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Ex Vivo Gene Delivery Using an Adenovirus Vector in Treatment for Dental Implant

使用腺病毒载体进行体外基因传递治疗牙种植体

基本信息

批准号：
12470419
负责人：
KANYAMA Manabu
金额：
$ 5.25万
依托单位：
OKAYAMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12470419/
关键词：
Adenovirus vector CTGF MC3T3-E1 cell Tooth extraction socket Titanium Ex vivo gene delivery MC3T3-El cell 抜歯創時癒過程 BMP遺伝子 CTGF遺伝子 MC3T3-E1細胞 TGF-β遺伝子

项目摘要

The purpose of this study was to contribute to shortening the healing period of the dental implant and expanding its application by ex vivo gene delivery using an adenovirus vector. It has been well-documented that connective tissue growth factor (CTGF) is up-regulated in the healing process at the bone fracture site in vivo and known as a potent stimulator for the proliferation and differentiation of osteoblasts in vitro. Therefore, CTGF gene transfer to the osteoblasts could be a promising strategy to accelerate bone formation. First, we investigated the possibility of recombinant adenovirus to transfer CTGF genes to a mouse osteoblastic (MC3T3-E1) cell line in vitro. Recombinant adenovirus encoding the LacZ gene and the human CTGF gene with CAG promotor (Ax1CACTGF) were applied to the MC3T3-E1 cells with 5, 10 and 50 multiplicity of infection (MOI). As the result, the MOI 50 transfection dosage produced almost 80% X-gal staining of the cells without any obvious cell damages and Ax1CACTGF transfection caused a marked up-regulation in CTGF mRNA expression and CTGF protein even 7 days after transfection. Second, we investigated the spatial and temporal expression of CTGF in the rat tooth extraction sockets to know the mechanisms of new alveolar bone formation. As the result, CTGF was expressed in the endothelial cells migrating into the granulation tissue at the sockets during 4 days after tooth extraction. Osteoblast-like cells proliferated in the sockets with CTGF expression at 4, 7, 10 and 14 days after extraction. Finally, we tried the ex vivo gene delivery, but we could not isolate osteoblasts from the defects of rat alveolar bone.Based on these findings, adenovirus-mediated CTGF gene transduction to the cultured osteoblast-like cells was highly successful. However, in order to do the ex vivo gene transfer, it is necessary to develop the new methods of isolating osteoblasts.

本研究的目的是通过腺病毒载体的体外基因传递，缩短种植体的愈合时间，扩大其应用范围。结缔组织生长因子（CTGF）在体内骨折部位的愈合过程中被上调，并且在体外被认为是成骨细胞增殖和分化的有效刺激物。因此，将CTGF基因转移到成骨细胞可能是一种很有希望的加速骨形成的策略。首先，我们在体外研究了重组腺病毒将CTGF基因转移到小鼠成骨细胞（MC3T3-E1）的可能性。将编码LacZ基因和含CAG启动子的人CTGF基因的重组腺病毒（Ax1CACTGF）分别以5、10和50倍感染（MOI）转染MC3T3-E1细胞。结果MOI 50转染剂量下，细胞X-gal染色率几乎达到80%，未见明显细胞损伤，Ax1CACTGF在转染后7天CTGF mRNA和CTGF蛋白表达明显上调。其次，我们研究了CTGF在大鼠拔牙槽内的时空表达，了解新牙槽骨形成的机制。结果显示，拔牙后4天，移入牙槽处肉芽组织的内皮细胞中表达CTGF。提取后4、7、10、14天，CTGF表达的成骨样细胞在牙槽内增殖。最后，我们尝试了体外基因传递，但我们无法从大鼠牙槽骨缺损中分离成骨细胞。基于这些发现，腺病毒介导的CTGF基因转导到培养的成骨细胞样细胞是非常成功的。然而，为了实现体外基因转移，有必要开发分离成骨细胞的新方法。