抑制mTOR信号通路在ALA-PDT杀伤皮肤鳞状细胞癌细胞A431细胞中的作用及机制的研究

发布时间：2018-06-18 07:10

本文选题：5-氨基酮戊酸 + 光动力疗法　；参考：《承德医学院》2017年硕士论文

【摘要】：背景及目的:皮肤鳞状细胞癌发病率在皮肤恶性肿瘤中位居第二,具有侵袭性较高、破坏性较大等特点。本病主要发生于老年人的暴露部位,发病原因多与紫外线照射、化学因素、病毒感染等有关。目前手术为治疗皮肤鳞状细胞癌的传统疗法,但对于一些身体耐受性差、皮损部位特殊的患者,光动力疗法(PDT)则为治疗皮肤鳞状细胞癌的首选治疗方法。5-氨基酮戊酸-光动力疗法(ALA-PDT)是一种治疗皮肤鳞状细胞癌的新型疗法,其优点为创伤性小,治疗后不留疤痕、不影响美观等特点,但不足之处是ALA-PDT治疗深度有限,对侵袭性皮肤鳞状细胞癌效果不佳。哺乳动物雷帕霉素蛋白(mTOR)是一种丝氨酸/苏氨酸激酶,在调节细胞生长与增殖方面起着重要作用,异常激活与多种恶性肿瘤的发生发展密切相关。研究表明mTOR在皮肤鳞状细胞癌中呈高表达状态。雷帕霉素是提取于吸水性链霉菌发酵液的一种大环内酯类免疫抑制剂,雷帕霉素及其衍生物为第一代mTOR抑制剂,具有很好的抗肿瘤活性。本实验通过采取ALA-PDT联合雷帕霉素作用于皮肤鳞状细胞癌细胞株A431后观察细胞增殖与凋亡情况,探讨抑制mTOR信号通路在ALA-PDT杀伤A431细胞中的作用及机制,为增强ALA-PDT治疗皮肤鳞状细胞癌的疗效提供新的实验室依据。方法:将对数生长期的A431细胞分为6个组,分别为:1.空白对照组,不做任何特殊处理;2.单纯ALA组,仅给予1mM/LALA避光孵育4h;3.单纯光照组,仅给予630nm红光照射(功率100mv/cm2,总能量为2J/cm2);4.ALA-PDT组,1mM/L ALA避光孵育4h后给予630nm红光照射(功率100mv/cm2,总能量为2J/cm2);5.mTOR阻断剂雷帕霉素组,给予浓度为100nmol/L的雷帕霉素处理细胞2h;6.雷帕霉素联合ALA-PDT组(联合治疗组),先应用ALA-PDT处理细胞后再给予100nmol/L雷帕霉素干预细胞。各组给予相应处理后,应用CCK-8检测各组A431细胞干预后12h,24h,48h的光密度值,计算各组A431细胞的存活率;选择Annexin V-FITC/PI双染流式细胞术检测各组A431细胞干预后12h,24h,48h的凋亡率;采用Western blot检测各组A431细胞干预后48h mTOR、活化的mTOR——磷酸化mTOR(Ser2448)及mTOR下游蛋白p70S6K的活化形式——磷酸化p70S6K(Thr389)蛋白产物的表达。数据分析采用SPSS 20.0统计软件,结果采用?x±s表示。多组间比较采用单因素方差分析,组间多重比较采用LSD-t检验,不同时间段均数之间比较采用重复测量选择。以α=0.05为检验标准,P0.05为差异有统计学意义。结果:1.CCK-8检测结果显示,联合治疗组干预细胞后12h,24h,48h细胞的存活率为45.28±0.90%,40.11±3.62%及36.98±2.06%,明显低于其余各组(P0.05),且在12-48h时限范围内呈时效关系(P0.05)。ALA-PDT干预细胞后12h,24h,48h细胞的存活率为51.76±1.74%,47.50±5.34%,42.43±2.22%,明显低于空白对照组、单纯ALA组(89.59±2.13%,87.88±3.87%,80.76±5.16%)及单纯光照组(91.14±4.45%,86.93±4.45%,81.90±3.00%)(P均0.05);雷帕霉素干预细胞后12h,24h,48h细胞存活率为53.00±6.59%,47.08±3.96%,41.31±2.21%,明显低于空白对照组(P均0.05)。2.流式细胞术发现,ALA-PDT作用于细胞后12h,24h,48h细胞的凋亡率为16.43±0.86%、19.37±0.85%、21.76±0.58%,较空白对照组(6.11±0.12%、6.36±0.15%、7.26±0.16%)、单纯ALA组(6.73±0.36%、7.46±0.10%、8.92±0.39%)、单纯光照组(7.03±0.18%、8.02±0.19%、7.73±1.58%)明显增高(P0.05)。雷帕霉素作用于细胞后,细胞的凋亡率为10.21±0.20%、10.64±0.54%、16.42±0.68%,也高于空白对照组,但差异无统计学意义(P0.05)。联合治疗组干预细胞后12h,24h,48h细胞的凋亡率为26.23±1.14%、32.53±1.58%、45.03±1.82%,明显高于其余各组,且在48h时细胞凋亡率最高,差异均有统计学意义(P0.05)。3.Western blot结果显示ALA-PDT作用于细胞48h后mTOR、p-mTOR(Ser2448)、p-p70S6K(Thr389)表达量分别为0.578±0.013、0.592±0.019、0.285±0.017,低于空白对照组(0.780±0.037、0.707±0.016、0.489±0.092)、单纯ALA组(0.811±0.018、0.717±0.090、0.482±0.011)及单纯光照组(0.768±0.016、0.700±0.020、0.473±0.010)(P均0.05);雷帕霉素作用于细胞48h后mTOR、p-mTOR(Ser2448)、p-p70S6K(Thr389)表达量分别为0.479±0.021、0.361±0.010、0.255±0.084,低于空白对照组(P均0.05);联合治疗组作用于细胞后48h mTOR、磷酸化mTOR(Ser2448)、磷酸化p70S6K(Thr389)表达量分别为0.230±0.016、0.191±0.088、0.175±0.009,低于其余各组(P均0.05)。结论:1.ALA-PDT作用于A431皮肤鳞状细胞癌细胞后可抑制A431细胞增殖,促进细胞凋亡。2.mTOR信号通路抑制剂雷帕霉素作用于A431皮肤鳞状细胞癌细胞后可抑制A431细胞增殖。3.ALA-PDT联合雷帕霉素作用于A431后与ALA-PDT相比可增强对A431细胞的杀伤作用,提示阻断mTOR信号通路可能成为增强ALA-PDT杀伤A431细胞新的治疗靶点。
