本文關(guān)鍵詞： UC-MSCs IDO 免疫調(diào)節(jié) 燒傷 炎癥　出處：《新疆醫(yī)科大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:比較體外誘導(dǎo)表達(dá)吲哚2,3-雙加氧酶(indoleamine2,3-dioxygenase,IDO)的臍帶間充質(zhì)干細(xì)胞)對(duì)T細(xì)胞增殖能力的影響,了解IDO高表達(dá)的UC-MSCs移植治療大鼠燒傷的免疫調(diào)節(jié)機(jī)制、促燒傷修復(fù)效果,探討UC-MSCs在燒傷治療的臨床應(yīng)用前景。方法:(1)體外實(shí)驗(yàn):建立穩(wěn)定的UC-MSCs系,用干擾素γ(Interferon-γ,IFN-γ)誘導(dǎo)UC-MSCs產(chǎn)生IDO,并分別用逆轉(zhuǎn)錄聚合鏈?zhǔn)椒磻?yīng)(Reverse Transcription Polymerase Chain Reaction RT-PCR)和Western Blot檢測(cè)誘導(dǎo)后UC-MSCs的IDO信使核糖核酸(Messenger ribonucleic acid,mRNA)表達(dá)水平和蛋白表達(dá)水平;將T細(xì)胞分別與不同比例及不同組別的UC-MSCs共培養(yǎng),檢測(cè)比較培養(yǎng)體系中T細(xì)胞的增殖能力。(2)動(dòng)物實(shí)驗(yàn):取體重200±5g SD(sprague-dawley)雄性大鼠294只隨機(jī)分為預(yù)實(shí)驗(yàn)組、鹽水對(duì)照組、空白對(duì)照組、MSCs組、Induced MSCs組、Induced MSCs+1-MT組。根據(jù)預(yù)實(shí)驗(yàn)組燒傷后炎癥介質(zhì)變化曲線,確定UC-MSCs移植時(shí)間點(diǎn)。將鹽水對(duì)照組、空白對(duì)照組、MSCs組、Induced MSCs組、Induced MSCs+1-MT組共計(jì)240只大鼠制備燒傷模型,在燒傷24h后對(duì)鹽水對(duì)照組進(jìn)行皮下鹽水注射,MSCs組進(jìn)行UC-MSCs移植,Induced MSCs組移植經(jīng)IFN-γ誘導(dǎo)表達(dá)IDO的UC-MSCs、Induced MSCs+1-MT組移植IFN-γ誘導(dǎo)表達(dá)IDO的UC-MSCs并添加IDO的拮抗劑1-MT,以上UC-MSCs的移植量均為2×106。并在燒傷后0d、1d、2d、3d、5d、7d、9d采集血樣,檢測(cè)其WBC、CRP及炎癥因子IFN-γ、TNF-α、IL-6、IL-10表達(dá)變化,通過圖像分析軟件比較14d、21d、28d時(shí)各組創(chuàng)面愈合率。結(jié)果:(1)通過RT-PCR和Western Blot檢測(cè),結(jié)果顯示未誘導(dǎo)前UC-MSCs的IDO表達(dá)較弱,經(jīng)過IFN-γ誘導(dǎo)后,其IDO的表達(dá)增強(qiáng),且隨著IFN-γ濃度的增加,UC-MSCs的IDO表達(dá)呈現(xiàn)出逐漸增強(qiáng)的趨勢(shì)。(2)共培養(yǎng)體系中,誘導(dǎo)產(chǎn)生IDO的UC-MSCs對(duì)T淋巴細(xì)胞的增殖與其他各組相比具有明顯的抑制作用(P0.01),未經(jīng)誘導(dǎo)的UC-MSCs組對(duì)T細(xì)胞的增殖抑制作用強(qiáng)于IFN-γ誘導(dǎo)后添加1-MT拮抗劑的UC-MSCs移植組,相同條件下加入IDO特異性的抑制劑1-MT的共培養(yǎng)體系中,UC-MSCs對(duì)T淋巴細(xì)胞的增殖抑制作用輕微。(3)在燒傷模型的炎癥因子檢測(cè)中,炎癥因子IFN-γ、TNF-α、IL-6、IL-10在燒傷后七天內(nèi)出現(xiàn)不同程度的升高。Induced MSCs組和MSCs組較兩個(gè)對(duì)照組及誘導(dǎo)后MSCs+1-MT組的WBC、CRP和炎癥因子TNF-α、IL-6、IL-10各項(xiàng)指標(biāo)均有不同程度的降低,但I(xiàn)nduced MSCs組的炎癥指標(biāo)較MSCs組下降幅度更為顯著,IFN-γ水平未見顯著差別。(4)創(chuàng)面愈合率。燒傷后14d,MSCs組、Induced MSCs組、Induced MSCs+1-MT組與兩組對(duì)照組相比,創(chuàng)面愈合率較高,但三組之間無統(tǒng)計(jì)學(xué)差異;燒傷后21d和28d,Induced MSCs組、MSCs組、Induced MSCs+1-MT組的創(chuàng)面愈合率顯著高于兩個(gè)對(duì)照組,且三組之間存在差異,即Induced MSCs組優(yōu)于MSCs組和Induced MSCs+1-MT組,而MSCs組優(yōu)于Induced MSCs+1-MT組。結(jié)論:經(jīng)IFN-γ誘導(dǎo)表達(dá)IDO的UC-MSCs對(duì)T淋巴細(xì)胞的增殖有明顯抑制作用。通過燒傷后移植UC-MSCs可降低燒傷后炎癥相關(guān)指標(biāo),而經(jīng)IFN-γ誘導(dǎo)后的UC-MSCs對(duì)炎癥免疫應(yīng)答水平的調(diào)節(jié)作用更為顯著,IDO是UC-MSCs發(fā)揮抑制炎癥作用的關(guān)鍵分子之一。在燒傷大鼠的組織修復(fù)過程中,UC-MSCs移植可促進(jìn)損傷部位血管形成、成纖維細(xì)胞的增殖以及后續(xù)的皮膚修復(fù)重建,促進(jìn)燒傷創(chuàng)面愈合,IDO參與了這一免疫調(diào)節(jié)作用且發(fā)揮重要作用。
[Abstract]:Objective: To compare the in vitro expression of 2,3- indole dioxygenase (indoleamine2,3-dioxygenase, IDO) of human umbilical cord mesenchymal stem cells) effect on T cell proliferation, immune to high expression of IDO UC-MSCs transplantation in the treatment of burn rats with adjustment mechanism, promote the burn repair effect of UC-MSCs in clinical application in the treatment of burn injury. Methods: (1) in vitro: to establish a stable UC-MSCs system with interferon gamma (Interferon- y, IFN- y) induced UC-MSCs production and IDO, respectively by reverse transcription polymerase chain reaction (Reverse Transcription Polymerase Chain Reaction RT-PCR) and Western Blot detected UC-MSCs IDO messenger RNA (Messenger ribonucleic, acid, mRNA) expression the expression level and protein; T cells were UC-MSCs with different proportion and different groups of co cultured T cell culture system, detection and comparison of proliferation ability (2) animal. Results: the weight of 200 + 5g SD (Sprague-Dawley) male rats 294 were randomly