喜树碱对HaCaT细胞、人原代角质形成细胞自噬及凋亡的影响

发布时间：2018-08-09 08:54

【摘要】：目的观察喜树碱对Ha Ca T细胞、人原代角质形成细胞(KCs)自噬和凋亡的影响,从而探讨喜树碱治疗银屑病的机制。方法(1)将Ha Ca T细胞分为对照组和实验组,对照组为0.1%二甲基亚砜(DMSO),实验组分别为5、10、25、50、100、200 nmol/L浓度的喜树碱。药物处理细胞24、48 h,用CCK8法检测细胞的增殖情况,流式细胞仪检测药物作用24 h的细胞凋亡情况,Western blot检测自噬相关蛋白微管相关蛋白1轻链3(LC3)、p62的变化,选取10 nmol/L喜树碱作用于Ha Ca T细胞24 h,间接免疫荧光法观察细胞自噬蛋白LC3的变化,透射电镜观察自噬体超微结构,确认喜树碱对Ha Ca T细胞自噬的诱导效应。(2)取行包皮环切术青少年的包皮组织,分离提取人原代角质形成细胞,取第三代细胞用于各项实验,实验组喜树碱浓度为200nmol/L、2μmol/L、6μmol/L,对照组为0.1%DMSO,药物处理细胞24h、48h后,采用CCK8法检测细胞增殖情况,流式细胞仪检测药物作用24 h的细胞凋亡情况,Western blot检测自噬相关蛋白LC3、p62的变化,选取2μmol/L浓度喜树碱作用于细胞24 h,间接免疫荧光法检测细胞自噬蛋白LC3的变化,透射电镜观察自噬体超微结构,进一步确认喜树碱对其自噬的诱导效应,免疫组化观察银屑病皮损部位和正常表皮LC3表达水平。(3)统计学分析:统计分析采用SPSS 17.0软件,计量资料以?x±s表示。各均数经Levene检验方差齐性。细胞的增殖抑制率以及凋亡率统计分析采用单因素方差分析,各组间的多重比较采用Dunnett t检验,间接免疫荧光检测自噬体阳性细胞率采用独立样本t检验。P0.05为差异有统计学意义。结果(1)5、10、25 nmol/L喜树碱对Ha Ca T细胞的增殖、凋亡无显著影响,50、100、200nmol/L喜树碱作用Ha Ca T细胞24 h的增殖抑制率分别为(31.23±1.00)%,(54.21±8.10)%,(66.75±10.70)%;25、50、100、200nmol/L喜树碱作用Ha Ca T细胞48h的增殖抑制率分别为(25.81±5.99)%、(44.35±5.32)%、(65.81±8.28)%、(73.23±9.59)%,与相同时段的对照组相比,差异有统计学意义(p均0.001)。50、100、200nmol/L喜树碱作用Ha Ca T细胞24 h的凋亡率分别为(14.46±2.38)%、(19.15±1.59)%、(29.88±1.37)%,与对照组(3.80±0.13)%比较,差异有统计学意义(P均0.001)。5、10 nmol/L喜树碱处理Ha Ca T细胞24 h后,LC3Ⅱ表达上调,p62蛋白表达下调。间接免疫荧光观察到10nmol/L喜树碱作用细胞24h后的绿色荧光聚集点明显增多,实验组与对照组的自噬体阳性细胞百分率分别为(36.67±4.55)%、6.23±0.92)%,两组差异有统计学意义(t=6.546,p=0.0028)。透射电镜观察到10nmol/L喜树碱作用细胞24h自噬体和自噬溶酶体的形成。(2)2μmol/L、6μmol/L喜树碱作用原代角质形成细胞24 h的增殖抑制率分别为(33.90±3.65)%、(51.72±5.06)%,与对照组(1.60±0.78)%相比差异有统计学意义,200nmol/L喜树碱作用原代角质形成细胞24 h的增殖抑制率为(7.59±1.82)%,与对照组比较差异无统计学意义。200nmol/L、2μmol/L、6μmol/L喜树碱作用原代角质形成细胞48 h的增殖抑制率分别为(19.21±5.74)%、(52.09±3.01)%、(63.37±5.45)%,与对照组(4.18±1.56)%相比,差异有统计学意义。200nmol/L、2μmol/L、6μmol/L喜树碱作用原代角质形成细胞24 h的凋亡率分别为(15.90±2.14)%、(29.33±3.51)%、(35.28±3.05)%,与对照组(2.30±1.68)%比较,差异有统计意义。2μmol/L、6μmol/L喜树碱处理原代角质形成细胞24 h后,LC3Ⅱ显著上调,p62蛋白表达下调。间接免疫荧光观察到2μmol/L喜树碱作用原代角质形成细胞24h后的绿色荧光聚集点,实验组与对照组的自噬体阳性细胞百分率分别为(60.16±8.78)%、(38.96±13.12)%,统计方法采用独立样本t检验,差异具有统计学意义(t=3.003,p=0.017)。透射电镜观察到2μmol/L喜树碱作用原代角质形成细胞24h自噬体和自噬溶酶体的形成;皮肤组织LC3免疫组化观察到银屑病皮损部位表皮LC3表达量较正常皮肤明显降低。结论低浓度喜树碱(5、10、25 nmol/L)可诱导Ha Ca T细胞产生自噬,对细胞增殖、凋亡无显著影响,高浓度喜树碱(50、100、200 nmol/L)抑制Ha Ca T细胞增殖,促使细胞发生凋亡,自噬水平降低。2μmol/L、6μmol/L喜树碱可诱导原代角质形成细胞自噬水平升高,同时诱导细胞凋亡。
[Abstract]:Objective To observe the effect of Camptothecin on the autophagy and apoptosis of Ha Ca T cells and human primary keratinocyte (KCs), and to explore the mechanism of camptothecin in the treatment of psoriasis. Methods (1) Ha Ca T cells were divided into control group and experimental group, and the control group was 0.1% two methyl sulfoxide (DMSO), and the test group was 5,10,25,50100200 nmol/L concentration of camptothecin. 