过表达SERPINB2上调RB1水平抑制白血病K562细胞的生长
发布时间：2018-08-07 12:50
【摘要】：目的慢性粒细胞白血病(chronic myeloid leukemia,CML)急变期的治疗是目前的重要问题,临床上亟需要找到新的分子治疗靶点。本研究意欲通过使用生物信息学方法分析CML基因表达谱数据并进行实验验证,找到对CML急变期白血病细胞起作用的潜在治疗靶点。方法本实验总共分为三个部分:1.使用基因集群富集分析软件(gene set enrichment analysis,GSEA)分析基因表达综合数据库(gene expression omnibus,GEO)中CML临床样本及小鼠细胞模型的基因表达谱数据,获得与CML急变期相关的基因集群,然后从中选择丝氨酸蛋白酶抑制剂家族成员B2(SERPINB2)作为研究对象。2.应用分子克隆技术在空载质粒p Adtrack-CMV的基础上构建p Adtrack-CMV-SERPINB2重组质粒,使用电穿孔方法将上述质粒分别转入CML急变细胞系K562细胞。运用细胞免疫荧光技术检测SERPINB2在细胞内的表达与分布。3.p Adtrack-CMV-SERPINB2重组质粒转染白血病K562细胞后,采用CCK-8实验和克隆形成实验检测细胞增殖和克隆形成能力,使用流式细胞术检测细胞周期,通过Western blot实验检测SERPINB2、BCR/ABL以及RB1的表达。结果1.使用GSEA软件分析,成功获取了CML急变期差异基因集群热力图以及富集信号通路。2.应用分子克隆技术以及电穿孔转染质粒的方法,成功构建了重组质粒p Adtrack-CMV-SERPINB2并转入K562细胞,转染效率在24小时达到75%,细胞免疫荧光实验显示SERPINB2在细胞中成功表达并主要在细胞浆中分布。3.与对照组相比,在K562细胞中过表达SERPINB2可抑制K562细胞的增殖(p0.001),明显降低K562细胞的克隆形成能力(p0.01)并导致G0/G1期的细胞比例增多(p0.001)。蛋白质免疫印迹实验显示BCR/ABL表达无明显变化,SERPINB2、RB1表达水平增高。结论综上所述,SERPINB2可通过上调细胞内RB1的水平,使细胞周期更多的停留在G1期,从而对K562细胞的增殖与克隆形成能力产生抑制作用。
[Abstract]:Objective at present, the treatment of (chronic myeloid leukemiaemia is an important problem, and it is urgent to find a new molecular therapy target in clinic. The purpose of this study was to analyze the CML gene expression profile data by using bioinformatics method and to find out the potential therapeutic targets for CML acute leukemia cells. Methods the experiment was divided into three parts: 1. Gene cluster analysis software (gene set enrichment analysis was used to analyze the gene expression profile data of CML clinical samples and mouse cell models in (gene expression omnibus-GEO (a comprehensive database of gene expression). B 2 (SERPINB2), a member of the serine protease inhibitor family, was selected as the object of study. The recombinant plasmid of p Adtrack-CMV-SERPINB2 was constructed on the basis of empty plasmid p Adtrack-CMV by molecular cloning technique. The above plasmids were transformed into K562 cell line by electroporation. The expression and distribution of SERPINB2 in K562 cells were detected by cell immunofluorescence technique. The ability of cell proliferation and clone formation was detected by CCK-8 assay and clone formation assay after transfection of Adtrack-CMV-SERPINB2 recombinant plasmid into K562 cells. The cell cycle was detected by flow cytometry and the expression of SERPINB2 BCR / ABL and RB1 was detected by Western blot assay. Result 1. The thermal map of differential gene cluster and the enrichment signal pathway. 2. 2 were successfully obtained by using GSEA software. The recombinant plasmid p Adtrack-CMV-SERPINB2 was successfully constructed and transferred into K562 cells by molecular cloning and electroporation. The transfection efficiency reached 75% in 24 hours. The cell immunofluorescence assay showed that SERPINB2 was expressed successfully and distributed mainly in the cytoplasm. Compared with the control group, overexpression of SERPINB2 in K562 cells inhibited the proliferation of K562 cells (p0.001), significantly decreased the clone forming ability of K562 cells (p0.01) and increased the proportion of K562 cells in G0/G1 phase (p0.001). Western blot analysis showed that the expression of BCR/ABL did not change significantly and the expression level of SERPINB2 / RB1 was increased. Conclusion SERPINB2 can inhibit the proliferation and clone formation of K562 cells by upregulating the level of intracellular RB1 and making the cell cycle stay in G1 phase.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.7
本文编号：2170075
[Abstract]:Objective at present, the treatment of (chronic myeloid leukemiaemia is an important problem, and it is urgent to find a new molecular therapy target in clinic. The purpose of this study was to analyze the CML gene expression profile data by using bioinformatics method and to find out the potential therapeutic targets for CML acute leukemia cells. Methods the experiment was divided into three parts: 1. Gene cluster analysis software (gene set enrichment analysis was used to analyze the gene expression profile data of CML clinical samples and mouse cell models in (gene expression omnibus-GEO (a comprehensive database of gene expression). B 2 (SERPINB2), a member of the serine protease inhibitor family, was selected as the object of study. The recombinant plasmid of p Adtrack-CMV-SERPINB2 was constructed on the basis of empty plasmid p Adtrack-CMV by molecular cloning technique. The above plasmids were transformed into K562 cell line by electroporation. The expression and distribution of SERPINB2 in K562 cells were detected by cell immunofluorescence technique. The ability of cell proliferation and clone formation was detected by CCK-8 assay and clone formation assay after transfection of Adtrack-CMV-SERPINB2 recombinant plasmid into K562 cells. The cell cycle was detected by flow cytometry and the expression of SERPINB2 BCR / ABL and RB1 was detected by Western blot assay. Result 1. The thermal map of differential gene cluster and the enrichment signal pathway. 2. 2 were successfully obtained by using GSEA software. The recombinant plasmid p Adtrack-CMV-SERPINB2 was successfully constructed and transferred into K562 cells by molecular cloning and electroporation. The transfection efficiency reached 75% in 24 hours. The cell immunofluorescence assay showed that SERPINB2 was expressed successfully and distributed mainly in the cytoplasm. Compared with the control group, overexpression of SERPINB2 in K562 cells inhibited the proliferation of K562 cells (p0.001), significantly decreased the clone forming ability of K562 cells (p0.01) and increased the proportion of K562 cells in G0/G1 phase (p0.001). Western blot analysis showed that the expression of BCR/ABL did not change significantly and the expression level of SERPINB2 / RB1 was increased. Conclusion SERPINB2 can inhibit the proliferation and clone formation of K562 cells by upregulating the level of intracellular RB1 and making the cell cycle stay in G1 phase.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.7
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相关期刊论文 前2条
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，本文编号：2170075
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