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

1)人牙髓成纤维细胞的分离及猿猴病毒40大T抗原cDNA的转染将牙髓组织切成小块后，用含0.1%胰蛋白酶和0.02%EDTA的PBS消化，再用含2 mg/ml胶原酶的PBS消化，得到单细胞悬液。将细胞传代几次，然后通过电穿孔转染。2）人牙髓成纤维细胞克隆的建立及克隆表型分析我们建立了传代100个以上PDL的细胞克隆，所有克隆均表现为正常永生化细胞的表型;因为细胞在琼脂糖中既没有显示出殖民地形成能力也没有在裸鼠中诱导肿瘤形成，可以认为它们不是肿瘤细胞。3）使用已建立的克隆通过用于牙科材料树脂（AP-X）的牙本质切片进行的细胞毒性试验使用已建立的细胞克隆，本研究探讨了牙用树脂通过牙本质小管时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。细胞毒试验后检测细胞存活数、白细胞介素1 β（IL-1 β）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达毒性作用，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。该毒性测试系统可显示牙科材料是否通过牙本质小管表达毒性作用。此外，该方法还可以进行多种分析，因此该系统具有多种实用性。

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.