24,48 h was used to detect cell proliferation by CCK8 method. Flow cytometry was used to detect the apoptosis of 24 h drugs. Western blot was used to detect the changes of autophagy related protein 1 light chain 3 (LC3), p62, and 10 nmol/L camptothecin was selected for Ha Ca T cells 24. Indirect immunofluorescence was used to observe the autophagic protein 3 changes, transmission electron microscope observation of autophagic ultrastructure, confirm the induction effect of Camptothecin on Ha Ca T cell autophagy. (2) take the circumcision of the circumcision of young adult circumcision tissue, isolation and extraction of human primary keratinocyte, third generation of cells used for various experiments, the experimental group of camptothecin concentration of 200nmol/L, 2 mu mol/L, 6 micron mol/L, the control group is 0. 1%DMSO, after the drugs were treated with 24h and 48h, the cell proliferation was detected by CCK8 method. The flow cytometry was used to detect the apoptosis of 24 h, Western blot was used to detect the autophagy related protein LC3, the change of p62, and 2 mu concentration of camptothecin was selected to act on the 24 h cell, and the change of the cell autophagy protein LC3 was detected by immunofluorescence. Electron microscope observation of autophagic ultrastructure, further confirm the induction effect of Camptothecin on its autophagy, immunohistochemical observation of psoriasis skin lesions and normal epidermis LC3 expression level. (3) statistical analysis: statistical analysis used SPSS 17 software, measurement data with x + s. The average number of all by Levene test variance homogeneity. Cell proliferation inhibition rate And the statistical analysis of apoptosis rate was analyzed by single factor analysis of variance. Dunnett t test was used for multiple comparison among each group. The positive cell rate of autophagic positive cells by indirect immunofluorescence was statistically significant with the independent sample t test of.P0.05. Results (1) 5,10,25 nmol/L camptothecin had no significant effect on the proliferation of Ha Ca T cells, 50100200n, 50100200n. The proliferation inhibition rate of mol/L Camptothecin on Ha Ca T cells 24 h was (31.23 + 1)%, (54.21 + 8.10)%, (66.75 + 10.70)%, and the proliferation inhibition rate of 25,50100200nmol/L camptothecin Ha Ca T cell 48h was (25.81 + 5.99)%, (44.35 + 5.32)%, (65.81 + 8.28)%, (65.81 + 8.28)%, and the difference was statistically significant compared with the control group at the same time period. Significance (P 0.001).50100200nmol/L camptothecin action of Ha Ca T cells 24 h apoptosis rate was (14.46 + 2.38)%, (19.15 + 1.59)%, (29.88 + 1.37)%, compared with the control group (3.80 + 0.13)%, the difference was statistically significant (P 0.001).5,10 nmol/L camptothecin Ha Ca T cells 24 The immunofluorescence showed that the green fluorescence aggregation point of 10nmol/L