喜樹堿對HaCaT細胞、人原代角質(zhì)形成細胞自噬及凋亡的影響
發(fā)布時間：2018-08-09 08:54
【摘要】：目的觀察喜樹堿對Ha Ca T細胞、人原代角質(zhì)形成細胞(KCs)自噬和凋亡的影響,從而探討喜樹堿治療銀屑病的機制。方法(1)將Ha Ca T細胞分為對照組和實驗組,對照組為0.1%二甲基亞砜(DMSO),實驗組分別為5、10、25、50、100、200 nmol/L濃度的喜樹堿。藥物處理細胞24、48 h,用CCK8法檢測細胞的增殖情況,流式細胞儀檢測藥物作用24 h的細胞凋亡情況,Western blot檢測自噬相關(guān)蛋白微管相關(guān)蛋白1輕鏈3(LC3)、p62的變化,選取10 nmol/L喜樹堿作用于Ha Ca T細胞24 h,間接免疫熒光法觀察細胞自噬蛋白LC3的變化,透射電鏡觀察自噬體超微結(jié)構(gòu),確認喜樹堿對Ha Ca T細胞自噬的誘導(dǎo)效應(yīng)。(2)取行包皮環(huán)切術(shù)青少年的包皮組織,分離提取人原代角質(zhì)形成細胞,取第三代細胞用于各項實驗,實驗組喜樹堿濃度為200nmol/L、2μmol/L、6μmol/L,對照組為0.1%DMSO,藥物處理細胞24h、48h后,采用CCK8法檢測細胞增殖情況,流式細胞儀檢測藥物作用24 h的細胞凋亡情況,Western blot檢測自噬相關(guān)蛋白LC3、p62的變化,選取2μmol/L濃度喜樹堿作用于細胞24 h,間接免疫熒光法檢測細胞自噬蛋白LC3的變化,透射電鏡觀察自噬體超微結(jié)構(gòu),進一步確認喜樹堿對其自噬的誘導(dǎo)效應(yīng),免疫組化觀察銀屑病皮損部位和正常表皮LC3表達水平。(3)統(tǒng)計學(xué)分析:統(tǒng)計分析采用SPSS 17.0軟件,計量資料以?x±s表示。各均數(shù)經(jīng)Levene檢驗方差齊性。細胞的增殖抑制率以及凋亡率統(tǒng)計分析采用單因素方差分析,各組間的多重比較采用Dunnett t檢驗,間接免疫熒光檢測自噬體陽性細胞率采用獨立樣本t檢驗。P0.05為差異有統(tǒng)計學(xué)意義。結(jié)果(1)5、10、25 nmol/L喜樹堿對Ha Ca T細胞的增殖、凋亡無顯著影響,50、100、200nmol/L喜樹堿作用Ha Ca T細胞24 h的增殖抑制率分別為(31.23±1.00)%,(54.21±8.10)%,(66.75±10.70)%;25、50、100、200nmol/L喜樹堿作用Ha Ca T細胞48h的增殖抑制率分別為(25.81±5.99)%、(44.35±5.32)%、(65.81±8.28)%、(73.23±9.59)%,與相同時段的對照組相比,差異有統(tǒng)計學(xué)意義(p均0.001)。50、100、200nmol/L喜樹堿作用Ha Ca T細胞24 h的凋亡率分別為(14.46±2.38)%、(19.15±1.59)%、(29.88±1.37)%,與對照組(3.80±0.13)%比較,差異有統(tǒng)計學(xué)意義(P均0.001)。5、10 nmol/L喜樹堿處理Ha Ca T細胞24 h后,LC3Ⅱ表達上調(diào),p62蛋白表達下調(diào)。間接免疫熒光觀察到10nmol/L喜樹堿作用細胞24h后的綠色熒光聚集點明顯增多,實驗組與對照組的自噬體陽性細胞百分率分別為(36.67±4.55)%、6.23±0.92)%,兩組差異有統(tǒng)計學(xué)意義(t=6.546,p=0.0028)。透射電鏡觀察到10nmol/L喜樹堿作用細胞24h自噬體和自噬溶酶體的形成。(2)2μmol/L、6μmol/L喜樹堿作用原代角質(zhì)形成細胞24 h的增殖抑制率分別為(33.90±3.65)%、(51.72±5.06)%,與對照組(1.60±0.78)%相比差異有統(tǒng)計學(xué)意義,200nmol/L喜樹堿作用原代角質(zhì)形成細胞24 h的增殖抑制率為(7.59±1.82)%,與對照組比較差異無統(tǒng)計學(xué)意義。200nmol/L、2μmol/L、6μmol/L喜樹堿作用原代角質(zhì)形成細胞48 h的增殖抑制率分別為(19.21±5.74)%、(52.09±3.01)%、(63.37±5.45)%,與對照組(4.18±1.56)%相比,差異有統(tǒng)計學(xué)意義。200nmol/L、2μmol/L、6μmol/L喜樹堿作用原代角質(zhì)形成細胞24 h的凋亡率分別為(15.90±2.14)%、(29.33±3.51)%、(35.28±3.05)%,與對照組(2.30±1.68)%比較,差異有統(tǒng)計意義。2μmol/L、6μmol/L喜樹堿處理原代角質(zhì)形成細胞24 h后,LC3Ⅱ顯著上調(diào),p62蛋白表達下調(diào)。間接免疫熒光觀察到2μmol/L喜樹堿作用原代角質(zhì)形成細胞24h后的綠色熒光聚集點,實驗組與對照組的自噬體陽性細胞百分率分別為(60.16±8.78)%、(38.96±13.12)%,統(tǒng)計方法采用獨立樣本t檢驗,差異具有統(tǒng)計學(xué)意義(t=3.003,p=0.017)。透射電鏡觀察到2μmol/L喜樹堿作用原代角質(zhì)形成細胞24h自噬體和自噬溶酶體的形成;皮膚組織LC3免疫組化觀察到銀屑病皮損部位表皮LC3表達量較正常皮膚明顯降低。結(jié)論低濃度喜樹堿(5、10、25 nmol/L)可誘導(dǎo)Ha Ca T細胞產(chǎn)生自噬,對細胞增殖、凋亡無顯著影響,高濃度喜樹堿(50、100、200 nmol/L)抑制Ha Ca T細胞增殖,促使細胞發(fā)生凋亡,自噬水平降低。2μmol/L、6μmol/L喜樹堿可誘導(dǎo)原代角質(zhì)形成細胞自噬水平升高,同時誘導(dǎo)細胞凋亡。
[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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R758.63
本文編號：2173572
[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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R758.63
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