本文關(guān)鍵詞：xCT缺陷的Sut黑色素細(xì)胞蛋白質(zhì)組學(xué)研究　出處：《天津醫(yī)科大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

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【摘要】：研究目的： 以xCT缺陷的Sut黑色素細(xì)胞為研究對象,以野生型Mela黑色素細(xì)胞為對照,應(yīng)用差異蛋白質(zhì)組學(xué)方法研究Sut細(xì)胞蛋白質(zhì)表達(dá)譜的變化,從蛋白質(zhì)組水平上尋找與xCT缺陷相關(guān)的蛋白質(zhì),為研究xCT缺陷引起的細(xì)胞生長抑制的機(jī)理提供實(shí)驗(yàn)支持。 研究方法： 1.Sut和Mela細(xì)胞的培養(yǎng)、收集與總蛋白質(zhì)的制備 Sut和Mela細(xì)胞用含10%胎牛血清,100units/ml雙抗的DMEM/F12培養(yǎng)液,于37℃,5%C02細(xì)胞培養(yǎng)箱培養(yǎng),收集對數(shù)生長期的細(xì)胞,細(xì)胞中加入適量裂解液裂解后以1,7000r/mmin高速離心,45min,取上清液即為細(xì)胞總蛋白。 2.Sut和Mela細(xì)胞總蛋白質(zhì)的二維凝膠電泳及差異蛋白質(zhì)的串聯(lián)質(zhì)譜分析 以Bradford方法測定Sut和Mela細(xì)胞的總蛋白濃度,對兩組樣品分別進(jìn)行二維凝膠電泳,應(yīng)用PDQuest軟件分析兩組樣品的二維凝膠電泳圖譜,挑選出Sut細(xì)胞中表達(dá)量明顯變化的蛋白質(zhì)點(diǎn),對這些蛋白質(zhì)點(diǎn)進(jìn)行串聯(lián)質(zhì)譜分析。 3.數(shù)據(jù)庫查詢鑒定差異蛋白質(zhì) 將串聯(lián)質(zhì)譜分析得到的數(shù)據(jù)輸入Mascot軟件,在SWISS-PROT數(shù)據(jù)庫中搜索,鑒定出差異蛋白質(zhì)。 4. RT-PCR方法檢測Calmodulin、Annexin A3、Vimentin和S100-A4的mRNA水平 提取Sut和Mela細(xì)胞的總RNA,反轉(zhuǎn)錄獲得細(xì)胞cDNA,通過PCR擴(kuò)增Calmoduln、Annexin A3、Vimentin和S100-A4的基因,擴(kuò)增產(chǎn)物進(jìn)行瓊脂糖凝膠電泳。凝膠掃描,保存圖像,通過繪制柱狀圖對比Sut細(xì)胞與Mela細(xì)胞mRNA表達(dá)量差異水平 研究結(jié)果： 1.Sut細(xì)胞表達(dá)差異蛋白質(zhì)的鑒定 比較Sut和Mela細(xì)胞總蛋白質(zhì)的二維凝膠電泳圖譜,發(fā)現(xiàn)Sut細(xì)胞有20個蛋白質(zhì)表達(dá)量發(fā)生明顯變化,其中10個蛋白質(zhì)表達(dá)量明顯上調(diào),另外10個明顯下調(diào)。對這20個蛋白質(zhì)點(diǎn)進(jìn)行串聯(lián)質(zhì)潛分析,將質(zhì)譜分析得到的數(shù)據(jù)在SWISS-PROT數(shù)據(jù)庫中搜索查詢,鑒定出這些蛋白質(zhì)。表達(dá)量上調(diào)的10個蛋白質(zhì)分別為：波形纖維蛋白(Vimentin),肌動蛋白alpha-1 b (Tubulin alpha-1 b),肌動蛋白beta-5 (Tubulin beta-5),鈣依賴磷脂結(jié)合蛋白A3 (Annexin A3),鈣調(diào)素(Calmodulin),鈣結(jié)合蛋白S100-A4 (protein S100-A4),分子量為11kDa的未知蛋白質(zhì),鈣結(jié)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);表達(dá)量下調(diào)的10個蛋白質(zhì)分別為：鈣腔蛋白(Calumenin), N-Myc下游調(diào)控基因1 (N-Myc downstream regulated genel,NDRGl)蛋白,鳥氨酸氨基轉(zhuǎn)移酶(Ornithine aminotransferase),二硫鍵異構(gòu)酶A3(protein disulfide isomerase A3，，PDIA3),二氫嘧啶酶相關(guān)蛋白2(Dihydropyrimidinase-related protein2，DPYSL2), ATP合成酶ATP5A1 (ATP5A1), H2B組蛋白(Histone H2B type 1-K, HISTLH2BK),分子量為14kDa的未知蛋白質(zhì),基因序列號為OTTMUSG00000007855的未知蛋白質(zhì)和分子量為47kDa的未知蛋白質(zhì)。 2. RT-PCR方法檢測Calmodulin、Annexin A3、Vimentin和S100-A4的1nRNA水平 通過RT-PCR方法驗(yàn)證Calmodulin、Annexin A3、Vimentin和S100-A4的mRNA水平。實(shí)驗(yàn)結(jié)果顯示,和Mela細(xì)胞相比,Calmodulin、Annexin A3、Vimentin和S100-A4的mRNA表達(dá)水平在Sut細(xì)胞中明顯上調(diào)。 研究結(jié)論： 1.與Mela細(xì)胞相比,Sut細(xì)胞中有20個蛋白質(zhì)點(diǎn)的表達(dá)量存在明顯變化,其中10個蛋白質(zhì)表達(dá)量上調(diào),10個蛋白質(zhì)表達(dá)量下調(diào)。有3個上調(diào)蛋白質(zhì)為細(xì)胞結(jié)構(gòu)相關(guān)蛋白(Vimentin, Tubulin alpha-lb, Tubulin beta-5),3個上調(diào)蛋白質(zhì)為鈣結(jié)合蛋白(Calmodulin, protein S100A-4, protein S100A-6),2個上調(diào)蛋白質(zhì)為蛋白酶(S-formylglutathione, Annexin A3);5個下調(diào)的蛋白質(zhì)為蛋白酶(protein NDRG1, PDIA3, ATP5A1, Ornithine aminotransfcrase aminotransferase, NME2),2個下調(diào)蛋白質(zhì)為信號轉(zhuǎn)導(dǎo)相關(guān)蛋白質(zhì)(DPYSL2, HISTLH2BK3) 2.鑒定出的xCT缺陷相關(guān)蛋白質(zhì)在細(xì)胞的自體吞噬、胞內(nèi)囊泡運(yùn)輸、MAP激酶信號通路,DNA復(fù)制等過程中起到重要作用,推測自體吞噬、MAP激酶信號通路可能在xCT缺陷引起的細(xì)胞生長抑制中起關(guān)鍵作用。自體吞噬、MAP激酶信號通路在Sut細(xì)胞生長抑制中的作用還有待于進(jìn)一步研究。 3.通過RT-PCR實(shí)驗(yàn)發(fā)現(xiàn),和Mela細(xì)胞相比,Sut細(xì)胞中Calmodulin、Annexin A3、Vimentin和S100-A4的mRNA表達(dá)水平明顯上調(diào),證明xCT缺陷的Sut細(xì)胞通過轉(zhuǎn)錄水平上調(diào)控Calmodulin、Annexin A3、Vimentin和S100-A4蛋白的表達(dá)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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R341

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