Development of a new cytotoxicity test method for dental materials

牙科材料细胞毒性测试新方法的开发

• 批准号: 01870080
• 负责人: SATO Atsushige
• 金额: $ 4.1万
• 依托单位: TOKYO MEDICAL and DENTAL UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B).
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01870080/
• 关键词: Cytotoxicity test method; SV-40 large T antigen; Human dental pulp; Mouse calvaria; Odontoblast; Osteoblast; Intracellular Ca^<2+>; Neutral red assay; 遺伝子導入; 骨芽細胞株の樹立; 石灰化; 細胞内カルシウムイオン; マウス頭頂骨細胞; ウサギ腎細胞; SV40DNA; 培養細胞; ヒト歯髄細胞

In order to develop a new cytotoxicity test method for assessing possible toxic effects of dental materials, the study was performed to establish new lines of cultured cells with highly susceptible to toxicants and quantitative indexes for cytotoxicity tests.1. The human dental pulp cells and mouse calvaria cells were transfected with the SV-40 large T antigen. Four immortalized cell lines, LSC cells from human pulp and DMCs LMCs and VMCs cells from mouse calvaria were obtained. These cell lines preserved some of the properties of the dental pulp or the osteoblast.2. The susceptibility of four cytotoxicity assays were compared. The MTT and NR assays showed higher susceptibility compared to the agar overlay assay and millipore filter method. The MTT assay showed the highest reproducibility among four tests.3. The intracellular Ca ion concentration ([Ca^<2+>]i) of JTC-12 cells was measured employing Fura 2 AM fluorescence. Exposing cells to low concentrations of HgCl_2, which had no apparent effect on viability, produced a rapid and prolonged increase in [Ca^<2+>]i. The [Ca^<2+>]i was a useful index to detect the initial lesion of cultured cells exposed to dental materials.4. These results suggest that the new cell lines and the [Ca^<2+>]i change may be useful in the future research for testing the toxicity of the dental materials.

为了建立一种新的细胞毒性试验方法，用于评价牙科材料可能产生的毒性作用，本研究建立了一种新的对毒物高度敏感的细胞培养系，并建立了细胞毒性试验的定量指标.用SV-40大T抗原转染人牙髓细胞和小鼠颅骨细胞。获得了四种永生化细胞系，人牙髓LSC细胞和小鼠颅骨DMCs LMCs和VMCs细胞。这些细胞系保留了牙髓或成骨细胞的一些特性。比较了四种细胞毒性试验的敏感性。MTT法和NR法的敏感性高于琼脂覆盖法和微孔滤膜法。四种方法中，MTT法重复性最好.采用Fura 2 AM荧光法测定JTC-12细胞内Ca ~（2+）浓度（[Ca ~（2+）]i）。低浓度HgCl_2对细胞活力无明显影响，但可使细胞内[Ca^<2+>]i迅速而持久地增加。[Ca^<2+>]i可作为检测牙体材料对培养细胞损伤的早期指标.这些结果表明，新的细胞系和[Ca^<2+>]i的变化可能是有用的，在未来的研究，以测试牙科材料的毒性。

项目成果

SATO,A.: "In vitro toxicity tests of biomaterials" J.Toxicol.Sci.15(3). 179 (1990)

SATO,A.：“生物材料的体外毒性测试”J.Toxicol.Sci.15(3)。

Ohmura T.: "A newly established cell line from mouse calraria" 歯基. 35. (1992)

Ohmura T.：“来自小鼠卡拉里亚的新建立的细胞系”Dentobase 35。（1992）

力武 康次: "ヒト歯髄由来美容細胞のプラスミドpMT1-neo遺伝子導入による不死化について" 口病誌. 56(4). 540-560 (1989)

Koji Rikitake：“通过引入质粒 pMT1-neo 基因实现人类牙髓来源的美容细胞的永生化”口腔医学杂志 56(4) (1989)。

RIKITAKE,Y.: "A Newly established cell line derived from the human dental pulp" Proc.Japan Acad.65B(8). 187 (1989)

RIKITAKE,Y.：“一种新建立的源自人牙髓的细胞系”Proc.Japan Acad.65B(8)。

SATO,A.: "A new in vitro cytotoxicity test method for dental materials" J.J.Dent.Mat.9(Suppl.). 110 (1990)

SATO,A.：“一种新的牙科材料体外细胞毒性测试方法”J.J.Dent.Mat.9（增刊）。