调控mTOR信号通路对婴幼儿血管瘤血管内皮细胞的影响

发布时间：2018-09-09 09:10

【摘要】：目的探讨m TOR信号通路在血管瘤消长中的作用,为临床以PI3K/AKT/m TOR信号通路及AMPK/m TOR信号通路为靶点治疗婴幼儿血管瘤提供实验参考。方法1.收集我院2008-2015年间存档的术前均未做任何辅助性治疗且病理诊断为小儿毛细血管瘤的病理标本50例,结合Mulliken分类法与Ki-67表达情况将标本进行分类和分期,免疫组织化学法检测并比较AMPK及m TOR在血管瘤增生期和消退期中的表达情况。2.手术切取婴幼儿增生期血管瘤组织,采用组织块法分离培养血管瘤血管内皮细胞,待单层细胞铺满培养瓶底75%以上后,去掉组织块,将细胞消化后予以传代培养。取第四代细胞予以第Ⅷ凝血因子相关抗原鉴定。3.取对数生长期细胞,运用无血清培养液同步化孵育24h,对照组加入完全培养基4m L、二甲双胍组加入含20mmol/L二甲双胍的培养液4m L、NVP-BEZ235组加入含0.5μmol/LNVP-BEZ235的培养液4ml、联合组加入含二甲双胍与NVP-BEZ235的培养液(培养液中二甲双胍终浓度为20mmol/L,NVP-BEZ235终浓度为0.5μmol/L)4ml,孵育24h后,流式细胞仪检测细胞凋亡情况,Western blot法检测血管瘤血管内皮细胞中AMPK、m TOR及p70s6k表达情况,比较各组AMPK、m TOR及p70s6k表达差异,分析血管瘤血管内皮胞凋亡与m TOR信号通路的关系。结果1.24例增生期血管瘤AMPK、m TOR的积分光密度分别为33.94±12.78,182.43±47.10;26例退化期血管瘤AMPK、m TOR的积分光密度分别为85.87±19.53,47.22±44.52。增生期血管瘤AMPK的积分光密度明显低于消退期,差异有显著性(t=11.02,P0.01);增生期血管瘤m TOR的积分光密度明显高于消退期,差异有显著性(t=10.44,P0.01);AMPK与m TOR的表达呈负相关关系(r=-0.78,P0.01)。2.血管瘤血管内皮细胞体外培养过程中,第一周见少量梭形细胞从细胞岛边缘爬出,第二周见细胞数量逐渐增加,且围绕细胞岛向周围呈放射状生长,第三周细胞开始不均匀分布生长,第四周见细胞铺满培养瓶底部。倒置相差显微镜下见单个细胞呈梭形或不规则多边形,细胞聚集处呈铺路石样或管形排列。第Ⅷ凝血因子相关抗原呈阳性表达。3.细胞总凋亡率:对照组为1.45±0.27,二甲双胍组为11.98±1.00,NVP-BEZ235组为16.12±1.88,联合组为25.96±0.96;二甲双胍组、NVP-BEZ235组、联合组与对照组比较,差异均有显著性(t=17.30、13.13、42.49,P0.01)。联合组与二甲双胍组、NVP-BEZ235组比较,差异均有显著性(t=3.90、8.17,P0.01)。二甲双胍组与NVP-BEZ235组比较,差异均有显著性(t=-18.53,P0.01)。4.AMPK表达情况:对照组为0.43±0.23,二甲双胍组为3.97±0.40,NVP-BEZ235组为0.55±0.23,联合组为3.99±1.10;二甲双胍组、联合组与照组比较,差异均有显著性(t=13.32、5.49,P0.01);二甲双胍组与NVP-BEZ235组比较,差异均有显著性(t=12.91,P0.01);联合组与NVP-BEZ235组比较,差异均有显著性(t=5.3,P0.01);NVP-BEZ235组与对照组比较,联合组与二甲双胍组比较,差异均无统计学意义(t=0.66、0.03,P0.05)。5.m TOR表达情况:对照组为6.35±1.81,二甲双胍组为2.47±0.69,NVP-BEZ235组为1.46±0.27,联合组为0.40±0.21;二甲双胍组、NVP-BEZ235组及联合组与对照组比较,差异均有统计学意义(t=-3.47、-4.63、-5.66,P0.05);联合组与二甲双胍组、NVP-BEZ235组比较,差异均有统计学意义(t=-4.99、-5.35,P0.05);二甲双胍组与NVP-BEZ235组比较,差异无统计学意义(t=2.37,P0.05)。6.p70S6K表达情况:对照组为0.65±0.22,二甲双胍组为2.49±0.19,NVP-BEZ235组为3.98±0.55,联合组为13.95±1.16;二甲双胍组、NVP-BEZ235组、联合组与对照组比较,差异均有显著性(t=11、9.78、19.54,P0.01);联合组与二甲双胍组、NVP-BEZ235组比较,差异均有显著性(t=16.9、13.46,P0.01);二甲双胍组与NVP-BEZ235组比较,差异具有统计学意义(t=-4.45,P0.05)。结论1.AMPK在血管瘤消退期表达高于增生期,m TOR在消退期表达低于增生期。2.激活AMPK或抑制PI3K均可使m TOR信号通路受抑,诱导体外培养血管瘤血管内皮细胞凋亡,尤以同时激活AMPK和抑制PI3K时效果最为明显。
[Abstract]:Objective To investigate the role of M TOR signaling pathway in the growth and decline of hemangioma, and to provide experimental reference for the clinical treatment of infantile hemangioma with PI3K/AKT/m TOR signaling pathway and AMPK/m TOR signaling pathway as targets. Methods 1. Collected from our hospital between 2008 and 2015, there was no preoperative adjuvant treatment and pathological diagnosis of infantile hemangioma. 50 cases of pathological specimens were classified and staged according to Mulliken's classification and Ki-67 expression. The expression of AMPK and m TOR in the proliferative and regressive stages of hemangioma was detected by immunohistochemistry. 2. The hemangioma tissues of infants and young children were excised and cultured by tissue block method. After the monolayer cells were covered with 75% or more of the bottom of the flask, the tissues were removed and digested and subcultured. The fourth generation cells were identified by factor_coagulation associated antigen. 