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標題: 應用反轉錄聚合酶鏈反應測定基質金屬蛋白酶及其抑制因子在人類乳癌石蠟組織中之表現
mRNA expressions of matrix metalloproteinases and tissue inhibitor metalloproteinases on formalin fixed paraffin embedded human breast tumor tissues using RT-PCR
作者: 謝昭漢
Hsieh, Chao-Han
關鍵字: 基質金屬蛋白西每;matrix metalloproteinases (MMP);基質金屬蛋白西每抑制因子;甲醛固定石蠟包埋組織;反轉錄聚合西每鏈反應;tissue inhibitors of metalloproteinases (TIMP);formalin fixation paraffin embedding (FFPE);reverse transcription polymerase chain reaction (RT-PCR)
出版社: 動物科學系所
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甲醛固定石蠟包埋 (formalin fixed paraffin embedded, FFPE) 組織是醫院及研究單位用於保存樣品最常使用之方式,其提供了大量之樣品可做後續研究。然而,FFPE這個處理保存了組織完整性卻造成其內核酸嚴重地被破壞。因此,本實驗初步目的是建立以 RT-PCR 測定FFPE組織中RNA表現之條件。本實驗比較不同蛋白酶 K處理時間、引子濃度、反轉錄套組、amplicon size之引子在新鮮冷凍 (fresh frozen) 組織與經過甲醛浸泡1, 2及3天之石蠟包埋乳癌組織中GAPDH mRNA表現之差異。研究確認甲醛固定時間超過一天會顯著減少RNA回收量及品質,蛋白酶K作用時間延長至24小時能顯著增加RNA回收率。理想之amplicon size為100 bp以下。引子濃度5 μM經電泳與呈色後有可辨識之條帶,而200 nM則否。Biomi Biotechnology反轉錄套組較Invitrogen Superscript反轉錄套組在測定小amplicon size時更為敏感。35個PCR cycle number與31個相比,其RNA表現較不受到甲醛固定天數之影響。

基質金屬蛋白酶 (matrix metalloproteinases, MMPs) 為分解細胞外基質之酵素,其活性受到內源性之金屬蛋白酶抑制因子 (tissue inhibitors of metalloproteinases, TIMPs) 所調節,因此這兩者常被認為與腫瘤之轉移及侵犯有關。本試驗應用前述RT-PCR條件進一步測定乳癌石蠟組織中MMP-2, -9, -14與TIMP-1, -2之RNA表現。實驗中使用三十例2009年及兩例2007年之石蠟組織來進行實驗。分別為11例乳頭狀瘤 (intraductal papilloma, IP),11例腺管原位癌 (ductal carcinoma in situ, DCIS),及10例侵襲性腺管癌 (invasive ductal carcinoma, IDC)。其RT-PCR之電泳呈色結果經影像擷取系統定量,並統計後顯示,MMP-9與TIMP-2之mRNA表現在DCIS顯著較低。所有FFPE樣品均未能測出TIMP-1之RNA表現。各類型樣品結果經相關性分析顯示,MMP-2與MMP-9、MMP-2與TIMP-2及MMP-9與TIMP-2間在IP、DCIS及IDC中具有高度之相關性。而在DCIS中,MMP-2、MMP-9、MMP-14及TIMP-2彼此間均具有高度之相關性,綜合以上結果,推論MMP-2、MMP-14與TIMP-2之交互作用在原位癌中細胞外基質之降解扮演重要角色,而MMP-9與侵犯性乳癌具有較密切的相關。

Formalin fixed paraffin embedded (FFPE) is the most commonly used method worldwide for tissue storage; this resource represents a vast repository of tissue material with a long-term clinical follow-up. Although, FFPE preserves the tissue integrity it may cause extensive damage to nucleic acids stored within the tissue. Hence, the primary goal of this experiment is to set up the best condition for detecting mRNA expression in FFPE tissues by RT-PCR. To optimize the RT-PCR condition, we compare the GAPDH mRNA expression in fresh frozen tissues and tissues with formalin fixation for 1, 2 and 3 days under different proteinase K reaction time, primer concentration, commercial RT kits, amplicon size of primer treatment. We concluded that a shorten fixation time to less than one day, and extended proteinase K reaction time to 24 hours produced better RNA quality and recovery. The appropriate primer amplicon size is smaller than 100 bp and the concentration of primer is better at 5 μM in 1.5 μl. Biomi Biotech RT kit is more sensitive than Invitrogen Superscript RT kit in detecting small amplicon size primer.

Matrix metalloproteinases (MMPs) can degrade extracellular matrix, which is regulated by tissue inhibitors of metalloproteinases (TIMPs). Hence, those two factors are considered to play an important role in cancer metastasis and invasion. We applied the above optimal condition for RT-PCR to measure the RNA expressions of matrix metalloproteinases (MMP) -2, -9, -14 and tissue inhibitor of metalloproteinases (TIMP) -1, -2 on FFPE human breast cancer tissues. Thirty cases of FFPE human breast tumor from 2009 and two cases of FFPE human breast tumor from 2007 were adopted in this experiment. FFPE of eleven cases of intraductal papilloma (IP), eleven cases of ductal carcinoma in situ (DCIS), and ten cases of invasive ductal carcinoma (IDC) were included in current study. The RT-PCR results were quantified. The statistics of the results show that RNA expressions of MMP-9 and TIMP-2 were significantly lower in DCIS. TIMP-1 RNA expression was not detected in all samples studied. Correlation analysis show that the expression between MMP-2 and MMP-9, MMP-2 and TIMP-2, MMP-9 and TIMP-2 are highly related. The correlation analysis of MMP-2, MMP-9, MMP-14 and TIMP-2 are highly related in DCIS. Our results suggest that MMP-2, MMP-14 and TIMP-2 are important in extracellular matrix degradation in DCIS and MMP-9 is more relevant with invasive ductal carcinoma.
其他識別: U0005-2508201118462700
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