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在本實驗室之前的研究顯示，TGF-β1(5ng/ml)會抑制OVCAR-3細胞株的生長，使其停留在G1期;TGF-β1處理15分鐘後，c-jum 、c-fos mRNA的表現開始增加，且在30分鐘後達最大量。本論文以mRNA differential display方法分析OVOCAR-3細胞株在TGF-β1處理15分鐘、30分鐘後其而mRNA表現的差異，及以西方墨點法，偵測OVCAR-3細胞株經TGF-β1作用後，其pRB蛋白質的含量變化。
在而mRNA differential diaplay實驗中，我們找到110個加入TGF-β1後會產生差異的訊號。將差異表現的DNA片段自膠中析出並經TA選殖後，得到43個菌株;挑選其中DNA嵌入物大於200個鹼基對者做DNA定序解讀，其中有8個具3'-polyA-tail的菌株，分別為:t8-1(3)、t8-4(2)、t18-1、t18-2、c18-2、g11-1、g16-2、及g16-4。在資料庫搜尋結果中，t8-1(3)和人類ferritin heavy chain高度相關;其餘菌株只搜尋到可能相關的序列。北方墨點偵測中，t8-l(3)、t8-4(2)、tl8-2、及g16-2的控制組及TGF-β1處理後之操作組的RNA表現並無差異;而t18-1、c18-2、g11-1、g16-4的控制組及TGF-β1處理後之操作組則均不見訊號。
在西方墨點的實驗中發現OVCAR-3細胞抹在TGF-β1處理72小時後，低磷酸化態(hyperphosphorylated)pRb約含量較高磷酸化態(hypophosphorylated)多，且低磷酸化pRb有隨處理時間增加而漸增的趨勢。此結果仍有待取得正控制組(positive control)以資確認。TGF-β1對OVCARR-3細胞株的生長抑制調控，其明確機制，仍需要更進一步的探討。
The growth of OVCAR-3 cell line was inhibited by 5 ng/ml TGF-β1 and arrested in G1 phase according to the previous study. Under the treatment of TGF-β1 for 15 minutes, the expression of c-jun, c-fos genes increased, and reached the maxirnun level after 30 minutes. The purpose of this thesis is to study the gene expression of the TGF-β1 treated OVCAR-3 cell line by mRNA differential display, and to detect the change of the translation level of the tumor suppressor gene, RB , by Western Blot.
In the mRNA differential display, we found 110 different signals after treated with TGF-β1, and 43 clones were obtained after DNA elution, TA cloning, and mini-prepared screening. After DNA sequencing, we found 8 3'-poly-A-tailed clones from 22 ones, in which the DNA inserts were larger than 200 base-pairs, and named them as t8-1(3), t8-4(2), t18-1, t18-2, c18-2, g11-1, g16-2, and g16-4 respectively. Refering to the database IGsuit, we found that the sequence of t8-1(3) is highly correlated with human feiritin heavy chain, and others show moderate correlation with some genes. From Northern blot analysis, there are no differences between the expression of control and TGF-β1-induced total RNA of the t8-1(3), t8-4(2), t18-2, and g16-2 clones; no signals could be detected for the other clones.
From Western blot analysis, pRb showed more hypophosphorylated than hyperphosphorylated form at 72 hours time point after TGF-β1 was added. Besides, the hypophosphorylated pRb seemed to increase gradually as longer as the time that the OVCAR-3 cell line was treated with TGF-β1. But, we still need to do some work with positive controls to confirm our findings.The mechanism of the TGF-β1-induced growth arrest of the OVCAR-3 cell line remains to investigate.
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