【摘要】：研究背景角膜移植手術是目前治療角膜盲最主要、最有效的復明手段,但術后發(fā)生免疫排斥反應仍然是導致移植失敗的最主要原因。雖然免疫抑制劑可以抑制免疫排斥反應,但價格昂貴且毒副作用大,限制了它的長期使用。因此近年來對角膜移植免疫排斥反應的研究方向主要集中在誘導受體產生針對移植角膜的特異性免疫耐受。已知樹突狀細胞(dendritic cells,DC)具有誘導移植免疫耐受的作用,同時研究表明,核因子-κB(NF-κB)與DC發(fā)育成熟及抗原呈遞功能的關鍵性調控基因的轉錄密切相關,p50/RelA是NF-κB最常見的二聚體,是其發(fā)揮生物學功能的最主要形式。因此,本研究欲通過低表達RelA基因從而下調NF-κB的轉錄活性,阻斷DC的成熟,并經尾靜脈注射該DC回輸給受體,通過建立大鼠同種異體角膜移植模型,觀察其在角膜移植免疫反應中的作用,以期為角膜移植免疫耐受提供新的理論和實驗依據(jù)。目的探討低表達RelA基因的樹突狀細胞在大鼠角膜移植免疫反應中的作用。方法(1)應用GM-CSF和IL-4體外培養(yǎng)、誘導分化供體大鼠骨髓源性DC,結合細胞形態(tài)、表型及功能加以鑒定。(2)應用高效抑制RelA表達的shRNA序列,行慢病毒包裝并感染未成熟DC(命名為RelA-DC),同時設立陰性對照病毒感染DC組(命名為SiNC-DC)及未感染慢病毒DC組(命名為Control-DC),分別給予1μg/ml的脂多糖刺激,利用RT-qPCR、Western Blot檢測DC中RelA基因表達;流式細胞儀分析DC表型;ELISA檢測DC上清IL-12和IFN-γ含量;MLR檢測DC刺激T淋巴細胞增殖的能力。(3)以SD大鼠為供體、Wistar大鼠為受體,建立大鼠同種異體角膜移植模型60例,隨機分成PBS組、mDC組、imDC組和RelA-DC組(n=15),分別于移植前7天經尾靜脈注射1ml PBS、mDC(5×106個)、imDC(5×106個)和 RelA-DC(5×106個);術后觀察角膜植片的存活時間并評分;第14天取角膜植片行HE切片觀察、通過RT-qPCR檢測Th1型細胞因子(IL-2、IFN-γ)和Th2型細胞因子(IL-4、IL-10)的mRNA表達水平,取脾臟通過流式細胞儀檢測CD4+CD25+Treg、CD4+FoxP3+Treg的陽性表達率及通過MLR觀察受體脾臟T細胞對供體抗原刺激的反應能力。結果(1)經形態(tài)學、表型和功能學鑒定,證實所培養(yǎng)細胞為DC。(2)RelA-DC的RelA基因轉錄水平和蛋白表達水平明顯低于SiNC-DC、Control-DC(P0.01);其表面分子(MHC Ⅱ、CD80、CD86)的表達水平、刺激 T淋巴細胞增殖能力及上清液細胞因子IL-12、IFN-y水平亦低于SiNC-DC、Control-DC(P0.05)。(3)RelA-DC組大鼠受體脾臟T細胞對供體抗原刺激的反應能力、Th1型細胞因子水平低于PBS組,mDC組和imDC組(P0.05),而角膜植片的存活時間、Th2型細胞因子水平、CD4+CD25+Treg及CD4+FoxP3+Treg陽性表達率高于PBS組,mDC組和imDC組(P0.05)。結論輸注低表達RelA基因的DC能抑制大鼠角膜移植術后免疫排斥反應,明顯延長角膜植片的存活時間,可能主要通過抑制T細胞增殖反應、誘導Th1/Th2偏移以及CD4+CD25+Treg和CD4+FoxP3+Treg的擴增參與免疫耐受的形成。
[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- 緯 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- 緯) 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，

本文編號：2382122