【摘要】： 研究背景和目的 臨床上同種異體皮膚移植是目前大面積深度燒傷患者早期創(chuàng)面覆蓋最直接、有效的治療方法,但是由于皮膚的強(qiáng)烈抗原特性,導(dǎo)致移植后3周左右外源皮膚就會(huì)發(fā)生不可逆的排斥反應(yīng),極大抑制了自體微粒皮混合大張異體皮移植效果。如何成功誘導(dǎo)出皮膚移植后的免疫耐受,臨床上至今未發(fā)現(xiàn)有效的措施。樹(shù)突狀細(xì)胞(dendritic cell ,DC)是目前已知的功能最強(qiáng)大的專職抗原提呈細(xì)胞,在免疫反應(yīng)中發(fā)揮了極其重要的作用。DC的分化發(fā)育過(guò)程伴隨著DC由未成熟的前體細(xì)胞分化發(fā)育為成熟細(xì)胞,細(xì)胞的形態(tài)、表面標(biāo)志以及生物學(xué)功能等都發(fā)生了相應(yīng)變化。作為DC未成熟的形式--- imDC(immature dendritic cell)因?yàn)槠涔δ苌献铒@著的特點(diǎn)就是可以誘導(dǎo)T淋巴細(xì)胞的特異性低應(yīng)答而成為當(dāng)前研究免疫耐受策略的熱點(diǎn),這在我們上一個(gè)國(guó)家自然科學(xué)基金項(xiàng)目(No.30271341)中也得到證實(shí)。利用imDC或者基因修飾DC進(jìn)行的聯(lián)合移植實(shí)驗(yàn)已經(jīng)在腎、肝、心臟、胰腺和小腸等器官中取得了令人較為滿意的效果,這為皮膚移植實(shí)驗(yàn)提供了非常有利的依據(jù)。 DC的發(fā)育成熟過(guò)程伴隨著其表型的變化,包括攝取能力減弱和MHC類分子、共刺激分子的表達(dá)上調(diào),最終變成強(qiáng)大的抗原提呈細(xì)胞。研究發(fā)現(xiàn)imDC在炎性介質(zhì)作用下逐漸遷移成熟的過(guò)程中,其表面趨化因子受體7(Chemokine receptor-7, CCR7)表達(dá)上調(diào)。通過(guò)CCR7與其配體MIP3β(即CCL19)和SLC(即CCL21)的相互作用,引導(dǎo)DC趨化遷移至淋巴結(jié),由此CCR7被認(rèn)為是成熟DC(mature DC,mDC)遷移歸巢完成抗原遞呈從而激活初始T細(xì)胞的主要受體之一。目前CCR7趨化遷移的能力在部分惡性腫瘤的轉(zhuǎn)移中得到證實(shí),表明CCR7嚴(yán)格調(diào)控了腫瘤細(xì)胞的轉(zhuǎn)移。腫瘤細(xì)胞這種非隨機(jī)的、有組織器官選擇性的轉(zhuǎn)移過(guò)程類似于免疫刺激過(guò)程中樹(shù)突狀細(xì)胞的定向遷移。基于CCR7具有非常重要的趨化遷移功能,若DC成熟度越高,CCR7表達(dá)更強(qiáng),則免疫系統(tǒng)呈現(xiàn)更為強(qiáng)勁的免疫應(yīng)答,反之則誘導(dǎo)免疫耐受,然而,imDC由于缺乏CCR7的表達(dá),向淋巴結(jié)的趨化遷移特性較弱,長(zhǎng)時(shí)間處于非T細(xì)胞區(qū)易受各種炎癥因子或者外來(lái)抗原的刺激而發(fā)育為mDC,嚴(yán)重影響其誘導(dǎo)免疫耐受的效果。 基于以上分析,本研究擬在國(guó)家自然科學(xué)基金的資助下,通過(guò)構(gòu)建含有小鼠CCR7的重組腺病毒,嘗試在imDC上建立CCR7的表達(dá),使imDC獲得原本mDC才具有的高效遷移能力,避免imDC在外周組織向成熟狀態(tài)的分化,有效地確保其誘導(dǎo)免疫耐受功能的發(fā)揮,為臨床上大面積深度燒傷患者早期創(chuàng)面覆蓋引起的免疫排斥提供新的解決方法和思路。 方法 1、提取小鼠胸腺總RNA,應(yīng)用逆轉(zhuǎn)錄PCR(RT-PCR)方法,以自行設(shè)計(jì)的帶有酶切位點(diǎn)的引物,擴(kuò)增獲得CCR7基因全部序列,經(jīng)過(guò)T-A克隆,酶切亞克隆到穿梭質(zhì)粒pAdTrack-CMV上,在BJ5183菌內(nèi)和pAdeasy-1同源重組,篩選陽(yáng)性克隆,酶切鑒定,線性化后脂質(zhì)體法轉(zhuǎn)染HEK293細(xì)胞進(jìn)行包裝、PCR鑒定及擴(kuò)增,得到含有CCR7基因的重組腺病毒,并根據(jù)報(bào)告基因GFP測(cè)定病毒滴度。 2、小劑量rmGM-CSF和rmIL-4聯(lián)合誘導(dǎo)培養(yǎng)BALB/c小鼠骨髓來(lái)源的imDC,光鏡下觀察并于第6天收獲;將上述構(gòu)建的重組腺病毒轉(zhuǎn)染imDC,根據(jù)報(bào)告基因綠色熒光蛋白(green fluorescence protein,GFP),檢測(cè)重組腺病毒的感染效率;以空病毒為對(duì)照,應(yīng)用逆轉(zhuǎn)錄PCR技術(shù)和免疫印跡(western blot)法,檢測(cè)轉(zhuǎn)染3天后imDC上CCR7基因在mRNA及蛋白水平表達(dá)的變化。 結(jié)果 1、成功構(gòu)建了小鼠CCR7重組腺病毒載體,重組腺病毒滴度可達(dá)1×109U /ml。 2、培養(yǎng)的imDC疏松貼壁生長(zhǎng),表面可見(jiàn)不規(guī)則的樹(shù)枝狀突起,體積較前增大;熒光顯微鏡下可見(jiàn)轉(zhuǎn)染腺病毒的細(xì)胞膜表面染有綠色熒光,呈明顯的不規(guī)則的毛刺樣突起。 3、重組腺病毒轉(zhuǎn)染imDC后,感染效率可達(dá)70%,CCR7在mRNA及蛋白表達(dá)上與空病毒組相比明顯增高。 結(jié)論 1、成功構(gòu)建小鼠CCR7基因重組腺病毒載體。獲得了高滴度的重組腺病毒。 2、小鼠骨髓來(lái)源的單核細(xì)胞在應(yīng)用低劑量rmGM-CSF和rmIL-4聯(lián)合刺激誘導(dǎo)后獲得具有典型形態(tài)特征的imDC。 3、構(gòu)建的重組腺病毒感染imDC后,CCR7的mRNA及蛋白表達(dá)明顯增高,成功地在imDC上建立了CCR7的表達(dá),并再次證明所構(gòu)建的腺病毒載體方法正確,轉(zhuǎn)染效率高,能夠有效表達(dá)目的基因,具有較高的感染力。這為進(jìn)一步研究轉(zhuǎn)染CCR7的imDC的趨化遷移功能改變及其誘導(dǎo)皮膚移植免疫耐受提供了實(shí)驗(yàn)基礎(chǔ)和依據(jù)。
