TO-2 DNA轉型之NIH-3T3細胞抗紫外線能力的研究

Abstract

吳郭魚卵巢細胞（TO-2 cells）建立於1983年，此細胞對紫外線（uv）的忍受性比哺乳類細胞還強，這一特性異於其他魚類細胞，因大多數的魚類細胞對紫外線是相當敏感的。NIH-3T3細胞株是老鼠胚胎的纖維原細胞 (fibroblast)，此細胞非常容易接受外來的DNA分子，而且對紫外線的抗性比TO-2細胞小很多。本實驗的目的在於建立TO-2的基因庫（genomic library)，以PSV2-neo為載體將NIH-3T3細胞轉型，並利用雙重篩選 (G418-resistance & UV-resistance selections）挑出一株具有較高抗紫外光能力的NIH-3T3轉型細胞(trans-format）。此外，我們也對此轉型細胞的DNA複製和uv修復能力做了初步的分析。 轉型細胞（3T-UV20-3,D37=7J/m）的UV存活率比TO-2（D37＝9.2J/m）稍差，但比NIH-3T3(D37=2.4J/m)高出許多。點墨雜合反應也顯示了3T-UV20-3的確併入了外來的TO-2基因。 以3H-thymidine細胞標誌方法所得DNA合成率的結果，和細胞存活率的結果有正相關。轉型細胞DNA合成速率受抑制的程度較少而回復的時間較快，顯示細胞受紫外線處理後，細胞功能被幹擾的程度較小。 此外，轉型細胞的複製後修補（Postreplication repair）能力，經實驗結果發現與NIH-3T3母細胞並無明顯的差別。

NIH-3T3 cell line is generally used in cell transformation assay, and it is more UV—sensitive than TO—2 cell which has been investigated for a long period in our laboratory. According to the difference in UV-resistance of these two cell lines, we tried to transfect 3T3 cells with pSV2neo based TO-2 genomic library, and to obtain a more UV-resistant transformed 3T3 cell line by selecting the transformant with G418 and UV irradiation. In addition, measurement of DNA synthesis and postreplication repair of UV-resistant clones have been done in this study. The results show that 3T-uv2O-3, a G418/UV-resistant NIH-3T3 transformant, is more uv-resistant than NIH-3T3 （D37＝7 J/m2 and 2.4 J/m2 ,respectively), but is slightly uv-sensitive compared with TO-2 (D37= 9.2 J/m2). Measurement of inhibition of DNA synthesis reveals a dose response in both 3T-uv2O-3 and NIH-3T3 cell lines, however the recovery of DNA synthesis after sublethal uv irradiation is faster in 3T—uv2O—3 cells. Moreover, there is no pronounced difference in the analysis of postreplication recovery, both two cell lines perform postreplication repair efficiently.