[Abstract]:Background and objective: the incidence of squamous cell carcinoma of the skin is second in the malignant tumor of the skin. It is characterized by high invasiveness and great destruction. This disease mainly occurs in the exposed parts of the elderly. The cause of the disease is mostly associated with ultraviolet radiation, chemical factors, and virus infection. The operation is a traditional treatment for the treatment of squamous cell carcinoma of the skin. But for some patients with poor physical tolerance and specific skin lesions, photodynamic therapy (PDT) is the first treatment for the treatment of squamous cell carcinoma of the skin..5- aminolevulinic acid photodynamic therapy (ALA-PDT) is a new therapy for the treatment of squamous cell carcinoma of the skin. Its advantages are small traumatic, no scar after treatment and no effect on beauty. But the deficiency is that the depth of ALA-PDT treatment is limited and the effect is not good for invasive squamous cell carcinoma of the skin. Mammalian rapamycin (mTOR) is a serine / threonine kinase, which plays an important role in regulating cell growth and proliferation. Abnormal activation is closely related to the development and development of various malignant tumors. MTOR is highly expressed in squamous cell carcinoma of the skin. Rapamycin is a macrolide immunosuppressant extracted from hygroscopic Streptomyces fermentation broth. Rapamycin and its derivatives are the first generation mTOR inhibitors, which have good antitumor activity. This experiment was conducted by using ALA-PDT combined with rapamycin in skin squamous cells. The cell proliferation and apoptosis were observed after A431, and the effect and mechanism of inhibiting the mTOR signaling pathway in ALA-PDT killing A431 cells were investigated to provide a new laboratory basis for enhancing the therapeutic effect of ALA-PDT in the treatment of squamous cell carcinoma of the skin. Methods: the logarithmic growth phase of A431 cells were divided into 6 groups, respectively: the blank control group, which were not used as the control group. 2. ALA group, only group ALA, only given 1mM/LALA to incubate 4H; 3. simple light group, only 630nm red light irradiation (power 100mv/cm2, total energy is 2J/cm2), 4.ALA-PDT group, 1mM/L ALA avoiding light incubating 4h after 4H to give 630nm red light irradiation (power 100mv/cm2, total energy); blocking agent rapamycin group, concentration for the concentration of 2H was treated with rapamycin, 6. rapamycin combined with ALA-PDT group (combined treatment group), and then ALA-PDT treated cells were first treated with 100nmol/L rapamycin. After corresponding treatment, CCK-8 was used to detect 12h, 24h, and 48h values of A431 cells in each group. The survival rate of A431 cells in each group was calculated and Annexin V was selected. -FITC/PI double dye flow cytometry was used to detect the apoptosis rate of 12h, 24h and 48h in each group of A431 cells. Western blot was used to detect the 48h mTOR, mTOR phosphorylation mTOR and the expression of phosphorylated protein products. The data analysis adopted 20 .0 statistical software, the results were x + s. Single factor analysis of variance was used in multiple