本文選題：RNA干擾 + 慢病毒載體　； 參考：《第三軍醫(yī)大學(xué)》2006年碩士論文

【摘要】： 人CCL20是近年來發(fā)現(xiàn)的一種CC亞族的趨化因子,經(jīng)與配體CCR6的作用直接參與了樹突狀細(xì)胞、T細(xì)胞的定向遷移。在皮膚組織中,CCL20主要由活化的角質(zhì)形成細(xì)胞產(chǎn)生,可強(qiáng)有力地誘導(dǎo)CD34+細(xì)胞造血祖細(xì)胞來源的朗格罕斯細(xì)胞(langerhans cell,LC)祖細(xì)胞向表皮內(nèi)遷移。因?yàn)楫惢虮砥づ囵B(yǎng)制作的組織工程皮膚本身無供體LC,受體LC所介導(dǎo)的間接抗原提呈途徑在啟動異基因組織工程皮膚移植物的排斥反應(yīng)中發(fā)揮著關(guān)鍵作用,因此,采用RNA干擾(RNA interference,RNAi)技術(shù)下調(diào)人角質(zhì)形成細(xì)胞的CCL20基因表達(dá),將有可能減少或消除受者的LC向組織工程皮膚移植物內(nèi)遷移,削弱或阻斷間接抗原提呈途徑,從而減輕受體的排斥反應(yīng),延長組織工程皮膚移植物的存活時間。本研究以人CCL20基因?yàn)榘谢?構(gòu)建含有人CCL20靶點(diǎn)序列的慢病毒載體,并利用包裝細(xì)胞293FT生產(chǎn)慢病毒顆粒,來感染人角質(zhì)形成細(xì)胞株HaCaT細(xì)胞,為進(jìn)一步篩選穩(wěn)定表達(dá)CCL20-siRNA的HaCaT細(xì)胞克隆奠定基礎(chǔ)。 目的: 1.利用RNAi技術(shù),以人CCL20基因?yàn)榘谢?設(shè)計(jì)CCL20基因特異性的小干擾RNA(small interference RNA,siRNA),構(gòu)建其短發(fā)夾RNA(short hairpin RNA, shRNA)重組慢病毒表達(dá)載體,并進(jìn)行測序鑒定。 2.利用重組成功的慢病毒表達(dá)載體轉(zhuǎn)染包裝細(xì)胞293FT,收集、濃縮病毒上清液,并用HeLa細(xì)胞測定其滴度;初步觀察了慢病毒顆粒對人角質(zhì)形成細(xì)胞株HaCaT細(xì)胞的感染情況。 方法: 1.設(shè)計(jì)并合成人CCL20基因特異性的DNA寡核苷酸,連接到經(jīng)Spe I和Sal I雙酶切線性化的pHSER-dsRNA-GFP-SIN載體質(zhì)粒上,轉(zhuǎn)化大腸桿菌(escherichia coli, E.coli) DH5α感受態(tài)細(xì)胞,篩選陽性菌落、擴(kuò)增后提取質(zhì)粒,進(jìn)行DNA測序鑒定。 2.重組慢病毒表達(dá)載體質(zhì)粒pHSER-CCL20-siRNA-GFP-SIN、慢病毒包裝質(zhì)粒Lentipack和慢病毒包膜蛋白質(zhì)粒Lentienv按照一定比例混合,與轉(zhuǎn)染試劑jetPEI一同轉(zhuǎn)染包裝細(xì)胞293FT,收集含有慢病毒顆粒的培養(yǎng)基上清液,4℃高速離心,濃縮病
[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 偽 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，

本文編號：2110537