[Abstract]:Study Background and Purpose The clinical allogenic skin graft is the most direct and effective treatment method for large-area deep-burn patients in the early stage, but due to the strong antigenic characteristics of the skin, irreversible discharge of the external skin at about 3 weeks after the transplantation can occur. The reprimand was greatly inhibited by the reprimand of the autograft. How to successfully induce the immune tolerance after skin graft has not been found in clinic until now Dendritic cells (DC) are the most powerful full-time antigen-presenting cells currently known and play a very important role in the immune response The differentiation and development of DC is accompanied by the development of the differentiation and development of the DC from the immature precursor cells into mature cells, the morphology of the cells, the surface markers, and the biological functions. The most significant feature of the DC immature form--imDC is that the specific low response of T-lymphocytes can be induced to become a hot spot in the current study of immune tolerance strategy, which is also available in a NSFC project (No. 30271341) The combination of the imDC or the genetically modified DC has proved to be a satisfactory effect in the kidneys, the liver, the heart, the pancreas, and the small intestine, which provides a very favorable effect for skin graft experiments The development and maturation process of DC is accompanied by the change of phenotype, including the decrease of uptake ability and MHC class, the expression of co-stimulatory molecules is up-regulated, and finally becomes powerful. It was found that the surface chemokine receptor 7 (Chemokine receptor-7, CC) in the process of gradual migration of imDC in the presence of an inflammatory mediator The expression of R7 is up-regulated. The migration of the DC to the lymph node is guided by the interaction of the CCR7 with its ligand MIP3 (i.e., the CCL19) and the SLC (i.e., CCL21), whereby the CCR7 is considered a mature DC (mDC) migration to the nest to complete the antigen presentation to activate the initial T cell One of the main receptors for CCR7 migration is confirmed in the transfer of some malignant tumors, indicating that CCR7 is strictly regulated. The transfer of tumor cells. The non-random, tissue-organ-selective transfer