groups, LSD-t test was used for multiple comparison between groups, and repeated measurements were used in different time periods. P0.05 was statistically significant with alpha =0.05 as the test standard. Results: the results of 1.CCK-8 test showed that the combined treatment group was 1 after the intervention of the cells. The survival rate of 2h, 24h and 48h cells was 45.28 + 0.90%, 40.11 + 3.62% and 36.98 + 2.06%, obviously lower than the other groups (P0.05), and the survival rate of 12h, 24h, 48h cells was 51.76 + 1.74%, 47.50 + 5.34%, 42.43 + 2.22%, in the 12-48h time limit, which was significantly lower than that in the blank control group (89.59 + 2.13). %, 87.88 + 3.87%, 80.76 + 5.16%) and simple light group (91.14 + 4.45%, 86.93 + 4.45%, 81.90 + 3%) (P mean 0.05). The survival rate of 12h, 24h, and 48h cells was 53 + 6.59% after the intervention of rapamycin, which was significantly lower than that in the blank control group (P all).2. flow cytometry, and ALA-PDT acts on 12h, 24h, 48h cells after cell. The rate of apoptosis was 16.43 + 0.86%, 19.37 + 0.85%, 21.76 + 0.58%, compared with the blank control group (6.11 + 0.12%, 6.36 + 0.15%, 7.26 + 0.16%). The simple ALA group was significantly higher (P0.05). 0.54%, 16.42 + 0.68%, but also higher than the blank control group, but the difference was not statistically significant (P0.05). The apoptosis rate of 12h, 24h and 48h cells in the combined treatment group was 26.23 + 1.14%, 32.53 + 1.58%, 45.03 + 1.82%, obviously higher than the other groups, and the apoptosis rate was the highest at 48h, the difference was statistically significant (P0.05).3.Western blot results showed significant results. The expression of mTOR, p-mTOR (Ser2448) and p-p70S6K (Thr389) was 0.578 + 0.013,0.592 + 0.019,0.285 + 0.017 after 48h, which was lower than that in the blank control group (0.780 + 0.037,0.707 + 0.092), simple ALA group (0.811 + 0.811 + 0.011) and simple light group (0.768 + 0.011 0.010). The expression of rapamycin, mTOR, p-mTOR (Ser2448) and p-p70S6K (Thr389) after 48h was 0.479 + 0.021,0.361 + 0.010,0.255 + 0.084 respectively, was lower than that in the blank control group (P 0.05), and the combined treatment group acted on 48h mTOR, phosphorylated mTOR (0.230 + + 0.088), respectively. 0.175 + 0.009, lower than the other groups (P 0.05). Conclusion: 1.ALA-PDT can inhibit the proliferation of A431 cells after the action of A431 skin squamous cell carcinoma cells, and promote the effect of rapamycin on A431 skin squamous cell carcinoma cells of.2.mTOR signaling pathway to inhibit A431 cell proliferation.3.ALA-PDT combined with rapamycin on A431 after A431. Compared with ALA-PDT, it can enhance the killing effect on A431 cells, suggesting that blocking mTOR signaling pathway may become a new therapeutic target for ALA-PDT to kill A431 cells.
【学位授予单位】：承德医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.5

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