本文選題：光動力療法 + 成纖維細胞��； 參考：《西南醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:探討5-氨基酮戊酸(5-aminolevulnilic acid,ALA)光動力療法(photodynamic therapy,PDT)對瘢痕疙瘩成纖維細胞(keloid fibroblasts,KFB)周期影響及調(diào)控機制的研究。方法:1.采用組織塊培養(yǎng)方法,分別體外培養(yǎng)KFB和正常皮膚成纖維細胞(human skin fibroblasts,HSFs),觀察其生物學(xué)特征;應(yīng)用抗波形蛋白抗體做免疫細胞化學(xué)鑒定,證實為成纖維細胞(fibroblast,FB)。2.KFB傳代培養(yǎng)后,選用第3-5代對數(shù)生長期的KFB進行實驗,分別加入含有ALA 0.25 mmol/L、0.5 mmol/L、1.0mmol/L、2.0mmol/L、4.0mmol/L、8.0mmol/L的無血清高糖DMEM培養(yǎng)液,避光培養(yǎng)4h后,光動力儀照射,光源距離15cm,波長630nm,光照密度為100m W/cm~2,光照時間分別為100s、200s、400s、800s、1200s(即光照能量分別為10J/cm~2、20J/cm~2、40J/cm~2、80J/cm~2、120J/cm~2)。繼續(xù)培養(yǎng)24h后,采用CCK8法檢測增殖活性,選出最優(yōu)光敏劑濃度+光照能量組合(ALA濃度為4.0mmol/L,PDT能量為80J/cm~2)用于實驗。3.將傳代培養(yǎng)的生長情況良好的KFB分為4組,即空白對照組,ALA+PDT組(AP組),p79350(p38 MAPK信號通路激動劑)+ALA+PDT組(PAP組),SB203580(p38 MAPK信號通路阻斷劑劑)+ALA+PDT組(SB組);正常皮膚成纖維細胞組標(biāo)記為HSFs組,以ALA濃度為4.0mmol/L,PDT能量為80J/cm~2(最優(yōu)光敏劑濃度+光照能量組合)進行干預(yù)KFB各分組,流式細胞儀檢測各組FB細胞周期中細胞分布比例;Western Blot法檢測各組中細胞周期蛋白D1(Cyclin D1)、磷酸化p38細胞絲裂原活化蛋白激酶(phospho-p38 mitogen-activated protein kinase,p-p38 MAPK)表達情況。結(jié)果:1.組織塊接種成功后約7天可見細胞形態(tài)呈長梭形或三角形,邊緣向外伸出數(shù)個突起;抗波形蛋白抗體免疫鑒定為陽性,證實其為成纖維細胞。2.CCK8檢測結(jié)果顯示:ALA-PDT干預(yù)后,KFB活性受到抑制,且在一定的范圍中,KFB的活性隨著ALA濃度、PDT能量的增大而降低。3.流式細胞儀檢測提示:五組細胞均以G0/G1期細胞為主峰,空白對照組中細胞S期峰值高于HSFs組,S期細胞比例顯著增高(49.32+2.8%),PI具有統(tǒng)計學(xué)差異(P0.05);AP組、PAP組、SB組中S期峰值均低于空白對照組,S期細胞比例顯著降低,分別為(20.26+2.5%、24.45+2.3%、16.97+2.6%),PI具有統(tǒng)計學(xué)差異(P0.05);AP組中S期細胞峰值高于SB組(16.97+2.6%),低于PAP組(24.45+2.3%),PI具有統(tǒng)計學(xué)差異(P0.05)。4.Western Blot法檢測提示:空白對照組中Cyclin D1表達明顯高于HSFs,AP組、PAP組、SB組中Cyclin D1、p-p38MAPK表達顯著低于空白對照組,AP組中Cyclin D1、p-p38MAPK表達高于SB組,低于PAP組,差異均具有統(tǒng)計學(xué)意義(p0.05)。結(jié)論:1.p-p38 MAPK/Cyclin D1在KFB中的異常表達可導(dǎo)致成纖維細胞的異常增殖;2.ALA-PDT對KFB增殖周期起抑制作用,其作用機制可能與抑制p38 MAPK信號通路及降低Cyclin D1表達,從而誘導(dǎo)KFB發(fā)生G1期阻滯有關(guān)。
[Abstract]:Aim: to investigate the effect of photodynamic therapeutic therapy (photodynamic) on the cycle of keloid fibroblasts (keloid fibroblasts) and its regulatory mechanism. Method 1: 1. The biological characteristics of KFB and normal skin fibroblast (human skin fibroblasts were observed by using tissue mass culture method, and the results of immunocytochemical identification with anti-vimentin antibody were confirmed as fibroblast FB. 2. KFB of the 3-5 generation logarithmic growth period was used to experiment, and 0.5 mmol / L (0.5 mmol / L) of Ala was added into 2.0 mmol / L 2.0 mmol / L (4.0 mmol / L) DMEM medium without serum and 8.0 mmol / L, respectively. The medium was irradiated by photodynamic instrument after 4 h of dark light culture. The light source distance is 15cm, wavelength is 630nm, light density is 100m W / cm ~ 2, illumination time is 100s ~ 200s ~ 200s ~ 200s ~ (400) s ~ (-1) ~ (800) s ~ (-1) (that is, light energy is 10J / cm ~ (2) ~ (2) ~ (20) J / cm ~ (2) ~ (20) J / cm ~ (2) ~ (80) J / cm ~ (2) / cm ~ (2120) J / cm ~ (2). After 24 hours of culture, the proliferative activity was detected by CCK8 method, and the optimum concentration of Guang Min was selected to be used in experiment .3.The optimum concentration of Guang Min was 4.0 mmol / L PDT energy of 80 J / cm ~ (2) (Ala concentration was 4.0 mmol 路L ~ (-1) 路L ~ (-1) 路L ~ (-1). KFB, which grew well in