Notch信号通路在普萘洛尔抑制婴幼儿血管瘤周细胞中的作用

摘要

目的 研究Notch信号通路在普萘洛尔抑制婴幼儿血管瘤周细胞中的作用,进一步探讨普萘洛尔治疗婴幼儿血管瘤的机制.方法 通过手术获取增生期血管瘤标本5例,酶解法和筛分法制成细胞悬液,以流式细胞仪分选出a-SMA阳性血管瘤周细胞传代培养,第4代细胞用于实验.加入不同浓度普萘洛尔(0、10、20、40、80、160、320 μmol/L)溶液,培养24、48及72 h后,分别加入CCK-8培养液和BrdU标记液,以酶标仪检测490 nm处吸光度,判定细胞活性和增殖情况;Real-time PCR检测普萘洛尔干预前、后血管瘤周细胞中Notch3及Hes1基因表达情况.所有数据应用SPSS13.0进行统计分析,组内、组间多组指标比较采用单因素方差分析,组内、组间2组指标比较采用配对资料t检验,P＜ 0.05为差异有统计学意义.结果 流式细胞仪分选a-SMA阳性细胞率达98.0％.加入普萘洛尔后,随着培养时间延长,细胞活性和增殖状态受到的抑制变得明显.当普萘洛尔浓度达40 μmol/L时,细胞活性和增殖开始明显受抑制,作用72 h时尤为显著,实验组细胞活性(0.17 ±0.05)与对照组(0.53±0.03)采用配对资料t检验进行比较,差异有统计学意义(t=3.502,P＜0.05),实验组细胞增殖(301.66 ±28.71)与对照组(498.66 ±56.09)比较差异有统计学意义(t=7.425,P＜0.05);Notch3及Hes1表达量随着普萘洛尔浓度的增加逐渐增多.结论 普萘洛尔可能通过活化的Notch信号通路参与血管瘤周细胞的分化和调控,进而治疗婴幼儿血管瘤.%Objective To explore the roles of Notch signal pathways in propranolol inhibiting hemangioma-derived pericyte (PCs) in order to improve the therapeutic mechanism of propranolol and provide new targets for its clinical treatment.Methods The specimens of proliferative hemangioma were obtained from operation.Cell suspension was prepared after incubation and digestion.Then a-SMA positive PCs were sorted out by flow cytometry and sub-cultured.Propranolol in different concentrations (0,10,20,40,80,160,320 μmol/L) was added into PCs solution and cultured for 24,48 and 72 h respectively.And CCK-8 culture and BrdU labeling solutions were added.Activity and proliferation of PCs before and after intervention of propranolol were respectively tested.The level of mRNA of Notch3 and Hes1 in PCs before and after intervention of propranolol were detected by RT-PCR.All data were analyzed by SPSS 13.0 statistical software.Single factor analysis of variance and paired data t test were used.Results The rate of a-SMA positive cells was 98.0％.The longer incubation time,the more marked inhibitions of cell activity and proliferation after propranolol dosing.The inhibition of cell activity and proliferation were significant at a propranolol concentration of 40 μ mol/L.Experimental group of cell activity(0.17 ± 0.05) is compared to control group(0.53 ± 0.03) with paired data t test,t value =3.502.Similarly,experimental group of cell proliferation(301.66 ± 28.71) is compared to control group (498.66 ± 56.09) with paired data t test,tvalue =7.425.And significant differences existed between them and those of control group (P ＜ 0.05),especially at 72 h.Gene expressions of Notch3 and Hesl increased with rising propranolol concentrations.Conclusions One therapeutic mechanism of propranolol to infantile hemangioma may be achieved by pericytes.Propranolol can inhibit the growth of infantile hemangioma by inhibiting proliferation of pericytes which is closely related to activated Notch signal pathways.