花生四烯酸对人增生性瘢痕成纤维细胞增殖的影响研究

发布时间：2018-03-11 16:43

本文选题：花生四烯酸　切入点：增生性瘢痕　出处：《南昌大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:观察花生四烯酸对体外培养的人增生性瘢痕(Hypertrophic Scar,HS)成纤维细胞(Fibroblast,FB)增殖的影响,并探讨花生四烯酸(Arachidonic acid,AA)在抑制人HS中FB增殖的可能作用机制。方法:(1)HS组织取自本院烧伤科行瘢痕整形手术患者,采用改良组织块联合胰酶消化法培养FB,取第4代FB用于实验;(2)倒置显微镜下观察含不同浓度(0μmol/L、5μmol/L、10μmol/L、20μmol/L、40μmol/L、80μmol/L)AA培养基培养一定时间FB的形态变化;(3)分别用含上述不同浓度AA的10%胎牛血清培养基培养FB 24h、48h、72 h后,CCK法检测FB增殖活力;(4)制作FB爬片,免疫细胞化学法检测加入上述含不同浓度AA培养基培养24小时后FB核中Ki-67蛋白的表达情况。结果:(1)组织块接种后第6d见细胞游出,呈单个散在长梭形,第10d组织块周围细胞明显增多,第15d左右组织块周围细胞生长密集,开始相互融合。细胞传代后7d左右基本长满瓶底,培养瓶瓶底细胞呈毛刷状、漩涡状或放射状生长,胞体呈长梭形,周围可见长短不一的突起,细胞排列整齐规律(2)在一定浓度的AA作用下,FB的生长形态出现明显变化。AA浓度为5μmol/L、10μmol/L时,FB细胞数量增加,细胞形态及生长走向未受到明显影响;AA浓度为20μmol/L、40μmol/L和80μmol/L时,FB的形状开始表现出不规整,细胞大小不均匀,形状改变,部分细胞呈圆钝形,数量减少,胞核浓缩。AA浓度越高,FB的形态改变表现越明显。(3)FB在5μmol/L、10μmol/L浓度的AA作用下,FB存活率未受到明显影响(p0.05),20-80μmol/L AA浓度范围内,FB随着AA作用浓度或时间的增加其抑制率均逐步增加(P0.05,且该浓度范围内各组间比较P0.01)。(4)免疫细胞化学显示AA浓度为20μmol/L、40μmol/L和80μmol/L时,FB核中Ki-67蛋白表达阳性细胞百分率均显著低于AA浓度为0μmol/L时(P0.05),且AA浓度越高,Ki-67蛋白阳性细胞百分率越低(P0.01);AA浓度为5μmol/L、10μmol/L时,Ki-67蛋白阳性细胞百分率低于AA浓度为0μmol/L时,但无统计学意义(P0.05)。结论:一定浓度的AA能抑制人HSFB的增殖,使处于分裂期细胞数量减少,这可能为其抑制HS形成作用机制之一。
[Abstract]:Aim: to observe the effect of arachidonic acid on proliferation of human hypertrophic scar ScarHSB fibroblasts in vitro. To explore the possible mechanism of arachidonic acidazide (AAA) in inhibiting FB proliferation in human HS. Methods the HS tissue was obtained from patients undergoing scar plastic surgery in burn department of our hospital. FB was cultured with modified tissue mass and trypsin digestion method. The 4th passage of FB was used in the experiment of FB 2) the morphological changes of FB were observed with different concentrations of 0 渭 mol / L (5 渭 mol / L ~ (10) 渭 mol / L ~ (10) 渭 mol / L ~ (10) 渭 mol / L ~ (10) 渭 mol / L ~ (10) 渭 mol / L ~ (10)) mol/L)AA medium of 40 渭 mol / L ~ (80) 渭 mol / L ~ (80) 渭 mol/L)AA). FB crawling tablets were made by CCK method after FB was cultured on FB 24 h medium for 48 h and 72 h later, FB proliferation activity was detected by CCK method. The expression of Ki-67 protein in FB nucleus was detected by immunocytochemistry after 24 hours of culture on the medium containing different concentrations of AA. At the 10th day, the cells around the tissue mass increased obviously, and the cells around the tissue mass around the 15th day grew densely and began to fuse with each other. After 7 days of passage, the cells were basically covered with the bottom of the bottle, and the cells at the bottom of the culture bottle were brush, whirlpool or radial. The cell body was spindle-shaped, with processes of different length and length around it. The cells were arranged regularly. (2) the growth morphology of FB was obviously changed when the concentration of AA was 5 渭 mol / L ~ (10 渭 mol/L), and the number of FB cells increased when the concentration of AA was 5 渭 mol / L ~ (10) 渭 mol / L ~ (10 渭 mol/L). When the concentration of AA was 20 渭 mol / L, 40 渭 mol/L and 80 渭 mol/L, the shape of FB began to show irregularity, cell size was uneven, shape changed, and some cells showed circular obtuse shape and decreased number. The higher the concentration of AA-A, the more obvious the morphological change of FB was. The survival rate of FB increased gradually with the increase of AA concentration or time when the concentration of AA was 5 渭 mol / L ~ (10 渭 mol/L). The survival rate of FB was not significantly affected in the range of 20 ~ 80 渭 mol/L AA. Immunocytochemistry showed that the percentage of positive cells for Ki-67 expression in FB nucleus at AA concentration of 20 渭 mol / L 40 渭 mol/L and 80 渭 mol/L was significantly lower than that at 0 渭 mol/L, and the higher the AA concentration was, the higher the concentration of Ki-67 protein was. The lower the percentage was, the lower the percentage of Ki-67 protein was when the concentration of AA was 5 渭 mol / L and the percentage of Ki-67 protein was lower than that at AA concentration of 0 渭 mol/L. Conclusion: AA at a certain concentration can inhibit the proliferation of human HSFB and reduce the number of cells in the mitotic phase, which may be one of its mechanisms of inhibiting HS formation.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R62

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