P53-MDM2在普萘洛尔诱导血管瘤内皮细胞凋亡中的作用
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摘要
目的初步研究P53、MDM2与普萘洛尔诱导血管瘤内皮细胞凋亡的关系,为血管瘤的治疗提供新的思路和靶点。方法体外培养血管瘤内皮细胞,实验设计为2组,加药组和空白对照组,加药组以100umol/L的普萘洛尔处理细胞,空白对照组不予以特殊处理。倒置显微镜、电镜下观察形态学变化,流式细胞术检测细胞凋亡率,RTPCR、Western-blot检测MDM2、P53 mRNA水平和蛋白水平上的变化。结果用普萘洛尔处理后,大部分细胞形态有紧密排列的"铺路石"形态逐渐变圆。透射电镜观察到细胞呈现凋亡相,对照组无明显变化;加药组细胞凋亡率为14.67%,空白对照组4.87%,具有统计学差异。RT-PCR、Western-blot结果一致,与空白对照组相比,P53、MDM2均升高,具有统计学差异。结论 100umol/L普萘洛尔能够诱发体外培养的血管瘤内皮细胞凋亡,能促使体外培养的血管瘤内皮细胞中P53与MDM2呈现高表达。
Objective To research new ideas and targets for the treatment of hemangioma, through a preliminary study of the relationship between P53, MDM2 and propranolol inducing apoptosis of hemangioma endothelial cells.Methods By Hemangioma endothelial cell culture, there are 2 groups, drug group and blank control group, the experimental group with 100 umol/L of propranolol treatment, while the blank control group will not be given special treatment. Observing the difference between the morphological changes of the cells under inverted microscope and transmission electron microscope.Detecteing cell apoptosis by flow cytometry rate, p53/MDM2 mRNA expression by using RT-PCR, and the protein level of p53/MDM2 by using Western-blot. Results Observing by the microscope, in vitro processing of hemangioma endothelial cells with propranolol, the shape of most cells change "paving stone" shape into becoming round gradually. Observing under transmission electron microscope, we can see that the cell membrane rupture,the nucleolus disappeared, a small crescent shaped patch of apoptotic bodies, dense, cells showed apoptotic phase, but the control group has no obvious change. Apoptosis rate: The apoptosis of propranolol treatment group was 14.67%, while in the blank control group, the apoptosis rate was 4.87%; It has the statistical significance. RT-PCR results: Comparing with the blank control group, P53, MDM2 were significantly increased at the m RNA level, and it has the statistical significance. Western-blot result: comparing with the blank control group, P53, MDM2 have an high expression at the protein levels, also. It has the statistical significance. Conclusion 100 umol/L propranolol can induce the apoptosis of human endothelial cells cultivated in vitro. High expression of P53 and MDM2 in 100 umol/L propranolol-induced apoptosis in Hemangioma Endothelial Cell. P53/MDM2 are involved in the process of apoptosis which can be caused by propranolol inducing the apoptosis of the hemangioma in vitro.
Objective To research new ideas and targets for the treatment of hemangioma, through a preliminary study of the relationship between P53, MDM2 and propranolol inducing apoptosis of hemangioma endothelial cells.Methods By Hemangioma endothelial cell culture, there are 2 groups, drug group and blank control group, the experimental group with 100 umol/L of propranolol treatment, while the blank control group will not be given special treatment. Observing the difference between the morphological changes of the cells under inverted microscope and transmission electron microscope.Detecteing cell apoptosis by flow cytometry rate, p53/MDM2 mRNA expression by using RT-PCR, and the protein level of p53/MDM2 by using Western-blot. Results Observing by the microscope, in vitro processing of hemangioma endothelial cells with propranolol, the shape of most cells change "paving stone" shape into becoming round gradually. Observing under transmission electron microscope, we can see that the cell membrane rupture,the nucleolus disappeared, a small crescent shaped patch of apoptotic bodies, dense, cells showed apoptotic phase, but the control group has no obvious change. Apoptosis rate: The apoptosis of propranolol treatment group was 14.67%, while in the blank control group, the apoptosis rate was 4.87%; It has the statistical significance. RT-PCR results: Comparing with the blank control group, P53, MDM2 were significantly increased at the m RNA level, and it has the statistical significance. Western-blot result: comparing with the blank control group, P53, MDM2 have an high expression at the protein levels, also. It has the statistical significance. Conclusion 100 umol/L propranolol can induce the apoptosis of human endothelial cells cultivated in vitro. High expression of P53 and MDM2 in 100 umol/L propranolol-induced apoptosis in Hemangioma Endothelial Cell. P53/MDM2 are involved in the process of apoptosis which can be caused by propranolol inducing the apoptosis of the hemangioma in vitro.
引文
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3 Jephson CG,Manunza F,Syed S,et al.Successful treatment of isolated subglottic haemangioma with propranolol alone.International journal of pediatric otorhinolaryngology,2009,73(12):1821-1823.
