建立過度表達類胰島素生長因子的NIH/3T3纖維母細胞株以應用在未來之治療

Abstract

類胰島素生長因子 (Insulin-like growth factor-1, IGF-1) 是一種多胜肽激素，在人體中有許多組織會生成製造。IGF-1被認為和發育、生長、分化和神經保護作用有關。IGF-1能夠通過血腦障壁 (Blood-brain barrier)，而因為這項特徵使得IGF-1被認為是一種具有潛力治療神經疾病的因子。另一方面來說，有許多類型的細胞像是纖維母細胞具有生成及釋放IGF-1。在我們實驗室先前的研究，轉染入腦衍生神經滋養因子 (brain-derived neurotrophic factor, BDNF) 或紅血球生成素 (Erythropoietin, EPO) 的NIH/3T3纖維母細胞株皆可以過量表達其轉染入的蛋白。而根據研究IGF-1具有的神經保護效果，本研究首先將嘗試建立過量表達IGF-1的NIH/3T3纖維母細胞株。 在本論文中，我們有兩個主要目的：(一) 建立可以過量表達IGF-1的纖維母細胞株；(二) 測試此過量表達IGF-1細胞株所釋放的IGF-1可能的生理功能。兩種分別帶有N端組氨酸標籤以及C端組氨酸標籤的IGF-1質體轉染入NIH/3T3纖維母細胞株。轉染的細胞經過濕黴素 (hygromycin) 篩選後，我們建立了兩株可以過量表達IGF-1的纖維母細胞株 (IGF1-3T3)。我們利用了逆轉錄聚合酶鏈式反應 (RT-PCR)、逆轉錄即時聚合酶鏈式反應 (RT-QPCR)、細胞免疫染色、西方墨點法 (Western-blot assay) 以及酶聯免疫吸附試驗 (ELISA) 等實驗方法對IGF1-3T3細胞株所釋放的IGF-1進行定性及定量分析。帶有組氨酸標籤的IGF-1 mRNA可經由RT-QPCR證實在細胞內表現量明顯增加。而在IGF1-3T3細胞株的培養液中也經由ELISA證實IGF-1分泌量亦明顯的增加。 為了測試IGF1-3T3釋放的IGF-1生物活性，我們利用IGF1-3T3的培養液和神經退化疾病細胞模式的細胞株，PC12-INT-EGFP。在利用神經生長因子 (Nerve growth factor, NGF) 誘導PC12-INT-EGFP細胞分化的第六天，給予含有IGF1-3T3培養液的條件培養基。經過培養48小時後，我們觀察到在含有IGF-1的條件培養基的組別中，可觀察到PC12-INT-EGFP細胞突起具有較高強度的中間絲蛋白綠色螢光，實驗顯示IGF-1具有影響神經細胞之生理功能。 本研究結果成功建立了兩株可以過量表達IGF-1的3T3纖維母細胞株，分別是N-His IGF1-3T3以及C-His IGF1-3T3。兩株細胞株經由實驗證實均可以釋放大量的IGF-1。因此，本研究所建立過量表達IGF-1的纖維母細胞株，將可以做為未來在研究神經疾病動物模式上一個可行測試的生物材料。

Insulin-like growth factor-1 (IGF-1) is a polypeptide hormone which is produced by multiple tissues in human. IGF-1 is considered to be related to development, growth, differentiation and neuroprotection. According to the characteristic of IGF-1 which is able to across the blood-brain barrier and have trophic effect on neuron, IGF-1 is regarded as a promising candidate to treat the neurological diseases. On the other hand, IGF-1 is suggested to be secreted from multiple cell types such as fibroblasts. In our laboratory, NIH/3T3 cells transfected with brain-derived neurotrophic factor (BDNF) or erythropoietin (EPO) could overexpress the protein respectively. Based on the neuroprotective effect of IGF-1 and the potential therapeutic effects of cell therapy on neurological diseases, we would first like to establish an IGF1-overexpressed 3T3 cell lines. In this study, we aimed to (1) establish IGF-1 overexpressed NIH/3T3 fibroblast cell lines and (2) examine the potential functional effects of IGF-1 secreted from IGF1-3T3 stable clones. Two plasmid DNA, N-terminal polyhistidine-tagged IGF-1 and C-terminal polyhistidine-tagged IGF-1, were transfected into NIH/3T3 cells respectively. Two stable IGF-1 overexpressed NIH/3T3 fibroblast cell lines were established after selected by hygromycin. IGF-1 secreted from two IGF-1 overexpressed cell lines were examined by reverse-transcriptase PCR (RT-PCR), reverse-transcriptase quantitative real-time PCR (RT-QPCR), immunocytochemistry, Western-blot assay and enzyme-linked immunosorbent assay (ELISA). The polyhistidine tagged IGF-1 cDNA fragment from transfected cells was detected by PCR and higher mRNA level could be demonstrated by RT-QPCR. The significantly higher concentration of secreted IGF-1 was also detected in the culture supernatants of two IGF1-3T3 cell lines by ELISA analysis. Subsequently, we examined the bioactivity of secreted IGF-1 via culturing the cell model of neurodegeneration, PC12-INT-EGFP cell line with conditioned media. The conditioned media, which were comprised of the culture supernatant of IGF1-3T3 cells, was supplied to PC12-INT-EGFP cells on the sixth day after induced by nerve growth factor (NGF). After culturing with conditioned media for 48 hours, stronger green fluorescence intensity in the processes of NGF-induced PC12-INT-EGFP cells could be observed in the groups supplied with IGF-1. From these observations, it is suggested that IGF-1 might have functional effects in the PC12-INT-EGFP cell model. In summary, we established two IGF1-overexpressed 3T3 cell lines, N-His IGF1-3T3 and C-His IGF1-3T3, and the abundant secreted IGF-1 was well demonstrated. The results of functional assay also suggest that the secreted IGF-1 exert bioactivity. Therefore, the IGF1-overexpressed cell lines we established in this study might be offered as potential biomaterials for the study of animal models with neurological diseases.