本文選題：花生四烯酸　切入點(diǎn)：增生性瘢痕　出處：《南昌大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:觀察花生四烯酸對體外培養(yǎng)的人增生性瘢痕(Hypertrophic Scar,HS)成纖維細(xì)胞(Fibroblast,FB)增殖的影響,并探討花生四烯酸(Arachidonic acid,AA)在抑制人HS中FB增殖的可能作用機(jī)制。方法:(1)HS組織取自本院燒傷科行瘢痕整形手術(shù)患者,采用改良組織塊聯(lián)合胰酶消化法培養(yǎng)FB,取第4代FB用于實驗;(2)倒置顯微鏡下觀察含不同濃度(0μmol/L、5μmol/L、10μmol/L、20μmol/L、40μmol/L、80μmol/L)AA培養(yǎng)基培養(yǎng)一定時間FB的形態(tài)變化;(3)分別用含上述不同濃度AA的10%胎牛血清培養(yǎng)基培養(yǎng)FB 24h、48h、72 h后,CCK法檢測FB增殖活力;(4)制作FB爬片,免疫細(xì)胞化學(xué)法檢測加入上述含不同濃度AA培養(yǎng)基培養(yǎng)24小時后FB核中Ki-67蛋白的表達(dá)情況。結(jié)果:(1)組織塊接種后第6d見細(xì)胞游出,呈單個散在長梭形,第10d組織塊周圍細(xì)胞明顯增多,第15d左右組織塊周圍細(xì)胞生長密集,開始相互融合。細(xì)胞傳代后7d左右基本長滿瓶底,培養(yǎng)瓶瓶底細(xì)胞呈毛刷狀、漩渦狀或放射狀生長,胞體呈長梭形,周圍可見長短不一的突起,細(xì)胞排列整齊規(guī)律(2)在一定濃度的AA作用下,FB的生長形態(tài)出現(xiàn)明顯變化。AA濃度為5μmol/L、10μmol/L時,FB細(xì)胞數(shù)量增加,細(xì)胞形態(tài)及生長走向未受到明顯影響;AA濃度為20μmol/L、40μmol/L和80μmol/L時,FB的形狀開始表現(xiàn)出不規(guī)整,細(xì)胞大小不均勻,形狀改變,部分細(xì)胞呈圓鈍形,數(shù)量減少,胞核濃縮。AA濃度越高,FB的形態(tài)改變表現(xiàn)越明顯。(3)FB在5μmol/L、10μmol/L濃度的AA作用下,FB存活率未受到明顯影響(p0.05),20-80μmol/L AA濃度范圍內(nèi),FB隨著AA作用濃度或時間的增加其抑制率均逐步增加(P0.05,且該濃度范圍內(nèi)各組間比較P0.01)。(4)免疫細(xì)胞化學(xué)顯示AA濃度為20μmol/L、40μmol/L和80μmol/L時,FB核中Ki-67蛋白表達(dá)陽性細(xì)胞百分率均顯著低于AA濃度為0μmol/L時(P0.05),且AA濃度越高,Ki-67蛋白陽性細(xì)胞百分率越低(P0.01);AA濃度為5μmol/L、10μmol/L時,Ki-67蛋白陽性細(xì)胞百分率低于AA濃度為0μmol/L時,但無統(tǒng)計學(xué)意義(P0.05)。結(jié)論:一定濃度的AA能抑制人HSFB的增殖,使處于分裂期細(xì)胞數(shù)量減少,這可能為其抑制HS形成作用機(jī)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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R62

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