divided into pre experimental group, saline control group, blank control group, MSCs group, Induced MSCs group, Induced MSCs+1-MT group. According to the curve changes of inflammatory mediators pre experimental group after burn, UC-MSCs transplantation time points. The saline control group and the blank control group, MSCs group, Induced MSCs group, Induced MSCs+1-MT group consisted of 240 rats in model group were prepared by burn, subcutaneous saline injection of saline in burn after 24h, MSCs group UC-MSCs Induced group MSCs after transplantation, transplantation of IFN- gamma induced expression of IDO UC-MSCs and 1-MT Induced antagonist MSCs+1-MT group transplantation of IFN- gamma induced expression of IDO UC-MSCs and adding IDO, transplantation above UC-MSCs were 2 * 106. and after burn 0d, 1D, 2D, 3D, 5D, 7d, 9D, blood samples were collected to detect the WBC, CRP and inflammatory factor IFN- gamma, TNF- alpha, IL-6, expression of IL-10, through the figure Image analysis software for comparison of 14d, 21d, 28d of the group wound healing rate. Results: (1) by RT-PCR and Western Blot detection results showed no UC-MSCs before induction of IDO expression is weak, after induced by IFN-, increase the expression of IDO, and with the increase of IFN- gamma concentration, UC-MSCs showed IDO expression the trend of a gradual increase. (2) co culture system, IDO induced UC-MSCs proliferation of T lymphocytes and the other groups have obvious inhibitory effect (P0.01), adding UC-MSCs transplantation group 1-MT antagonist group without UC-MSCs induced proliferation of T cells inhibited by IFN- gamma induced after under the same conditions, adding IDO specific inhibitors of 1-MT co culture system, UC-MSCs on proliferation of T lymphocytes inhibited. (3) in the detection of inflammatory cytokines in burn model, inflammatory factor IFN- gamma, alpha TNF-, IL-6, IL-10 in seven days after burn in Different degrees of elevated.Induced MSCs group and MSCs group compared with the control group two and group MSCs+1-MT after induction of WBC, CRP and inflammatory factor TNF- alpha, IL-6, lower IL-10 indicators have different degrees of inflammation, but the index of Induced MSCs group than in MSCs group decreased more significantly, IFN- levels were no significant difference. (4) the rate of wound healing after burn. 14d, MSCs group, Induced MSCs group, Induced MSCs+1-MT group compared with two control groups, the wound healing rate is high, but there is no significant difference between the three groups; after burn 21d and 28d, Induced, MSCs group, MSCs group, Induced MSCs+1-MT group wound healing rate was significantly higher than that in two a control group, and the differences between the three groups, namely Induced MSCs group than in MSCs group and Induced MSCs+1-MT group, and MSCs group than in Induced group. Conclusion: MSCs+1-MT can significantly inhibit the expression of IDO by IFN- induced UC-MSCs proliferation of T lymphocytes. Burn after transplantation of UC-MSCs can reduce the inflammation related indicators after burn, while the IFN- gamma induced UC-MSCs on inflammatory immune response regulation is more significant, IDO UC-MSCs is one of the key molecules play inhibit inflammation. In the repair of burn rat tissues, UC-MSCs transplantation can promote the formation of vascular injury. A subsequent reconstruction and repair of skin fibroblast proliferation, promote wound healing, IDO are involved in the immune regulation and play an important role.

【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R644

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