本文選題：人骨形成蛋白 + 成熟肽��； 參考：《廣州醫(yī)學院》2009年碩士論文

【摘要】： 骨的愈合有三個重要的因素:一為骨質再生細胞(骨間質干細胞,定向造骨細胞),二為骨質誘導蛋白(生長因子),三為愈合區(qū)域的環(huán)境(載體和來源組織)。在目前的整形外科手術中,組織工程學技術可以利用一些生物活性物質,如骨形成蛋白(bone morphogenetic proteins, BMPs),來改變骨移植過程的模式。BMPs是一類酸性糖蛋白,屬于乙型轉化生長因子(transforming growth factor-bate, TGF-βs)超家族,能夠誘導促進軟骨和骨的形成,在胚胎發(fā)育過程中發(fā)揮重要的作用,包括腦的發(fā)育和骨的形成。BMPs在體外與多種不同性質的載體連接,可以組成各種類型的有生物誘骨活性的骨修復材料,在矯形外科等領域有廣泛的應用前景及重要的應用意義。目前最少有20種BMPs被確認,這些BMPs被廣泛應用于骨的再生、牙質的生長和腎臟細胞的生長發(fā)育等研究中。 BMP-2是BMPs中生物學活性最高的蛋白之一,也是研究最為廣泛的生長因子之一,其主要生物學作用是誘導未分化的間質細胞分化增殖,形成軟骨和新生骨。直接局部應用BMP-2,因為藥物的彌散和免疫反應,療效較低。故應用轉基因技術將編碼BMP-2的基因導入恰當?shù)陌屑毎麅?使之連續(xù)釋放BMP-2,并以自分泌或旁分泌的形式作用于靶細胞,則可以大大彌補外源性細胞因子局部應用的缺陷。BMP-7最初從骨基質中分離純化得到,其功能是調控骨和軟骨的發(fā)生,它與早前從牛成骨蛋白提取物中分離的成骨蛋白1(osteogenetic protein,OP-1)是同一種蛋白。BMP-7具有細胞因子的特點,用量少而能發(fā)揮很大的作用,但它進入體內后易隨體液流失降解,且半衰期短,不能局限在骨缺損局部發(fā)揮作用,因此必須尋找更有效的替代方法,使其穩(wěn)定表達,基因治療成為備選的方法之一。 BMP-2和-7基因開放讀碼框(open reading frame, ORF)均可編碼由信號肽、前肽和成熟肽組成的多肽,由蛋白酶切去信號肽和前肽后可得到有功能的成熟肽。在本研究將利用基因重組技術,從BMP-2和-7基因表達量豐的骨肉瘤細胞中克隆兩基因的成熟肽的基因序列,并構建它們的真核表達載體。實驗中應用的真核表達載體為pcDNA3.1(+)。 結果顯示:本研究已成功克隆和構建了BMP-2和-7成熟肽的真核表達載體,重組子序列與Genebank中登錄的序列完全一致。實驗結果可為在骨修復和細胞分化增殖等方面得到廣泛應用,可為應用BMP-2和-7進行基因治療等提供有力的實驗支持。
[Abstract]:There are three important factors in bone healing: one is bone regeneration cell (bone interstitial stem cell), the other is bone inducible protein (growth factor), and the third is environment (carrier and source tissue) of healing area. In current orthopedic surgery, tissue engineering techniques can use some bioactive substances, such as bone morphogenetic protein morphogenetic, BMPsN, to change the pattern of bone transplantation. BMPs are a class of acidic glycoproteins. TGF- 尾, which belongs to the growth transforming factor-bate (TGF- 尾) superfamily, can induce the formation of cartilage and bone and play an important role in embryonic development, including brain development and bone formation. It can form various kinds of bone repair materials with biological osteoinductive activity, and has a wide application prospect and important application significance in orthopaedic surgery and other fields. At least 20 BMPs have been identified, which have been widely used in bone regeneration, odontoplasmic growth and renal cell growth, etc. BMP-2 is one of the most bioactive proteins in BMPs. It is also one of the most widely studied growth factors, whose main biological role is to induce the differentiation and proliferation of undifferentiated interstitial cells, forming cartilage and new bone. Direct local application of BMP-2, because of the drug dispersion and immune response, the efficacy is low. Therefore, the gene encoding BMP-2 was introduced into the appropriate target cells by transgenic technology, and the BMP-2 was continuously released and acted on the target cells in the form of autocrine or paracrine. BMP-7 was originally isolated and purified from bone matrix and its function was to regulate the occurrence of bone and cartilage. It is the same protein, BMP-7, which was isolated from bovine osteogenic protein extract (OP-1), has the characteristics of cytokine, and can play a great role in less dosage. However, it is easy to degrade with the loss of body fluid and has a short half-life after entering the body. It is important to find more effective alternative methods to stabilize the expression of bone defects. Gene therapy has become one of the alternative methods. BMP-2 and -7 genes can encode peptides composed of signal peptide, prepeptide and mature peptide by open reading frame (ORF). A functional mature peptide can be obtained by digesting signal peptide and propeptide from protease. In this study, gene sequences of mature peptides of BMP-2 and -7 genes were cloned from osteosarcoma cells with abundant expression of BMP-2 and -7 genes by gene recombination technique, and their eukaryotic expression vectors were constructed. The eukaryotic expression vector was pcDNA3.1. The results showed that the eukaryotic expression vectors of BMP-2 and -7 mature peptides were cloned and constructed successfully in this study. The experimental results can be widely used in bone repair and cell differentiation and proliferation, and can provide powerful experimental support for gene therapy with BMP-2 and -7.
【學位授予單位】：廣州醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R341

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相關期刊論文 前2條

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本文編號：2032406