论文标题:VEGF基因转染的骨髓基质细胞复合磷酸钙陶瓷成骨能力研究
Osteogenesis of Marrow Stroma Cells with VEGF Gene Transfection and Porous Calcium Phosphate
论文作者 高全文
论文导师 董绍忠；杨连甲,论文学位 硕士,论文专业 口腔基础医学
论文单位 中国人民解放军第四军医大学,点击次数 96,论文页数 69页File Size5589k
2003-04-01论文网 http://www.lw23.com/lunwen_2090252/ 组织工程;骨髓基质细胞;β－磷酸三钙;基因转染;血管内皮生长因于
tissue engineering,bone marrow stroma cells,β-tricalcium;phosphate,gene transfection,vascular endothelial growth factor
组织工程是应用细胞生物学和工程学原理，对病损组织结构和功能的修复与重建进行研究开发的一门新兴学科。其目的是研究和开发组织替代物。本研究对用组织工程方法再造骨组织进行了一系列的研究。 1．骨髓基质细胞成骨能力的实验研究 方法：体外分离、培养MSCs，观察其生长特点并进行碱性磷酸酶(ALP)组织化学染色；用矿化液连续培养，观察其骨形成能力。结果：倒置显微镜下观察，在矿化条件下，细胞传代培养5—6天后，长至铺满，细胞连续培养30天，Von-Kassa染色显示有局灶状钙盐沉积。结论：MSCs易于分离培养，体外扩增速度快，在矿化条件下，骨髓基质细胞在体外即具有很强的成骨能力，可以形成矿化结节。因而可以作为种子细胞，用于骨组织工程。 2．人血管内皮生长因子165真核表达载体的构建及鉴定 方法：利用基因克隆技术，将位于原核克隆载体pSP73上的目的基因VEGF_(165)克隆到真核表达载体pcDNA3.1上。将获得的真核表达质粒pcDNA3.1进一步用限制性内切酶酶切鉴定及基因测序，证明人VEGF_(165)基因正确克隆到pcDNA3.1真核表达载体上。 3．人血管内皮生长因子165基因转染骨髓基质细胞 方法：利用基因转移技术，将构建重组质粒pcDNA3.1-VEGF_(165)通过脂质体介导转染SD大鼠骨髓基质细胞，然后以G418筛选阳性克隆，用免疫细胞化学方法鉴定。免疫细胞化学证实，重组质粒转染的骨髓基质细胞中有VEGF_(165)基因的表达。 4．骨髓基质细胞诱导培养后与磷酸钙陶瓷复合的实验研究 方法：分离的骨髓基质细胞用含10％胎牛血清的DMEM培养，经矿化液诱导培养后分化为成骨细胞。将该细胞与预备处理后的磷酸钙陶瓷复合，通过细胞计数检测附着后的细胞生长增殖特性，并在扫描电镜下观察细胞 第四军医大学硕士学位论文在磷酸钙陶瓷表面的贴附情况。结论：磷酸钙陶瓷的生物相容性良好，细胞贴附后继续保持成骨细胞的表型，可以作为骨组织工程的支架材料。 5．基因转染的自体细胞与磷酸钙陶瓷复合异位成骨的研究 方法：成年雄性 SD大鼠 12只，全麻及无菌条件下取其左侧股骨、胜骨。将骨髓细胞接种入培养瓶中体外扩增培养，第sd开始用矿化液诱导培养持续1周，骨髓基质细胞向成骨细胞分化，诱导后的细胞行传代培养。l周后将骨髓基质细胞消化并在脂质体介导下进行VEGF；6s转染及G－418连续筛选 14d。然后将转染的和末转染的自体细胞混合一同接种于磷酸钙陶瓷上体外继续培养7d。最后将细胞一陶瓷复合体植入大鼠背部皮下和肌肉内。结论：应用以磷酸钙陶瓷为支架的VEGF165基因转染自体骨髓基质细胞种植，在组织工程骨周围有较多的血管形成，异位骨形成能力较好。
Tissue engineering is an interdisciplinary science of co-development and combination of modern cell biology, biomaterial science and engineering. The aim of tissue engineering is to investigate and restore tissue and organ substitutes. In this study, bone formation was conducted using the method of tissue engineering.1. The experiment of bone Marrow stroma cells of cultured in vitro.Method: SD rat MSCs were cultured in vitro, cell proliferation and ALP activity were observed. Bone formation potentiality of MSCs in mineralized condition was investigated by ALP staining. Results: Cells occupied the whole bottom of cell culture bottle in about 5-6 days. When cultured up to 30 days, von Kossa staining showed there was calcium deposition in the nodules.2. Construction and identification of eukaryotic expression plasmid of humanVEGF,65.Methods: In this experiment, the human VEGFies gene in pSP73 plamsid was restricted by BamH I and Xho I, then the VEGFies gene was cloned into eukaryotic expression pcDNA3.l. The eukaryotic expression plamsid pcDNA3.1 was transformed into E.colidh5a which was cultured overnight, then plamsid pcDNA3.l-VEGFies was substracted as well as purificated largely. Results: The positive clone by identification of recombinant and sequencing showed that the human VEGF165 was cloned correctly into the eukaryotic expression plamsid.3. Construction and expression of eukaryotic expression vector containinggene in rat bone marrow stroma cells.Methods: The recombinant eukaryotic expression vector pcDNA3.1-VEGFi65 was transfected into the rMASCs by and positive clones were screened with G418. The expression of VEGFies gene in the transfected cells was detected by immunocytochemical staining. Results: The expression of hVEGF165 gene in the transfected cells had been demonstrated by immunocytochemical staining. Therefore, it is possible to use the rMSCs expressing hVEGF^s gene as seeded cells in the bone tissue engineering. 4. The experiment of porous calcium phosphate ceramic combined with SD rat bone marrow stroma cells in vitro.Methods: Bone marrow stroma cells were cultured with DMEM containing 10%FBS. After inoculating the cells onto the surface of porous calcium phosphate ceramic, we surveyed the characteristic of proliferation by cell counting. Using scanning electronic microscope, we observed cellular morphology. Results: Rat bone marrow stroma cell could be attached to and extended on the surface of porous calcium phosphate ceramic, and normally grown, proliferated. Porous calcium phosphate ceramic could promote cell proliferation. Conclusion: The results show that porous calcium phosphate ceramic has good biocompatibility.With rat bone marrow stroma cells, they can be used as biomaterials in bone tissue engeering.5. SD rat marrow stroma cells with VEGF)65 gene transfection loading on porous calcium phosphate ceramic scaffolds.Methods: Autogenous marrow stromal cells were obtained from femurs and tibias of 12 male adult SD rats under general anesthesia and sterile condition and cultured in DMEM supplemented with 10%FBS. VEGFi65 gene was transfectedinto stroma cells by means of LipofectAMINE?2000 reagent transfection. The stably gene expressive cells were screened with G-418 for fourteen days. The cell mixture were seeded in porous calcium phosphate bioceramic and cultured in vitro for another 7 days. The cell-ceramic compound was implanted subcutaneously and intramuscularly in the corresponding rat.
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