CCL20-siRNA慢病毒载体构建及其感染人角质形成细胞株的初步研究

发布时间：2018-07-09 20:18

  本文选题：RNA干扰 + 慢病毒载体　； 参考：《第三军医大学》2006年硕士论文

【摘要】： 人CCL20是近年来发现的一种CC亚族的趋化因子,经与配体CCR6的作用直接参与了树突状细胞、T细胞的定向迁移。在皮肤组织中,CCL20主要由活化的角质形成细胞产生,可强有力地诱导CD34+细胞造血祖细胞来源的朗格罕斯细胞(langerhans cell,LC)祖细胞向表皮内迁移。因为异基因表皮培养制作的组织工程皮肤本身无供体LC,受体LC所介导的间接抗原提呈途径在启动异基因组织工程皮肤移植物的排斥反应中发挥着关键作用,因此,采用RNA干扰(RNA interference,RNAi)技术下调人角质形成细胞的CCL20基因表达,将有可能减少或消除受者的LC向组织工程皮肤移植物内迁移,削弱或阻断间接抗原提呈途径,从而减轻受体的排斥反应,延长组织工程皮肤移植物的存活时间。本研究以人CCL20基因为靶基因,构建含有人CCL20靶点序列的慢病毒载体,并利用包装细胞293FT生产慢病毒颗粒,来感染人角质形成细胞株HaCaT细胞,为进一步筛选稳定表达CCL20-siRNA的HaCaT细胞克隆奠定基础。 目的: 1.利用RNAi技术,以人CCL20基因为靶基因,设计CCL20基因特异性的小干扰RNA(small interference RNA,siRNA),构建其短发夹RNA(short hairpin RNA, shRNA)重组慢病毒表达载体,并进行测序鉴定。 2.利用重组成功的慢病毒表达载体转染包装细胞293FT,收集、浓缩病毒上清液,并用HeLa细胞测定其滴度;初步观察了慢病毒颗粒对人角质形成细胞株HaCaT细胞的感染情况。 方法: 1.设计并合成人CCL20基因特异性的DNA寡核苷酸,连接到经Spe I和Sal I双酶切线性化的pHSER-dsRNA-GFP-SIN载体质粒上,转化大肠杆菌(escherichia coli, E.coli) DH5α感受态细胞,筛选阳性菌落、扩增后提取质粒,进行DNA测序鉴定。 2.重组慢病毒表达载体质粒pHSER-CCL20-siRNA-GFP-SIN、慢病毒包装质粒Lentipack和慢病毒包膜蛋白质粒Lentienv按照一定比例混合,与转染试剂jetPEI一同转染包装细胞293FT,收集含有慢病毒颗粒的培养基上清液,4℃高速离心,浓缩病
[Abstract]:Human CCL20 is a chemokine of CC subfamily discovered in recent years. It is directly involved in the directional migration of dendritic cell T cells through the interaction of CCR6 with the ligand CCR6. CCL20 is mainly produced by activated keratinocytes in skin tissue, which can strongly induce the migration of langerhans cells derived from hematopoietic progenitor cells of CD34 cells into the epidermis. Because allogeneic epidermal culture does not have donor LCs, the indirect antigen presentation pathway mediated by receptor LC plays a key role in initiating rejection of allogeneic tissue engineered skin grafts. The down-regulation of CCL20 gene expression in human keratinocytes by RNA interference (RNAi) may reduce or eliminate the LC migration to tissue engineered skin grafts and weaken or block the indirect antigen presentation pathway. So as to alleviate the rejection of the receptor and prolong the survival time of tissue engineered skin graft. In this study, human CCL20 gene was used as target gene to construct lentivirus vector containing human CCL20 target sequence, and lentivirus particles were produced by packaging cell 293FT to infect human keratinocyte line HaCaT cells. The results laid a foundation for further screening of HaCaT cells expressing CCL20-siRNA stably. Objective: 1. Using human CCL20 gene as the target gene, the short hairpin (short hairpin RNAs (shRNAs) were designed by using RNAi technique, and the recombinant lentivirus expression vector was constructed. And sequenced. 2. The recombinant lentivirus expression vector was used to transfect the packaging cell line 293FT. the virus supernatant was collected and concentrated, and its titer was determined by HeLa cells, and the infection of lentivirus particles on human keratinocyte line HaCaT was preliminarily observed. Methods: 1. The specific DNA oligonucleotides of human CCL20 gene were designed and synthesized, and ligated to pHSER-dsRNA-GFP-SIN vector plasmid linearized by SPE I and Sal I, and transformed into E. coli (escherichia coli, E.coli) DH5 伪 competent cells. DNA sequencing. 2. Recombinant lentivirus expression vector pHSER-CCL20-siRNA-GFP-SIN.The lentivirus packaging plasmid Lentipack was mixed with Lentienv in a certain proportion. The package cells 293FT were transfected with jetPEI, the supernatant of culture medium containing lentivirus particles was collected and centrifuged at 4 鈩，

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