過(guò)表達(dá)SERPINB2上調(diào)RB1水平抑制白血病K562細(xì)胞的生長(zhǎng)
發(fā)布時(shí)間：2018-08-07 12:50
【摘要】：目的慢性粒細(xì)胞白血病(chronic myeloid leukemia,CML)急變期的治療是目前的重要問(wèn)題,臨床上亟需要找到新的分子治療靶點(diǎn)。本研究意欲通過(guò)使用生物信息學(xué)方法分析CML基因表達(dá)譜數(shù)據(jù)并進(jìn)行實(shí)驗(yàn)驗(yàn)證,找到對(duì)CML急變期白血病細(xì)胞起作用的潛在治療靶點(diǎn)。方法本實(shí)驗(yàn)總共分為三個(gè)部分:1.使用基因集群富集分析軟件(gene set enrichment analysis,GSEA)分析基因表達(dá)綜合數(shù)據(jù)庫(kù)(gene expression omnibus,GEO)中CML臨床樣本及小鼠細(xì)胞模型的基因表達(dá)譜數(shù)據(jù),獲得與CML急變期相關(guān)的基因集群,然后從中選擇絲氨酸蛋白酶抑制劑家族成員B2(SERPINB2)作為研究對(duì)象。2.應(yīng)用分子克隆技術(shù)在空載質(zhì)粒p Adtrack-CMV的基礎(chǔ)上構(gòu)建p Adtrack-CMV-SERPINB2重組質(zhì)粒,使用電穿孔方法將上述質(zhì)粒分別轉(zhuǎn)入CML急變細(xì)胞系K562細(xì)胞。運(yùn)用細(xì)胞免疫熒光技術(shù)檢測(cè)SERPINB2在細(xì)胞內(nèi)的表達(dá)與分布。3.p Adtrack-CMV-SERPINB2重組質(zhì)粒轉(zhuǎn)染白血病K562細(xì)胞后,采用CCK-8實(shí)驗(yàn)和克隆形成實(shí)驗(yàn)檢測(cè)細(xì)胞增殖和克隆形成能力,使用流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期,通過(guò)Western blot實(shí)驗(yàn)檢測(cè)SERPINB2、BCR/ABL以及RB1的表達(dá)。結(jié)果1.使用GSEA軟件分析,成功獲取了CML急變期差異基因集群熱力圖以及富集信號(hào)通路。2.應(yīng)用分子克隆技術(shù)以及電穿孔轉(zhuǎn)染質(zhì)粒的方法,成功構(gòu)建了重組質(zhì)粒p Adtrack-CMV-SERPINB2并轉(zhuǎn)入K562細(xì)胞,轉(zhuǎn)染效率在24小時(shí)達(dá)到75%,細(xì)胞免疫熒光實(shí)驗(yàn)顯示SERPINB2在細(xì)胞中成功表達(dá)并主要在細(xì)胞漿中分布。3.與對(duì)照組相比,在K562細(xì)胞中過(guò)表達(dá)SERPINB2可抑制K562細(xì)胞的增殖(p0.001),明顯降低K562細(xì)胞的克隆形成能力(p0.01)并導(dǎo)致G0/G1期的細(xì)胞比例增多(p0.001)。蛋白質(zhì)免疫印跡實(shí)驗(yàn)顯示BCR/ABL表達(dá)無(wú)明顯變化,SERPINB2、RB1表達(dá)水平增高。結(jié)論綜上所述,SERPINB2可通過(guò)上調(diào)細(xì)胞內(nèi)RB1的水平,使細(xì)胞周期更多的停留在G1期,從而對(duì)K562細(xì)胞的增殖與克隆形成能力產(chǎn)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R733.7
本文編號(hào)：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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R733.7
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 ;Interaction of Plasminogen Activator Inhibitor-2 and Proteasome Subunit,Beta Type 1[J];Acta Biochimica et Biophysica Sinica;2004年01期
2 張宇清,李平,侯敏,王霞,樊靜,譚理,朱運(yùn)松;PAI-2與IRF-3相互作用的鑒定(英文)[J];生物化學(xué)與生物物理學(xué)報(bào);2003年07期
，本文編號(hào)：2170075
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