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目的探讨靶向卡氏肺孢子菌(Pneumocystis carinii,PC)主要表面糖蛋白基因(Major surface glycoprotein gene,MSG)上游保守序列(Upstream conserved sequence,UCS)的microRNA重组质粒对大鼠卡氏肺孢子菌肺炎(Pneumocystis cariniipneumonia,PCP)的治疗作用。方法将SD大鼠经腹股沟皮下注射地塞米松磷酸钠7周,制备大鼠PCP感染模型。将模型大鼠随机分为5组:microRNA重组质粒治疗组(M组)、磺胺药物治疗组(H组)、生理盐水对照组(C1组)、空载体质粒对照组(C2组)和模型对照组(C3组),其中M组和C1、C2组经尾静脉分别注射含microRNA重组质粒pPC-UCS、生理盐水和空载体,H组用磺胺药物灌胃治疗,C3组不做处理,每日1次,疗程为7 d。1周后吉姆萨染色,油镜下计数大鼠肺组织液印片中PC包囊数;HE染色,光镜观察肺组织病理改变;ELISA法检测大鼠血清中IL-10和IFNγ的表达水平;RT-PCR法检测肺组织中PC MSG-UCSmRNA的表达水平;电镜观察PC的超微结构。结果与C1、C2和C3组相比,M组和H组大鼠肺组织PC包囊数均明显减少(P<0.05),肺组织炎症较轻,大鼠血清IL-10的表达水平均显著降低(P<0.05),而IFNγ的表达水平显著升高(P<0.05),大鼠肺组织PC MSG-UCS mRNA的表达水平均显著降低(P<0.05);电镜观察显示,M组和H组PC包囊表面均出现破损,C1、C2、C3组未发现破损。结论 microRNA重组质粒pPC-UCS对大鼠PCP有明显的治疗作用,为治疗PCP提供了新的思路。
Objective To investigate the effect of microRNA recombinant plasmids targeting upstream sequence (UCS) of major surface glycoprotein gene (MSG) of Pneumocystis carinii (PC) Pneumocystis cariniipneumonia (PCP). Methods SD rats were inoculated subcutaneously with dexamethasone sodium phosphate for 7 weeks to prepare rat model of PCP infection. The model rats were randomly divided into 5 groups: microRNA recombinant plasmid treatment group (M group), sulfa drug treatment group (H group), saline control group (C1 group), empty vector plasmid control group (C2 group) and model control (Group C3). The mice in group M and groups C1 and C2 were injected with pPC-UCS containing microRNA, saline and empty vector through caudal vein respectively. Group H was treated with sulfonamide intragastrically. Group C3 was not treated daily. 1 times, treatment for 7 d. 1 week after Giemsa staining, counting the amount of PC cytoplasm in the lung tissue of rats under oil microscope; the pathological changes of lung tissue were observed by HE staining and light microscope; the levels of IL-10 and IFNγ in serum were detected by ELISA; The expression of PC-MSG-UCS mRNA in lung tissue was detected by RT-PCR. The ultrastructure of PC was observed by electron microscope. Results Compared with C1, C2 and C3 groups, the numbers of PC cysts in lung tissue of M group and H group were significantly decreased (P <0.05), lung tissue inflammation was lighter, and serum IL-10 level was significantly higher (P <0.05), while the expression of IFNγ was significantly increased (P <0.05). The expression of PC-MSG-UCS mRNA in lung tissue of rats was significantly decreased (P <0.05) Group PC cyst surface were damaged, C1, C2, C3 group found no damage. Conclusion The microRNA recombinant plasmid pPC-UCS has a significant therapeutic effect on PCP in rats and provides a new idea for the treatment of PCP.