本文選題：rhddADAM15 + 小鼠黑色素瘤細(xì)胞(B16)��； 參考：《江南大學(xué)》2008年博士論文

【摘要】： ADAM (A Disintegrin And Metalloproteinase)是一類含有七個功能結(jié)構(gòu)域并在細(xì)胞間相互識別和相互作用中發(fā)揮重要作用的跨膜糖蛋白家族。ADAM15作為該家族唯一在去整合素結(jié)構(gòu)域(disintegrin)含有RGD(Arg-Gly-Asp)基序的成員,在細(xì)胞外基質(zhì)降解、細(xì)胞粘附、細(xì)胞內(nèi)信號轉(zhuǎn)導(dǎo)和腫瘤的病理變化等過程中發(fā)揮重要作用,但是其抗腫瘤機理需進一步闡釋。本文在成功獲取高表達(dá)水平的重組人ADAM15去整合素結(jié)構(gòu)域蛋白片段(rhddADAM15)的基礎(chǔ)上,以小鼠黑色素瘤細(xì)胞(B16)為研究模型,研究rhddADAM15對B16細(xì)胞體外增殖、粘附和遷移等細(xì)胞行為的影響,通過篩選rhddADAM15特異結(jié)合的12肽以及rhddADAM15在B16細(xì)胞上相互作用的靶點蛋白,探討rhddADAM15干預(yù)腫瘤細(xì)胞行為的生理機制。主要研究結(jié)果如下: 1)為獲得大量具有生物活性的rhddADAM15蛋白,在詳盡分析目標(biāo)蛋白的cDNA序列的基礎(chǔ)上,(1)選擇能為大腸桿菌稀有密碼子提供額外tRNA的大腸桿菌Escherichia coli Rosetta(DE3)作為宿主菌,將目標(biāo)蛋白以GST(Glutathione-S-transferase)融合蛋白形式表達(dá),在最佳誘導(dǎo)表達(dá)條件下融合蛋白GST-ADAM15濃度為298 mg/L,最佳凝血酶酶切條件下,rhddADAM15濃度為42 mg/L; (2)采用PCR體外定點突變技術(shù)將凝血酶識別序列附近稀有密碼子GGA(Gly)替換為GGC,同時消除Pro-Glu-Phe殘基,構(gòu)建突變表達(dá)質(zhì)粒,提高凝血酶酶切效率。在相同的表達(dá)和酶切條件下獲得GST-△-ADAM15和rhddADAM15蛋白濃度分別為326 mg/L和68mg/L,比野生型分別提高9.4%和61.9%。這一結(jié)果表明,在充分認(rèn)識目標(biāo)蛋白特性的基礎(chǔ)上定向選擇表達(dá)宿主并改造表達(dá)質(zhì)粒能實現(xiàn)外源蛋白高水平表達(dá); 2)采用MTT法測定rhddADAM15對B16、MCF-7/W以及HMEC-1細(xì)胞體外增殖的作用,采用創(chuàng)傷愈合實驗、Matrigel基質(zhì)膠以及Transwell侵襲小室法觀察rhddADAM15對B16細(xì)胞體外遷移、粘附和侵襲的影響;采用臺盼藍(lán)染色法分析細(xì)胞活力、流式細(xì)胞儀檢測rhddADAM15對B16細(xì)胞周期的影響。結(jié)果表明rhddADAM15對腫瘤細(xì)胞和正常內(nèi)皮細(xì)胞生長均有一定的抑制作用(其IC50分別為9.5、11.8和14.9μg/mL),且明顯抑制B16細(xì)胞的體外增殖、遷移、粘附和侵襲。臺盼藍(lán)染色法表明低劑量的rhddADAM15并不能導(dǎo)致B16細(xì)胞出現(xiàn)壞死,且rhddADAM15并不導(dǎo)致B16細(xì)胞出現(xiàn)典型的凋亡峰,而是將細(xì)胞阻滯于G2/ M期,并且這種作用呈劑量依賴關(guān)系; 3)采用親和甄別磁珠技術(shù)篩選rhddADAM15在B16細(xì)胞上的結(jié)合蛋白。將rhddADAM15采用蛋白生物素化方法標(biāo)記生物素,借助生物素-親和素系統(tǒng),將生物素化的rhddADAM15固定于親和素標(biāo)記的免疫磁珠。從B16細(xì)胞裂解液中釣出與之相互作用的蛋白,經(jīng)濃縮富集后,采用雙向電泳分離技術(shù)分離獲得主要的結(jié)合蛋白點,然后對這些蛋白點進行電噴霧串聯(lián)質(zhì)譜技術(shù)(ESI-MS)分析,在蛋白數(shù)據(jù)庫中檢索,共鑒定了8個蛋白質(zhì),按功能可分為如下三類:(1)臨床應(yīng)用的腫瘤標(biāo)志物(蘋果酸脫氫酶、磷酸甘油醛脫氫酶和谷氨酸脫氫酶);(2)能量代謝相關(guān)蛋白(烯醇酶、磷酸丙糖異構(gòu)酶、原肌球蛋白和血紅蛋白);(3)信號轉(zhuǎn)導(dǎo)通路成員(p38MAPK激酶)等。在此基礎(chǔ)上通過分子對接軟件molsoft ICM模擬計算獲得rhddADAM15與p38激酶相互作用的最佳模式和重要的結(jié)合位點; 4)采用噬菌體展示技術(shù)篩選與rhddADAM15特異性相互作用的多肽序列。采用噬菌體隨機十二肽庫對固定有rhddADAM15的親和磁珠系統(tǒng)進行生物淘洗,獲得4條與rhddADAM15特異結(jié)合的12肽。將獲得的12肽與蛋白質(zhì)庫進行序列比對,發(fā)現(xiàn)細(xì)胞周期相關(guān)蛋白如Cdc25和p53,信號通路相關(guān)蛋白/受體,如整合素αvβ3的胞外區(qū),絲裂原活化蛋白激酶p38α等與所獲得的4條12肽具有高度同源性; 5)基于上述研究所獲得的rhddADAM15在B16細(xì)胞上潛在的靶點蛋白,進一步研究rhddADAM15與p38MAPK激酶的體外相互作用,結(jié)合rhddADAM15對B16細(xì)胞行為的抑制作用,采用蛋白點雜交和激酶活性測定方法分析rhddADAM15在體外與重組p38激酶蛋白的結(jié)合情況,同時研究不同劑量rhddADAM15對B16細(xì)胞中p38MAPK激酶磷酸化水平的影響,并觀察rhddADAM15對p38激酶活性受到部分抑制的B16細(xì)胞的增殖和細(xì)胞周期抑制作用的變化。發(fā)現(xiàn):(1)rhddADAM15在離體低溫條件下能夠和重組p38激酶蛋白結(jié)合,但rhddADAM15(0-10μg/mL)在體外對p38MAPK激酶活性沒有影響;(2)同樣劑量的rhddADAM15作用于B16細(xì)胞24 h后,細(xì)胞中p38MAPK激酶的磷酸化水平提高(被激活);(3)p38激酶的特異性抑制劑SB203580(0.5 mmol/L)預(yù)處理B16細(xì)胞30 min后,rhddADAM15對細(xì)胞增殖的抑制作用減弱(IC50由未經(jīng)處理的9.5μg/mL增加到11.4μg/mL),對細(xì)胞周期的阻滯作用也減弱(G2/M期細(xì)胞比率從未經(jīng)處理的的28.77%降到16.59%)。上述結(jié)果表明,p38MAPK信號轉(zhuǎn)導(dǎo)通路參與了rhddADAM15對B16細(xì)胞增殖和細(xì)胞周期的抑制作用,同時也表明生理條件下rhddADAM15與p38MAPK激酶相互作用的復(fù)雜性。
