本文選題：自噬 + 凋亡�。� 參考：《天津醫(yī)科大學》2017年碩士論文

【摘要】：研究目的:本研究通過應用自噬誘導劑雷帕霉素(RAPA)聯(lián)合順鉑(DDP)檢測其對舌鱗狀細胞癌p160細胞凋亡、增殖、遷移能力的影響,結(jié)合自噬相關蛋白MAP1LC3、PTEN、m TOR在人舌癌中表達及其與舌癌臨床病理特征間的相關性,探討自噬相關m TOR信號通路與凋亡的可能相關機制,旨在為自噬相關蛋白及通路作為舌癌治療的新靶點提供理論依據(jù)。研究方法:1、應用CCK-8法檢測不同濃度RAPA、DDP作用于舌癌p160細胞不同時間后的細胞生長抑制率,并計算DDP半數(shù)抑制濃度(IC50),篩選RAPA藥物濃度,檢測RAPA、DDP、RAPA+DDP分別作用于p160細胞24 h后細胞生長抑制情況;2、應用Western-blot法檢測不同濃度RAPA作用于p160細胞后其自噬相關蛋白LC3的表達情況;3、應用流式細胞儀檢測RAPA、DDP、RAPA+DDP分別作用于p160細胞24 h后細胞的凋亡情況;4、應用劃痕實驗檢測RAPA、DDP、RAPA+DDP分別作用于p160細胞6 h、18 h、24 h后細胞的遷移距離;5、隨機選取2011年1月-2015年1月期間于天津市口腔醫(yī)院行手術切除的人舌鱗狀細胞癌石蠟標本46例、癌旁組織13例、癌前病變組織13例,轉(zhuǎn)移淋巴結(jié)標本39例,應用免疫組織化學染色法檢測MAP1LC3、PTEN、m TOR蛋白的表達;并分析其表達水平與各臨床病理指標間的相關性及其相互之間的關系。研究結(jié)果:1、RAPA單純作用于p160細胞時,24 h及48 h時:細胞生長表現(xiàn)為先增加后抑制;72 h時:細胞生長受到抑制,且隨濃度增加而增加。RAPA作用于p160細胞后,低濃度時起保護作用,高濃度時起抑制作用,而且,低濃度作用時間足夠長時,也可引起自噬性死亡;2、DDP單純作用于p160細胞時,細胞生長受到抑制,且隨時間及濃度的增加而增加,具有時間及藥物濃度依賴性;3、采用重復測量方差分析及LSD-t兩兩比較,CCK-8法檢測對照組、RAPA組、DDP組、RAPA+DDP組四組不同加藥實驗組細胞活力,對照組及RAPA組細胞活力無差別,DDP組及DDP+RAPA組細胞出現(xiàn)生長抑制,且DDP+RAPA組(OD值=0.83±0.13)細胞活力明顯小于DDP組(OD值=1.14±0.16,P=0.008);4、流式細胞儀檢測四組細胞凋亡情況,結(jié)果顯示對照組及RAPA組細胞基本無凋亡,DDP組存在明顯凋亡(22.3%),RAPA+DDP聯(lián)合用藥組細胞凋亡則更加明顯,由22.3%增加至47.0%,且主要表現(xiàn)為早期凋亡細胞的增加;5、采取劃痕實驗比較對照組、RAPA組、DDP組、DDP+RAPA四組之間細胞在6 h、18 h、24 h時與0 h遷移距離差值的大小,結(jié)果顯示:6 h后對照組的劃痕距離為0.56±0.07 cm,RAPA組劃痕距離為0.54±0.09 cm,DDP組劃痕距離為0.18±0.14 cm,DDP+RAPA組劃痕距離為0.09±0.07 cm;18 h后對照組的劃痕距離為0.86±0.18 cm,RAPA組劃痕距離為0.84±0.11 cm,DDP組劃痕距離為0.52±0.23cm,DDP+RAPA組劃痕距離為0.42±0.05 cm;24 h后對照組的劃痕距離為1.29±0.21 cm,RAPA組劃痕距離為1.16±0.04 cm,DDP組劃痕距離為0.64±0.26 cm,DDP+RAPA組劃痕距離為0.54±0.03 cm。采用方差分析及多個樣本均數(shù)兩兩比較,DDP組及RAPA+DDP組遷移距離與對照組相比,差異均有統(tǒng)計學意義(P0.05),而三個時間段中,只有24 h時,DDP組與RAPA+DDP組的遷移距離差值相比,差異有統(tǒng)計學意義(P0.05);6、免疫組化結(jié)果顯示:PTEN、LC3、m TOR主要表達于細胞漿,46例舌癌病灶標本中,PTEN在舌癌組織中的表達率(34.78%)低于癌前病變及癌旁組織中的陽性表達(69.23%,76.92%,P0.05);MAP1LC3在舌癌組織中的陽性表達率(28.26%)低于癌前病變及癌旁組織中的陽性表達(61.53%,69.23%,P0.05);m TOR在舌癌組織中的表達率(65.22%)高于癌前病變及癌旁組織中的陽性表達(23.08%,31.77%,P0.05)。卡方檢驗顯示:PTEN、MAP1LC3、m TOR在年齡、性別、吸煙、飲酒、組織學類型中的表達差異均無統(tǒng)計學意義(P(29)0.05);MAP1LC3、PTEN及m TOR的表達與舌癌的有無淋巴結(jié)轉(zhuǎn)移、臨床分期有關(P0.05),隨著淋巴結(jié)轉(zhuǎn)移、臨床分期越晚,PTEN及LC3的陽性表達率降低,m TOR的陽性表達率越高,差異有統(tǒng)計學意義(P0.05)。結(jié)論:1、雷帕霉素可誘導細胞發(fā)生自噬,一定程度的自噬具有細胞保護作用,而過度自噬則可引起細胞凋亡,且這種凋亡可能為自噬性死亡;2、雷帕霉素可能作為化療增敏劑應用于臨床,仍需動物實驗及臨床試驗進一步研究;3、PTEN、LC3、m TOR蛋白表達水平與患者的臨床分期、淋巴結(jié)轉(zhuǎn)移有關,PTEN、LC3蛋白低表達、m TOR蛋白高表達時提示可能預后不良;4、m TOR信號通路參與細胞自噬過程,且與細胞凋亡相關,可以作為舌癌治療新的研究方向及靶點。
[Abstract]:Objective: To investigate the effect of autophagic inducer rapamycin (RAPA) combined with cisplatin (DDP) on the apoptosis, proliferation and migration of p160 cells in tongue squamous cell carcinoma, the expression of autophagy related protein MAP1LC3, PTEN, m TOR in human tongue cancer and its correlation with the clinicopathological features of tongue cancer, and to explore the autophagy related TOR letter of M The possible mechanism of number pathway and apoptosis is designed to provide a theoretical basis for the autophagy related proteins and pathways as a new target for the treatment of tongue cancer. 