本文選題：腺病毒　切入點(diǎn)：CTLA4Ig　出處：《西南大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 異基因(異體、異種)皮膚移植后的排斥反應(yīng)主要是由活化T細(xì)胞主導(dǎo)的急性細(xì)胞性排斥反應(yīng),此過程中T細(xì)胞共刺激信號(hào)的參與對(duì)于T細(xì)胞活化和免疫排斥的發(fā)生是必需的,阻斷共刺激信號(hào),可導(dǎo)致T細(xì)胞無反應(yīng)、細(xì)胞凋亡或克隆丟失,從而誘導(dǎo)免疫耐受。在已發(fā)現(xiàn)的多種T細(xì)胞共刺激信號(hào)中,公認(rèn)最為重要的是B7-CD28信號(hào)和CD40-CD40L信號(hào)。利用蛋白或抗體可有效對(duì)其阻斷,誘導(dǎo)免疫耐受,但存在制備復(fù)雜、成本較高、半衰期短和需要長期施用等弊端。腺病毒載體安全性高、制備容易以其介導(dǎo)基因治療的方式將共刺激分子基因?qū)氚屑?xì)胞中,使治療性蛋白在特定區(qū)域表達(dá),阻斷共刺激信號(hào),有望降低成本、延長效應(yīng)時(shí)間。皮膚成纖維細(xì)胞易獲取、培養(yǎng)及大量擴(kuò)增,也是常用的組織工程皮膚種子細(xì)胞和皮膚基因治療理想的靶細(xì)胞。因此本研究以體外培養(yǎng)的人皮膚成纖維細(xì)胞為靶細(xì)胞,用腺病毒介導(dǎo)CTLA4Ig和CD40Ig基因轉(zhuǎn)染阻斷公認(rèn)最為重要的B7/CD28和CD40/CD40L信號(hào),通過對(duì)Adv-CTLA4Ig、Adv-CD40Ig感染人皮膚成纖維細(xì)胞的效率檢測、感染后細(xì)胞形態(tài)和增殖能力檢測、目的蛋白表達(dá)檢測及對(duì)其免疫性能影響檢測,研究、評(píng)價(jià)腺病毒基因治療方式介導(dǎo)CTLA4Ig和CD40Ig阻斷雙共刺激信號(hào)應(yīng)用于人皮膚成纖維細(xì)胞修飾和治療的效果和可行性,為其應(yīng)用提供基礎(chǔ)資料。其主要內(nèi)容如下: 1.采用流式細(xì)胞分析技術(shù)檢測腺病毒對(duì)體外培養(yǎng)人皮膚成纖維細(xì)胞的轉(zhuǎn)染效率。結(jié)果表明:Adv-EGFP在感染復(fù)數(shù)(MOI)為50,100時(shí)對(duì)人皮膚成纖維細(xì)胞轉(zhuǎn)染效率約為51.7±7.24%、69.99±3.26%,同時(shí)對(duì)細(xì)胞形態(tài)和增殖能力無明顯影響。因此本研究選用100為腺病毒感染人皮膚成纖維細(xì)胞的MOI。 2.采用形態(tài)學(xué)觀察和MTT法細(xì)胞活力檢測重組腺病毒轉(zhuǎn)染對(duì)體外培養(yǎng)人皮膚成纖維細(xì)胞生長形態(tài)和增殖能力的影響。結(jié)果表明:重組病毒Adv-CTLA4Ig、Adv-CD40Ig在MOI為100時(shí)單獨(dú)和混合轉(zhuǎn)染人皮膚成纖維細(xì)胞后,實(shí)驗(yàn)組與對(duì)照組細(xì)胞形態(tài)均呈長梭形、不規(guī)則三角形,平行、放射狀或漩渦狀分布,有較長胞質(zhì)突起,輪廓清晰,并且各組細(xì)胞增殖能力無顯著差異(P＞0.05)。 3.采用酶聯(lián)免疫吸附試驗(yàn)(ELISA)檢測重組腺病毒介導(dǎo)CTLA4Ig和CD40Ig在體外培養(yǎng)人皮膚成纖維細(xì)胞培養(yǎng)上清中的表達(dá)情況。結(jié)果顯示:重組病毒Adv-CTLA4Ig、Adv-CD40Ig在MOI為100單獨(dú)和混合轉(zhuǎn)染人皮膚成纖維細(xì)胞后,在培養(yǎng)上清中均檢測到相應(yīng)目的蛋白的表達(dá),其表達(dá)量分別是:Adv-CTLA4Ig轉(zhuǎn)染組:CTLA4Ig 2.79±0.08ug/ml;Adv-CD40Ig轉(zhuǎn)染組:CD40Ig 1.25±0.11ug/ml;Adv-CTLA4Ig+Adv-CD40Ig轉(zhuǎn)染組:CTLA4Ig2.14±0.21ug/ml,CD40Ig 1.00±0.04ug/ml。 4.采用重組腺病毒Adv-CTLA4Ig、Adv-CD40Ig轉(zhuǎn)染后的體外培養(yǎng)人皮膚成纖維細(xì)胞與人外周血單個(gè)核細(xì)胞混合培養(yǎng)檢測對(duì)人皮膚成纖維細(xì)胞免疫性能的影響。結(jié)果顯示:重組病毒Adv-CTLA4Ig、Adv-CD40Ig以MOI為100單獨(dú)和混合轉(zhuǎn)染的體外培養(yǎng)人皮膚成纖維細(xì)胞對(duì)人外周血單個(gè)核細(xì)胞增殖抑制均優(yōu)于對(duì)照組,有顯著性差異(單處理組P＜0.05,混合組P＜0.01),Adv-CTLA4Ig和Adv-CD40Ig混合處理組抑制效果優(yōu)于各單處理組(P＜0.05)達(dá)到31.67±2.25%,Adv-CTLA4Ig處理組(23.41±1.23%)優(yōu)于Adv-CD40Ig(19.83±4.71%)(P＜0.05)。 綜上所述,重組腺病毒Adv-CTLA4Ig、Adv-CD40Ig可以有效轉(zhuǎn)染體外培養(yǎng)人皮膚成纖維細(xì)胞;介導(dǎo)CTLA4Ig和CD40Ig表達(dá);對(duì)細(xì)胞生長形態(tài)和增殖能力無明顯影響;通過腺病毒介導(dǎo)方式能有效阻斷B7/CD28、CD40/CD40L共刺激信號(hào),抑制體外培養(yǎng)人外周血單個(gè)核細(xì)胞增殖,阻斷B7/CD28信號(hào)誘導(dǎo)抑制效果優(yōu)于CD40/CD40L信號(hào),阻斷雙信號(hào)可更大程度誘導(dǎo)免疫抑制,因共刺激信號(hào)多樣、免疫反應(yīng)復(fù)雜性及施用量、處理時(shí)間的原因,并不能完全抑制。結(jié)論:腺病毒基因治療方式介導(dǎo)CTLA4Ig和CD40Ig阻斷B7/CD28、CD40/CD40L共刺激信號(hào)可應(yīng)用于人皮膚成纖維細(xì)胞修飾和治療。
