類胰島素生長因子及其細胞產物之生物活性測定

Abstract

本研究乃以對 Balb/3T3 老鼠胚胎細胞株之生長促進效果，作為類胰島素生長因子之生物活性指標。採用比較不耗費藥物、樣品且快速之 MTT 法測定細胞增生情形。當小牛血清濃度為 1%，DMEM 中葡萄糖及麩胺醯胺濃度分別為 15 mM 與 2 mM 時，細胞生長於 72 小時內無顯著性變化 (p> 0.05)。當於此細胞培養條件下，分別添加 5-120 nM IGF1 及 5-90 nM IGF2，在 37oC、5% CO2 操作條件下，對 Balb/3T3 細胞株之生長促進效果 [growth stimulation ratio (%), GSR] 有顯著之差異。且添加濃度與生長促進效果呈現 dose-dependent 關係，兩者間的線性迴歸方程式分別為 y = 0.5903x + 20.503，R2 = 0.9669；y = 0.7987x + 34.336，R2 = 0.871。顯示此培養條件已具備使 Balb/3T3 細胞株增生之足夠養分。此條件下，相同 IGF 濃度之細胞生長促進效果，其測定靈敏度分別可提高 2 倍 (IGF1) 及 1.25 倍 (IGF2)。利用所建立之測定方法探討 IGF1、IGF2 之穩定性，顯示 IGF1 及 IGF。於 37oC 下可穩定保存 24 小時，且分別於 pH 6-7 和 pH 7-8 的範圍內活性維持穩定。蔗糖可增加 IGF1 及 IGF2 於 37oC 下之活性穩定性，麥芽糖和乳糖於測定濃度範圍無明顯影響；分析 IGF大腸菌轉形株細胞萃取物之經尿素重溶之樣品，也有顯著促進生長之效果，顯示此方法可直接用於非純化樣品之生物活性測試。

Bioactivity of IGFs was evaluated by its growth-promoting activation on Balb/3T3 fibroblast cells. The MTT colorimetrical assay in 96-well flat bottom plate was used to determine growth-promoting activity due to less amounts of reagents and samples required, cost-effective and rapid. When Balb/3T3 cell lines were grown in a medium consisted of 1% calf serum and 99% DMEM (15 mM glucose and 2 mM L-glutamine), there were no significant difference in cell numbers in 72 hours (p> 0.05). To initiate cell proliferation, different concentrations of IGF1 and IGF2 in the range of 5-120 nM and 5-90 nM, respectively were added to the culture medium. The cells were then cultured in an incubator supplied with 5% CO2 at 37oC and an apparent difference in growth stimulation ratio (%) was observed. Moreover, it is dose-dependent between growth stimulation and adding concentration, the linear regression curves in both are y = 0.5903x + 20.503，R2 = 0.9669 and y = 0.7987x + 34.336，R2 = 0.871 respectively. This outcome proved that in such nutrition status can provide enough nutrients for Balb/3T3 growing. To compare to the predecessors’ research, the study outcome shows that the IGF1 growth stimulation ratio (%), GSR is double higher and IGF2 is 1.25 higher in same concentration. According to this, the Medium in this study provides cells to maintain its balance and it is apply to IGF1 and IGF2, in the meantime, the IGF growth is more obviously. To investigate the process of IGF1 and IGF2 stability, IGF1 and IGF2 is stability at 37oC in 24 hours. When adding sugar in IGF1 and IGF2 working in 37oC. The bioactivity is better than none adding. In this assay culture, adding crude IGF1 and IGF2 in concentration range 5-120 nM, has made an significant different in GSR%. Through simply step can measure IGF bioactivity, and it will benefit the future applications in industrial production.