本文選題：癌細胞 + U2-OS　； 參考：《濟南大學》2017年碩士論文

【摘要】：人骨形態(tài)發(fā)生蛋白-7(Human bone morphogenetic protein-7,hBMP-7),又名成骨蛋白-1(Osteogenic Protein,OP-1),屬于轉(zhuǎn)化生長因子-β(Transforming growth factor-β,TGF-β)超家族。hBMP-7能夠誘導動物或人間充質(zhì)干細胞分化為骨、軟骨、韌帶、肌腱和神經(jīng)組織,在臨床骨科領域中具有廣闊的應用前景,可用于促進脊柱融合和治療骨折、骨缺損、骨不連、股骨頭缺血性壞死等。但是,在基因工程表達該蛋白的過程中遇到了一些難以克服的難題:原核表達系統(tǒng)由于缺乏翻譯后加工、修飾機制,使表達的蛋白復性困難;畢赤酵母的糖基化形式與人類存在差異亦不利于該蛋白的表達;CHO細胞表達的重組人骨形態(tài)發(fā)生蛋白-7(rhBMP-7)雖有活性,但是表達量很低,尚不具備產(chǎn)業(yè)化條件,因此,國內(nèi)外生產(chǎn)此蛋白的公司極少,價格昂貴。所以,能否克服如上困難是rhBMP-7能否真正實現(xiàn)產(chǎn)業(yè)化的關鍵。目的:人源癌細胞具有完整的翻譯后加工機制、可無限增殖和體外易培養(yǎng)的特點,能否讓其“棄惡從善”用于表達具有臨床應用價值、結(jié)構(gòu)復雜的蛋白質(zhì)是一個具有挑戰(zhàn)性的課題。本研究擬利用人源癌細胞U2-OS進行rhBMP-7蛋白的表達,以探討人骨肉瘤U2-OS細胞作為表達系統(tǒng)表達rhBMP-7的可行性,本研究有可能為復雜結(jié)構(gòu)蛋白質(zhì)的高效表達提供一種新的解決方案。方法:(1)rhBMP-7在U2-OS細胞中的表達:構(gòu)建真核表達質(zhì)粒pcDNA3.1-rhBMP-7,利用Lipofectamine2000將真核表達質(zhì)粒轉(zhuǎn)染至U2-OS細胞中,采用實時熒光定量PCR法檢測rhBMP-7基因在mRNA水平上的表達;Western Blot法檢測rhBMP-7的表達。(2)分泌性rhBMP-7的表達:分析三種信號肽(hBMP-7自身信號肽B7、人基質(zhì)金屬蛋白酶-9信號肽M9、人纖溶酶原信號肽SP)對U2-OS細胞分泌表達rhBMP-7的影響。構(gòu)建真核表達質(zhì)粒pcDNA3.1-rhB7-h BMP7、pcDNA3.1-rhSP-hBMP7、pcDNA3.1-rhM9-hBMP7。利用Lipofectamine2000將真核表達質(zhì)粒轉(zhuǎn)染至U2-OS細胞中,采用實時熒光定量PCR法檢測rhBMP-7基因在mRNA水平上的表達,Western Blot法檢測U2-OS細胞內(nèi)與其培養(yǎng)基中分泌表達的rhBMP-7。在Western Blot檢測分泌表達的rhBMP-7的基礎上進一步利用ELISA檢測U2-OS細胞內(nèi)與細胞外分泌表達的rhBMP-7。(3)rhBMP-7的純化與分析:選用親和層析法純化細胞分泌表達的rhBMP-7蛋白;通過糖苷內(nèi)切酶酶切樣品rhBMP-7,對其進行糖基化分析;純化的rhBMP-7作用于NIH3T3細胞,通過檢測NIH3T3細胞的ALP活性,分析rhBMP-7的生物活性。結(jié)果:(1)Western Blot結(jié)果顯示rhBMP-7蛋白分子量為65-75KDa,實驗組細胞中rhBMP-7表達量高于陰性對照組。結(jié)果表明rhBMP-7可在U2-OS細胞中表達。(2)研究分泌性rhBMP-7的表達中,通過Western Blot與ELISA檢測U2-OS細胞內(nèi)與細胞外分泌表達的rhBMP-7,結(jié)果表明U2-OS細胞內(nèi)與細胞外均有表達rhBMP-7。ELISA結(jié)果顯示細胞外表達的rh BMP-7蛋白量高于細胞內(nèi)表達的rhBMP-7,說明rhBMP-7實現(xiàn)了分泌表達,而且hBMP-7自身信號肽引導的U2-OS細胞分泌表達的rhBMP-7蛋白量較高。(3)經(jīng)鎳柱純化得到rhBMP-7蛋白,通過BCA法測得rhBMP-7蛋白濃度為28.3mg/L,在緩沖液中可完全溶解。rhBMP-7經(jīng)SDS-PAGE電泳顯示條帶單一,純度較好。(4)通過糖苷內(nèi)切酶(Endo H)酶切rhBMP-7對其進行糖基化分析。未經(jīng)Endo H酶切的rhBMP-7單體rhBMP-7蛋白分子量為34-40KDa,二聚體rhBMP-7蛋白分子量為65-75KDa。經(jīng)Endo H酶切的rhBMP-7單體蛋白分子量為17-20KDa,二聚體rh BMP-7蛋白分子量為34-40KDa。根據(jù)Endo H酶切rhBMP-7的結(jié)果得出:U2-OS細胞對表達rhBMP-7蛋白進行了糖基化修飾。(5)rhBMP-7對NIH3T3細胞堿性磷酸酶(ALP)的影響結(jié)果顯示:含rhBMP-7培養(yǎng)基使NIH3T3細胞的ALP活性明顯增強,而且呈現(xiàn)出明顯的量、效關系,表明U2-OS細胞表達的rhBMP-7具有生物活性。結(jié)論:(1)本實驗結(jié)果表明,可以利用U2-OS細胞表達rhBMP-7,而且信號肽會影響細胞對表達蛋白的分泌效率;hBMP-7自身信號肽引導U2-OS細胞分泌表達rhBMP-7的蛋白量較高。(2)U2-OS細胞對表達rhBMP-7蛋白進行了糖基化修飾,蛋白可溶。生物活性檢測結(jié)果表明U2-OS細胞表達的rhBMP-7具有生物活性。(3)本實驗的結(jié)果表明:U2-OS細胞可作為表達系統(tǒng)進行復雜結(jié)構(gòu)蛋白質(zhì)的制備,此為今后大規(guī)模表達和制備結(jié)構(gòu)復雜的細胞因子、人源抗體、疫苗等奠定了實驗基礎。