4 秦中平,刘学键,李克雷,等.小剂量普萘洛尔口服治疗婴儿血管瘤的近期疗效与安全性评价.中华医学杂志,2009,89(44):3130-3134.
5 刘文英.血管瘤治疗的进展及普萘洛尔临床应用问题.中华妇幼临床医学杂志(电子版),2010,6(3):156-159.
6 Ji Y,Li K,Xiao X,et al.Effects of propranolol on the proliferation and apoptosis of hemangioma-derived endothelial cells.Journal of Pediatric Surgery,2012,47(12):2216-2223.
7 Haupt Y,Maya R,Kazaz A,et al.Mdm2 promotes the rapid degradation of p53.Nature,1997,387(6630):296-299.
8 Vassilev LT,Vu BT,Graves B,et al.In vivo activation of the p53 pathway by small-molecule antagonists of MDM2.Science,2004,303(5659):844-848.
9 Kubbutat MH,Jones SN,Vousden KH.Regulation of p53 stability by Mdm2.Nature,1997,387(6630):299-303.
10 Momand J,Wu H-H,Dasgupta G.MDM2-master regulator of the p53 tumor suppressor protein.Gene,2000,242(1):15-29.
11 Wong A,Hardy KL,Kitajewski AM,et al.Propranolol accelerates adipogenesis in hemangioma stem cells and causes apoptosis of hemangioma endothelial cells.Plastic and reconstructive surgery,2012,130(5):1012.
12 Greenberger S,Boscolo E,Adini I,et al.Corticosteroid suppression of VEGF-A in infantile hemangioma-derived stem cells.New England Journal of Medicine,2010,362(11):1005-1013.
13 Bielenberg DR,Bucana CD,Sanchez R,et al.Progressive growth of infantile cutaneous hemangiomas is directly correlated with hyperplasia and angiogenesis of adjacent epidermis and inversely correlated with expression of the endogenous angiogenesis inhibitor,IFN-beta.International journal of oncology,1999,14(3):401-409.
14 Tu J-B,Ma R-Z,Dong Q,et al.Induction of apoptosis in infantile hemangioma endothelial cells by propranolol.Experimental and Therapeutic Medicine,2013,6(2):574-578.
2 Sans V,de la Roque ED,Berge J,et al.Propranolol for severe infantile hemangiomas:follow-up report.Pediatrics,2009,124(3):e423-e431.
3 Jephson CG,Manunza F,Syed S,et al.Successful treatment of isolated subglottic haemangioma with propranolol alone.International journal of pediatric otorhinolaryngology,2009,73(12):1821-1823.
4 秦中平,刘学键,李克雷,等.小剂量普萘洛尔口服治疗婴儿血管瘤的近期疗效与安全性评价.中华医学杂志,2009,89(44):3130-3134.
5 刘文英.血管瘤治疗的进展及普萘洛尔临床应用问题.中华妇幼临床医学杂志(电子版),2010,6(3):156-159.
6 Ji Y,Li K,Xiao X,et al.Effects of propranolol on the proliferation and apoptosis of hemangioma-derived endothelial cells.Journal of Pediatric Surgery,2012,47(12):2216-2223.
7 Haupt Y,Maya R,Kazaz A,et al.Mdm2 promotes the rapid degradation of p53.Nature,1997,387(6630):296-299.
8 Vassilev LT,Vu BT,Graves B,et al.In vivo activation of the p53 pathway by small-molecule antagonists of MDM2.Science,2004,303(5659):844-848.
9 Kubbutat MH,Jones SN,Vousden KH.Regulation of p53 stability by Mdm2.Nature,1997,387(6630):299-303.
10 Momand J,Wu H-H,Dasgupta G.MDM2-master regulator of the p53 tumor suppressor protein.Gene,2000,242(1):15-29.
11 Wong A,Hardy KL,Kitajewski AM,et al.Propranolol accelerates adipogenesis in hemangioma stem cells and causes apoptosis of hemangioma endothelial cells.Plastic and reconstructive surgery,2012,130(5):1012.
12 Greenberger S,Boscolo E,Adini I,et al.Corticosteroid suppression of VEGF-A in infantile hemangioma-derived stem cells.New England Journal of Medicine,2010,362(11):1005-1013.
13 Bielenberg DR,Bucana CD,Sanchez R,et al.Progressive growth of infantile cutaneous hemangiomas is directly correlated with hyperplasia and angiogenesis of adjacent epidermis and inversely correlated with expression of the endogenous angiogenesis inhibitor,IFN-beta.International journal of oncology,1999,14(3):401-409.
14 Tu J-B,Ma R-Z,Dong Q,et al.Induction of apoptosis in infantile hemangioma endothelial cells by propranolol.Experimental and Therapeutic Medicine,2013,6(2):574-578.
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