[Abstract]:ADAM (A Disintegrin And Metalloproteinase) is a class of transmembrane glycoprotein family.ADAM15 that contains seven functional domains and plays an important role in intercellular recognition and interaction as the only member of the family that contains RGD (Arg-Gly-Asp) motif in the domain of the integrin domain (disintegrin), and is degraded in the extracellular matrix. Adhesion, intracellular signal transduction and tumor pathological changes play an important role, but their anti-tumor mechanisms need to be further explained. On the basis of successful acquisition of recombinant human ADAM15 domain protein fragment (rhddADAM15), a rat melanoma cell (B16) was used as a model to study rhddADA. The effects of M15 on the proliferation, adhesion and migration of B16 cells in vitro, and to explore the physiological mechanism of rhddADAM15 intervention in tumor cell behavior by screening the specific binding 12 peptide of rhddADAM15 and the target protein of rhddADAM15 interaction on B16 cells. The main results are as follows:
1) in order to obtain a large number of bioactive rhddADAM15 proteins, on the basis of detailed analysis of the cDNA sequence of the target protein, (1) select the Escherichia coli Escherichia coli Rosetta (DE3) which can provide extra tRNA for the rare codon of Escherichia coli as the host bacteria, and the target egg white is expressed in the form of GST (Glutathione-S-transferase) fusion protein. Under the optimal induction condition, the concentration of fusion protein GST-ADAM15 was 298 mg/L, and the concentration of rhddADAM15 was 42 mg/L under the optimum thrombin enzyme digestion condition. (2) the rare codon GGA (Gly) near the thrombin recognition sequence was replaced with GGC in vitro by PCR, and the Pro-Glu-Phe residue was eliminated and the mutant expression plasmid was constructed to improve the coagulation. The concentration of GST- Delta -ADAM15 and rhddADAM15 protein was 326 mg/L and 68mg/L respectively under the same expression and enzyme cutting conditions, respectively, which was 9.4% and 61.9%. respectively higher than that of wild type. The results showed that the selective expression of the host and the expression plasmid on the basis of fully understanding the characteristics of the target protein could achieve the high water content of the exogenous protein. Flat expression;
2) the effect of rhddADAM15 on the proliferation of B16, MCF-7/W and HMEC-1 cells in vitro was measured by MTT, and the effects of rhddADAM15 on the migration, adhesion and invasion of B16 cells in vitro were observed by the wound healing experiment, Matrigel matrix gel and Transwell invasion, and the cell viability was analyzed by trypan blue staining, and the flow cytometry was used to detect rhddADAM15 pairs. The effect of B16 cell cycle shows that rhddADAM15 inhibits the growth of tumor cells and normal endothelial cells (IC50 is 9.5,11.8 and 14.9 mu g/mL respectively), and obviously inhibits the proliferation, migration, adhesion and invasion of B16 cells in vitro. Trypan blue staining shows that low dose rhddADAM15 does not cause B16 cells to appear bad. RhddADAM15 did not cause typical apoptotic peak in B16 cells, but blocked G2/ M phase.
