RelA修饰的树突状细胞诱导大鼠角膜移植免疫耐受的实验研究
发布时间：2018-12-16 09:18
【摘要】：研究背景角膜移植手术是目前治疗角膜盲最主要、最有效的复明手段,但术后发生免疫排斥反应仍然是导致移植失败的最主要原因。虽然免疫抑制剂可以抑制免疫排斥反应,但价格昂贵且毒副作用大,限制了它的长期使用。因此近年来对角膜移植免疫排斥反应的研究方向主要集中在诱导受体产生针对移植角膜的特异性免疫耐受。已知树突状细胞(dendritic cells,DC)具有诱导移植免疫耐受的作用,同时研究表明,核因子-κB(NF-κB)与DC发育成熟及抗原呈递功能的关键性调控基因的转录密切相关,p50/RelA是NF-κB最常见的二聚体,是其发挥生物学功能的最主要形式。因此,本研究欲通过低表达RelA基因从而下调NF-κB的转录活性,阻断DC的成熟,并经尾静脉注射该DC回输给受体,通过建立大鼠同种异体角膜移植模型,观察其在角膜移植免疫反应中的作用,以期为角膜移植免疫耐受提供新的理论和实验依据。目的探讨低表达RelA基因的树突状细胞在大鼠角膜移植免疫反应中的作用。方法(1)应用GM-CSF和IL-4体外培养、诱导分化供体大鼠骨髓源性DC,结合细胞形态、表型及功能加以鉴定。(2)应用高效抑制RelA表达的shRNA序列,行慢病毒包装并感染未成熟DC(命名为RelA-DC),同时设立阴性对照病毒感染DC组(命名为SiNC-DC)及未感染慢病毒DC组(命名为Control-DC),分别给予1μg/ml的脂多糖刺激,利用RT-qPCR、Western Blot检测DC中RelA基因表达;流式细胞仪分析DC表型;ELISA检测DC上清IL-12和IFN-γ含量;MLR检测DC刺激T淋巴细胞增殖的能力。(3)以SD大鼠为供体、Wistar大鼠为受体,建立大鼠同种异体角膜移植模型60例,随机分成PBS组、mDC组、imDC组和RelA-DC组(n=15),分别于移植前7天经尾静脉注射1ml PBS、mDC(5×106个)、imDC(5×106个)和 RelA-DC(5×106个);术后观察角膜植片的存活时间并评分;第14天取角膜植片行HE切片观察、通过RT-qPCR检测Th1型细胞因子(IL-2、IFN-γ)和Th2型细胞因子(IL-4、IL-10)的mRNA表达水平,取脾脏通过流式细胞仪检测CD4+CD25+Treg、CD4+FoxP3+Treg的阳性表达率及通过MLR观察受体脾脏T细胞对供体抗原刺激的反应能力。结果(1)经形态学、表型和功能学鉴定,证实所培养细胞为DC。(2)RelA-DC的RelA基因转录水平和蛋白表达水平明显低于SiNC-DC、Control-DC(P0.01);其表面分子(MHC Ⅱ、CD80、CD86)的表达水平、刺激 T淋巴细胞增殖能力及上清液细胞因子IL-12、IFN-y水平亦低于SiNC-DC、Control-DC(P0.05)。(3)RelA-DC组大鼠受体脾脏T细胞对供体抗原刺激的反应能力、Th1型细胞因子水平低于PBS组,mDC组和imDC组(P0.05),而角膜植片的存活时间、Th2型细胞因子水平、CD4+CD25+Treg及CD4+FoxP3+Treg阳性表达率高于PBS组,mDC组和imDC组(P0.05)。结论输注低表达RelA基因的DC能抑制大鼠角膜移植术后免疫排斥反应,明显延长角膜植片的存活时间,可能主要通过抑制T细胞增殖反应、诱导Th1/Th2偏移以及CD4+CD25+Treg和CD4+FoxP3+Treg的扩增参与免疫耐受的形成。
[Abstract]:Background Corneal transplantation is the most important and effective method in the treatment of corneal blindness at present, but the immune rejection is still the main reason for the failure of corneal transplantation. Although immunosuppressants can inhibit immune rejection, their long-term use is limited by their high cost and side effects. Therefore, in recent years, the research direction of corneal transplantation immune rejection is mainly focused on inducing the receptor to produce specific immune tolerance for corneal transplantation. Dendritic cells (dendritic cells,DC) have been known to induce transplanted immune tolerance. Studies have also shown that nuclear factor- 魏 B (NF- 魏 B) is closely related to the transcription of key genes that regulate the development, maturation and antigen presentation of DC. P50/RelA is the most common dimer of NF- 魏 B and the main form of its biological function. Therefore, the aim of this study was to down-regulate the transcriptional activity of NF- 魏 B by low expression of RelA gene, block the maturation of DC, and inject the DC back to the receptor via caudal vein to establish a rat corneal allograft model. In order to provide new theoretical and experimental basis for corneal transplantation immune tolerance, we observed its role in corneal transplantation immune response. Objective to investigate the role of dendritic cells with low expression of RelA gene in corneal transplantation in rats. Methods (1) GM-CSF and IL-4 were used to induce bone marrow-derived DC, binding cells in vitro to identify the morphology, phenotype and function of the cells. (2) shRNA sequence was used to inhibit the expression of RelA. Lentivirus was packaged and infected with immature DC (named RelA-DC). The negative control virus infected DC group (named SiNC-DC) and the non-infected lentivirus DC group (named Control-DC) were given 1 渭 g/ml lipopolysaccharide stimulation respectively. RelA gene expression in DC was detected by RT-qPCR,Western Blot. DC phenotype was analyzed by flow cytometry, IL-12 and IFN- 纬 contents in DC supernatant were detected by ELISA. MLR was used to detect the ability of DC to stimulate T lymphocyte proliferation. (3) 60 cases of rat corneal allograft model were established with SD rat as donor and Wistar rat as receptor. 60 cases were randomly divided into PBS group, mDC group, imDC group and RelA-DC group (n = 15). 