| Objective To investigate the role of m TOR signaling pathway in the growth and regression 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.Methods1.50 pathological specimens were collected in our hospital during 2008-2015,which were archived preoperatively without any adjuvant therapy and pathologically diagnosed as infantile capillary hemangioma,and were classified and staged by the classification of Mulliken combined with the expressin of Ki-67.The expression of AMPK and m TOR in both proliferative and regressive phase of these specimens were detected and compared by immunohistochemistry.2.Proliferative phase infant hemangioma specimens were obtained by operation,and these hemangioma endothelial cells were isolated by tissue explant method and cultured.Tissues were removed and cells were digested and subcultured after the single cells overspreaded more than 75% of the bottom of culture bottle.The fourth generation cells were identified by antigen of coagulation factor ?.3.The logarithmic growth phase cells were synchronously incubated with serum-free medium for 24 h,the control group was added 4m L complete medium,the merformin group was added 4m L culture medium containing metformin with a concentration of 20mmol/L,the NVP-BEZ235 group was added 4m L culture medium containing NVP-BEZ235 with a concertration of 0.5?mol/L,the combined group was added 4m L culture medium containing both metformin and NVP-BEZ235(the final concentration of metformin was 20mmol/L,that of NVPBEZ235 was 0.5?mol/L),after 24 h incubation,the cell apoptosis rate was detected by flow cytometrythe,and the expression of AMPK,m TOR and p70s6 k in the vascular endothelial cells was detected by Western blot.Differences of AMPK,m TOR and p70s6 k in each group were compared,and the relationship between the apoptosis and m TOR signaling pathway was analyzed.Results1.Twenty four cases of hemangioma specimens were classified as in proliferative phase,and their integral optical density of AMPK and m TOR were 33.94± 12.78 and 182.43± 47.10 respectively.While other twenty six specimens were in the involutive phase,their integral optical density of AMPK and m TOR were 85.87±19.53 and 47.22±44.52 respectively.Integral optical density of AMPK in the involutive phase hemangioma was significantly higher than that in the proliferative phase(t=11.02,P<0.01).Integral optical density of m TOR in the proliferative phase hemangioma was significantly higher than that in the involutive phase(t=10.44,P<0.01).The expression of AMPK and m TOR was negatively correlated(r=-0.78,P<0.01).2.A few of spindle cells extended out from the edge of cell islands which were cultured in vitro in the 1st week,and a gradual increasing number of cells with a radial growth in the 2nd week,and with unevenly distribution of growth in the 3ird week,and then covering more than 75% bottom of the culture bottle in the 4th week.Single cells were fusiform or irregular polygonal,and aggregated cells were arranging like cobblestones or tubular observed by a inverted phase contrast microscope.The expression of coagulation factor VIII was positive.3.The total apoptosis rate of cells: the control group was 1.45±0.27,the metformin group was 11.98±1.00,NVP-BEZ235 group was 16.12±1.88,and the combined group was 25.96±0.96.Compared the control group with the metformin group,the NVP-BEZ235 group and the combined group,these differences were significant(t=17.30,13.13,42.49,P<0.01).Compared the combined group with the metformin group and the NVP-BEZ235 group,these differences were significant(t=3.90,8.17,P<0.01).Compare the metformind group with the NVP-BEZ235 group,the difference was significant(t=18.53,P<0.01).4.The expression of AMPK: the control group was 0.43±0.23,metformin group was3.97±0.40,NVP-BEZ235 group was 0.55±0.23,and the combined group was3.99 ± 1.10.Compared the control group with the metformin group and the combined group,these differences were significant(t=13.32,5.49,P<0.01).Compared the NVP-BEZ235 group with the combined group,the difference was significant(t=5.3,P<0.01).Compared the metformin group with the NVP-BEZ235 group,the difference was significant(t=12.91,P<0.01).Compared the control group with the NVP-BEZ235 group,and compared the combined group with metformin group,these differences were not statistically significant(t=-0.66,-0.03,P>0.05).5.The expression of m TOR: the control group was 6.35±1.81,metformin group was2.47±0.69,NVP-BEZ235 group was 1.46±0.27,and the combined group was0.40±0.21.Compared the control group with the metformin group,NVP-BEZ235 group and combination group,these differences were statistically significant(t=-3.47,-4.63,-5.66,P<0.05).Compared the combined group with the metformin group,NVP-BEZ235 group,these differences were statistically significant(t=-4.99,-5.3,P<0.05).Compared the metformin group with NVP-BEZ235 group,the difference was not significant(t=2.37,P>0.05).6.The expression of p70s6k: the control group was 0.65±0.22,metformin group was2.49±0.19,NVP-BEZ235 group was 3.98±0.55,and the combined group was13.95±1.16.Compared the control group with the metformin group,NVP-BEZ235 group and combined group,these differences were significant(t=11,9.78,19.54,P<0.01).Compared the combined group with the metformin group and NVP-BEZ235 group,these differences were statistically significant(t=16.9,13.46,P<0.01).Compared the metformin group with NVP-BEZ235 group,the difference was not significant(t=-4.45,P>0.05).Conclusions1.The expression of AMPK in involutive phase is higher than that in proliferative phase,and the expression of m TOR in involutive phase is lower than that in proliferative phase.2.Activation of AMPK or inhibition of PI3 K can inhibit the m TOR signaling pathway,and induce the apoptosis of vascular endothelial cells of infantile hemangioma in vitro,especially when activated AMPK and inhibit PI3 K at the same time. |
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