3) the binding protein of rhddADAM15 on B16 cells was screened by affinity screening magnetic beads. Biotin was used to mark biotin by protein biotin, and biotin avidin system was used to immobilization of biotinylated rhddADAM15 on avidin labeled immunomagnetic beads. The proteins interacting with them from the B16 cell lysate were found. After concentration and enrichment, the main binding protein points were obtained by two-dimensional electrophoresis separation technology, and then the protein spots were analyzed by electrospray ionization tandem mass spectrometry (ESI-MS), and 8 proteins were identified in the protein database. According to their functions, they can be divided into three categories: (1) the clinical application of the tumor markers (malate dehydrogenase, phosphoric acid) Glyceraldehyde dehydrogenase and glutamate dehydrogenase (2) energy metabolism related proteins (enolase, phosphosin isomerase, promyosin and hemoglobin); (3) signal transduction pathway members (p38MAPK kinase), and so on. On this basis, the best model of the interaction between rhddADAM15 and p38 kinase is obtained by molecular docking software molsoft ICM. Important binding sites;
4) using phage display technique to screen the polypeptide sequence with rhddADAM15 specific interaction. Using the phage random twelve peptide library, 4 rhddADAM15 affinity magnetic beads system was washed, and 4 12 peptides specifically binding to rhddADAM15 were obtained. The sequence alignment of the obtained 12 peptide and the protein pool was compared, and the cell cycle correlation was found. Proteins such as Cdc25 and p53, signaling pathway related proteins / receptors, such as the extracellular domain of integrin alpha v beta 3, and mitogen activated protein kinase p38 alpha are highly homologous to the 4 12 peptides obtained.
5) based on the potential target protein of rhddADAM15 on B16 cells obtained from the above study, the interaction of rhddADAM15 and p38MAPK kinase in vitro was further studied, and the inhibition action of rhddADAM15 on B16 cell behavior was combined, and the binding of protein point hybridization and kinase activity was used to analyze the binding of rhddADAM15 in vitro and the recombinant p38 kinase protein. At the same time, the effects of different doses of rhddADAM15 on the phosphorylation of p38MAPK kinase in B16 cells and the changes in the proliferation and cell cycle inhibition of B16 cells partially inhibited by p38 kinase activity were observed. (1) rhddADAM15 can be combined with the recombinant kinase protein under the isolated cryogenic strip, but rhddADAM, but rhddADAM 15 (0-10 g/mL) had no effect on the activity of p38MAPK kinase in vitro; (2) after the same dose of rhddADAM15 acted on B16 cells 24 h, the level of phosphorylation of p38MAPK kinase in the cells was increased (activated); (3) the inhibitory effect of rhddADAM15 on cell proliferation was weakened after the specific inhibitor SB203580 (0.5 mmol/L) of p38 kinase (0.5 mmol/L). C50 was increased to 11.4 UU g/mL from untreated 9.5 mu g/mL), and the cell cycle retardation was also reduced (the ratio of G2/M cell ratio never treated by 28.77% to 16.59%). The results showed that the p38MAPK signal transduction pathway was involved in the inhibition of rhddADAM15 on the proliferation and cell cycle of B16 cells, and also indicated rhddAD under physiological conditions. The complexity of the interaction between AM15 and p38MAPK kinase.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R341

【引證文獻】

中國碩士學(xué)位論文全文數(shù)據(jù)庫 前1條

1 金雄華;剛性陶瓷/瓊脂糖復(fù)合蛋白質(zhì)層析介質(zhì)的制備及應(yīng)用研究[D];江南大學(xué);2012年

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本文編號：2105666