5-氨基酮戊酸光動力療法對正常人角質(zhì)形成細胞的影響
發(fā)布時間：2018-08-15 11:42
【摘要】： 目的： 5-氨基酮戊酸光動力療法(5-Aminolevulinic acid Photodynamic Therapy, ALA-PDT)對體外培養(yǎng)的正常人角質(zhì)形成細胞(keratinocyte, KC)的凋亡的影響及機制,為臨床用ALA-PDT治療銀屑病提供理論依據(jù)。 方法： 1.采用Dispase分離酶、胰蛋白酶兩次消化法制備和培養(yǎng)正常人包皮組織角質(zhì)形成細胞。 2.HE染色法、生長曲線鑒定細胞及其活力。 3.MTT法、流式細胞儀和熒光顯微鏡檢測角質(zhì)形成細胞的ALA-PDT最佳藥物濃度和最適避光時間。光源波長為635nm的半導(dǎo)體激光,輸出功率30mW,能量密度12J／cm2。 4. Hoechst33342/PI雙染色法、吖啶橙染色、Annexin V/PI雙染法檢測ALA-PDT對角質(zhì)形成細胞的凋亡情況。 5.檢測ALA-PDT對角質(zhì)形成細胞細胞生長周期的影響。 6.結(jié)果采用SPSS16.0進行統(tǒng)計學(xué)分析。 結(jié)果： 1.角質(zhì)形成細胞在光學(xué)顯微鏡下形態(tài)學(xué)觀察顯示,細胞呈典型上皮樣特征,高核漿比例,細胞緊密排列,輪廓清楚折光性好,為所需角質(zhì)形成細胞。細胞在接種后活性較好的細胞即迅速貼壁。初期細胞以圓形為主,隨著時間的延長,逐步變?yōu)椴灰?guī)則形或多角形。3天左右可見許多角質(zhì)形成細胞形成的小集落,四周有衛(wèi)星樣角質(zhì)形成細胞細胞增殖,細胞的均質(zhì)性和透明度加強。一周左右細胞融合可達90%,細胞活力較佳。HE染色中,光鏡下觀察,可見細胞質(zhì)呈粉紅色,細胞核呈藍色。 2.MTT法中,與對照組相比,0.1 mmol/L、0.6 mmol/L、1.2mmol/L、1.8mmol/L、 3.6mmol/LALA組角質(zhì)形成細胞在37℃避光作用0.5h、1h、3h和5h后照光,結(jié)果以避光1h中0.6 mmol/L ALA-PDT組光密度(optical density, OD)值最低,差異有統(tǒng)計學(xué)意義(P0.05)。熒光顯微鏡下觀察熒光細胞數(shù),0.6 mmol/L的ALA避光1h吸收發(fā)光的陽性細胞數(shù)最多(P0.05)。FCM檢測0.6mmol/L的ALA在避 光作用1h及3h時吸收ALA情況較佳,兩組之間差異無統(tǒng)計學(xué)意義(P0.05)。3.與空白組相比,0.6mmol/L ALA-PDT組可明顯促進角質(zhì)細胞的凋亡,Hoechst33342/PI和AO檢測顯示凋亡率分別為(27.3±1.9)%,P0.05和(28.2±1.0)%, P0.05。Annexin V/PI雙染法顯示其早期凋亡率為(16.2±0.8)%,P0.05。 4.經(jīng)0.6mmol/L ALA-PDT作用后,角質(zhì)細胞細胞周期中G1期DNA含量增多,S期與G2期DNA含量減少,PI值減少。 結(jié)論： 1. 0.6mmol/L的ALA在37℃避光條件下孵育1h后進行635nm的半導(dǎo)體激光照射為最佳藥物濃度和最適宜孵育時間。 2. 0.6mmol/L的ALA在37℃避光孵育1h后進行635nm的半導(dǎo)體激光照射可明顯促進角質(zhì)細胞的凋亡,同時能改變角質(zhì)細胞的細胞周期,G1期DNA含量增多,S期與G2期DNA含量減少,PI值減少,抑制角質(zhì)細胞的增殖。
[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 鹵1.9) and (28.2 鹵1.0), respectively. The early apoptotic rate of P0.05.Annexin V/PI was (16.2 鹵0.8) and (16.2 鹵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，
本文編號：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 鹵1.9) and (28.2 鹵1.0), respectively. The early apoptotic rate of P0.05.Annexin V/PI was (16.2 鹵0.8) and (16.2 鹵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，
本文編號：2184093
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