黄荆子乙酸乙酯提取物对翼状胬肉增殖的影响

发布时间：2018-02-27 21:22

  本文关键词： 翼状胬肉 成纤维细胞 黄荆子乙酸乙酯提取物 增殖　出处：《南华大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：观察黄荆子乙酸乙酯提取物（the acetoacetate extract ofVitex negundo seed,Evn-50）对于体外培养的人翼状胬肉成纤维细胞（human pterygium fiborblasts,HPF）增殖的影响，寻找药物辅助治疗和预防术后翼状胬肉复发的新途径。 方法：取临床手术切除的人翼状胬肉标本无菌条件剪成1×1×1mm~3组织块，采用组织块双酶消化法（胰蛋白酶和胶原酶）原代培养人翼状胬肉成纤维细胞，传至第3或4代时，以抗α-SMA蛋白抗体行免疫荧光染色，结合细胞学形态鉴定为人翼状胬肉成纤维细胞。用0.25μg/ml、0.5μg/ml、1μg/ml、2μg/ml、4μg/ml的Evn-50作用于体外培养的HPF,同时设不加药的阴性对照组，，48h后观察Evn-50对HPF细胞形态的改变，免疫荧光法检测HPF中细胞增殖核抗原（Ki-67）的变化，流式细胞仪测定细胞周期时相变化。 结果：（1）体外培养的细胞倒置相差显微镜可见细胞形态一般为长梭形，胞核位于细胞中央，呈椭圆形；经免疫荧光染色后，α-SMA表达阳性，可以鉴定为成纤维细胞。 （2）Evn-50作用48h后，流式细胞仪检测结果发现，随着Evn-50浓度增大，翼状胬肉成纤维细胞发生周期阻滞，大量的细胞停留在G0/G1期，而S期，G2/M期的细胞逐渐减少。计算其增殖率（S%+G2/M%），结果表明细胞增殖能力下降。 （3）Evn-50作用48h后，免疫荧光法检测HPF中Ki-67表达发现当Evn-50的浓度在l-4μg/ml范围内能呈浓度依赖性地抑制HPF表达细胞增殖核抗原（Ki-67）(P0.05)。 结论:黄荆子乙酸乙酯提取物能抑制HPF的增殖，流式细胞仪检测显示Evn-50阻滞细胞在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 伪 - 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 ) .

【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R779.6

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