[Abstract]:Human bone morphogenetic protein -7 (Human bone morphogenetic protein-7, hBMP-7), also known as osteogenic protein -1 (Osteogenic Protein, OP-1), belongs to transforming growth factor beta (Transforming growth beta, beta) superfamily can induce animal or human mesenchymal stem cells to differentiate into bone, cartilage, ligaments, tendons and nerve tissue, in clinical practice. There are broad applications in the field of Department of orthopedics, which can be used to promote spinal fusion and the treatment of fractures, bone defects, bone nonunion and avascular necrosis of the femoral head. However, there are some difficult problems to be overcome in the process of gene engineering expression of this protein: the prokaryotic expression system is due to the lack of post-translational processing, modification mechanism, and expression of protein. The glycosylation form of Pichia pastoris and human existence are not good for the expression of the protein. Although the recombinant human bone morphogenetic protein -7 (rhBMP-7) expressed by CHO cells is active, the expression is very low and there is no industrial condition. Therefore, there are few companies producing this protein at home and abroad, and the price is expensive. Difficulty is the key to the real industrialization of rhBMP-7. Objective: human cancer cells have a complete post-translational processing mechanism, which can grow infinitely and easily in vitro. It is a challenging task to make its "abandoned evil and good" to express the value of clinical application. The expression of rhBMP-7 protein in human cancer cell U2-OS is used to explore the feasibility of human osteosarcoma U2-OS cells as expression system for expression of rhBMP-7. This study may provide a new solution for the efficient expression of complex structural proteins. Method: (1) the expression of rhBMP-7 in U2-OS cells: Construction of eukaryotic expression plasmid pcDNA3.1-rhBMP- 7, the eukaryotic expression plasmid was transfected into U2-OS cells by Lipofectamine2000, and the expression of rhBMP-7 gene at mRNA level was detected by real-time fluorescence quantitative PCR; Western Blot method was used to detect the expression of rhBMP-7. (2) the expression of secretory rhBMP-7: analysis of three signal peptides (hBMP-7 itself signal peptide B7, human matrix metalloproteinase -9 signal peptide, human The effect of plasminogen signal peptide SP on the expression of rhBMP-7 in U2-OS cells. Construction of eukaryotic expression plasmid pcDNA3.1-rhB7-h BMP7, pcDNA3.1-rhSP-hBMP7, pcDNA3.1-rhM9-hBMP7. using Lipofectamine2000 to transfect eukaryotic expression plasmid into U2-OS cells, and detect the expression of rhBMP-7 gene on mRNA level by real-time fluorescent quantitative PCR. Ern Blot assay was used to detect the expression of rhBMP-7. secreted in U2-OS cells and the expression of rhBMP-7 in Western Blot by Western Blot to further purify and analyze rhBMP-7. (3) rhBMP-7 in U2-OS cells and exocrine expression by ELISA: affinity chromatography was used to purify the secretory expression of the rhBMP-7 protein; The glycoside endonuclease was cut to the