1, the CCK-8 method was used to detect the growth inhibition rate of different concentrations of RAPA, DDP on the p160 cells of tongue cancer after different time, and to calculate the DDP half inhibitory concentration (IC50), and to screen the RAPA drugs. Concentration, detection of RAPA, DDP, RAPA+DDP respectively on the cell growth inhibition of p160 cells after 24 h; 2, Western-blot method was used to detect the expression of autophagy related protein LC3 in p160 cells with different concentrations of RAPA; 3, using flow cytometry to detect RAPA, DDP, RAPA+ cells, respectively, on the apoptosis of cells after 24 cells; 4 RAPA, DDP and RAPA+DDP were used to detect the migration distance of p160 cells 6 h, 18 h and 24 h, respectively. 5, 46 cases of human tongue squamous cell carcinoma, 13 cases of paracancerous tissue, 13 precancerous tissue and 39 metastatic lymph nodes, were selected at random in Tianjin Stomatological Hospital during January 2011 -2015 years. The expression of MAP1LC3, PTEN, m TOR protein was detected by immunohistochemical staining, and the correlation between the expression level and the clinicopathological indexes and the relationship between them were analyzed. The results were as follows: 1, when RAPA acts on p160 cells, 24 h and 48 h: the cell growth table is first increased after inhibition, and at 72 h: cell growth is inhibited when 72 h. And with the increase of concentration, the increase of.RAPA effect on p160 cells, the low concentration plays a protective role, the high concentration plays a inhibition effect, and the low concentration time is long enough, it can also cause autophagic death. 2, the cell growth is inhibited when DDP is acting on p160 cells, and increases with time and concentration, with time and medicine. 3, 3, using repeated measurement of variance analysis and LSD-t 22 comparison, CCK-8 method was used to detect the cell viability of the control group, group RAPA, DDP group and RAPA+DDP group, the cell viability of the control group and the RAPA group was not different, the cell viability of the DDP group and the DDP+RAPA group was inhibited, and the DDP+RAPA group (OD =0.83 + 0.13) cell vitality was obvious. Less than DDP group (=1.14 + 0.16, P=0.008); 4, flow cytometry was used to detect the apoptosis in four groups, the results showed that the cells in the control group and the RAPA group had no apoptosis, and the DDP group had obvious apoptosis (22.3%), and the apoptosis of the combined RAPA+DDP group was more obvious, from 22.3% to 47%, and the main expression was the increase of early apoptotic cells; 5, mining. The scratch test was taken to compare the difference between the control group, RAPA group, DDP group and DDP+RAPA