發(fā)布時(shí)間：2018-01-09 08:37

  本文關(guān)鍵詞：hVEGF-165和hBMP-2真核共表達(dá)載體的構(gòu)建及其轉(zhuǎn)染對(duì)人骨髓間充質(zhì)干細(xì)胞成骨能力的影響　出處：《中國(guó)協(xié)和醫(yī)科大學(xué)》2005年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 骨缺損 骨形態(tài)發(fā)生蛋白 血管內(nèi)皮細(xì)胞生長(zhǎng)因子 重組質(zhì)粒 骨髓間充質(zhì)干細(xì)胞 基因治療

【摘要】：顱頜面部骨缺損是平、戰(zhàn)時(shí)意外事故的常見(jiàn)傷類(lèi)。骨形態(tài)發(fā)生蛋白(Bone Morphogenetic Proteins，BMPs)對(duì)骨原細(xì)胞的分化起主要的決定性作用，是促進(jìn)成骨的重要因子；血管內(nèi)皮生長(zhǎng)因子(vascular endothelial growth factor，VEGF)是新近發(fā)現(xiàn)的在創(chuàng)傷愈合過(guò)程中促進(jìn)血管再生發(fā)揮著重要作用的生長(zhǎng)因子。本研究在創(chuàng)建顱骨缺損動(dòng)物模型的基礎(chǔ)上，采用免疫組化及原位雜交技術(shù)，動(dòng)態(tài)觀察了創(chuàng)傷后組織中BMP2、4及VEGF的變化。應(yīng)用分子克隆和轉(zhuǎn)基因技術(shù)，構(gòu)建了hVEGF165與hBMP2真核細(xì)胞共表達(dá)載體——pIRES-hBMP2-hVEGF165，并應(yīng)用組織工程技術(shù)進(jìn)行人骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells，BMSCs)的分離和培養(yǎng)，在傳代后采用陽(yáng)離子脂質(zhì)體介導(dǎo)的轉(zhuǎn)染方式，將外源VEGF165及hBMP2 cDNA導(dǎo)入BMSCs，使其正常表達(dá)，以提高BMSCs的成骨能力，提供改良骨組織工程種子細(xì)胞，從而促進(jìn)骨創(chuàng)傷愈合過(guò)程。本實(shí)驗(yàn)通過(guò)免疫組化、免疫印記、RT-PCR等方法檢測(cè)基因轉(zhuǎn)染后BMSC中的VEGF165、BMP2蛋白質(zhì)骨鈣素mRNA的表達(dá)程度，同時(shí)也觀察了對(duì)BMSCI型膠原表達(dá)及堿性磷酸酶活性的影響。 結(jié)果發(fā)現(xiàn)：VEGF在顱骨缺損后骨缺損3天時(shí)，骨缺損端內(nèi)軟骨細(xì)胞和成骨細(xì)胞呈陽(yáng)性表達(dá)，基質(zhì)內(nèi)呈弱陽(yáng)性表達(dá)，1周表達(dá)逐漸減弱；2周時(shí)表達(dá)增強(qiáng)，3周時(shí)軟骨細(xì)胞分裂增生，呈團(tuán)狀排列，表達(dá)達(dá)到高峰，呈強(qiáng)陽(yáng)性，4周時(shí)減弱，5周時(shí)仍可見(jiàn)部分細(xì)胞呈陽(yáng)性。BMP2在骨缺損3天后，骨缺損端可見(jiàn)散在分布的未分化間充質(zhì)細(xì)胞及細(xì)胞間質(zhì)呈陽(yáng)性。1周時(shí)原始骨痂成骨細(xì)胞、骨端骨細(xì)胞、新形成的軟骨基質(zhì)呈陽(yáng)性。肉芽組織間質(zhì)呈陽(yáng)性。2周呈弱陽(yáng)性，3周時(shí)，新形成得軟骨細(xì)胞出現(xiàn)陽(yáng)性染色，軟骨基質(zhì)也呈陽(yáng)性。幼稚軟骨細(xì)胞、骨痂表面成骨細(xì)胞呈陽(yáng)性，4周時(shí)減弱，5周時(shí)仍可見(jiàn)部分細(xì)胞呈陽(yáng)性。BMP4 mRNA在缺損后1周，骨折間充質(zhì)細(xì)胞呈陽(yáng)性，3周時(shí)表達(dá)明顯增強(qiáng)，，4周時(shí)減弱，其余時(shí)間點(diǎn)未見(jiàn)表達(dá)。 構(gòu)建的重組質(zhì)粒pIRES-hBMP2-hVEGF165經(jīng)酶切和電泳及測(cè)序鑒定后證明構(gòu)建正確。基因轉(zhuǎn)染后BMSC能檢測(cè)到BMP2和hVEGF165蛋白分泌量明顯升高，促進(jìn)BMSC合成I型膠原，提高其堿性磷酸酶活性以及骨鈣素mRNA的表達(dá)程度與對(duì)照組相比，非常顯著。這說(shuō)明： 1．VEGF在顱骨缺損創(chuàng)傷后的表達(dá)與創(chuàng)傷愈合過(guò)程中新生血管形成的時(shí)間相似，說(shuō)明VEGF參與調(diào)節(jié)了骨創(chuàng)傷愈合過(guò)程中的血管再生階段，對(duì)毛細(xì)血管的發(fā)生有積極促進(jìn)作
[Abstract]:Craniofacial bone defect is a common injury in wartime accidents. Bone morphogenetic protein (Bone
Morphogenetic Proteins, BMPs) on the differentiation of osteoprogenitor cells play a decisive role in the main, is an important factor in promoting osteogenesis; vascular endothelial growth factor (vascular endothelial, growth factor, VEGF) is to promote angiogenesis plays an important role in the growth factor in wound healing process of newly discovered. In this study the skull defect animal model, immunohistochemistry and in situ hybridization, observed the changes of BMP2,4 and VEGF after trauma in the tissue. The application of molecular cloning and transgenic technology, constructed vector pIRES-hBMP2-hVEGF165 co expression of hVEGF165 and hBMP2 in eukaryotic cells, and the application of tissue engineering of human bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs) were isolated and cultured, using cationic liposome mediated transfection method in the passage, the exogenous VEGF165 and hBMP2 cDN A into BMSCs, the normal expression, in order to improve the osteogenic ability of BMSCs, provides improved seed cells for bone tissue engineering, so as to promote the process of bone wound healing. This experiment by immunohistochemistry, Western blot and RT-PCR were used to detect gene transfection in BMSC VEGF165. The expression level of BMP2 protein osteocalcin mRNA, at the same time we also observed the effect on the expression of type BMSCI collagen and alkaline phosphatase activity.
The results showed that: VEGF in skull bone defect after 3 days, in the end of chondrocyte bone defect and osteoblasts showed positive expression of stroma showed weak positive expression, the expression decreased gradually 1 weeks; 2 weeks was enhanced at 3 weeks, the proliferated chondrocytes arranged in clusters, reaching a peak expression that was strongly positive, decreased at 4 weeks, 5 weeks still visible part of the cells were.BMP2 positive in bone defect 3 days after the end of bone defect scattered in the distribution of undifferentiated mesenchymal cells and stromal cells were positive for.1 weeks when the primary callus bone cells, bone bone cells, cartilage matrix the formation of granulation tissue stroma was positive. Positive.2 weeks were weakly positive at week 3, the newly formed cartilage cells appeared positive staining of cartilage matrix is also positive. Immature cartilage cells, bone callus surface cells were positive, decreased at 4 weeks, 5 weeks still visible part of the cells were positive for.BMP4 in the mRNA defect After 1 weeks, the mesenchymal cells in the fracture were positive, the expression was obviously enhanced at 3 weeks and decreased at 4 weeks, and no expression was found at the rest of the time.
The recombinant plasmid pIRES-hBMP2-hVEGF165 was verified by enzyme digestion and electrophoresis and sequencing proved correctly constructed. After transfection of BMSC gene can be detected BMP2 and hVEGF165 protein secretion increased significantly, BMSC promote the synthesis of collagen type I, increase the activity of alkaline phosphatase and osteocalcin mRNA expression level compared with the control group that is very significant:
The expression of 1.VEGF in traumatic skull defect is similar to the time of neovascularization during wound healing. It indicates that VEGF is involved in regulating the stage of angiogenesis in the process of bone wound healing, and has a positive effect on the occurrence of capillaries.

【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2005
【分類(lèi)號(hào)】：R346

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前2條

1 何春耒;腺病毒介導(dǎo)的BMP-2基因轉(zhuǎn)染BMSCs復(fù)合納米羥基磷灰石體外構(gòu)建組織工程骨的實(shí)驗(yàn)研究[D];廣州醫(yī)學(xué)院;2010年

2 尹康;不同時(shí)機(jī)轉(zhuǎn)染基因?qū)ν孟骂M骨牽引區(qū)細(xì)胞周期蛋白表達(dá)的影響[D];瀘州醫(yī)學(xué)院;2012年

本文編號(hào)：1400745