本文關(guān)鍵詞： 翼狀胬肉 成纖維細胞 黃荊子乙酸乙酯提取物 增殖　出處：《南華大學》2012年碩士論文　論文類型：學位論文

【摘要】：目的：觀察黃荊子乙酸乙酯提取物（the acetoacetate extract ofVitex negundo seed,Evn-50）對于體外培養(yǎng)的人翼狀胬肉成纖維細胞（human pterygium fiborblasts,HPF）增殖的影響，尋找藥物輔助治療和預防術(shù)后翼狀胬肉復發(fā)的新途徑。 方法：取臨床手術(shù)切除的人翼狀胬肉標本無菌條件剪成1×1×1mm~3組織塊，采用組織塊雙酶消化法（胰蛋白酶和膠原酶）原代培養(yǎng)人翼狀胬肉成纖維細胞，傳至第3或4代時，以抗α-SMA蛋白抗體行免疫熒光染色，結(jié)合細胞學形態(tài)鑒定為人翼狀胬肉成纖維細胞。用0.25μg/ml、0.5μg/ml、1μg/ml、2μg/ml、4μg/ml的Evn-50作用于體外培養(yǎng)的HPF,同時設(shè)不加藥的陰性對照組，，48h后觀察Evn-50對HPF細胞形態(tài)的改變，免疫熒光法檢測HPF中細胞增殖核抗原（Ki-67）的變化，流式細胞儀測定細胞周期時相變化。 結(jié)果：（1）體外培養(yǎng)的細胞倒置相差顯微鏡可見細胞形態(tài)一般為長梭形，胞核位于細胞中央，呈橢圓形；經(jīng)免疫熒光染色后，α-SMA表達陽性，可以鑒定為成纖維細胞。 （2）Evn-50作用48h后，流式細胞儀檢測結(jié)果發(fā)現(xiàn)，隨著Evn-50濃度增大，翼狀胬肉成纖維細胞發(fā)生周期阻滯，大量的細胞停留在G0/G1期，而S期，G2/M期的細胞逐漸減少。計算其增殖率（S%+G2/M%），結(jié)果表明細胞增殖能力下降。 （3）Evn-50作用48h后，免疫熒光法檢測HPF中Ki-67表達發(fā)現(xiàn)當Evn-50的濃度在l-4μg/ml范圍內(nèi)能呈濃度依賴性地抑制HPF表達細胞增殖核抗原（Ki-67）(P0.05)。 結(jié)論:黃荊子乙酸乙酯提取物能抑制HPF的增殖，流式細胞儀檢測顯示Evn-50阻滯細胞在DNA合成前期（G0/G1期）。
[Abstract]:Objective : To investigate the effect of ethyl acetate extract ofvitexin ( Evn - 50 ) on the proliferation of human Pterygium fibroblasts ( HPF ) cultured in vitro , and to find a new way for the adjuvant treatment and prevention of recurrence of Pterygium . Methods : The specimens of Pterygium were cut into 1 脳 1 脳 1 - 1 mm ~ 3 tissue pieces by using tissue - block double - enzyme digestion method ( trypsin and collagenase ) . The cells were cultured in vitro . The results showed that the cells were cultured in vitro by using 0.25 渭g / ml , 0.5 渭g / ml , 1 渭g / ml , 2 渭g / ml , 4 渭g / ml Evn - 50 . The changes of the morphology of the cells were observed after 48 h . The changes of the cell proliferation nuclear antigen ( Ki - 67 ) in HPF were detected by immunofluorescence assay . Results : ( 1 ) The cell morphology of the cells cultured in vitro was generally spindle - shaped , the nucleus was located in the center of the cell , and the nucleus was oval ; after the immunofluorescence staining , the expression of 偽 - SMA was positive , which could be identified as fibroblasts . ( 2 ) After 48 hours of Evn - 50 , the flow cytometry showed that with the increase of the concentration of Evn - 50 and the cycle arrest of Pterygium fibroblasts , a large number of cells remained in G0 / G1 phase , while the S phase and G2 / M phase decreased gradually . The proliferation rate of cells was calculated ( S % + G2 / M % ) , and the results showed that the cell proliferation ability was decreased . ( 3 ) After 48 hours of Evn - 50 , the expression of Ki - 67 in HPF was detected by immunofluorescence assay . When the concentration of Evn - 50 was in the range of 1 - 4 渭g / ml , the expression of Ki - 67 was inhibited in a concentration - dependent manner ( P < 0.05 ) . Conclusion : The extract of ethyl acetate could inhibit the proliferation of HPF . Flow cytometry showed that Evn - 50 blocked cells were in the early stage of DNA synthesis ( G0 / G1 phase ) .

【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R779.6

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