【摘要】 为探究旋毛虫肌幼虫谷氨酸脱羧酶（GAD）在肌幼虫侵入肠道中发挥的作用，本研究分别将设计与合成的siRNA-GAD、siRNA-Control转染旋毛虫肌幼虫（siRNA-GAD组、siRNA-Control组、后者为阳性对照组NC）并设未转染siRNA的阳性对照组（PC组），于转染后12 h分别采用荧光定量PCR(qPCR)和western blot检测各组旋毛虫肌幼虫GAD基因的转录及其蛋白表达水平。结果显示，siRNA-GAD组相对PC组肌幼虫GAD基因的转录水平和蛋白表达水平分别降低63.7%(P<0.001)和62%(P<0.0001)，表明siRNA-GAD能够干扰旋毛虫肌幼虫GAD基因的转录及表达。将上述各组旋毛虫肌幼虫分别感染BALB/c小鼠，并设未感染的小鼠为阴性对照，感染10 d时对小鼠采样，采用ELISA检测小鼠血清中促炎细胞因子TNF-α、IFN-γ和抗炎细胞因子IL-4、IL-10的分泌水平；通过流式细胞术检测小鼠脾脏CD4~+/CD8~+T细胞比值；制备各组小鼠小肠组织病理切片观察其病理变化；采用qPCR检测小鼠小肠调节性T细胞（Treg细胞）活化标志物GARP、CD62L、OX40和趋化因子受体CXCR3、CCR4、CCR5、CCR10 mRNA的相对转录水平；采用免疫组化法检测各组小鼠小肠Foxp3的表达情况。结果显示，与阴性对照组相比，旋毛虫感染引起宿主血清细胞因子TNF-α、IFN-γ、IL-4和IL-10的分泌水平均显著上升（P<0.05），相比PC组siRNA-GAD组小鼠血清中IL-4和IL-10的分泌水平分别下降7.249%和8.460%(P<0.05);siRNA-GAD组小鼠脾脏中CD4~+/CD8~+T细胞比值升高34.72%，差异显著（P<0.05）；组织病理切片观察可见，PC组小鼠小肠上皮受损，局部炎性浸润，siRNA-GAD组小鼠肠绒毛水肿严重；相较PC组，siRNA-GAD组小鼠小肠Treg细胞活化标志物GARP和OX45的转录水平分别降低37.30%和13.06%(P<0.05);Treg细胞趋化因子受体CXCR3和CCR4的转录水平分别降低77.26%和61.06%(P<0.05)；免疫组化结果显示，与阴性对照组相比，旋毛虫感染后小鼠小肠Treg细胞中的黄色颗粒增多，而siRNA-GAD组小鼠小肠Treg细胞中的黄色颗粒沉着相对较少，表明干扰GAD基因能够降低旋毛虫成虫期诱导Treg细胞标志蛋白Foxp3的表达。上述结果表明，干扰旋毛虫GAD基因能够降低小鼠小肠Treg细胞的活化水平，抑制宿主Th2型免疫反应，提高宿主非特异性免疫反应水平，也表明GAD参与旋毛虫诱导的宿主免疫反应，本研究为探究旋毛虫的致病机制及旋毛虫病的防治提供新思路。更多还原

【Abstract】 In order to investigate the role of glutamic acid decarboxylase(GAD) in Trichinella spiralis muscle larvae during intestinal invasion, in this study, siRNA-GAD and siRNA-Control were transfected into T. spiralis muscle larvae respectively(designated as siRNA-GAD group, siRNA-Control group), along with a positive control group that was not transfencted with siRNA(PC group). At 12 hours post-transfection, qPCR and western blot were used to detect the transcriptional levels of the GAD gene and its protein expression levels in T. spiralis muscle larvae in each group. The results showed that, compared to the PC group, the transcription and protein expression levels of GAD gene were reduced by 63.7% and 62%, respectively, in the siRNA-GAD group(P<0.01), indicating that siRNA-GAD successfully interfered with the transcription and expression of GAD gene in T. spiralis muscle larvae. BALB/c mice were infected with Trichinella spiralis muscle larvae from the respective experimental groups, with uninfected mice serving as negative controls(NC group). At 10 days post-infection, samples were collected for comprehensive analysis: serum levels of pro-inflammatory cytokines(TNF-α, IFN-γ) and anti-inflammatory cytokines(IL-4, IL-10) were measured by ELISA;mRNA transcription of Treg cell activation markers(GARP, CD62L, OX40) and chemokine receptors(CXCR3, CCR4, CCR5,CCR10) in small intestine tissues were quantified using quantitative real-time PCR(qPCR); the CD4~+/CD8~+T cell ratio in spleen was determined by flow cytometry; small intestinal pathological changes were evaluated through hematoxylin-eosin(H.E.) staining of tissue sections, while Foxp3 expression was detected by immunohistochemistry(IHC). Results showed that Trichinella infection caused a significant increase in the secretion levels of host serum cytokines TNF-α, IFN-γ, IL-4 and IL-10 compared to the negative control group(P<0.05), and compared to PC group, IL-4 and IL-10 in the siRNA-GAD group decreased by 7.249% and 8.460%,respectively(P<0.05); the ratio of CD4~+/CD8~+T cells in the siRNA-GAD group was significantly elevated by 34.72%(P<0.05);histopathological sections showed that T. spiralis infection induced intestinal epithelial damage in the mouse, and localized inflammatory infiltration. In the siRNA-GAD group, the edema of intestinal villi was severe. The transcript levels of Treg cell activation markers GARP and OX45 in the siRNA-GAD group, compared to those in the PC group, were reduced significantly by37.30% and 13.06%, respectively; compare to that of the PC group, the ratio of the Treg cell chemotaxis level in the siRNA-GAD group was increased significantly by 34.72%. The transcript levels of Treg cell chemokine receptors CXCR3 and CCR4 in GAD group were decreased by 77.26% and 61.06%, respectively. Immunohistochemistry results showed that yellow particles in Treg cells increased in the small intestine after Trichinella infection compared to the negative control group, while the siRNA-GAD group had the least amount of yellow particles deposited. These results demonstrate that silencing the GAD gene significantly downregulates the expression of T cell(Treg) marker protein Foxp3 during the adult stage of Trichinella spiralis infection. The above results indicate that interference with the GAD gene of Trichinella reduces the activation level of small intestinal Treg cells in mice, inhibits the host Th2-type immune response, and increases the level of host nonspecific immunity, suggesting that GAD is involved in Trichinellainduced host immune response, which provides a new idea for investigating the mechanism of pathogenesis of Trichinella as well as the prevention and control of Trichinellosis.更多还原