高毒力肺炎克雷伯菌分子特征、血流感染危险因素及其延迟中性粒细胞凋亡的调控机制

发布时间：2018-06-10 02:16

本文选题：高毒力肺炎克雷伯菌 + 分子特征　；参考：《南昌大学》2017年硕士论文

【摘要】：目的:1.研究高毒力肺炎克雷伯菌血流感染的流行特征及危险因素分析,为临床合理治疗和防治高毒力肺炎克雷伯菌血流感染提供科学依据。2.探究血流感染中高毒力肺炎克雷伯菌毒力、分子流行病学特征及其对中性粒细胞功能的影响。3.初步探究高毒力肺炎克雷伯菌延迟中性粒细胞凋亡的调控机制。方法:1.收集南昌大学第一附属医院2014年1月-2016年12月住院患者血液标本分离出的肺炎克雷伯菌178株。采用肠杆菌科基因间重复一致序列聚合酶链反应(ERIC-PCR)与多位点序列分析法(MLST)确定主要基因分型及其病区分布。2.聚合酶链式反应(PCR)及测序技术确定其血清荚膜分型及毒力基因携带,本研究定义凡是符合以下任何一项:携带K1、K2、rmpA/rmpA2基因或者高黏液表型阳性的菌株为高毒力菌株,否则为非高毒力菌株;查询统计分析这178例患者的临床资料,实验分为高毒力菌株组与非高毒力菌株组,运用单因素分析和多因素logistic回归分析高毒力肺炎克雷伯菌血流感染的危险因素。3.收集南昌大学第一附属医院2016年6月-2016年12月住院患者血标本分离出的肺炎克雷伯菌55株,PCR扩增及测序确定其wzi分型,PCR扩增确定其ompK36等位基因分群(A、B、C、D),用血清抗性、抗中性粒细胞吞噬试验来研究肺炎克雷伯菌的体外毒力性;采用流式细胞仪检测患者外周血中性粒细胞表面分子表达的变化,采用ELISA检测患者体内炎症因子的释放量。4.建立体外中性粒细胞感染模型和体内腹腔感染脓毒症动物模型,采用Western-Blot检测中性粒细胞凋亡调控蛋白的表达情况。结果1.178株血流感染肺炎克雷伯菌,主要基因分型是ST11型,其中HvKP菌株以ST23型为主。主要见于消化科、烧伤科、神经外科、重症医学科,且呈现克隆性传播。2.伴有糖尿病基础疾病、引流、血型(A型)、使用喹诺酮类及碳青霉烯类抗菌药物是HvKP血流感染的独立危险因素。3.在55株血流感染KP中,有20株HvKP菌株,35株non-HvKP菌株。HvKP菌株的MLST分型以ST23和ST65为主,而non-HvKP菌株则以ST11型为主;HvKP菌株的ompK36等位基因以C群为主,而non-HvKP菌株则以A群和D群为主;HvKP菌株的荚膜血清分型以wzi2-K2和wzi1-K1为主,而non-HvKP菌株则以wzi64-K64分型为主。4.在20株血流感染HvKP中,有16株表现出高水平的血清抗性能力,而在35株non-HvKP中,仅有4株表现出高水平的血清抗性能力;HvKP菌株携带毒力相关基因数多于non-HvKP菌株。5.在抗中性粒细胞吞噬方面,大部分non-HvKP菌株的中性粒细吞噬率均在55-80%左右,而大部分HvKP菌株的中性粒细胞吞噬率在30%左右。6.相比较non-HvKP组,HvKP组患者体内PD-1、CD86表达明显下调,而PD-L1、CD80明显变化;HvKP组患者体内IL-2、IL-8、IL-1β释放量明显增多,而TNF-α释放量明显降低。7.在HvKP感染的早期,HvKP感染组NF-κB P65蛋白含量显著高于non-HvKP感染组;HvKP感染组Bax/Bcl-2的比值显著低于non-HvKP感染组;HvKP感染组Caspase-3,8,9蛋白含量显著低于non-HvKP感染组。结论:1.在本院血流感染KP菌株的MLST分型以ST11为主,而血流感染HvKP菌株以ST23、ST65型为主。2.通过本文对HvKP血流感染的危险因素分析,提示我们要特别重视合并有慢性内科基础疾病,特别是伴有糖尿病的血流感染KP患者;其次要注意引流操作的无菌意识及引流时间和碳青霉烯类、喹诺酮类抗菌药物的合理使用。3.HvKP菌株的ompK36等位基因以C群为主,荚膜血清分型以wzi2-K2和wzi1-K1为主。4.相比较non-HvKP菌株,HvKP菌株携带毒力相关基因数较多,血清抗性能力和对中性粒细胞吞噬能力均较高。5.HvKP血流感染患者体内炎症因子的释放量改变以及PMN表面分子的表达紊乱,提示HvKP感染可能使PMN的功能发生了变化。6.我们推测在HvKP感染的早期,HvKP诱导PMN凋亡延迟的作用可能是通过调节Bax/Bcl-2比值使Caspase-3,8,9的表达下调,进而激活NF-κB,从而延迟中性粒细胞的凋亡。
[Abstract]:Objective: 1. to study the epidemiological characteristics and risk factors of high virulence Klebsiella pneumoniae blood flow infection and to provide scientific basis for the rational treatment and prevention of Klebsiella pneumoniae blood flow infection by.2. to explore the virulence of Klebsiella pneumoniae, the characteristics of molecular epidemic and the effect on neutrophils function in the blood flow infection. .3. preliminarily explored the regulatory mechanism of delayed neutrophils apoptosis of Klebsiella pneumoniae. Methods: 1. collect 178 Klebsiella pneumoniae isolated from the blood specimens of hospitalized patients in the First Affiliated Hospital of Nanchang University in December January 2014. Using INTERGENE repeat sequence polymerase chain reaction (ERIC-PCR) and multiple sites in the Enterobacteriaceae. The point sequence analysis (MLST) determines the main genotyping and its endemic area distribution.2. polymerase chain reaction (PCR) and sequencing technology to determine its sera pods and virulence genes. This study defines any of the following: strains with K1, K2, rmpA/rmpA2, or high mucous phenotypes are highly virulent, otherwise Non high virulence strains; the clinical data of these 178 patients were analyzed and analyzed. The experiment was divided into high virulent and non virulence strains. Single factor analysis and multiple factor Logistic regression analysis were used to analyze the risk factor of Klebsiella pneumoniae blood flow infection.3.. The First Affiliated Hospital of Nanchang University was hospitalized in December -2016 June 2016. 