免疫抑制对ST-239型MRSA毒力因子表达的影响效应研究
发布时间：2018-09-03 15:58
【摘要】：目的研究不同免疫状态下ST-239型MRSA毒力因子的表达情况,为结合宿主免疫状态对金黄色葡萄球菌毒力因子基因表达的影响提供新的思路和依据。方法将SPF清洁级Wistar大鼠随机分成对照组和模型组,每组12只,雌雄各半。以40mg/kg的环磷酰胺连续肌肉注射3天,造成大鼠免疫抑制,在此基础上通过滴鼻方式滴入小剂量高浓度的新鲜MRSA菌悬液,连续滴注3天,造成免疫抑制MRSA定植模型。对照组将正常大鼠直接鼻腔滴入小剂量高浓度的新鲜MRSA菌悬液。细菌定植一定时间后取大鼠鼻粘膜组织,运用实时荧光定量PCR技术分析体内试验中,不同免疫状态下大鼠鼻粘膜组织中分离的MRSA毒力因子在基因水平上的表达差异;同时处死各组动物后,取模型组和对照组动物的血清分别加入TSB培养基,以普通TSB培养基作对照,将MRSA在不同培养基中培养后,运用实时荧光定量PCR技术分析不同培养条件下不同培养时间点的MRSA毒力因子在基因水平上的表达差异。结果1.处于免疫抑制状态的大鼠较正常大鼠,MRSA更容易在鼻粘膜定植;2.与肌注环磷酰胺前比较,肌注环磷酰胺后3天、7天时,大鼠血液学检查可见外周血WBC、PLT数量迅速下降,同时RBC、HGB亦明显降低(P0.01);外周血中CD3+T,CD3+CD4+T,CD3+CD8+T淋巴细胞比例明显降低并且B淋巴细胞以及NK细胞的比例也明显降低(P0.01)。外周血血清中免疫球蛋白IgG、IgM、IgA的含量明显下降(P0.01);3.连续3次向大鼠鼻腔滴注小剂量高浓度的新鲜MRSA菌悬液,细菌定植一段时间后可稳定存在鼻粘膜组织细胞内外;4.体内试验毒力因子检测结果表明,定植在模型组大鼠鼻粘膜组织中的MRSA毒力因子hla和spa的表达水平要高于对照组,(P0.05),其中目的基因spa的表达水平显著升高(P0.01);5.体外试验毒力因子检测结果表明,MRSA在不同环境下培养不同时间后,细菌毒力因子的表达也存在一定的差异。培养至90min时,模型组与对照组毒力因子hla和spa的表达水平相对于培养0min时变化不明显(P0.05);而在细菌培养至120min时,模型组hla基因的表达水平较对照组显著升高(P0.01),且spa基因的表达水平较对照组升高(P0.05)。结论1.当宿主处于免疫抑制状态时,可能通过促进金黄色葡萄球菌毒力因子的表达增强金黄色葡萄球菌的致病力;2.当MRSA感染宿主时,细菌的生长情况和在培养基中的生长情况完全不同,可能导致金黄色葡萄球菌的不同毒力因子的表达存在一定的差异。
[Abstract]:Objective to study the expression of ST-239 type MRSA virulence factor in different immune states, and to provide a new idea and basis for combining host immune state with the expression of staphylococcus aureus virulence factor gene. Methods SPF clean grade Wistar rats were randomly divided into control group and model group. The immunosuppression of 40mg/kg was induced by intramuscular injection of cyclophosphamide in rats for 3 days. On this basis, the immunosuppressive MRSA model was established by infusing a small dose of fresh MRSA suspension by nasal drip for 3 days. In the control group, the normal rats were directly dripped into the nasal cavity with a small dose of fresh MRSA suspension. The nasal mucosa of rats was harvested after bacterial colonization for a certain time. The expression of MRSA virulence factors in nasal mucosa of rats in vivo was analyzed by real-time fluorescence quantitative PCR technique. At the same time, the serum of the model group and the control group were added to the TSB medium, and the normal TSB medium was used as the control. The MRSA was cultured in different culture medium. The expression differences of MRSA virulence factors at different culture time points in different culture conditions were analyzed by real-time fluorescence quantitative PCR. Result 1. MRSA was more easily colonized in nasal mucosa in immunosuppressive rats than in normal rats. Compared with that before cyclophosphamide injection, the number of WBC,PLT in peripheral blood of rats was decreased rapidly at 7 days after intramuscular injection of cyclophosphamide. At the same time, RBC,HGB was also significantly decreased (P0.01), and the proportion of CD3 T T T CD4 T T T tou CD 3 CD8 T lymphocytes and B lymphocytes and NK cells in peripheral blood was significantly decreased (P0.01). The content of immunoglobulin IgG,IgM,IgA in peripheral blood decreased significantly (P0.01). A small dose of fresh MRSA suspension was injected into the nasal cavity of rats for three consecutive times. After a period of bacterial colonization, there was a stable presence of the nasal mucosal cells in and out of the nasal mucosa. The results of virulence factor test in vivo showed that the expression levels of MRSA virulence factor hla and spa in the nasal mucosa of the model group were higher than those in the control group (P0.05), and the expression level of the target gene spa was significantly increased (P0.01). The results of virulence factor test in vitro showed that the expression of virulence factor in MRSA was different after different culture time. When cultured to 90min, the expression levels of hla and spa in model group and control group were not significantly different from those in 0min group (P0.05), but when the bacteria were cultured to 120min, the expression level of hla and spa in the model group and the control group was not significantly different from that in the control group (P0.05). The expression level of hla gene in model group was significantly higher than that in control group (P0.01), and the expression level of spa gene in model group was higher than that in control group (P0.05). Conclusion 1. When the host is in immunosuppressive state, it may enhance the pathogenicity of Staphylococcus aureus by promoting the expression of virulence factor of Staphylococcus aureus. When MRSA infected the host, the bacterial growth was completely different from that in the culture medium, which may lead to different expression of different virulence factors of Staphylococcus aureus.
