Cloning, Expression and Identification of Specific Genes related Osseointegration to Titanium.

本研究的目的是阐明钛诱导的细胞行为，并分离促进钛种植体-骨整合的特异性基因。为了鉴定与钛骨整合相关的关键基因，我们在钛涂层玻璃上培养成骨细胞系MC3T3-E1细胞，并分析其基因表达。1)在高真空条件下，将钛和铬涂层在抛光玻璃表面（直径33 mm，厚度1.5 mm）上。将涂有Ti和Cr的玻璃盘置于6孔板中，用硅胶环固定。无涂层玻璃盘作为对照。2)细胞附着、增殖和分化评估。mts法测定MC3T3-E1细胞的初始细胞附着和增殖情况(CellTiter 96【〇！R】水溶液，Promega， USA)，碱性磷酸酶活性测定法评价细胞分化。播种后3 h， Ti-coa . More ted玻璃上附着细胞的平均相对数量显著高于未涂布和cr涂布玻璃。同样，在ti涂层玻璃上播种后3 d的平均细胞数显著高于其他条件。三种条件下，播种后成骨细胞碱性磷酸酶（ALP）活性均随时间增加而升高，其中播种后第14天钛涂层玻璃上的碱性磷酸酶活性显著高于其他条件。结果表明，与未涂布和Cr涂布玻璃板相比，Ti涂布玻璃板促进了MC3T3-E1细胞的附着、增殖和分化。3)成骨细胞基因表达对钛的影响将成骨细胞培养在Ti、Cr和未涂布玻璃板上。然后用cDNA减法杂交鉴定差异表达基因。分离得到20个独立克隆，通过核苷酸测序鉴定出7个克隆，其中包括EST基因；xab-2,sod-1，半乳糖凝集素-1，肌动蛋白相关蛋白2/3 mRNA， RIKEN cDNA 2210013021基因，EST 601086505F1, EST 01439。ti涂层玻璃上galectin-1、xab-2、sod-1基因的表达高于未涂层玻璃和cr涂层玻璃。这些结果表明，ti包覆比Cr或未包覆更有利于成骨细胞的附着、增殖和分化，而galectin-1、xab-2和sod-1的上调可能与Ti-bone整合有关。少

The aim of this study is to clarify the cell behavior induced by titanium and to isolate specific genes that promote the titanium implant-bone integration. Especially, to identify the master key genes related osseointegration to titanium, the osteoblastic cell line, MC3T3-E1 cells were cultured on titanium coated glasses and the gene expression were analyzed.1)Titanium and chrome coated glassesTi and Cr were coated to polished glass surfaces (33 mm in diameter by 1.5 mm in thickness) in a high vacuum condition. Ti and Cr coated glass disks were placed in a 6 well plate and fixed by silicone rings. Non-coated glass disks were served as controls.2)Cell attachment, proliferation and differentiation assessments.Initial cell attachment and proliferation of MC3T3-E1 cells were estimated by MTS-assay (CellTiter 96【○!R】 AQueous One Solution, Promega, USA), and cell differentiation was evaluated by alkaline phosphatase activity assay. The mean relative amount of the attached cells on the Ti-coa … More ted glass was significantly higher than those on non-coated and Cr-coated glass at 3 hours after seeding. On the same way the mean cell number on Ti-coated glass at 3 days after seeding was significantly higher than those of other conditions. The mean alkaline phosphatase(ALP) activity of the seeded osteoblasts also increased with time in these three conditions, while the ALP activity level on the Ti-coated glass at 14 days after seeding was significantly higher than those of other conditions. These results suggest that, Ti-coated glass plate accelerated the cell attachment, proliferation and differentiation of the MC3T3-E1 cells, compared to the non- and Cr-coated glass plates3)Effect of gene expression of osteoblast on titaniumOsteoblasts were cultured on Ti coated, Cr coated and non-coated glass plates. And then differentially expressed genes were identified by cDNA subtractive hybridization. Twenty independent clones were isolated and by nucleotide sequencing of these clones, seven clones were identified including EST genes ; xab-2,sod-1,galectin-1,actin related protein 2/3 mRNA, RIKEN cDNA 2210013021 gene, EST 601086505F1, and EST 01439. And galectin-1,xab-2,sod-1 gene expression on Ti-coated glass were higher than non-coated, Cr-coated glass.These results suggest that Ti-coating is more advantageous for osteoblastic cell attachment, proliferation and differentiation than Cr or non-coating and galectin-1,xab-2 and sod-1 up-regulation could be related to the Ti-bone integration. Less