【摘要】：目的研究不同免疫狀態(tài)下ST-239型MRSA毒力因子的表達(dá)情況,為結(jié)合宿主免疫狀態(tài)對金黃色葡萄球菌毒力因子基因表達(dá)的影響提供新的思路和依據(jù)。方法將SPF清潔級Wistar大鼠隨機分成對照組和模型組,每組12只,雌雄各半。以40mg/kg的環(huán)磷酰胺連續(xù)肌肉注射3天,造成大鼠免疫抑制,在此基礎(chǔ)上通過滴鼻方式滴入小劑量高濃度的新鮮MRSA菌懸液,連續(xù)滴注3天,造成免疫抑制MRSA定植模型。對照組將正常大鼠直接鼻腔滴入小劑量高濃度的新鮮MRSA菌懸液。細(xì)菌定植一定時間后取大鼠鼻粘膜組織,運用實時熒光定量PCR技術(shù)分析體內(nèi)試驗中,不同免疫狀態(tài)下大鼠鼻粘膜組織中分離的MRSA毒力因子在基因水平上的表達(dá)差異;同時處死各組動物后,取模型組和對照組動物的血清分別加入TSB培養(yǎng)基,以普通TSB培養(yǎng)基作對照,將MRSA在不同培養(yǎng)基中培養(yǎng)后,運用實時熒光定量PCR技術(shù)分析不同培養(yǎng)條件下不同培養(yǎng)時間點的MRSA毒力因子在基因水平上的表達(dá)差異。結(jié)果1.處于免疫抑制狀態(tài)的大鼠較正常大鼠,MRSA更容易在鼻粘膜定植;2.與肌注環(huán)磷酰胺前比較,肌注環(huán)磷酰胺后3天、7天時,大鼠血液學(xué)檢查可見外周血WBC、PLT數(shù)量迅速下降,同時RBC、HGB亦明顯降低(P0.01);外周血中CD3+T,CD3+CD4+T,CD3+CD8+T淋巴細(xì)胞比例明顯降低并且B淋巴細(xì)胞以及NK細(xì)胞的比例也明顯降低(P0.01)。外周血血清中免疫球蛋白IgG、IgM、IgA的含量明顯下降(P0.01);3.連續(xù)3次向大鼠鼻腔滴注小劑量高濃度的新鮮MRSA菌懸液,細(xì)菌定植一段時間后可穩(wěn)定存在鼻粘膜組織細(xì)胞內(nèi)外;4.體內(nèi)試驗毒力因子檢測結(jié)果表明,定植在模型組大鼠鼻粘膜組織中的MRSA毒力因子hla和spa的表達(dá)水平要高于對照組,(P0.05),其中目的基因spa的表達(dá)水平顯著升高(P0.01);5.體外試驗毒力因子檢測結(jié)果表明,MRSA在不同環(huán)境下培養(yǎng)不同時間后,細(xì)菌毒力因子的表達(dá)也存在一定的差異。培養(yǎng)至90min時,模型組與對照組毒力因子hla和spa的表達(dá)水平相對于培養(yǎng)0min時變化不明顯(P0.05);而在細(xì)菌培養(yǎng)至120min時,模型組hla基因的表達(dá)水平較對照組顯著升高(P0.01),且spa基因的表達(dá)水平較對照組升高(P0.05)。結(jié)論1.當(dāng)宿主處于免疫抑制狀態(tài)時,可能通過促進(jìn)金黃色葡萄球菌毒力因子的表達(dá)增強金黃色葡萄球菌的致病力;2.當(dāng)MRSA感染宿主時,細(xì)菌的生長情況和在培養(yǎng)基中的生長情況完全不同,可能導(dǎo)致金黃色葡萄球菌的不同毒力因子的表達(dá)存在一定的差異。
[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.
【學(xué)位授予單位】：內(nèi)蒙古醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R446.5

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