of tumor cells is similar to that of the dendritic cells in the process of immunostimulation. The directional migration of the cells has a very important chemotaxis function based on the CCR7. If the higher the DC maturity, the CCR7 expression is stronger, the immune system exhibits a stronger immune response, whereas the immune tolerance is induced, however, the imDC is directed towards the lymph node due to the lack of CCR7 expression The characteristics of migration and migration are weak, and the non-T cell region is easy to be stimulated by various inflammatory factors or foreign antigens for a long time, and is developed into mDC, which has a serious effect on the induction. Based on the above analysis, this study is to establish the expression of CCR7 on imDC by constructing the recombinant adenovirus containing the CCR7 of the mouse under the support of the National Natural Science Foundation of China, so that the imDC can obtain the high-efficiency migration ability of the original mDC, and the imDC is avoided. The differentiation of the peripheral tissue to the mature state effectively ensures the function of the immune tolerance, and is an immune rejection caused by the early wound covering of the patients with large-area deep burn. provide Method 1, the total RNA of the thymus of the mouse is extracted, a reverse transcription PCR (RT-PCR) method is applied, the whole sequence of the CCR7 gene is obtained by using a self-designed primer with the enzyme cutting site, the whole sequence of the CCR7 gene is obtained through amplification, the whole sequence of the CCR7 gene is obtained through T-A cloning, the enzyme is subcloned into the shuttle plasmid pAdTrack-CMV, and the whole sequence of the BJ5183 is and p Adeasy-1 homologous recombination, screening positive clone, enzyme digestion identification, linearized liposome method to transfect HEK293 cells to carry out packaging, PCR identification and amplification to obtain the recombinant gland containing the CCR7 gene. The virus was detected by the reporter gene GFP. The low dose of rmGM-CSF and rmIL-4 were combined to induce the imDC of the bone marrow of the BALB/ c mice and then harvested at the 6th day. The constructed recombinant adenovirus was transfected to imDC, and the green fluorescent protein (green fluorescent protein) was used as the reporter gene. The infection efficiency of recombinant adenovirus was detected by the method of RT-PCR and Western blot, and the imd after 3 days was detected by RT-PCR and western blot. C涓，

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