ALA-PDT对瘢痕疙瘩成纤维细胞周期影响及调控机制的研究

发布时间：2018-06-29 07:46

本文选题：光动力疗法 + 成纤维细胞　；参考：《西南医科大学》2017年硕士论文

【摘要】：目的:探讨5-氨基酮戊酸(5-aminolevulnilic acid,ALA)光动力疗法(photodynamic therapy,PDT)对瘢痕疙瘩成纤维细胞(keloid fibroblasts,KFB)周期影响及调控机制的研究。方法:1.采用组织块培养方法,分别体外培养KFB和正常皮肤成纤维细胞(human skin fibroblasts,HSFs),观察其生物学特征;应用抗波形蛋白抗体做免疫细胞化学鉴定,证实为成纤维细胞(fibroblast,FB)。2.KFB传代培养后,选用第3-5代对数生长期的KFB进行实验,分别加入含有ALA 0.25 mmol/L、0.5 mmol/L、1.0mmol/L、2.0mmol/L、4.0mmol/L、8.0mmol/L的无血清高糖DMEM培养液,避光培养4h后,光动力仪照射,光源距离15cm,波长630nm,光照密度为100m W/cm~2,光照时间分别为100s、200s、400s、800s、1200s(即光照能量分别为10J/cm~2、20J/cm~2、40J/cm~2、80J/cm~2、120J/cm~2)。继续培养24h后,采用CCK8法检测增殖活性,选出最优光敏剂浓度+光照能量组合(ALA浓度为4.0mmol/L,PDT能量为80J/cm~2)用于实验。3.将传代培养的生长情况良好的KFB分为4组,即空白对照组,ALA+PDT组(AP组),p79350(p38 MAPK信号通路激动剂)+ALA+PDT组(PAP组),SB203580(p38 MAPK信号通路阻断剂剂)+ALA+PDT组(SB组);正常皮肤成纤维细胞组标记为HSFs组,以ALA浓度为4.0mmol/L,PDT能量为80J/cm~2(最优光敏剂浓度+光照能量组合)进行干预KFB各分组,流式细胞仪检测各组FB细胞周期中细胞分布比例;Western Blot法检测各组中细胞周期蛋白D1(Cyclin D1)、磷酸化p38细胞丝裂原活化蛋白激酶(phospho-p38 mitogen-activated protein kinase,p-p38 MAPK)表达情况。结果:1.组织块接种成功后约7天可见细胞形态呈长梭形或三角形,边缘向外伸出数个突起;抗波形蛋白抗体免疫鉴定为阳性,证实其为成纤维细胞。2.CCK8检测结果显示:ALA-PDT干预后,KFB活性受到抑制,且在一定的范围中,KFB的活性随着ALA浓度、PDT能量的增大而降低。3.流式细胞仪检测提示:五组细胞均以G0/G1期细胞为主峰,空白对照组中细胞S期峰值高于HSFs组,S期细胞比例显著增高(49.32+2.8%),PI具有统计学差异(P0.05);AP组、PAP组、SB组中S期峰值均低于空白对照组,S期细胞比例显著降低,分别为(20.26+2.5%、24.45+2.3%、16.97+2.6%),PI具有统计学差异(P0.05);AP组中S期细胞峰值高于SB组(16.97+2.6%),低于PAP组(24.45+2.3%),PI具有统计学差异(P0.05)。4.Western Blot法检测提示:空白对照组中Cyclin D1表达明显高于HSFs,AP组、PAP组、SB组中Cyclin D1、p-p38MAPK表达显著低于空白对照组,AP组中Cyclin D1、p-p38MAPK表达高于SB组,低于PAP组,差异均具有统计学意义(p0.05)。结论:1.p-p38 MAPK/Cyclin D1在KFB中的异常表达可导致成纤维细胞的异常增殖;2.ALA-PDT对KFB增殖周期起抑制作用,其作用机制可能与抑制p38 MAPK信号通路及降低Cyclin D1表达,从而诱导KFB发生G1期阻滞有关。
[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.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R751

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