camptothecin activated cells 24h increased obviously. The percentage of autophagic positive cells in the experimental group and the control group were (36.67 + 4.55)%, 6.23 + 0.92% respectively. The two groups were statistically significant (t=6.546, p=0.0028). The transmission electron microscope observed the 24h autophago and autophago of 10nmol/L acuminate cells. The formation of lysosomes. (2) the proliferation inhibition rate of 2 mu mol/L, 6 mu mol/L camptothecin acting primary keratinocyte 24 h was (33.90 + 3.65)%, (51.72 + 5.06)%, compared with the control group (1.60 + 0.78)%, the difference was statistically significant. The proliferation inhibition rate of 200nmol/L camptothecin in primary keratinocytes was (7.59 + 1.82)%, and the control group. The difference was not statistically significant.200nmol/L, 2 mu mol/L, 6 mol/L camptothecin acting primary keratinocytes 48 h proliferation inhibition rate was (19.21 + 5.74)%, (52.09 + 3.01)%, (63.37 + 5.45)%, compared with the control group (4.18 + 1.56)%, the difference was statistically meaning.200nmol/L, 2 mu mol/L, 6 mu mol/L camptothecin acting primary keratinocyte 24 The apoptosis rate of h was (15.90 + 2.14)%, (29.33 + 3.51)%, (35.28 + 3.05)%, compared with the control group (2.30 + 1.68)%, the difference was statistically significant.2 mol/L, and 6 u mol/L camptothecin treated the original keratinocyte 24 h, LC3 II was significantly up-regulated, and the expression of p62 protein was downregulated. Indirect immunofluorescence observed that 2 mol/L camptothecin acted as the primary cutin formation fine. The percentage of green fluorescent aggregation after 24h was (60.16 + 8.78)% and (38.96 + 13.12)% respectively in the experimental group and the control group (38.96 + 13.12)%. The statistical method was statistically significant (t=3.003, p=0.017) by independent sample t test. The transmission electron microscope observed that 2 u mol/L camptothecin acted as the autophago and autophago of the keratinocyte of the primary keratinocyte. The formation of lysosomes; LC3 immunohistochemical staining of skin tissue observed that the expression of LC3 in the epidermis of psoriatic lesions was significantly lower than that of normal skin. Conclusion low concentration camptothecin (5,10,25 nmol/L) can induce autophagy in Ha Ca T cells and have no significant effect on cell proliferation and apoptosis. High concentration camptothecin (50100200 nmol/L) inhibits Ha Ca T cells. Colonization can induce apoptosis and decrease the level of autophagy by.2 mu mol/L. 6 u mol/L camptothecin can induce the increase of autophagy and induce apoptosis in the primary keratinocytes.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R758.63

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