5-氨基酮戊酸光动力疗法对正常人角质形成细胞的影响
发布时间：2018-08-15 11:42
【摘要】： 目的： 5-氨基酮戊酸光动力疗法(5-Aminolevulinic acid Photodynamic Therapy, ALA-PDT)对体外培养的正常人角质形成细胞(keratinocyte, KC)的凋亡的影响及机制,为临床用ALA-PDT治疗银屑病提供理论依据。 方法： 1.采用Dispase分离酶、胰蛋白酶两次消化法制备和培养正常人包皮组织角质形成细胞。 2.HE染色法、生长曲线鉴定细胞及其活力。 3.MTT法、流式细胞仪和荧光显微镜检测角质形成细胞的ALA-PDT最佳药物浓度和最适避光时间。光源波长为635nm的半导体激光,输出功率30mW,能量密度12J／cm2。 4. Hoechst33342/PI双染色法、吖啶橙染色、Annexin V/PI双染法检测ALA-PDT对角质形成细胞的凋亡情况。 5.检测ALA-PDT对角质形成细胞细胞生长周期的影响。 6.结果采用SPSS16.0进行统计学分析。 结果： 1.角质形成细胞在光学显微镜下形态学观察显示,细胞呈典型上皮样特征,高核浆比例,细胞紧密排列,轮廓清楚折光性好,为所需角质形成细胞。细胞在接种后活性较好的细胞即迅速贴壁。初期细胞以圆形为主,随着时间的延长,逐步变为不规则形或多角形。3天左右可见许多角质形成细胞形成的小集落,四周有卫星样角质形成细胞细胞增殖,细胞的均质性和透明度加强。一周左右细胞融合可达90%,细胞活力较佳。HE染色中,光镜下观察,可见细胞质呈粉红色,细胞核呈蓝色。 2.MTT法中,与对照组相比,0.1 mmol/L、0.6 mmol/L、1.2mmol/L、1.8mmol/L、 3.6mmol/LALA组角质形成细胞在37℃避光作用0.5h、1h、3h和5h后照光,结果以避光1h中0.6 mmol/L ALA-PDT组光密度(optical density, OD)值最低,差异有统计学意义(P0.05)。荧光显微镜下观察荧光细胞数,0.6 mmol/L的ALA避光1h吸收发光的阳性细胞数最多(P0.05)。FCM检测0.6mmol/L的ALA在避 光作用1h及3h时吸收ALA情况较佳,两组之间差异无统计学意义(P0.05)。3.与空白组相比,0.6mmol/L ALA-PDT组可明显促进角质细胞的凋亡,Hoechst33342/PI和AO检测显示凋亡率分别为(27.3±1.9)%,P0.05和(28.2±1.0)%, P0.05。Annexin V/PI双染法显示其早期凋亡率为(16.2±0.8)%,P0.05。 4.经0.6mmol/L ALA-PDT作用后,角质细胞细胞周期中G1期DNA含量增多,S期与G2期DNA含量减少,PI值减少。 结论： 1. 0.6mmol/L的ALA在37℃避光条件下孵育1h后进行635nm的半导体激光照射为最佳药物浓度和最适宜孵育时间。 2. 0.6mmol/L的ALA在37℃避光孵育1h后进行635nm的半导体激光照射可明显促进角质细胞的凋亡,同时能改变角质细胞的细胞周期,G1期DNA含量增多,S期与G2期DNA含量减少,PI值减少,抑制角质细胞的增殖。
[Abstract]:Objective: to study the effect and mechanism of 5-aminolevulinic acid photodynamic therapy (5-Aminolevulinic acid Photodynamic Therapy, ALA-PDT) on the apoptosis of normal human keratinocytes (keratinocyte, KC) in vitro, and to provide a theoretical basis for the treatment of psoriasis with ALA-PDT. Methods: 1. Keratinocytes of normal prepuce tissue were prepared and cultured by Dispase isolating enzyme and trypsin twice digestion method. 2.HE staining and growth curve were used to identify the cells and their activity. 3.MTT method was used. Flow cytometry and fluorescence microscopy were used to detect the optimal concentration of ALA-PDT in keratinocytes. A semiconductor laser with a wavelength of 635nm, with an output power of 30 MW and an energy density of 12 J / cm 2. 4. Hoechst33342/PI double staining and acridine orange staining Annexin V/PI double staining were used to detect the apoptosis of keratinocytes induced by ALA-PDT. To detect the effect of ALA-PDT on the growth cycle of keratinocytes. 6. 6. Results SPSS16.0 was used for statistical analysis. Results: 1. The morphological observation of keratinocytes under optical microscope showed that the keratinocytes had typical epithelial-like characteristics, high nuclear cytoplasm ratio, tight arrangement of cells, and clear outline and good refraction, which were the required keratinocytes. The cells that had better activity after inoculation quickly adhered to the wall. At the beginning, the cells were mainly round, and gradually became irregular or polygonal. 3 days or so, many small colonies formed by keratinocytes were observed, and there were satellite like keratinocytes proliferating around them. Cell homogeneity and transparency are enhanced. The cell fusion can reach 90% in a week or so. In HE staining, the cytoplasm is pink and the nucleus is blue. 2.MTT method. Compared with the control group, 0.1 mmol / L ~ (0.6) mmol / L ~ (1.2) mmol 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) mg 路L ~ (-1) 路L ~ (-1) 路min ~ (-1) 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) and 0.1 mmol / L ~ (0.2) mmol / L ~ (1.2) mmol / L Under fluorescence microscope, the number of ALA positive cells with 0. 