细胞自噬与舌鳞状细胞癌生物学行为相关性的初步研究

发布时间：2018-05-22 11:39

本文选题：自噬 + 凋亡　；参考：《天津医科大学》2017年硕士论文

【摘要】：研究目的:本研究通过应用自噬诱导剂雷帕霉素(RAPA)联合顺铂(DDP)检测其对舌鳞状细胞癌p160细胞凋亡、增殖、迁移能力的影响,结合自噬相关蛋白MAP1LC3、PTEN、m TOR在人舌癌中表达及其与舌癌临床病理特征间的相关性,探讨自噬相关m TOR信号通路与凋亡的可能相关机制,旨在为自噬相关蛋白及通路作为舌癌治疗的新靶点提供理论依据。研究方法:1、应用CCK-8法检测不同浓度RAPA、DDP作用于舌癌p160细胞不同时间后的细胞生长抑制率,并计算DDP半数抑制浓度(IC50),筛选RAPA药物浓度,检测RAPA、DDP、RAPA+DDP分别作用于p160细胞24 h后细胞生长抑制情况;2、应用Western-blot法检测不同浓度RAPA作用于p160细胞后其自噬相关蛋白LC3的表达情况;3、应用流式细胞仪检测RAPA、DDP、RAPA+DDP分别作用于p160细胞24 h后细胞的凋亡情况;4、应用划痕实验检测RAPA、DDP、RAPA+DDP分别作用于p160细胞6 h、18 h、24 h后细胞的迁移距离;5、随机选取2011年1月-2015年1月期间于天津市口腔医院行手术切除的人舌鳞状细胞癌石蜡标本46例、癌旁组织13例、癌前病变组织13例,转移淋巴结标本39例,应用免疫组织化学染色法检测MAP1LC3、PTEN、m TOR蛋白的表达;并分析其表达水平与各临床病理指标间的相关性及其相互之间的关系。研究结果:1、RAPA单纯作用于p160细胞时,24 h及48 h时:细胞生长表现为先增加后抑制;72 h时:细胞生长受到抑制,且随浓度增加而增加。RAPA作用于p160细胞后,低浓度时起保护作用,高浓度时起抑制作用,而且,低浓度作用时间足够长时,也可引起自噬性死亡;2、DDP单纯作用于p160细胞时,细胞生长受到抑制,且随时间及浓度的增加而增加,具有时间及药物浓度依赖性;3、采用重复测量方差分析及LSD-t两两比较,CCK-8法检测对照组、RAPA组、DDP组、RAPA+DDP组四组不同加药实验组细胞活力,对照组及RAPA组细胞活力无差别,DDP组及DDP+RAPA组细胞出现生长抑制,且DDP+RAPA组(OD值=0.83±0.13)细胞活力明显小于DDP组(OD值=1.14±0.16,P=0.008);4、流式细胞仪检测四组细胞凋亡情况,结果显示对照组及RAPA组细胞基本无凋亡,DDP组存在明显凋亡(22.3%),RAPA+DDP联合用药组细胞凋亡则更加明显,由22.3%增加至47.0%,且主要表现为早期凋亡细胞的增加;5、采取划痕实验比较对照组、RAPA组、DDP组、DDP+RAPA四组之间细胞在6 h、18 h、24 h时与0 h迁移距离差值的大小,结果显示:6 h后对照组的划痕距离为0.56±0.07 cm,RAPA组划痕距离为0.54±0.09 cm,DDP组划痕距离为0.18±0.14 cm,DDP+RAPA组划痕距离为0.09±0.07 cm;18 h后对照组的划痕距离为0.86±0.18 cm,RAPA组划痕距离为0.84±0.11 cm,DDP组划痕距离为0.52±0.23cm,DDP+RAPA组划痕距离为0.42±0.05 cm;24 h后对照组的划痕距离为1.29±0.21 cm,RAPA组划痕距离为1.16±0.04 cm,DDP组划痕距离为0.64±0.26 cm,DDP+RAPA组划痕距离为0.54±0.03 cm。采用方差分析及多个样本均数两两比较,DDP组及RAPA+DDP组迁移距离与对照组相比,差异均有统计学意义(P0.05),而三个时间段中,只有24 h时,DDP组与RAPA+DDP组的迁移距离差值相比,差异有统计学意义(P0.05);6、免疫组化结果显示:PTEN、LC3、m TOR主要表达于细胞浆,46例舌癌病灶标本中,PTEN在舌癌组织中的表达率(34.78%)低于癌前病变及癌旁组织中的阳性表达(69.23%,76.92%,P0.05);MAP1LC3在舌癌组织中的阳性表达率(28.26%)低于癌前病变及癌旁组织中的阳性表达(61.53%,69.23%,P0.05);m TOR在舌癌组织中的表达率(65.22%)高于癌前病变及癌旁组织中的阳性表达(23.08%,31.77%,P0.05)。卡方检验显示:PTEN、MAP1LC3、m TOR在年龄、性别、吸烟、饮酒、组织学类型中的表达差异均无统计学意义(P(29)0.05);MAP1LC3、PTEN及m TOR的表达与舌癌的有无淋巴结转移、临床分期有关(P0.05),随着淋巴结转移、临床分期越晚,PTEN及LC3的阳性表达率降低,m TOR的阳性表达率越高,差异有统计学意义(P0.05)。结论:1、雷帕霉素可诱导细胞发生自噬,一定程度的自噬具有细胞保护作用,而过度自噬则可引起细胞凋亡,且这种凋亡可能为自噬性死亡;2、雷帕霉素可能作为化疗增敏剂应用于临床,仍需动物实验及临床试验进一步研究;3、PTEN、LC3、m TOR蛋白表达水平与患者的临床分期、淋巴结转移有关,PTEN、LC3蛋白低表达、m TOR蛋白高表达时提示可能预后不良;4、m TOR信号通路参与细胞自噬过程,且与细胞凋亡相关,可以作为舌癌治疗新的研究方向及靶点。
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.86

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