xCT缺陷的Sut黑色素细胞蛋白质组学研究

发布时间：2018-01-15 01:17

本文关键词：xCT缺陷的Sut黑色素细胞蛋白质组学研究　出处：《天津医科大学》2011年硕士论文　论文类型：学位论文

更多相关文章： xCT Sut黑色素细胞 蛋白质组学 RT-PCR

【摘要】：研究目的：以xCT缺陷的Sut黑色素细胞为研究对象,以野生型Mela黑色素细胞为对照,应用差异蛋白质组学方法研究Sut细胞蛋白质表达谱的变化,从蛋白质组水平上寻找与xCT缺陷相关的蛋白质,为研究xCT缺陷引起的细胞生长抑制的机理提供实验支持。研究方法： 1.Sut和Mela细胞的培养、收集与总蛋白质的制备 Sut和Mela细胞用含10%胎牛血清,100units/ml双抗的DMEM/F12培养液,于37℃,5%C02细胞培养箱培养,收集对数生长期的细胞,细胞中加入适量裂解液裂解后以1,7000r/mmin高速离心,45min,取上清液即为细胞总蛋白。 2.Sut和Mela细胞总蛋白质的二维凝胶电泳及差异蛋白质的串联质谱分析以Bradford方法测定Sut和Mela细胞的总蛋白浓度,对两组样品分别进行二维凝胶电泳,应用PDQuest软件分析两组样品的二维凝胶电泳图谱,挑选出Sut细胞中表达量明显变化的蛋白质点,对这些蛋白质点进行串联质谱分析。 3.数据库查询鉴定差异蛋白质将串联质谱分析得到的数据输入Mascot软件,在SWISS-PROT数据库中搜索,鉴定出差异蛋白质。 4. RT-PCR方法检测Calmodulin、Annexin A3、Vimentin和S100-A4的mRNA水平提取Sut和Mela细胞的总RNA,反转录获得细胞cDNA,通过PCR扩增Calmoduln、Annexin A3、Vimentin和S100-A4的基因,扩增产物进行琼脂糖凝胶电泳。凝胶扫描,保存图像,通过绘制柱状图对比Sut细胞与Mela细胞mRNA表达量差异水平研究结果： 1.Sut细胞表达差异蛋白质的鉴定比较Sut和Mela细胞总蛋白质的二维凝胶电泳图谱,发现Sut细胞有20个蛋白质表达量发生明显变化,其中10个蛋白质表达量明显上调,另外10个明显下调。对这20个蛋白质点进行串联质潜分析,将质谱分析得到的数据在SWISS-PROT数据库中搜索查询,鉴定出这些蛋白质。表达量上调的10个蛋白质分别为：波形纤维蛋白(Vimentin),肌动蛋白alpha-1 b (Tubulin alpha-1 b),肌动蛋白beta-5 (Tubulin beta-5),钙依赖磷脂结合蛋白A3 (Annexin A3),钙调素(Calmodulin),钙结合蛋白S100-A4 (protein S100-A4),分子量为11kDa的未知蛋白质,钙结合蛋白S100-A6 (protein S100-A6),核苷二磷酸激酶B(Nucleoside diphosphate kinase B,NME2),甲酰谷胱甘肽(S-formylglutathione);表达量下调的10个蛋白质分别为：钙腔蛋白(Calumenin), N-Myc下游调控基因1 (N-Myc downstream regulated genel,NDRGl)蛋白,鸟氨酸氨基转移酶(Ornithine aminotransferase),二硫键异构酶A3(protein disulfide isomerase A3，，PDIA3),二氢嘧啶酶相关蛋白2(Dihydropyrimidinase-related protein2，DPYSL2), ATP合成酶ATP5A1 (ATP5A1), H2B组蛋白(Histone H2B type 1-K, HISTLH2BK),分子量为14kDa的未知蛋白质,基因序列号为OTTMUSG00000007855的未知蛋白质和分子量为47kDa的未知蛋白质。 2. RT-PCR方法检测Calmodulin、Annexin A3、Vimentin和S100-A4的1nRNA水平通过RT-PCR方法验证Calmodulin、Annexin A3、Vimentin和S100-A4的mRNA水平。实验结果显示,和Mela细胞相比,Calmodulin、Annexin A3、Vimentin和S100-A4的mRNA表达水平在Sut细胞中明显上调。研究结论： 1.与Mela细胞相比,Sut细胞中有20个蛋白质点的表达量存在明显变化,其中10个蛋白质表达量上调,10个蛋白质表达量下调。有3个上调蛋白质为细胞结构相关蛋白(Vimentin, Tubulin alpha-lb, Tubulin beta-5),3个上调蛋白质为钙结合蛋白(Calmodulin, protein S100A-4, protein S100A-6),2个上调蛋白质为蛋白酶(S-formylglutathione, Annexin A3);5个下调的蛋白质为蛋白酶(protein NDRG1, PDIA3, ATP5A1, Ornithine aminotransfcrase aminotransferase, NME2),2个下调蛋白质为信号转导相关蛋白质(DPYSL2, HISTLH2BK3) 2.鉴定出的xCT缺陷相关蛋白质在细胞的自体吞噬、胞内囊泡运输、MAP激酶信号通路,DNA复制等过程中起到重要作用,推测自体吞噬、MAP激酶信号通路可能在xCT缺陷引起的细胞生长抑制中起关键作用。自体吞噬、MAP激酶信号通路在Sut细胞生长抑制中的作用还有待于进一步研究。 3.通过RT-PCR实验发现,和Mela细胞相比,Sut细胞中Calmodulin、Annexin A3、Vimentin和S100-A4的mRNA表达水平明显上调,证明xCT缺陷的Sut细胞通过转录水平上调控Calmodulin、Annexin A3、Vimentin和S100-A4蛋白的表达水平。
[Abstract]:The purpose of the study is:
In xCT deficient Sut melanoma cells as the research object, using the wild-type Mela melanoma cells as control, the expression method of proteomics study of Sut cell protein by looking for differences, associated with defective xCT protein from the protein level of xCT, due to defects in cell growth inhibition mechanism to provide experimental support.
Research methods:
Culture of 1.Sut and Mela cells, collection and preparation of total protein
Sut and Mela cells containing 10% fetal bovine serum, 100units/ml double antibody DMEM/F12 medium at 37 DEG C, 5%C02 cell culture incubator, collect the logarithmic growth phase were added to 17000r/mmin lysis after high speed centrifugation, 45min cells, the supernatant is the total cell protein.
Two-dimensional gel electrophoresis and differential protein tandem mass spectrometry analysis of the total protein of 2.Sut and Mela cells
The total protein concentration of Sut and Mela cells was assayed by Bradford method, two groups of samples were subjected to two-dimensional gel electrophoresis, two-dimensional gel electrophoresis analysis of two samples using the PDQuest software, select Sut cells expression changes of protein, the protein spots were identified by tandem mass spectrometry analysis.
3. database query and identification of differential proteins
The data obtained by tandem mass spectrometry are entered into the Mascot software, and the differential proteins are identified in the SWISS-PROT database.