6 mmol/L ALA at 1 h was the highest (P0.05). ALA of 0.6mmol/L had better absorption of ALA at 1 h and 3 h after light avoidance (P0.05). There was no significant difference between the two groups (P0.05). Compared with the control group, 0.6 mmol / L ALA-PDT could significantly promote the apoptosis of keratinocytes. The apoptotic rates of Hoechst33342 / Pi and AO were (27.3 卤1.9) and (28.2 卤1.0), respectively. The early apoptotic rate of P0.05.Annexin V/PI was (16.2 卤0.8) and (16.2 卤0.8) respectively. After treated with 0.6mmol/L ALA-PDT, DNA content in G 1 phase increased and DNA content in S phase and G 2 phase decreased in keratinocyte cycle. Conclusion: 1. ALA of 0.6mmol/L was incubated at 37 鈩，
本文编号：2184093
[Abstract]:Objective: to study the effect and mechanism of 5-aminolevulinic acid photodynamic therapy (5-Aminolevulinic acid Photodynamic Therapy, ALA-PDT) on the apoptosis of normal human keratinocytes (keratinocyte, KC) in vitro, and to provide a theoretical basis for the treatment of psoriasis with ALA-PDT. Methods: 1. Keratinocytes of normal prepuce tissue were prepared and cultured by Dispase isolating enzyme and trypsin twice digestion method. 2.HE staining and growth curve were used to identify the cells and their activity. 3.MTT method was used. Flow cytometry and fluorescence microscopy were used to detect the optimal concentration of ALA-PDT in keratinocytes. A semiconductor laser with a wavelength of 635nm, with an output power of 30 MW and an energy density of 12 J / cm 2. 4. Hoechst33342/PI double staining and acridine orange staining Annexin V/PI double staining were used to detect the apoptosis of keratinocytes induced by ALA-PDT. To detect the effect of ALA-PDT on the growth cycle of keratinocytes. 6. 6. Results SPSS16.0 was used for statistical analysis. Results: 1. The morphological observation of keratinocytes under optical microscope showed that the keratinocytes had typical epithelial-like characteristics, high nuclear cytoplasm ratio, tight arrangement of cells, and clear outline and good refraction, which were the required keratinocytes. The cells that had better activity after inoculation quickly adhered to the wall. At the beginning, the cells were mainly round, and gradually became irregular or polygonal. 3 days or so, many small colonies formed by keratinocytes were observed, and there were satellite like keratinocytes proliferating around them. Cell homogeneity and transparency are enhanced. The cell fusion can reach 90% in a week or so. In HE staining, the cytoplasm is pink and the nucleus is blue. 2.MTT method. Compared with the control group, 0.1 mmol / L ~ (0.6) mmol / L ~ (1.2) mmol 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) mg 路L ~ (-1) 路L ~ (-1) 路min ~ (-1) 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) and 0.1 mmol / L ~ (0.2) mmol / L ~ (1.2) mmol / L Under fluorescence microscope, the number of ALA positive cells with 0. 6 mmol/L ALA at 1 h was the highest (P0.05). ALA of 0.6mmol/L had better absorption of ALA at 1 h and 3 h after light avoidance (P0.05). There was no significant difference between the two groups (P0.05). Compared with the control group, 0.6 mmol / L ALA-PDT could significantly promote the apoptosis of keratinocytes. The apoptotic rates of Hoechst33342 / Pi and AO were (27.3 卤1.9) and (28.2 卤1.0), respectively. The early apoptotic rate of P0.05.Annexin V/PI was (16.2 卤0.8) and (16.2 卤0.8) respectively. After treated with 0.6mmol/L ALA-PDT, DNA content in G 1 phase increased and DNA content in S phase and G 2 phase decreased in keratinocyte cycle. Conclusion: 1. ALA of 0.6mmol/L was incubated at 37 鈩，
本文编号：2184093
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