本文選題：巴德-畢氏綜合征　切入點：KIF2A重組質(zhì)粒　出處：《昆明理工大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:探索KIF2A基因與BBS1、BBS7、A]NXA1、KIF3A、β-Tublin等纖毛方法:KIF2A重組質(zhì)粒和干擾RNA瞬時轉(zhuǎn)染293T和3T3-L1細胞,提取轉(zhuǎn)染細胞的總RNA及總蛋白,運用熒光定量PCR、western blotting等方法檢測KIF2A的表達情況并分析KIF2A基因與BBS1、BBS7、ANXA1、KIF3A、β-Tublin等纖毛相關(guān)基因之間的關(guān)系以及KIF2A基因與細胞增殖的關(guān)系,并嘗試利用Crispr-Cas9技術(shù)對KIF2A基因進行敲除;苗族BBS家系進行致病基因篩選首先提取苗族BBS患者及其母親(無BBS表型)的全血DNA,送由華大基因公司外顯子測序以及生物信息學(xué)分析,篩查出該苗族BBS患者基因突變具體位置,隨后提取300例傣族、300例哈尼族、300例瑤族及76例苗族及BBS家系成員全血DNA,PCR擴增出含突變序列的BBS7片段由華大基因Sanger法測序,對這一篩查結(jié)果進行驗證。相關(guān)基因之間的關(guān)聯(lián)性以及對細胞增殖的影響;對收集的苗族巴德-畢氏綜合(Bardet-Biedl syndrome,BBS)家系進行致病基因的篩查并對篩查結(jié)果用Sanger測序法驗證。結(jié)果:KIF2A基因能夠在293T及3T3-L1細胞內(nèi)過表達或敲除;初步發(fā)現(xiàn)KIF2A與BBS1、BBS7、ANXA1、KIF3A、β-Tublin等纖毛相關(guān)基因之間的關(guān)系,即在293T細胞中若KIF2A基因過表達,BBS7基因表達量相對于對照組表達量下降,KIF3A基因表達量相對于對照組表達量升高,在3T3-L1細胞KIF2A基因過表達模型中,ANXA1基因與對照組相比表達量升高;利用Crispr-Cas9技術(shù)初步構(gòu)建KIF2A基因敲除載體,對KIF2A基因的敲除效果還需進一步驗證;在細胞增殖實驗中發(fā)現(xiàn),KIF2A基因過表達在一定程度上促進293T及3T3-L1細胞增殖;在苗族BBS家系致病基因篩查發(fā)現(xiàn)該苗族BBS患者BBS7基因第5外顯子563-564位置缺失堿基AC,并通過300例傣族、300例哈尼族、300例瑤族及76例苗族及BBS家系成員進行Sanger測序?qū)@一結(jié)果進行了驗證。結(jié)論:在本次實驗中,293T細胞KIF2A基因的過表達可能調(diào)控BBS7、KIF3A基因的表達,3T3-L1細胞中KIF2A基因的過表達可能影響ANXA1基因的表達,但還需要進一步進行相關(guān)實驗對這些基因的表達情況進行驗證;成功篩查并驗證苗族BBS家系致病基因,這對BBS致病基因的研究具有促進意義。
[Abstract]:Objective: to explore the methods of KIF2A gene and BBS1hBBS7A] NXA1KIF3A, 尾 -Tublin ciliated plasmid and interference RNA to instantly transfect 293T and 3T3-L1 cells, and extract the total RNA and total protein of transfected cells. Fluorescence quantitative PCR western blotting was used to detect the expression of KIF2A and to analyze the relationship between the KIF2A gene and the ciliated genes such as BBS1, BBS7ANXA1, KIF3A, 尾 -Tublin, and the relationship between KIF2A gene and cell proliferation. Crispr-Cas9 technique was used to knockout the KIF2A gene. The whole blood DNA of the Miao BBS patients and their mothers (without BBS phenotype) was first extracted from the BBS pedigree of the Miao nationality, and then sent to the exon sequencing and bioinformatics analysis of Huada gene company to screen out the specific location of the gene mutation in the patients with BBS in the Miao nationality. After that, 300 Dai cases of Hani nationality, 300 cases of Yao nationality, 76 cases of Miao nationality and family members of BBS were amplified by whole blood DNA polymerase chain reaction. The BBS7 fragment containing mutation sequence was sequenced by Sanger method of Huada gene. The results of the screening were verified. The correlation between the genes involved and the effect on cell proliferation; The pathogenic genes of Bard Biedl syndromesis-BBSs of Miao nationality were screened and the results were verified by Sanger sequencing. Results: KIF2A gene could be overexpressed or knocked out in 293T and 3T3-L1 cells. The relationship between KIF2A and other ciliated genes such as BBS1, BBS7, ANXA1, 尾 -Tublin and other ciliated genes was preliminarily found. In 293T cells, if the expression of KIF2A gene over-expressed was lower than that of the control group, the expression of KIF3A gene was higher than that of the control group. In the overexpression model of KIF2A gene in 3T3-L1 cells, the expression of ANXA1 gene was increased compared with the control group, and the knockout vector of KIF2A gene was constructed by using Crispr-Cas9 technique, and the effect of KIF2A gene knockout still needs to be further verified. The overexpression of KIF2A gene promoted the proliferation of 293T and 3T3-L1 cells to some extent. In the BBS pedigree of Miao nationality, the deletion of BBS7 gene at exon 563-564 was found in the BBS patients of Miao nationality, and Sanger sequencing was carried out in 300 cases of Dai nationality, 300 cases of Hani nationality, 300 cases of Yao nationality and 76 cases of Miao nationality and BBS family members. Conclusion: the overexpression of KIF2A gene may regulate the expression of KIF2A gene in BBS7t3-L1 cells and may affect the expression of ANXA1 gene. However, further experiments are needed to verify the expression of these genes, and to screen and verify the pathogenicity genes of BBS family of Miao nationality successfully, which is of great significance for the study of the pathogenic genes of BBS.
【學(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R596.1

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本文編號：1557106