1ml PBS,mDC (5 脳 106), imDC (5 脳 106) and RelA-DC (5 脳 106) were injected via caudal vein 7 days before transplantation. The survival time and score of corneal grafts were observed. On the 14th day, the corneal grafts were taken for HE section observation. The mRNA expression levels of Th1 type cytokines (IL-2,IFN- 纬) and Th2 type cytokines (IL-4,IL-10) were detected by RT-qPCR. The spleen was taken to detect CD4 CD25 Treg, by flow cytometry. The positive expression rate of CD4 FoxP3 Treg and the ability of T cells to respond to donor antigen stimulation were observed by MLR. Results (1) by morphological, phenotypic and functional identification, the transcriptional level and protein expression level of RelA gene in cultured cells with DC. (2) RelA-DC were significantly lower than those in SiNC-DC,Control-DC (P0.01). The expression level of MHC 鈪，
本文编号：2382122
[Abstract]:Background Corneal transplantation is the most important and effective method in the treatment of corneal blindness at present, but the immune rejection is still the main reason for the failure of corneal transplantation. Although immunosuppressants can inhibit immune rejection, their long-term use is limited by their high cost and side effects. Therefore, in recent years, the research direction of corneal transplantation immune rejection is mainly focused on inducing the receptor to produce specific immune tolerance for corneal transplantation. Dendritic cells (dendritic cells,DC) have been known to induce transplanted immune tolerance. Studies have also shown that nuclear factor- 魏 B (NF- 魏 B) is closely related to the transcription of key genes that regulate the development, maturation and antigen presentation of DC. P50/RelA is the most common dimer of NF- 魏 B and the main form of its biological function. Therefore, the aim of this study was to down-regulate the transcriptional activity of NF- 魏 B by low expression of RelA gene, block the maturation of DC, and inject the DC back to the receptor via caudal vein to establish a rat corneal allograft model. In order to provide new theoretical and experimental basis for corneal transplantation immune tolerance, we observed its role in corneal transplantation immune response. Objective to investigate the role of dendritic cells with low expression of RelA gene in corneal transplantation in rats. Methods (1) GM-CSF and IL-4 were used to induce bone marrow-derived DC, binding cells in vitro to identify the morphology, phenotype and function of the cells. (2) shRNA sequence was used to inhibit the expression of RelA. Lentivirus was packaged and infected with immature DC (named RelA-DC). The negative control virus infected DC group (named SiNC-DC) and the non-infected lentivirus DC group (named Control-DC) were given 1 渭 g/ml lipopolysaccharide stimulation respectively. RelA gene expression in DC was detected by RT-qPCR,Western Blot. DC phenotype was analyzed by flow cytometry, IL-12 and IFN- 纬 contents in DC supernatant were detected by ELISA. MLR was used to detect the ability of DC to stimulate T lymphocyte proliferation. (3) 60 cases of rat corneal allograft model were established with SD rat as donor and Wistar rat as receptor. 60 cases were randomly divided into PBS group, mDC group, imDC group and RelA-DC group (n = 15). 1ml PBS,mDC (5 脳 106), imDC (5 脳 106) and RelA-DC (5 脳 106) were injected via caudal vein 7 days before transplantation. The survival time and score of corneal grafts were observed. On the 14th day, the corneal grafts were taken for HE section observation. The mRNA expression levels of Th1 type cytokines (IL-2,IFN- 纬) and Th2 type cytokines (IL-4,IL-10) were detected by RT-qPCR. The spleen was taken to detect CD4 CD25 Treg, by flow cytometry. The positive expression rate of CD4 FoxP3 Treg and the ability of T cells to respond to donor antigen stimulation were observed by MLR. Results (1) by morphological, phenotypic and functional identification, the transcriptional level and protein expression level of RelA gene in cultured cells with DC. (2) RelA-DC were significantly lower than those in SiNC-DC,Control-DC (P0.01). The expression level of MHC 鈪，
本文编号：2382122
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