小鼠CCR7重组腺病毒转染未成熟树突状细胞的实验研究
发布时间：2018-11-11 14:58
【摘要】： 研究背景和目的 临床上同种异体皮肤移植是目前大面积深度烧伤患者早期创面覆盖最直接、有效的治疗方法,但是由于皮肤的强烈抗原特性,导致移植后3周左右外源皮肤就会发生不可逆的排斥反应,极大抑制了自体微粒皮混合大张异体皮移植效果。如何成功诱导出皮肤移植后的免疫耐受,临床上至今未发现有效的措施。树突状细胞(dendritic cell ,DC)是目前已知的功能最强大的专职抗原提呈细胞,在免疫反应中发挥了极其重要的作用。DC的分化发育过程伴随着DC由未成熟的前体细胞分化发育为成熟细胞,细胞的形态、表面标志以及生物学功能等都发生了相应变化。作为DC未成熟的形式--- imDC(immature dendritic cell)因为其功能上最显著的特点就是可以诱导T淋巴细胞的特异性低应答而成为当前研究免疫耐受策略的热点,这在我们上一个国家自然科学基金项目(No.30271341)中也得到证实。利用imDC或者基因修饰DC进行的联合移植实验已经在肾、肝、心脏、胰腺和小肠等器官中取得了令人较为满意的效果,这为皮肤移植实验提供了非常有利的依据。 DC的发育成熟过程伴随着其表型的变化,包括摄取能力减弱和MHC类分子、共刺激分子的表达上调,最终变成强大的抗原提呈细胞。研究发现imDC在炎性介质作用下逐渐迁移成熟的过程中,其表面趋化因子受体7(Chemokine receptor-7, CCR7)表达上调。通过CCR7与其配体MIP3β(即CCL19)和SLC(即CCL21)的相互作用,引导DC趋化迁移至淋巴结,由此CCR7被认为是成熟DC(mature DC,mDC)迁移归巢完成抗原递呈从而激活初始T细胞的主要受体之一。目前CCR7趋化迁移的能力在部分恶性肿瘤的转移中得到证实,表明CCR7严格调控了肿瘤细胞的转移。肿瘤细胞这种非随机的、有组织器官选择性的转移过程类似于免疫刺激过程中树突状细胞的定向迁移。基于CCR7具有非常重要的趋化迁移功能,若DC成熟度越高,CCR7表达更强,则免疫系统呈现更为强劲的免疫应答,反之则诱导免疫耐受,然而,imDC由于缺乏CCR7的表达,向淋巴结的趋化迁移特性较弱,长时间处于非T细胞区易受各种炎症因子或者外来抗原的刺激而发育为mDC,严重影响其诱导免疫耐受的效果。 基于以上分析,本研究拟在国家自然科学基金的资助下,通过构建含有小鼠CCR7的重组腺病毒,尝试在imDC上建立CCR7的表达,使imDC获得原本mDC才具有的高效迁移能力,避免imDC在外周组织向成熟状态的分化,有效地确保其诱导免疫耐受功能的发挥,为临床上大面积深度烧伤患者早期创面覆盖引起的免疫排斥提供新的解决方法和思路。 方法 1、提取小鼠胸腺总RNA,应用逆转录PCR(RT-PCR)方法,以自行设计的带有酶切位点的引物,扩增获得CCR7基因全部序列,经过T-A克隆,酶切亚克隆到穿梭质粒pAdTrack-CMV上,在BJ5183菌内和pAdeasy-1同源重组,筛选阳性克隆,酶切鉴定,线性化后脂质体法转染HEK293细胞进行包装、PCR鉴定及扩增,得到含有CCR7基因的重组腺病毒,并根据报告基因GFP测定病毒滴度。 2、小剂量rmGM-CSF和rmIL-4联合诱导培养BALB/c小鼠骨髓来源的imDC,光镜下观察并于第6天收获;将上述构建的重组腺病毒转染imDC,根据报告基因绿色荧光蛋白(green fluorescence protein,GFP),检测重组腺病毒的感染效率;以空病毒为对照,应用逆转录PCR技术和免疫印迹(western blot)法,检测转染3天后imDC上CCR7基因在mRNA及蛋白水平表达的变化。 结果 1、成功构建了小鼠CCR7重组腺病毒载体,重组腺病毒滴度可达1×109U /ml。 2、培养的imDC疏松贴壁生长,表面可见不规则的树枝状突起,体积较前增大;荧光显微镜下可见转染腺病毒的细胞膜表面染有绿色荧光,呈明显的不规则的毛刺样突起。 3、重组腺病毒转染imDC后,感染效率可达70%,CCR7在mRNA及蛋白表达上与空病毒组相比明显增高。 结论 1、成功构建小鼠CCR7基因重组腺病毒载体。获得了高滴度的重组腺病毒。 2、小鼠骨髓来源的单核细胞在应用低剂量rmGM-CSF和rmIL-4联合刺激诱导后获得具有典型形态特征的imDC。 3、构建的重组腺病毒感染imDC后,CCR7的mRNA及蛋白表达明显增高,成功地在imDC上建立了CCR7的表达,并再次证明所构建的腺病毒载体方法正确,转染效率高,能够有效表达目的基因,具有较高的感染力。这为进一步研究转染CCR7的imDC的趋化迁移功能改变及其诱导皮肤移植免疫耐受提供了实验基础和依据。
[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，
本文编号：2325196
[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，
本文编号：2325196
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