3. The logarithmic growth phase cells were incubated in serum-free medium for 24 hours. The control group was added with 4 mL of complete medium, and the metformin group was added with 20 mmol/L. Metformin culture medium 4mL, NVP-BEZ235 group added 0.5 micromol/LNVP-BEZ235 culture medium 4ml, combined group added Metformin and NVP-BEZ235 culture medium (the final concentration of metformin in culture medium is 20 mmol/L, NVP-BEZ235 final concentration is 0.5 micromol/L) 4ml, 24 hours after incubation, flow cytometry detection of apoptosis, Western blot detection The expressions of AMPK, m TOR and P70S6K in hemangioma endothelial cells were compared, and the relationship between apoptosis of hemangioma endothelial cells and m TOR signaling pathway was analyzed. The integral optical density of AMPK in proliferative hemangioma was significantly lower than that in regressive hemangioma (t = 11.02, P 0.01); the integral optical density of M TOR in proliferative hemangioma was significantly higher than that in regressive hemangioma (t = 10.44, P 0.01); the expression of AMPK was negatively correlated with that of M TOR (r = - 0.78, P 0.0). 1) 2. During the in vitro culture of hemangioma endothelial cells, a small number of spindle cells emerged from the edge of the cell island in the first week, and the number of cells increased gradually in the second week. The cells grew radially around the cell island. At the third week, the cells began to grow unevenly, and at the fourth week, the cells covered the bottom of the culture bottle. The total cell apoptosis rate was 1.45 (+ 0.27) in the control group, 11.98 (+ 1.00) in the metformin group, 16.12 (+ 1.88) in the NVP-BEZ235 group, and 25.96 (+ 0.96) in the combined group; in the metformin group, the NVP-BEZ235 group, the conjunction group. Compared with the control group, the difference was significant (t = 17.30, 13.13, 42.49, P 0.01). Compared with metformin group, NVP-BEZ235 group, the difference was significant (t = 3.90, 8.17, P 0.01). Compared with the NVP-BEZ235 group, the difference was significant (t =-18.53, P 0.01). 4. AMPK expression in the control group was 0.43 + 0.23, and the metformin group was 0.43 + 0.23. There were significant differences between the combined group and the control group (t = 13.32, 5.49, P 0.01), the metformin group and the NVP-BEZ235 group (t = 12.91, P 0.01), and the combined group and the NVP-BEZ235 group (t = 5.3, P 0.01). Compared with the control group, there was no significant difference between the combined group and the metformin group (t = 0.66, 0.03, P 0.05). 5. MTR expression: the control group was 6.35 (+ 1.81), the metformin group was 2.47 (+ 0.69), the NVP-BEZ235 group was 1.46 (+ 0.27), the combined group was 0.40 (+ 0.21); the metformin group, the NVP-BEZ235 group and the combined group were all different from the control group. Significance of accounting (t = - 3.47, - 4.63, - 5.66, P 0.05); There were significant differences between the combined group and the metformin group, NVP-BEZ235 group (t = - 4.99, - 5.35, P 0.05); there was no significant difference between the metformin group and the NVP-BEZ235 group (t = 2.37, P 0.05). 6. p70S6K expression: the control group was 0.65 + 0.22, the metformin group was 2.49 + 0.19, and the NVP-BEZ235 group was 2.49 + 0.19, NVP-BEZ235. Metformin group, NVP-BEZ235 group, combined group and control group, the difference was significant (t = 11, 9.78, 19.54, P 0.01); combined group and metformin group, NVP-BEZ235 group, NVP-BEZ235 group, the difference was significant (t = 16.9, 13.46, P 0.01); metformin group and NVP-BEZ235 group, the difference was statistically significant (t = 16.9, 13.46, P 0.01); metformin group and NVP-BEZ235 group, the difference was statistically significant. (t = - 4.45, P 0.05). Conclusion 1. The expression of AMPK is higher in the regressive phase than in the proliferative phase, and the expression of MTOR is lower in the regressive phase than in the proliferative phase.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R732.2

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