subculture, was divided into 4 groups. The normal skin fibroblasts group was labeled as HSFs group, the normal skin fibroblast group was labeled as HSFs group, the normal skin fibroblast group was labeled as HSFs group, and the ALA PDT group (PAP group) was treated with ALA PDT (p38 MAPK signal pathway blocker), the normal skin fibroblast group was labeled as HSFs group, and the normal skin fibroblast group was labeled as HSFs group, while the normal skin fibroblast group was labeled as HSFs group, and the normal skin fibroblast group was labeled as HSFs group. When Ala concentration was 4.0 mmol 路L ~ (-1) PDT energy was 80 J / cm ~ (2) (optimal Guang Min concentration light energy combination), KFB groups were treated with Ala concentration of 4.0 mmol 路L ~ (-1) / L ~ (-1). The expression of cyclin D1 and phospho-p38 mitogen-activated protein kinase- p-p38 MAPK were detected by flow cytometry and Western blot. The result is 1: 1. About 7 days after the successful inoculation of the tissue mass, the cells were found to be fusiform or triangular in shape, with several protrusions protruding outward from the edges, and the anti-vimentin antibody was identified as positive by immunoassay. The results of CCK8 assay showed that the activity of KFB was inhibited after the intervention of w ALA-PDT, and the activity of KFB decreased with the increase of Ala concentration and PDT energy in a certain range. The flow cytometry showed that the G0 / G1 phase cells were the main peak in all the five groups. The peak value of S phase in the control group was significantly higher than that in the HSFs group (49.322.8%). There was significant difference in Pi (P0.05). The peak value of S phase in the AP group was significantly lower than that in the control group, and the percentage of S phase cells in the control group was significantly lower than that in the control group. Pi was significantly higher in AP group than that in SB group (16.972.6%) and lower than that in PAP group (24.452.3%) (P0.05). Western blot analysis showed that the expression of Cyclin D1 in the blank control group was significantly higher than that in the HSFsAP-PAP group (P < 0.05). 4. Western blot analysis showed that the expression of Cyclin D1 in the control group was significantly higher than that in the control group (P < 0.05). 4. Western blot analysis showed that the expression of Cyclin D1 in the control group was significantly higher than that in the control group (P < 0.05). The expression of p-p38 MAPK in the middle Cyclin D1P p-p38 MAPK was significantly lower than that in the control group (P < 0.05), and the expression of p-p38 MAPK in the AP group was significantly higher than that in the SB group. The difference was statistically significant compared with PAP group (p0.05). Conclusion the abnormal expression of p-p38 MAPK / Cyclin D1 in KFB can induce the abnormal proliferation of fibroblasts. 2. ALA-PDT can inhibit the proliferation of KFB. The mechanism may be related to the inhibition of p38 MAPK signaling pathway and the decrease of Cyclin D1 expression, thus inducing G1 arrest of KFB.
【學(xué)位授予單位】：西南醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R751

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