55 strains of Klebsiella pneumoniae isolated from blood samples, PCR amplification and sequencing to determine their WZI typing, and PCR amplification to determine their ompK36 allele groups (A, B, C, D). Serum resistance and anti neutrophil phagocytosis test were used to study the virulence of Klebsiella pneumoniae in vitro. Flow cytometry was used to detect the surface of neutrophils in peripheral blood of patients. The changes in the expression of the sub expression, using ELISA to detect the release of inflammatory factors in the patient's body, to establish an in vitro neutrophils infection model and an animal model of sepsis in the body, and to detect the expression of the regulation of the apoptosis of neutrophils by Western-Blot. Results 1.178 strains of Klebsiella pneumoniae were infected with the main genotyping of ST1. Type 1, of which HvKP strain is mainly ST23, mainly in the digestive, burn, Department of Neurosurgery, and severe medical sciences, and presents clonically transmitted.2. with diabetes based diseases, drainage, blood group (A), the use of quinolones and carbapenems as an independent risk factor for HvKP blood flow infection,.3. in 20 blood stream infection KP, there are 20 strains of blood flow infection. The MLST genotyping of 35 strains of non-HvKP strain.HvKP was dominated by ST23 and ST65, while the non-HvKP strain was dominated by ST11 type, and the ompK36 allele of the HvKP strain was dominated by C group, while the non-HvKP strain was the dominant group. Of the main.4. in 20 blood flow infection HvKP, 16 showed high level of serum resistance, but only 4 of 35 strains of non-HvKP showed high level of serum resistance. The number of Virulence Related Genes of strain HvKP was more than that of non-HvKP strain.5. in the anti neutrophil phagocytosis, and the neutrophil phagocytosis rate of most of the strains of non-HvKP strains were all At about 55-80%, the neutrophil phagocytosis rate of most HvKP strains was compared to that of non-HvKP group at about 30%.6., and PD-1, CD86 expression in HvKP group was obviously down, while PD-L1 and CD80 were obviously changed, IL-2, IL-8, and beta release in the HvKP group were significantly increased. The content of NF- kappa B P65 protein was significantly higher than that in non-HvKP infection group, and the ratio of Bax/Bcl-2 in HvKP infection group was significantly lower than that in non-HvKP infection group, and the Caspase-3,8,9 protein content in HvKP infection group was significantly lower than that in non-HvKP infection group. Conclusion: 1. Through the analysis of the risk factors of HvKP blood flow infection, we suggest that we should pay special attention to the patients with chronic basic diseases, especially the KP patients with blood flow infection with diabetes; secondly, we should pay attention to the aseptic consciousness of the drainage operation, the time of drainage and the carbapenems, the rational use of the ompK36 of the.3.HvKP strain of the quinolone antibiotics. The allele was mainly C group, and the capsule serotyping was compared with the non-HvKP strain of wzi2-K2 and wzi1-K1 as the main.4. phase. The HvKP strains carried more virulence related genes. The serum resistance ability and the neutrophil phagocytosis were all higher in the patients with.5.HvKP blood flow infection, and the expression of PMN surface molecules was disordered. It is suggested that HvKP infection may change the function of PMN.6.. We speculate that in the early stage of HvKP infection, HvKP induced PMN apoptosis delayed by regulating the Bax/Bcl-2 ratio to decrease the expression of Caspase-3,8,9, and then activate NF- kappa B to delay the apoptosis of neutrophils.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446.5

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