[Abstract]:Allogeneic (allogeneic, xenogeneic) skin graft rejection after rejection is mainly by acute cellular activation of T cells, this process involved in T cell costimulatory signal for T cell activation and immune rejection is required, blocking costimulatory signals can lead to T cell reaction, cell cloning of apoptosis or loss, thereby inducing immune tolerance. T cells have been found in a variety of costimulatory signals, recognized as the most important is the B7-CD28 and CD40-CD40L signals. To be effective on the block by proteins or antibodies, induced immune tolerance, but in the preparation of complex, high cost, short half-life and need long-term application other drawbacks. Adenovirus vector has high safety, easy preparation for its mediated gene therapy to costimulatory molecule gene into the target cells, the expression of therapeutic proteins in specific areas, blocking costimulatory signals, is expected to drop Low cost, prolong the effect time. Skin fibroblasts were easy to obtain, culture and amplification of target cells, is also commonly used as seed cells for skin tissue engineering skin and ideal gene therapy. Therefore in this study, human skin fibroblasts cultured in vitro as target cells with adenovirus mediated CTLA4Ig gene transfection and CD40Ig blocking recognized the most important B7/CD28 and CD40/CD40L signal, through the Adv-CTLA4Ig, Adv-CD40Ig infection of human skin fibroblasts detection efficiency, after infection, cell morphology and proliferation ability of detection, protein expression and detection of its immune effect, performance detection, evaluation of adenovirus mediated CTLA4Ig gene therapy and CD40Ig double blocking costimulatory signal applied to human skin fibroblasts and the effect and feasibility of modified treatment, provide the basis for its application. The main contents are as follows:
1. by flow cytometry analysis technique for detection of adenovirus in human skin fibroblasts transfection efficiency in vitro. The results showed that Adv-EGFP infection in the complex (MOI) 50100 on human skin fibroblasts transfection efficiency is about 51.7 + 7.24%, 69.99 + 3.26%, but has no obvious effect on the morphology and proliferation of cells. Therefore, this study selected 100 adenovirus infection of human skin fibroblasts MOI.
