- 學位論文
建立過量表達血管內皮生長因子之纖維母細胞株研究
The study of VEGFA-overexpressed NIH/3T3 fibroblast cell lines
摘要
血管內皮生長因子A（Vascular Endothelial Growth Factor-A, VEGF-A）是一種具有促進血管新生、神經新生及神經保護功能的生長因子。小鼠的VEGF-A具有許多同功型（isoform），在本研究中使用最常見的兩種：VEGF120與VEGF164。根據過去的文獻，VEGF120對於促進血管新生的臨床應用以及VEGF164在腦損傷後的血管新生、神經新生與神經保護方面可能十分重要。此外，有研究指出給予帶有VEGF121的腺病毒載體能改善心肌缺血模型中的側支血管發育及心肌的灌注與功能，而給予VEGF165重組蛋白則能減少中風恢復期間的神經功能缺損。因此，本研究嘗試先建立能過度表達VEGF-A的纖維母細胞株，做為未來可能之應用。 在本研究中我們將帶有組氨酸標籤（His-tag）的小鼠VEGF-A（VEGF120、VEGF164）互補核酸序列（cDNA）分別轉植入NIH/3T3纖維母細胞株。在經過藥物的篩選之後，我們建立了能過度表達VEGF-A的細胞株（3T3-neo-V120-NH、3T3-neo-V120-CH、3T3-His-V164）。接著我們利用逆轉錄聚合酶鏈式反應（RT-PCR）、即時聚合酶鏈式反應（Q-PCR）、細胞免疫染色、西方墨點法以及酶聯免疫吸附試驗（ELISA）等方法對3T3-neo-V120-NH、3T3-neo-V120-CH、3T3-His-V164細胞進行VEGF-A產物分析。由RT-PCR和Q-PCR的分析結果顯示，在3T3-neo-V120-CH細胞中，VEGF120的mRNA表現量相較於控制組有明顯地上升，而較多的VEGF-A產物也在細胞免疫染色及西方墨點法分析中被證實。在ELISA實驗結果中，我們發現3T3-neo-V120-CH細胞的VEGF-A分泌量在第1天到第3天顯著地高於控制組。而3T3-neo-V120-NH細胞與3T3-His-V164細胞的RT-PCR、細胞免疫染色和西方墨點法分析結果則顯示，我們轉植入3T3細胞株中的cDNA均未能成功表達。 由本研究的結果，我們推論3T3-neo-V120-CH細胞可以在前幾天高度表達VEGF120，而這些VEGF-A能否應用在實驗動物模式中仍需進一步的實驗來證實。
並列摘要
Vascular endothelial growth factor A (VEGF-A), one of the VEGF family, is the well-known molecule in this family due to its ability to promote angiogenesis, neurogenesis and neuroprotection. VEGF-A in mice are expressed as several isoforms, and two common isoforms, VEGF120 and VEGF164, are applied in this study. Previous studies indicated that VEGF120 could be important for angiogenesis of clinical applications, and VEGF164 might be eminent in angiogenesis, neurogenesis and neuroprotection after brain injury. Besides, treatment with adenovirus vectors encoding VEGF121 had been reported to improve development of collateral vessels and the function as well as perfusion of myocardium in porcine myocardial ischemia model. Recombinant human VEGF165 administration was able to reduce neurological deficits while stroke recovery. Therefore, we would like to establish VEGFA-overexpressed NIH/3T3 fibroblast cell lines for further in vivo study. In this study, mouse VEGF-A cDNAs (VEGF120 and VEGF164) with poly-histidine tag (His-tag) were transfected into NIH/3T3 cells separately. After stable clone selection, stable VEGFA-overexpressed cell lines (3T3-neo-V120-NH, 3T3-neo-V120-CH and 3T3-His-V164) were established. The expression of VEGF120 or VEGF164 was analyzed by reverse-transcriptase polymerase chain reaction (RT-PCR), RT-quantitative real-time PCR (RT-qPCR), immunocytochemistry (ICC), western blot analysis and enzyme-linked immunosorbent assay (ELISA). We found that VEGF120 mRNA expression level was elevated in 3T3-neo-V120-CH cells analyzed by RT-PCR and Q-PCR, while more cytosolic and extra-cellular VEGF-A product from 3T3-neo-V120-CH cells was demonstrated by ICC and western blot assays. More secreted VEGF-A from 3T3-neo-V120-CH cells was also found from day 1 to day 3 by ELISA analysis. However, transfected VEGF-A cDNA was failed to express in 3T3-neo-V120-NH cells or 3T3-His-V164 cells. In conclusion, our results showed that a VEGFA-overexpressed NIH/3T3 cell line, which could produce abundant amount of VEGF120 in first few days, was established and could be investigated for further in vivo study.
參考文獻
延伸閱讀
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