Establishment of Human Fibroblasts deribed from Dental Pulp Tissues of Primary Teeth and Permanent Teeth, and Development of Systems of Cytotoxicity Tests for Dental Materials

乳牙和恒牙牙髓组织中人成纤维细胞的建立以及牙科材料细胞毒性测试系统的开发

基本信息

批准号：
08672378
负责人：
HASEGAWA Tomokazu
金额：
$ 1.66万
依托单位：
Nagasaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

1)Separation of Human Fibroblasts Derived from Pulp Tissue and Transfection of Simian Virus 40 large T antigen cDNA For this research, some pulp tissues were used. In order to get single cell suspension, after cutting the pulp tissue into small pieces, digestion was accomplished by using PBS containing 0.1% trypsin and 0.02% EDTA.Then second digestion was caried out by using PBS containing 2mg/ml collagenase. The cells were subcultured few times, and then transfected by electroporation. Stable transfected cells were selecled, and further subcultured to obtain the population doubling level (PDL).2)Establishment of Fibroblasts' Clones derived from human Pulp Tissue and Analysis of Phenotypes of Clones We established cell clones which were subcultured more than 100 PDL.All clones showed the phenotype of normal immortalized cells ; because the cells neither showed the colony forming abilities in agarose nor induced tumor formation when used in nude mice, it could be considered that they are not tumor cells.3)Cytotoxity Test through the Dentin Slices for Dental Materials, Resin (AP-X) using Established Clones Using established cell clones, we investigated whether dental resin through the dentin tubles would express the cell toxity. We also compared the responses to dental materials of clones derived from primary and permanent teeth pulps. Survival cell number, synthesis of Interleukin-1beta (ILL beta), determination of apoptosis were analyzed after cytotoxity test. Unporimerised resin could express toxic effects on all clones through the dentin tubles, and did not show any difference in response to these materials among primary and permanent teeth clones. This system of toxic test can show whether or not dental materials express toxic effects through the dentin tubules. Moreover, various analysis can be carried out by using this method, so that this system is vary useful.

1)从牙髓组织中分离人成纤维细胞并转染猿猴病毒40大T抗原cDNA 在本研究中，使用了一些牙髓组织。为了得到单细胞悬液，将牙髓组织切成小片后，用含有0.1％胰蛋白酶和0.02％EDTA的PBS进行消化。然后用含有2mg/ml胶原酶的PBS进行第二次消化。将细胞传代培养数次，然后通过电穿孔转染。选择稳定转染的细胞，进一步传代以获得群体倍增水平（PDL）。2）人牙髓组织来源的成纤维细胞克隆的建立和克隆表型分析我们建立了传代培养超过100 PDL的细胞克隆。克隆显示正常永生化细胞的表型；由于细胞在琼脂糖中既没有表现出集落形成能力，也没有在裸鼠中诱导肿瘤形成，因此可以认为它们不是肿瘤细胞。3）通过牙科材料树脂牙本质切片（AP-X）进行细胞毒性测试使用已建立的克隆使用已建立的细胞克隆，我们研究了牙科树脂通过牙本质小管是否会表达细胞毒性。我们还比较了来自乳牙和恒牙牙髓的克隆对牙科材料的反应。细胞毒性试验后分析存活细胞数、白细胞介素1β（ILL beta）合成、细胞凋亡测定。未聚合的树脂可以通过牙本质小管对所有克隆表现出毒性作用，并且在乳牙和恒牙克隆中对这些材料的反应没有表现出任何差异。该毒性测试系统可以显示牙科材料是否通过牙本质小管表现出毒性作用。而且，利用该方法还可以进行多种分析，因此该系统具有一定的实用性。