2. by morphological observation and MTT method to detect the cell viability of recombinant adenovirus transfected into cultured human skin fibroblasts influence cell morphology and proliferation in vitro. The results showed that the recombinant virus Adv-CTLA4Ig, Adv-CD40Ig at MOI 100 alone and mixed transfected human skin fibroblast cells, the experimental group and the control group showed cell morphology long fusiform, irregular triangle, parallel, radial or whorled distribution, long cytoplasmic processes, clear outline, and no significant difference between the groups of cell proliferation (P > 0.05).
3. by enzyme-linked immunosorbent assay (ELISA) detection of recombinant adenovirus mediated CTLA4Ig and CD40Ig expression in the supernatant of cultured human cultured skin fibroblasts in vitro. The results showed that the recombinant virus Adv-CTLA4Ig, Adv-CD40Ig in MOI 100 alone and mixed into human skin fibroblasts, in culture supernatant were detected to express the corresponding protein and its expression are: Adv-CTLA4Ig transfection group: CTLA4Ig 2.79 + 0.08ug/ml; Adv-CD40Ig transfection group: CD40Ig 1.25 + 0.11ug/ml; Adv-CTLA4Ig+Adv-CD40Ig group: CTLA4Ig2.14 + 0.21ug/ml, CD40Ig + 0.04ug/ml. 1
4. using the recombinant adenovirus Adv-CTLA4Ig in cultured human skin fibroblasts and mixed peripheral blood mononuclear cells cultured fibroblasts cell immunity detection on human skin Adv-CD40Ig after transfection in vitro. The results showed that the recombinant virus Adv-CTLA4Ig, cultured human skin fibroblasts on human peripheral blood mononuclear cell proliferation inhibition better than the control group Adv-CD40Ig with MOI as 100 separate and mixed transfection in vitro, there was significant difference (P < 0.05 single treatment group, mixed group P < 0.01), Adv-CTLA4Ig and Adv-CD40Ig mixed treatment group the inhibitory effect is better than the single treatment group (P < 0.05) to 31.67 + 2.25%, 23.41 + 1.23% (Adv-CTLA4Ig treatment group) is better than that of Adv-CD40Ig (19.83 + 4.71%) (P < 0.05).
In summary, the recombinant adenovirus Adv-CTLA4Ig Adv-CD40Ig skin fibroblasts were cultured in vitro mediated transfection effectively; the expression of CD40Ig and CTLA4Ig; the cell growth morphology and proliferation ability has no effect; can effectively block B7/CD28 mediated by adenovirus, CD40/ CD40L costimulatory signal suppression in cultured human peripheral blood mononuclear cell proliferation cells, B7/CD28 signaling induced inhibitory effect than CD40/CD40L signal blocking, blocking double signal can be a greater degree of immunosuppression induced by costimulatory signals, because of diversity, complexity and application amount of the immune response causes the processing time, and can not be completely inhibited. Conclusion: adenovirus mediated CTLA4Ig gene therapy and CD40Ig blocking B7/CD28 costimulatory CD40/CD40L the signal can be applied to human skin fibroblasts and modification treatment.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392;Q78

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