sample rhBMP-7 to carry out the glycosylation analysis. The purified rhBMP-7 acted on the NIH3T3 cells and analyzed the bioactivity of rhBMP-7 by detecting the ALP activity of NIH3T3 cells. Results: (1) the Western Blot results showed that the rhBMP-7 protein molecular weight was 65-75KDa, and the rhBMP-7 expression in the tested group was higher than that of the negative control group. The results showed that rhBMP-7 could be expressed in U2-OS cells. (2) to study the expression of secretory rhBMP-7, the expression of rhBMP-7 in U2-OS cells and cell exocrine in U2-OS cells was detected by Western Blot and ELISA. The results showed that the RH BMP-7 protein in the U2-OS cell and outside cells expressed the RH BMP-7 protein amount of the cell appearance of the cells higher than that of the intracellular expression. 7, the expression of rhBMP-7 was expressed, and the expression of rhBMP-7 protein secreted by U2-OS cells guided by hBMP-7 self signal peptide was higher. (3) the rhBMP-7 protein was purified by the nickel column, the concentration of rhBMP-7 protein was 28.3mg/L by the BCA method, and the.RhBMP-7 was completely dissolved in the buffer solution, and the purity of the band was single and the purity was better. (4) The glycosylation analysis was carried out by the glucoside endonuclease (Endo H) enzyme cut rhBMP-7. The molecular weight of rhBMP-7 monomer rhBMP-7 protein was 34-40KDa without Endo H enzyme cut, and the molecular weight of the two polymer rhBMP-7 protein was 65-75KDa. through the Endo H enzyme. The results of cutting rhBMP-7 showed that the expression of rhBMP-7 protein was glycosylated by U2-OS cells. (5) the effect of rhBMP-7 on the alkaline phosphatase (ALP) of NIH3T3 cells showed that the ALP activity of NIH3T3 cells was obviously enhanced by the rhBMP-7 culture medium, and showed a significant amount and effect relationship, indicating that rhBMP-7 of the U2-OS cell expression was bioactive. Conclusions: (1) the experimental results show that the expression of rhBMP-7 can be expressed by U2-OS cells, and the signal peptide can affect the secretion efficiency of the expressed protein, and the hBMP-7 self signal peptide leads U2-OS cells to secrete a higher protein expression of rhBMP-7. (2) U2-OS cells have glycosylated, soluble protein and bioactivity detection for the expression of rhBMP-7 protein. The results showed that the rhBMP-7 expressed by U2-OS cells had biological activity. (3) the results of this experiment showed that U2-OS cells could be used as an expression system to prepare complex structural proteins, which laid the experimental basis for large-scale expression and preparation of complex cytokine, human antibody and vaccine in the future.
【學位授予單位】：濟南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R915

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