本文選題：移植排斥反應(yīng)　切入點(diǎn)：Genistein　出處：《山東大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：背景與目的隨著手術(shù)技術(shù)的不斷發(fā)展、免疫抑制劑的推廣應(yīng)用及綜合醫(yī)療水平的提高,心臟移植技術(shù)得到了快速的發(fā)展,患者移植術(shù)后的生存率也得到了明顯的提升。但在實(shí)際臨床中,患者仍需面臨超急性排斥反應(yīng)、急性排斥反應(yīng)和慢性排斥反應(yīng)等免疫反應(yīng)的危險(xiǎn),且其作用直接影響患者的預(yù)后及生存,其中對(duì)急性排斥反應(yīng)的早期診斷和干預(yù)治療尤為重要。眾多研究表明,趨化因子受體及配體間的相互作用,在免疫細(xì)胞的誘導(dǎo)活化、特異性募集和宿主抗移植物移植排斥反應(yīng)中發(fā)揮著重要作用,在移植免疫的急性排斥反應(yīng)中異�；钴S。因此趨化因子受體的拮抗劑有望在移植免疫抑制方面發(fā)揮重要作用。趨化因子受體CXCR3是G蛋白超家族的成員,其與配體間的特異性結(jié)合可誘導(dǎo)以T淋巴細(xì)胞為主的多種免疫細(xì)胞的活化和增殖,是導(dǎo)致急性排斥反應(yīng)中炎癥細(xì)胞浸潤和組織損傷的重要因素。如果能阻斷CXCR3的活化過程,抑制相關(guān)免疫細(xì)胞的增殖,就可能對(duì)移植心臟起到保護(hù)作用。前期研究證實(shí),CXCR3活化過程中的下游級(jí)聯(lián)反應(yīng)依賴?yán)野彼岬鞍准っ?PTK)的激活,而Genistein具有抑制酪氨酸蛋白激酶的作用。本實(shí)驗(yàn)通過建立大鼠急性排斥反應(yīng)及免疫耐受模型,觀察CXCR3與心臟移植急性排斥反應(yīng)的相關(guān)性,并探討Genistein聯(lián)合環(huán)孢素A對(duì)大鼠同種異體急性排斥反應(yīng)中CXCR3表達(dá)的影響,為心臟移植急性排斥反應(yīng)的免疫抑制治療提供新的方向。方法選取近交系Wistar大鼠為供者,SD大鼠為受者,使用“套管連接”技術(shù)將供者心臟移植至受者大鼠右側(cè)頸部皮下。移植后的大鼠隨機(jī)分為3組,每組12只,急性排斥反應(yīng)(AR)組：心臟移植術(shù)后未采取任何治療；環(huán)孢素A(CsA)組：受者鼠移植術(shù)后接受CsA 10mg/kg/d治療；CsA+Genistein (C+G)組：術(shù)后接受CsA 10mg/kg/d及Genistein lOmg/kg/d的聯(lián)合治療。術(shù)后每天通過視診或觸診受者鼠右側(cè)頸部,觀察移植心臟存活情況。術(shù)后第7天,采集3組移植心臟,制備成組織切片,HE染色觀察移植心臟的病理學(xué)改變,免疫組織化學(xué)染色及Western blotting法檢鋇CXCR3受體的表達(dá),采用圖像分析軟件對(duì)其表達(dá)程度進(jìn)行半定量分析。結(jié)果CsA組和C+G組的移植心臟存活的情況要明顯好于AR組,移植心臟免疫耐受性良好。HE染色結(jié)果可見,AR組中的移植心臟組織可見的彌漫的炎性細(xì)胞浸潤和心肌細(xì)胞變性壞死,呈現(xiàn)急性排斥反應(yīng)的典型表現(xiàn),C+G組的排斥反應(yīng)強(qiáng)度明顯弱于前兩組。免疫組化染色中CXCR3在各組中的IOD值為：AR組(1.05±0.20)×105,CsA組(0.53±0.16)×105,C+G組(0.24±0.08)×105。CsA組的表達(dá)水平明顯低于AR組；C+G組的表達(dá)水平明顯低于前2組(P0.05)。Western blotting中檢測(cè)各組的灰度比值的趨勢(shì)與免疫組化檢測(cè)結(jié)果一致,C+G組移植心臟組織中CXCR3蛋白的表達(dá)水平明顯低于前2組(P0.05)。結(jié)論CXCR3的表達(dá)程度與心臟移植急性排斥反應(yīng)的程度呈正相關(guān)；Genistein聯(lián)合CsA可明顯減輕移植心臟組織中CXCR3的表達(dá),能進(jìn)一步減輕急性排斥反應(yīng)。
[Abstract]:Background and objective with the development of surgical techniques, immunosuppression and application of comprehensive health care level, heart transplantation technology has been rapid development, the survival rate of patients after transplantation has been significantly improved. But in clinical practice, patients still need to face the danger of hyperacute rejection, acute rejection chronic rejection and immune response, and its effect directly affect the prognosis and survival of patients, the early diagnosis and treatment of acute allograft rejection is very important. Many studies have shown that chemokine receptor interactions and ligand, in induced activation of immune cells, plays an important role in raising specific and host versus graft rejection, very active in the acute rejection of transplantation immune reaction. Therefore chemokine receptor antagonists in transplantation immune suppression is expected to Play an important role. The chemokine receptor CXCR3 is a member of the G protein superfamily, which is combined with the specific ligand can induce a variety of immune cells in the T lymphocyte activation and proliferation, is an important factor leading to inflammatory cell infiltration and tissue injury in acute rejection. If we can block the activation process of