【学位授予单位】：内蒙古医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5
本文编号：2220462
[Abstract]:Objective to study the expression of ST-239 type MRSA virulence factor in different immune states, and to provide a new idea and basis for combining host immune state with the expression of staphylococcus aureus virulence factor gene. Methods SPF clean grade Wistar rats were randomly divided into control group and model group. The immunosuppression of 40mg/kg was induced by intramuscular injection of cyclophosphamide in rats for 3 days. On this basis, the immunosuppressive MRSA model was established by infusing a small dose of fresh MRSA suspension by nasal drip for 3 days. In the control group, the normal rats were directly dripped into the nasal cavity with a small dose of fresh MRSA suspension. The nasal mucosa of rats was harvested after bacterial colonization for a certain time. The expression of MRSA virulence factors in nasal mucosa of rats in vivo was analyzed by real-time fluorescence quantitative PCR technique. At the same time, the serum of the model group and the control group were added to the TSB medium, and the normal TSB medium was used as the control. The MRSA was cultured in different culture medium. The expression differences of MRSA virulence factors at different culture time points in different culture conditions were analyzed by real-time fluorescence quantitative PCR. Result 1. MRSA was more easily colonized in nasal mucosa in immunosuppressive rats than in normal rats. Compared with that before cyclophosphamide injection, the number of WBC,PLT in peripheral blood of rats was decreased rapidly at 7 days after intramuscular injection of cyclophosphamide. At the same time, RBC,HGB was also significantly decreased (P0.01), and the proportion of CD3 T T T CD4 T T T tou CD 3 CD8 T lymphocytes and B lymphocytes and NK cells in peripheral blood was significantly decreased (P0.01). The content of immunoglobulin IgG,IgM,IgA in peripheral blood decreased significantly (P0.01). A small dose of fresh MRSA suspension was injected into the nasal cavity of rats for three consecutive times. After a period of bacterial colonization, there was a stable presence of the nasal mucosal cells in and out of the nasal mucosa. The results of virulence factor test in vivo showed that the expression levels of MRSA virulence factor hla and spa in the nasal mucosa of the model group were higher than those in the control group (P0.05), and the expression level of the target gene spa was significantly increased (P0.01). The results of virulence factor test in vitro showed that the expression of virulence factor in MRSA was different after different culture time. When cultured to 90min, the expression levels of hla and spa in model group and control group were not significantly different from those in 0min group (P0.05), but when the bacteria were cultured to 120min, the expression level of hla and spa in the model group and the control group was not significantly different from that in the control group (P0.05). The expression level of hla gene in model group was significantly higher than that in control group (P0.01), and the expression level of spa gene in model group was higher than that in control group (P0.05). Conclusion 1. When the host is in immunosuppressive state, it may enhance the pathogenicity of Staphylococcus aureus by promoting the expression of virulence factor of Staphylococcus aureus. When MRSA infected the host, the bacterial growth was completely different from that in the culture medium, which may lead to different expression of different virulence factors of Staphylococcus aureus.
【学位授予单位】：内蒙古医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5
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