本文選題：5-氨基酮戊酸 + 光動力療法��； 參考：《承德醫(yī)學院》2017年碩士論文

【摘要】：背景及目的:皮膚鱗狀細胞癌發(fā)病率在皮膚惡性腫瘤中位居第二,具有侵襲性較高、破壞性較大等特點。本病主要發(fā)生于老年人的暴露部位,發(fā)病原因多與紫外線照射、化學因素、病毒感染等有關(guān)。目前手術(shù)為治療皮膚鱗狀細胞癌的傳統(tǒng)療法,但對于一些身體耐受性差、皮損部位特殊的患者,光動力療法(PDT)則為治療皮膚鱗狀細胞癌的首選治療方法。5-氨基酮戊酸-光動力療法(ALA-PDT)是一種治療皮膚鱗狀細胞癌的新型療法,其優(yōu)點為創(chuàng)傷性小,治療后不留疤痕、不影響美觀等特點,但不足之處是ALA-PDT治療深度有限,對侵襲性皮膚鱗狀細胞癌效果不佳。哺乳動物雷帕霉素蛋白(mTOR)是一種絲氨酸/蘇氨酸激酶,在調(diào)節(jié)細胞生長與增殖方面起著重要作用,異常激活與多種惡性腫瘤的發(fā)生發(fā)展密切相關(guān)。研究表明mTOR在皮膚鱗狀細胞癌中呈高表達狀態(tài)。雷帕霉素是提取于吸水性鏈霉菌發(fā)酵液的一種大環(huán)內(nèi)酯類免疫抑制劑,雷帕霉素及其衍生物為第一代mTOR抑制劑,具有很好的抗腫瘤活性。本實驗通過采取ALA-PDT聯(lián)合雷帕霉素作用于皮膚鱗狀細胞癌細胞株A431后觀察細胞增殖與凋亡情況,探討抑制mTOR信號通路在ALA-PDT殺傷A431細胞中的作用及機制,為增強ALA-PDT治療皮膚鱗狀細胞癌的療效提供新的實驗室依據(jù)。方法:將對數(shù)生長期的A431細胞分為6個組,分別為:1.空白對照組,不做任何特殊處理;2.單純ALA組,僅給予1mM/LALA避光孵育4h;3.單純光照組,僅給予630nm紅光照射(功率100mv/cm2,總能量為2J/cm2);4.ALA-PDT組,1mM/L ALA避光孵育4h后給予630nm紅光照射(功率100mv/cm2,總能量為2J/cm2);5.mTOR阻斷劑雷帕霉素組,給予濃度為100nmol/L的雷帕霉素處理細胞2h;6.雷帕霉素聯(lián)合ALA-PDT組(聯(lián)合治療組),先應用ALA-PDT處理細胞后再給予100nmol/L雷帕霉素干預細胞。各組給予相應處理后,應用CCK-8檢測各組A431細胞干預后12h,24h,48h的光密度值,計算各組A431細胞的存活率;選擇Annexin V-FITC/PI雙染流式細胞術(shù)檢測各組A431細胞干預后12h,24h,48h的凋亡率;采用Western blot檢測各組A431細胞干預后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)蛋白產(chǎn)物的表達。數(shù)據(jù)分析采用SPSS 20.0統(tǒng)計軟件,結(jié)果采用?x±s表示。多組間比較采用單因素方差分析,組間多重比較采用LSD-t檢驗,不同時間段均數(shù)之間比較采用重復測量選擇。以α=0.05為檢驗標準,P0.05為差異有統(tǒng)計學意義。結(jié)果:1.CCK-8檢測結(jié)果顯示,聯(lián)合治療組干預細胞后12h,24h,48h細胞的存活率為45.28±0.90%,40.11±3.62%及36.98±2.06%,明顯低于其余各組(P0.05),且在12-48h時限范圍內(nèi)呈時效關(guān)系(P0.05)。ALA-PDT干預細胞后12h,24h,48h細胞的存活率為51.76±1.74%,47.50±5.34%,42.43±2.22%,明顯低于空白對照組、單純ALA組(89.59±2.13%,87.88±3.87%,80.76±5.16%)及單純光照組(91.14±4.45%,86.93±4.45%,81.90±3.00%)(P均0.05);雷帕霉素干預細胞后12h,24h,48h細胞存活率為53.00±6.59%,47.08±3.96%,41.31±2.21%,明顯低于空白對照組(P均0.05)。2.流式細胞術(shù)發(fā)現(xiàn),ALA-PDT作用于細胞后12h,24h,48h細胞的凋亡率為16.43±0.86%、19.37±0.85%、21.76±0.58%,較空白對照組(6.11±0.12%、6.36±0.15%、7.26±0.16%)、單純ALA組(6.73±0.36%、7.46±0.10%、8.92±0.39%)、單純光照組(7.03±0.18%、8.02±0.19%、7.73±1.58%)明顯增高(P0.05)。雷帕霉素作用于細胞后,細胞的凋亡率為10.21±0.20%、10.64±0.54%、16.42±0.68%,也高于空白對照組,但差異無統(tǒng)計學意義(P0.05)。聯(lián)合治療組干預細胞后12h,24h,48h細胞的凋亡率為26.23±1.14%、32.53±1.58%、45.03±1.82%,明顯高于其余各組,且在48h時細胞凋亡率最高,差異均有統(tǒng)計學意義(P0.05)。3.Western blot結(jié)果顯示ALA-PDT作用于細胞48h后mTOR、p-mTOR(Ser2448)、p-p70S6K(Thr389)表達量分別為0.578±0.013、0.592±0.019、0.285±0.017,低于空白對照組(0.780±0.037、0.707±0.016、0.489±0.092)、單純ALA組(0.811±0.018、0.717±0.090、0.482±0.011)及單純光照組(0.768±0.016、0.700±0.020、0.473±0.010)(P均0.05);雷帕霉素作用于細胞48h后mTOR、p-mTOR(Ser2448)、p-p70S6K(Thr389)表達量分別為0.479±0.021、0.361±0.010、0.255±0.084,低于空白對照組(P均0.05);聯(lián)合治療組作用于細胞后48h mTOR、磷酸化mTOR(Ser2448)、磷酸化p70S6K(Thr389)表達量分別為0.230±0.016、0.191±0.088、0.175±0.009,低于其余各組(P均0.05)。結(jié)論:1.ALA-PDT作用于A431皮膚鱗狀細胞癌細胞后可抑制A431細胞增殖,促進細胞凋亡。2.mTOR信號通路抑制劑雷帕霉素作用于A431皮膚鱗狀細胞癌細胞后可抑制A431細胞增殖。3.ALA-PDT聯(lián)合雷帕霉素作用于A431后與ALA-PDT相比可增強對A431細胞的殺傷作用,提示阻斷mTOR信號通路可能成為增強ALA-PDT殺傷A431細胞新的治療靶點。
[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.
【學位授予單位】：承德醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R739.5

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