【作者】 吕欣；

【导师】 俞松；

【作者基本信息】 遵义医学院 ， 小儿外科， 2010， 硕士

【摘要】 目的探讨mTOR/p70S6K信号通路在小儿血管瘤的发生、发展及消退过程中的作用。方法1.收集遵义医学院附属医院2005年-2009年未经其他治疗、单纯行手术切除并经病理诊断为血管瘤的标本31例,结合Mulliken分类法与增殖细胞核抗原(PCNA)表达对血管瘤进行分类和分期,免疫组化检测并比较增生期血管瘤和消退期血管瘤组织中mTOR、p70S6K-α的表达水平。2.用组织块法培养血管瘤内皮细胞,待长成单层细胞以后传代,建立血管瘤血管内皮细胞系,观察其增殖特征,并用第Ⅷ凝血因子相关抗原进行鉴定。同步化培养血管瘤内皮细胞,采用Western blot法检测血管瘤内皮细胞p70S6K-α的表达水平,同期用流式细胞仪检测血管瘤血管内皮细胞周期的变化；并分析mTOR、p70S6K-α的表达水平与血管瘤血管内皮细胞周期的分布及细胞凋亡的相关性。3.待细胞处于对数生长期,换无血清培养液孵育48h使细胞同步化,然后分别加入0,雷帕霉素10nmol/L,继续孵育24h,细胞刮刀收集细胞,检测血管瘤内皮细胞mTOR、p70S6K-α的表达水平,同时检测血管瘤血管内皮细胞周期的分布。结果1.18例增殖期血管瘤mTOR、p70S6K-α的积分光密度分别为6336.48±1655.89,588.72±223.87；13例消退期血管瘤mTOR、p70S6K-α的积分光密度分别为846.22±297.09,3235.64±947.77；血管瘤增殖期p70S6K-α的积分光密度明显低于消退期,差异有显著性(P<0.01)；血管瘤增殖期mTOR的积分光密度明显高于消退期,差异有显著性(P<0.01)。2.培养的血管瘤内皮细胞于4天后开始贴壁生长,一周后只有少量细胞铺展贴壁生长,两周后开始生长分裂,三周后细胞开始快速生长,四周后细胞铺满板底。用相差倒置显微镜观察,发现所分离的内皮细胞呈上皮细胞形态。培养细胞第Ⅷ凝血因子相关抗原表达为阳性。3.mTOR蛋白表达水平较高,p70S6K-α蛋白表达水平较低时,处于G0/G1期的细胞较少(55.95±4.38)%；mTOR蛋白表达水平下降,p70S6K-α蛋白表达水平升高,处于Go/Gl期的细胞明显增多(77.86±8.18)%。4.雷帕霉素作用于体外培养血管瘤血管内皮细胞24小时后,细胞周期变阻滞在G1期,雷帕霉素作用前后差异具有显著性(P<0.01);mTOR蛋白表达水平下降(P<0.01)；p70S6K-α蛋白表达水平升高(P<0.01)。结论1.在血管瘤增生期mTOR高表达可能导致p70S6K-α的活化磷酸化,促进血管瘤血管内皮细胞由G0/G1进入S期,血管瘤呈快速增殖状态。而在血管瘤消退期,在某种机制作用下,mTOR表达减少,抑制了p70S6K-α的磷酸化,使细胞周期阻滞在G0/G1期,进而促进血管瘤血管内皮细胞凋亡,血管瘤表现出自然消退的趋向。2.体外培养的血管瘤内皮细胞中,mTOR高表达导致p70S6K的活化磷酸化,促进血管瘤血管内皮细胞由G0/G1进入S期,血管内皮细胞快速增殖；mTOR表达减少,抑制了p70S6K的磷酸化,细胞周期阻滞在G0/G1期,进而促进血管内皮细胞凋亡。3.雷帕霉素可将体外培养的血管瘤内皮细胞滞留于G0/G1。更多还原

【Abstract】 Objective To study the effect of mTOR/p70S6K signal pathways in the different phases of children hemangioma.Methods1.31 hemangioma specimens were classified by Mulliken’s classification and the expres-sion of proliferating cell nuclear antigen (PCNA).The expressions of PCNA, mTOR,p70S6K-a were detected by immunohistochemistry method.2. The hemangioma vascular endothelial cells which came from the sterile hemangioma were cultured by using the tissue culture method.The hemangioma vascular endothelial cell was identified by the immunohistochemistry technique observing the expression of the FactorⅧantigen which only existed in endothelial cells. Synchronized cultured endothelial cells,mTOR and p70S6K-a of them were detected by Western blot mean-while cell cycle were examined by flow cytometry technique,and then analysis of mTOR,p70S6K-a in expression level of hemangioma vascular endothelial cell cycle and apoptosis related.3. Repamycin was added to observed the effects on the expressions of mTOR,p70S6K-αand the cell cycle distribution with the hemangioma vascular endothelial cell.Results1.31 hemagioma specimens were classified as 18 hemagioma proliferating phase speci-mens,13 hemagioma involuting phase specimens.In the hemagioma proliferating ph-ase specimens, the IOD of mTOR,p70S6K-a expressed were 6336.48±1655.89, 588.72±223.87 respectively. In the hemagioma involuting phase specimens, the IOD of mTOR, p70S6K-a expressed were 846.22±297.09,3235.64±947.77 respectively. In the hemagioma proliferating phase specimens,the IOD of p70S6K-a expressed were obviously lower than the IOD in the proliferating phase specimens (P<0.01).In the hemagioma proliferating phase specimens,the IOD of mTOR expressed was obviously higher than the IOD in the involuting phase specimens (P<0.01).2. The hemangioma vascular endothelial cells migrated from the tissue to the postcultural after four days, only a small number of cells spread after one week adherent growth, growth and division after two weeks, three weeks after the cells began to grow fast, and around, the cells covered the bottom plate.Inverted phase contrast microscope and found that the isolated endothelial cultured epithelial cell morphology. Cultured cells of FactorⅧrelated antigen was positive.3. When higher expression level of mTOR protein, lower of p70S6K-a, the cells in G0/G1 phase were less (55.95±4.38)%,when mTOR protein expression level decreased,and p70S6K-a protein improved,cells in the G0/G1 phase increased (77.86±8.18)%.4. After rapamycin acts on the cultured hemangioma vascular endothelial cells for 24 hour, inducing G1 cell cycle arrest (P<0.01); the expression of mTOR protein decreased(P <0.01),the expression of p70S6K-αprotein rised(P<0.01).Conclusions1. mTOR high expressed in proliferation may lead to the phosphorylation of p70S6k, promot hemangiomas vascular endothelial cells from G0/G1 into S phase and show a rapid proliferation state. under the action of some mechanism, the expression of mTOR was decreased,which inhibited p70S6k phosphorylation,the cell cycle arrestted in G0/G1 phase, thus accelerated the apoptosis of hemangiomas vascular endothelial cells and the hemangioma vascular tumors demonstrated natural regres sion trend.2. In cultured vascular endothelial cells,the high expression of mTOR led to the phosphory-lation of p70S6k, promot hemangioma vascular endothelial cells from G0/G1 into S phase, the proliferation of endothelial cells was rapid,on the contrary.The expression of mTOR decreased, inhibited the phosphorylation of p70S6K,cell cycle was arrest in G1/G2 phase, promoting the apoptosis of vascular endothelial cells.3. Rapamycin might remain the cultured hemangioma vascular endothelial cells in the G0/G1 phase.更多还原