination of normal dairy cows. At the same time, the majority of mastitis source of Staphylococcus aureus shows multiple drug resistance due to the abuse of antibiotics, which poses a serious threat to public health. In recent years, people have improved the management of milk cow's dry milk period, regulate nutrition and prepare biological agents in recent years. To prevent cow mastitis, but the effect is not ideal. The prevention and cure of Staphylococcus aureus mastitis is difficult, the basic reason is that the mechanism of the mammary gland infection of Staphylococcus aureus is not clear at the present stage, and it seriously affects its prevention and control process. The invasion of the original bacteria can lead to the apoptosis of the mammary epithelial cells,.NF- kappa B, a protein molecule with multidirectional regulation, and participates in the regulation of gene expression of various factors. At the same time, NF- kappa B is a cross point of multiple signal transduction pathways, and plays a very important role in apoptosis. The study of how NF- kappa B regulates the epithelial cells of the mammary gland in S.aurues mastitis Apoptosis, the mechanism of NF- kappa B regulating the apoptosis of mammary epithelial cells in the process of S.aureus breast infection, will be helpful to further understand the mechanism of mammary gland infection and prognosis of cow S.aureus, and lay a theoretical foundation for the new strategy of cow mastitis prevention and control. This experiment has established the infection model of S.aurues mastitis in vivo and in vitro, through the determination of the sense of the infection. The apoptosis of Mouse mammary epithelial cell (MMEC) MMEC and the transcription and expression of NF- kappa B related pathway proteins in the infected mouse mammary gland in order to elucidate the mechanism of NF- kappa B regulating the apoptosis of mammary epithelial cells, and to provide a theoretical basis for clarifying the mechanism of mammary gland injury induced by S.aureus infection of dairy cows and to find new drugs to interfere with mammary inflammation. The target lay a theoretical basis. MMEC was obtained by trypsin digestion. MMEC was spread to 6 orifice plates, and the cells grew to 80%. The infection of MMEC mastitis in vitro was divided into four groups, PBS control group (NC group), PDTC treatment group (PDTC group), S.aureus infection group (S.aureus group), PDTC pretreated S.aureus infection group (PDTC+S.aureus group). Flow finer was used. The apoptosis rate of the mammary epithelial cells after infection was measured by cytosolic method, the apoptosis morphology was observed by Hoechst33342 staining, and the m RNA and protein levels of NF- kappa B P65 and P50 and its I kappa B were detected by fluorescence quantitative PCR and Western blot. The main results were as follows: 1.1 The apoptosis rate of mouse mammary epithelial cells was induced to collect the cells after 3H infection. The apoptosis rate of MMEC was detected by Annex V and PI double staining. It was found that S.aureus could induce apoptosis of mammary epithelial cells of mice (P0.01), while PDTC+S.aureus group, the apoptosis rate of mammary epithelium was significantly lower than that of the S.aureus infection group (P0.01), while there was no significant difference between the PBS group and the PDTC group. The apoptotic morphology of the mammary epithelial cells of the.1.2 mice was observed. After the cell apoptosis was observed, the nucleus crinkled, and after Hoechst33342 staining, the cell showed bright blue.S.aurues infection group showing bright blue apoptotic cells, and PPDTC+S.aureus group, the apoptotic cells obviously decreased the.1.3 Reeal Time PCR to detect NF- kappa B P65, P50 and transcription level transcription level The transcriptional level of P65, P50, and I kappa B was measured. The transcriptional level of the three genes of P65, P50, I kappa B and the PBS control group increased and the difference was very significant (P0.01). The levels of B P65, I kappa B, P-P65, P-I kappa B were measured with beta -actin as the internal reference. The levels of P65 and I kappa B protein and phosphorylation were measured, and there was no significant difference in the level of P65 protein in the four groups. The expression of P-I kappa B in the urues infection group was higher than that in the PBS control group. The expression of PDTC+S.aureus in the PDTC+S.aureus group and the S.aurues infection group were decreased by.1.5 ELISA, and the secretion quantity of the S.aurues infection group was significantly higher than that in the PBS group. Group comparison decreased significantly (P0.05), the secretion of IL-6 was less than that of S.aurues infection group, but there was no significant difference. The establishment of mastitis model in Kunming mice after 1 weeks of childbirth, the use of signal pathway inhibitor PDTC was intraperitoneal injection, and the experiment was divided into four groups: PBS perfusion Breast Group (NC group); PDTC preconditioning group (PDTC group); S.aurues infection. Group S.aurues infection group (group PDTC+S.aureus) after PDTC pretreatment. The whole blood cell count was carried out in different treatments, the bacterial isolation, recovery identification and counting of breast tissue were used to evaluate the changes of clinical symptoms and pathological changes of breast in different treatment groups, and Tunel was used to evaluate the effects of NF- kappa B signaling pathway on breast epithelial cell withering. The results were as follows: 2.1 the whole blood cell count of different treatment groups took blood cell count, platelet and red blood cell, hemoglobin, hematocrit, mean red blood cell volume, average red blood cell hemoglobin content, and Ping Junhong cell hemoglobin concentration index between different groups, S.aurues infection group and PDTC+S.aure The difference of leucocyte in US group was significant (P? 0.05), the difference of neutrophils between S.aureus infection group and PDTC+S.aureus group was significant. The difference of lymphocyte between S.aurues infection group and PDTC+S.aureus group was significant.2.2 different processing group and clinical symptom changes of S.aureus division results in different treatment groups were expressed LOG10, PBS group and PDTC group did not separate bacteria, S.a The CFU in the UREUS infection group was 8.4 + 0.05, and the CFU in the PDTC+S.aureus group was 6.27 + 0.048. The two groups were extremely significant (P0.01).PBS and PDTC group. The mammary tissues of the mice were milk white, S.aureus infection group and PDTC+S.aureus group had bleeding phenomenon, and the clinical symptoms were not different. The pathological tissue section score and the S.aureus infection group score were 3.4 + 0.. 54, the PDTC+S.aureus group was 2.4 + 0.54, and the difference was significant (P0.05). The apoptosis of mammary gland epithelial cells was detected by.2.3 Tunel in situ apoptosis. The results showed that the structure of the mammary gland was clear in the PBS group and the PDTC group, the number of apoptotic cells was less, and the structure of the mammary gland was destroyed in the S.aureus infection group. A large number of MMEC apoptosis, the number of apoptotic cells in group PDTC+S.aureus decreased. In vivo animal experiments showed similar regularity with in vitro experiments. Results: 1 the apoptosis of mammary epithelial cells was caused by Staphylococcus aureus, and the signal pathway inhibitor PDTC did not cause the apoptosis of mammary epithelial cells by.NF- kappa B signaling pathway involved in the mammary gland The apoptosis of.2 Staphylococcus aureus induced the apoptosis of mammary epithelial cells by making NF- kappa B signaling pathway and its inhibitory protein I kappa B transcriptional level rising and P-P65 and P-I kappa B expression increased to play a role in the regulation of.3 TNF- and IL-6 secretion by NF- kappa signaling pathway. The regulation of signal pathway in.4 animal experiment HE staining, S.aurueus can cause mastitis in mice. After PDTC suppression signal pathway, it can reduce the inflammatory response of mastitis, and also reduce the number of apoptotic cells, which is in accordance with the experimental results in vitro.
【学位授予单位】:华中农业大学
【学位级别】:硕士
【学位授予年份】:2015
【分类号】:S858.23
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