建立及特徵分析持續表達腦部神經滋養因子的3T3纖維母細胞株

Abstract

腦衍生神經滋養因子(BDNF)在中樞神經系統扮演許多重要的角色。最近有研究發現，BDNF具有誘導腦部成體幹細胞分化的功能，且被認為具有治療腦部損害的潛力。目前，由外加小型滲透幫浦長期導入腦中的BDNF仍有許多待解決的問題。但這些問題可選擇移植能夠持續表達BDNF的細胞而獲得解決之道。 在本研究中，我們同時轉殖小鼠BDNF和綠色螢光蛋白(EGFP)的互補核酸序列(cDNA)進入3T3纖維母細胞株中。在經過neomycin的相似藥物G418的篩選之後，我們建立了兩株穩定獨立表達BDNF和EGFP的細胞株，並命名為3T3-BDNF-EGFP細胞。本實驗中利用免疫染色、西方墨點法和酵素連結抗體吸附法(ELISA)對3T3-BDNF-EGFP細胞進行基因產物分析。另外也透過分析雞胚背根神經結(dorsal root ganglia, DRG)的存活率，來測試由3T3-BDNF-EGFP細胞所分泌的BDNF之生物活性。 從我們得到的免疫染色以及西方墨點法的結果發現，BDNF在3T3-BDNF-EGFP細胞的表達量比控制組來得多。在ELISA的實驗中，也可以發現由3T3-BDNF-EGFP細胞所分泌出BDNF的量具有統計上顯著的增加。而我們進一步利用雞胚的DRG神經元測試BDNF的生物活性，發現由這些3T3-BDNF-EGFP細胞分泌的BDNF能使DRG神經元擁有較高的存活率。 由本研究的結果，我們可以推論3T3-BDNF-EGFP細胞可以穩定表達具有功能性的BDNF。我們認為這些大量表達BDNF，且具有EGFP螢光的細胞，將可以應用在細胞移植治療腦部損傷的動物模式中。

Brain-derived neurotrophic factor (BDNF) influences almost all aspects in central nervous system (CNS). Recently, BDNF was discovered as an inducer for adult stem cells and considered as a potential therapeutic agent for the brain injury. However, there are several drawbacks about using injections or minipumps for long-term BDNF treatment. Alternatively, cell mediated BDNF delivery could be one of solutions for it. In this study, we transfected cDNAs of mouse BDNF and enhanced green fluorescent protein (EGFP) into Swiss albino mouse 3T3 cell line. After selection of neomycin analogue G418, two stable 3T3-BDNF-EGFP cell clones constitutively expressing BDNF and EGFP independently were established. Stable 3T3-BDNF-EGFP cell clones were analyzed by immunocytochemistry, Western blot, and Enzyme-Linked Immunosorbent Assay (ELISA). Besides, the functional test of secreted BDNF activity was also assayed via the viability of chicken DRG neurons. Immunostaining patterns and Western blots of 3T3-BDNF-EGFP cells that we obtained showed more BDNF products than controls. In the ELISA study, the amount of BDNF secreted from 3T3-BDNF-EGFP cells (339 ± 50 pg/ml) was also showed significantly larger than others. Furthermore, we found the higher survival rate in the functional assay of embryonic chicken DRG neurons with BDNF secreted from 3T3-BDNF-EGFP cells. From the present study, we conclude that the stable 3T3-BDNF-EGFP cells could constitutively express functional BDNF in vitro. These BDNF-enriched cells with strong green fluorescence will be useful for further studies, such as the intracerebral cell grafting for brain injuries.