four groups in the distance difference between the 6 h, 18 h, 24 h and 0 h. The results showed that the scratch distance of the control group was 0.56 + 0.07 cm after 6 h, the scratch distance of the RAPA group was 0.54 + 0.09 cm, the scratch distance of the DDP group was 0.18 + 0.14, and the distance of the scratch group was 0.09 + 0.07; The scratch distance of the H group was 0.86 + 0.18 cm, the scratch distance in the RAPA group was 0.84 + 0.11 cm, the scratch distance in the DDP group was 0.52 + 0.23cm, the scratch distance in the group DDP+RAPA was 0.42 + 0.05 cm, and the scratch distance of the control group was 1.29 + 0.21 cm, and the scratch distance of the RAPA group was 1.16 + 0.04 cm. For 0.54 + 0.03 cm., the migration distance between group DDP and RAPA+DDP group was statistically significant (P0.05) compared with the control group (P0.05), while the difference between group DDP and RAPA+DDP group was statistically significant (P0.05) in three time periods, and 6, immunohistochemical results showed P. TEN, LC3, m TOR were mainly expressed in cytoplasm. In 46 cases of tongue cancer, the expression rate of PTEN in tongue carcinoma (34.78%) was lower than that in precancerous lesions and adjacent tissues (69.23%, 76.92%, P0.05), and the positive expression rate of MAP1LC3 in tongue carcinoma (28.26%) was lower than that in precancerous lesions and paracancerous tissues (61.53%, 69.23%, P0). .05); the expression rate of M TOR in tongue carcinoma (65.22%) was higher than that in precancerous lesions and adjacent tissues (23.08%, 31.77%, P0.05). Chi square test showed that there was no significant difference in the expression of PTEN, MAP1LC3, m TOR in age, sex, smoking, drinking, and histology (P (29) 0.05); MAP1LC3, PTEN, and M. No lymph node metastasis, clinical staging (P0.05), with lymph node metastasis, the more late the clinical stage, the positive expression rate of PTEN and LC3 decreased, the positive expression rate of M TOR was higher, the difference was statistically significant (P0.05). Conclusion: 1, rapamycin can induce autophagy in cells, a certain degree of autophagy has cell protection, and excessive autophagy can be used. The apoptosis may be caused by autophagic death. 2, rapamycin may be used as a chemosensitizer in the clinic and still needs further study in animal and clinical trials. 3, the expression level of PTEN, LC3, m TOR protein is related to the clinical stages of the patients, the lymph node transfer, the low expression of PTEN, LC3 protein, and the high expression of M TOR protein. The prognosis may be poor. 4, the m TOR signaling pathway is involved in autophagy and is related to apoptosis. It can be used as a new research direction and target for the treatment of tongue cancer.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R739.86

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