CXCR3, related to the inhibition of the proliferation of immune cells. It may protect the heart transplantation. Previous studies demonstrated that the downstream cascade activation of CXCR3 dependent protein tyrosine kinase (PTK) activation, and Genistein can inhibit the role of protein tyrosine kinase. This experiment establish and acute rejection in rat model of immune tolerance correlation, observation of CXCR3 and acute rejection of heart transplantation, and investigate the effect of Genistein combined with cyclosporine A on the expression of CXCR3 in rat acute allograft rejection effect, Immune to acute rejection of heart transplantation rejection and provide a new direction of treatment. Methods of inbred Wistar rats as donors and SD rats as recipients, the use of "connection" technology of the donor heart transplantation to recipient rats subcutaneously on the right side of the neck. The rats were randomly divided into 3 groups after transplantation, 12 rats in each group, acute rejection (AR) group: no treatment after heart transplantation; cyclosporine A (CsA) group: transplantation rats underwent CsA after 10mg/kg/d treatment; CsA+Genistein (C+G) group: after receiving combination therapy and Genistein lOmg/kg/d CsA 10mg/kg/d. Every day after operation by visual examination or palpation of recipients on the right side of the neck, to observe the cardiac allograft survival. After seventh days, collecting 3 groups of heart transplantation, preparation of tissue sections to observe the pathological changes of the transplanted heart HE staining, immunohistochemical staining and Western blotting method for detecting CXCR3 receptor The expression of image analysis software was used for semi quantitative analysis of the degree of expression. Results CsA group and C+G group of cardiac allograft survival is significantly better in the AR group, heart transplantation immune tolerance.HE good dyeing result shows that in the AR group of heart tissue transplantation visible diffuse infiltration of inflammatory cells and myocardial cells the typical performance showed degeneration and necrosis, acute rejection, rejection of the strength of C+G group was significantly lower than the former two groups. Immunohistochemical staining of CXCR3 in each group of the IOD value is: AR group (1.05 + 0.20) * 105, CsA group (0.53 + 0.16) * 105, C+G group (0.24 + 0.08) * 105.CsA group the expression level was significantly lower than that of AR group; the expression level of C+G group was significantly lower than the former 2 groups (P0.05) were detected in.Western gray level ratio in the blotting trend and immunohistochemical detection results, CXCR3 protein C+G in heart tissue transplantation group was significantly lower In the first 2 groups (P0.05). Conclusion the degree of CXCR3 expression is positively correlated with the degree of acute rejection in heart transplantation. Genistein combined with CsA can significantly reduce the expression of CXCR3 in transplanted heart tissue, and further reduce the acute rejection.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R654.2

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本文編號(hào)：1644541