The 4. RT-PCR method detects the mRNA levels of Calmodulin, Annexin A3, Vimentin, and S100-A4
The total RNA extracted from Sut and Mela cells, reverse transcription for cDNA cells, Calmoduln, amplified by PCR Annexin A3, Vimentin and S100-A4 genes were observed by agarose gel electrophoresis of PCR products. Gel scanning, save the image, by drawing histogram comparison between Sut cells and Mela cells mRNA expression level difference
Research results:
Identification of differential protein expression in 1.Sut cells
Two dimensional gel electrophoresis of total protein between Sut and Mela cells, Sut cells have 20 protein expression changed significantly, among which 10 proteins were up-regulated, and 10 were down regulated. These 20 proteins by tandem mass spectrometry analysis potential analysis, the data obtained in the SWISS-PROT database search the query, identified these proteins. 10 proteins upregulated respectively: vimentin (Vimentin), B (Tubulin alpha-1 B alpha-1 actin, beta-5 actin) (Tubulin beta-5), a calcium dependent phospholipid binding protein A3 (Annexin A3), calmodulin (Calmodulin), S100-A4 (calcium binding protein protein S100-A4), the molecular weight of unknown protein 11kDa, calcium binding protein S100-A6 (protein S100-A6), two B (Nucleoside nucleoside phosphate kinase diphosphate kinase B, NME2, formylglutathione (S-form) Ylglutathione); expression of 10 proteins were down regulated respectively: calumenin (Calumenin), N-Myc 1 (N-Myc downstream downstream gene regulated Genel protein, NDRGl), ornithine aminotransferase (Ornithine aminotransferase), two disulfide isomerase A3 (protein disulfide isomerase A3, PDIA3), dihydropyrimidinase related protein two 2 (Dihydropyrimidinase-related, protein2, DPYSL2), ATP synthase ATP5A1 (ATP5A1), group H2B (Histone H2B type 1-K protein, HISTLH2BK), the molecular weight of unknown protein 14kDa, gene sequence number for unknown protein and molecular weight of OTTMUSG00000007855 was unknown protein 47kDa.
The 2. RT-PCR method detects the 1nRNA levels of Calmodulin, Annexin A3, Vimentin, and S100-A4
The mRNA level of Calmodulin, Annexin A3, Vimentin and S100-A4 was verified by RT-PCR method. The experimental results showed that the expression level of Calmodulin, Annexin A3, Annexin and Calmodulin in the cells increased significantly compared with Mela cells.
The conclusions are as follows:
1. compared with Mela cells, there are obvious changes in expression levels of 20 protein spots in Sut cells, among which 10 proteins were up-regulated and 10 protein expression. 3 up-regulated protein related protein (Vimentin, Tubulin, alpha-lb cells, Tubulin beta-5), 3 up-regulated proteins for calcium binding protein (Calmodulin, protein S100A-4, protein S100A-6), 2 up-regulated protein protease (S-formylglutathione, Annexin A3); 5 down regulated protein protease (protein NDRG1, PDIA3, ATP5A1, Ornithine aminotransfcrase aminotransferase, NME2), 2 down regulated proteins signal transduction related proteins (DPYSL2, HISTLH2BK3)
XCT defect related protein 2. identified in cellular autophagy, vesicle transport, MAP kinase pathway, DNA replication process plays an important role that autophagy, MAP kinase signaling pathway may be caused by defects in xCT cell growth inhibition plays a key role. Autophagy, MAP kinase signaling pathway in growth inhibition the role of Sut in cells remains to be further studied.
3., through the RT-PCR experiment, we found that compared with Mela cells, the mRNA expression level of Calmodulin, Annexin A3, Vimentin and S100-A4 in Sut cells was significantly up-regulated, indicating that the xCT deficient Sut cells regulate the expression level of S100-A4, S100-A4, and protein at the transcriptional level.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R341

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