补肾活血调营化痰方干预哮喘MSCs归巢免疫及其对Treg-STAT6通路的影响

According to the theory of traditional Chinese medicine "Kidney storing essence, promoting bone and producing pulp" and ""Jing - blood - Jin - phlegm ", starting from the bone marrow response mechanism of airway inflammation in asthma, the ovalbumin sensitized rat asthma model, amplification cultured bone marrow mesenchymal stem cells (MSCs）, CFSE double labeled MSCs, body and tail vein transplantation of asthma rats. FCM analysis of lung tissue in CFSE-MSCs, MSCs-CXCR4, SDF-1mRNA expression in lung tissue of rats with detection of fqRT-PCR, detection of Western-blot expression in lung tissue of rats with SDF-1. FCM detection of CD4+CD25+Treg, detection of STAT6 protein expression in lung tissue of rats with Western-blot, STAT6mRNAs expression with fqRT-PCR.Study on effect of Bushen Huoxue Huatan Decoction for regulating effect of camp of MSCs homing immunity, and to explore the mechanism of asthma inflammation in Th2 from the Treg-ATAT6 pathway, tries to reveal the effects of compound Chinese Medicine has a way of airway inflammation in asthma, for Bushen-huoxue-tiaoying-hatan formula on the treatment of asthma and provide objective basis, to provide a scientific basis for further optimizing asthma characteristic therapy.

本课题依据"肾藏精，主骨生髓"及"精-血-津-痰"密切相关的中医理论，从哮喘气道炎症的骨髓反应机制出发，拟采用卵蛋白致敏激发哮喘大鼠模型，体外扩增培养大鼠同源异体骨髓间充质细胞（MSCs），CFSE荧光双标MSCs，并尾静脉移植哮喘大鼠体内。FCM分析肺组织CFSE-MSCs、MSCs-CXCR4，fqRT-PCR、Western-blot检测大鼠肺组织SDF-1mRNA及蛋白表达。FCM检测CD4+CD25+Treg，fqRT-PCR、Western-blot检测大鼠肺组织STAT6 mRNA及蛋白表达。研究补肾活血调营化痰方对MSCs肺组织归巢的干预作用，探讨MSCs对哮喘Th2炎症Treg-STAT6通路的影响，寻找补肾活血调营化痰方可能的作用环节和靶点，试图揭示中药有效复方控制哮喘气道炎症的可能途径，为进一步优化哮喘特色制剂及提取有效组分获取证据。

骨髓间充质干细胞（BMSCs）是机体内的成体干细胞，参与了对哮喘气道炎症的免疫调控。本课题依据“肾藏精，主骨生髓”及“精-血-津-痰”密切相关的中医理论，从哮喘气道炎症的骨髓反应机制出发，采用卵清白蛋白（OVA）致敏和激发制备哮喘大鼠模型，体外扩增培养大鼠同源异体骨髓间充质干细胞（BMSCs），尾静脉移植哮喘大鼠体内，并予以阳和平喘颗粒药物干预，探寻阳和平喘颗粒对BMSCs移植向肺组织归巢及气道炎症的干预作用，从而进一步为探讨中医补肾活血化痰治法对哮喘内源性BMSCs增殖分化及功能的促进作用奠定基础。结果发现：1、在哮喘大鼠模型中，通过予以静脉注射BMSCs后，能显著降低哮喘大鼠BALF中炎性细胞数目、血清IL-4、IL-5、IL-13及肺组织STAT6、SDF-1含量，提高外周血CD4+CD25+调节性T细胞比例，减缓气道炎症。2、通过予以阳和平喘颗粒药物干预哮喘大鼠模型，能进一步促进降低BMSCs移植哮喘大鼠BALF中炎性细胞数目、血清IL-4、IL-5、IL-13含量，调节CD4+CD25+调节性T细胞、STAT6、SDF-1的表达，从而进一步改善气道炎症。3、在采用抗CD25+单克隆抗体的哮喘大鼠模型中，BMSCs调节哮喘气道炎症的作用下降，而予以阳和平喘颗粒联合干预后，其气道炎症较单独予以BMSCs干预改善明显。这些研究结果提示：BMSCs能够通减少哮喘气道炎性细胞浸润，提高Treg表达，改善哮喘气道炎症，是治疗哮喘的重要方向；中药阳和平喘颗粒能够上调Treg表达，通过介导Treg-STAT6通路促进BMSCs对哮喘气道炎症的干预作用。