KIF2A对BBS家系表达差异基因的影响及苗族BBS家系致病基因的筛选

发布时间：2018-03-02 15:33

本文选题：巴德-毕氏综合征　切入点：KIF2A重组质粒　出处：《昆明理工大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:探索KIF2A基因与BBS1、BBS7、A]NXA1、KIF3A、β-Tublin等纤毛方法:KIF2A重组质粒和干扰RNA瞬时转染293T和3T3-L1细胞,提取转染细胞的总RNA及总蛋白,运用荧光定量PCR、western blotting等方法检测KIF2A的表达情况并分析KIF2A基因与BBS1、BBS7、ANXA1、KIF3A、β-Tublin等纤毛相关基因之间的关系以及KIF2A基因与细胞增殖的关系,并尝试利用Crispr-Cas9技术对KIF2A基因进行敲除;苗族BBS家系进行致病基因筛选首先提取苗族BBS患者及其母亲(无BBS表型)的全血DNA,送由华大基因公司外显子测序以及生物信息学分析,筛查出该苗族BBS患者基因突变具体位置,随后提取300例傣族、300例哈尼族、300例瑶族及76例苗族及BBS家系成员全血DNA,PCR扩增出含突变序列的BBS7片段由华大基因Sanger法测序,对这一筛查结果进行验证。相关基因之间的关联性以及对细胞增殖的影响;对收集的苗族巴德-毕氏综合(Bardet-Biedl syndrome,BBS)家系进行致病基因的筛查并对筛查结果用Sanger测序法验证。结果:KIF2A基因能够在293T及3T3-L1细胞内过表达或敲除;初步发现KIF2A与BBS1、BBS7、ANXA1、KIF3A、β-Tublin等纤毛相关基因之间的关系,即在293T细胞中若KIF2A基因过表达,BBS7基因表达量相对于对照组表达量下降,KIF3A基因表达量相对于对照组表达量升高,在3T3-L1细胞KIF2A基因过表达模型中,ANXA1基因与对照组相比表达量升高;利用Crispr-Cas9技术初步构建KIF2A基因敲除载体,对KIF2A基因的敲除效果还需进一步验证;在细胞增殖实验中发现,KIF2A基因过表达在一定程度上促进293T及3T3-L1细胞增殖;在苗族BBS家系致病基因筛查发现该苗族BBS患者BBS7基因第5外显子563-564位置缺失碱基AC,并通过300例傣族、300例哈尼族、300例瑶族及76例苗族及BBS家系成员进行Sanger测序对这一结果进行了验证。结论:在本次实验中,293T细胞KIF2A基因的过表达可能调控BBS7、KIF3A基因的表达,3T3-L1细胞中KIF2A基因的过表达可能影响ANXA1基因的表达,但还需要进一步进行相关实验对这些基因的表达情况进行验证;成功筛查并验证苗族BBS家系致病基因,这对BBS致病基因的研究具有促进意义。
[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.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R596.1

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