microRNA沉默卡氏肺孢子菌主要表面糖蛋白基因对大鼠卡氏肺孢子菌肺炎的治疗作用

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。

Curative effect of microRNA silencing major surface glycoprotein gene on Pneumocystis carinii pneumonia in rats

Objective To investigate the curative effect of microRNA targeting the upstream conserved sequence of major surface glycoprotein(MSG)gene of Pneumocystis carinii(PC) on Pneumocystis carinii pneumonia(PCP)in rats.Methods SD rat model of PCP was established by subcutaneous injection with dexamethasone sodium phosphate for 7 weeks.Model rats were randomly divided into microRNA(M),sulfa drug(H),physiological saline contro(l C1),empty vector control(C2)and model control(C3) groups.The rats in M,C1 and C2 groups were injected i.v.with recombinant plasmid pPC-UCS containing microRNA,physiological saline and empty vector respectively,while those in H group with sulfa drug by gastric lavage,once a day for 7 d.However,the rats in C3 group were untreated.The PCs in lung tissues of rats were counted under oil immersion lens by Giemsa staining,while the pathological changes of lung tissues were observed under light microscope by HE staining,the IL-10 and IFNγ expression levels in sera were determined by ELISA,and the expression levels of PC MSG-UCS mRNA in lung tissue by RT-PCR.The ultrastructure of PCs were observed under electron microscope.Results As compared with those in C1,C2 and C3 groups,the numbers of PCs in lung tissues and IL-10 expression levels in sera of rats in M and H groups decreased significantly(P < 0.05),while the IFNγ expression levels increased significantly(P < 0.05),and the PC-MSG mRNA expression levels in lung tissue decreased significantly(P < 0.05).Electron microscopy showed breakages on surface of PCs in M and H groups,while no breakages on those in C1,C2 and C3 groups.Conclusion Recombinant plasmid pPC-UCS containing microRNA showed significantly curative effect on PCP in rats,which provided a novel approach for therapy of PCP.