β肾上腺素受体信号通路对婴幼儿血管瘤内皮细胞增殖与凋亡的调控作用

发布时间：2018-06-30 07:25

本文选题：婴幼儿血管瘤 + 内皮细胞　；参考：《复旦大学》2013年博士论文

【摘要】：婴幼儿血管瘤(infantile hemangiomas, IH)是一种十分常见的良性肿瘤,在新生儿中发病率可高达10%,以颜面部最为好发。常是在患儿出生时或出生后不久,皮肤上出现蚊咬状红点,并迅速增长,以3～4月时最为显著。约有80%左右的血管瘤可以自行消退,另有20%左右称为复杂性血管瘤,常生长在要害部位。在出生后头几周即开始系统性地运用皮质类固醇类药物,曾被认为是治疗婴幼儿血管瘤(IHs)的首选。研究发现,此类药物可阻碍IH的进一步增殖,但并不能诱导IH萎缩消退,因而仅对早期、处于增殖状态的IH有效。虽然80%的IH发病于生后前6月,但许多患儿到医院治疗时往往已较晚,从而限制了皮质类固醇类药物的使用。此外,许多IH患儿、甚至是新生儿期的IH患儿对甾类药物的治疗并不敏感,同时此类药物的副作用又非常明显。二线治疗药物包括长春新碱与干扰素-α,但药物的毒副作用仍然非常严重。盐酸普萘洛尔是非选择性的p受体阻滞剂,大量的临床病例研究已经证实其对IH患儿有较好的疗效。最近,临床随机对照研究也证实盐酸普萘洛尔运用于婴幼儿及儿童局灶性及节段性IHs,疗效肯定且毒副作用小。目前,有研究者已将普萘洛尔作为治疗IH的一线药物,而其它β受体阻滞剂如醋丁洛尔等也被运用于临床治疗,均取得了较好的疗效。不过,β受体阻滞剂对IH的作用机制尚不明确。故本课题拟观察β受体阻滞剂对体外培养的血管瘤内皮细胞(HemECs)增殖与凋亡等生物学特性的影响,并深入探讨β受体信号通路在调控过程中的作用及分子机制,从而为β受体阻滞剂治疗IH提供重要的理论依据,并为早期干预IH提供新的治疗靶点。第一部分普萘洛尔对血管瘤内皮细胞增殖与凋亡的影响及其作用机制目的研究普萘洛尔对体外培养的婴幼儿血管瘤内皮细胞(HemECs)增殖、凋亡的影响及其作用机制。方法组织块消化、流式分选婴幼儿HemECs。采用Cell Counting Kit (CCK-8)法检测细胞活性变化,BrdU标记法检测细胞增殖状态。采用Annexin V-FITC/PI双染流式细胞仪术与Hoechst33342荧光染色检测观察细胞凋亡情况。应用western blot检测VEGF、Caspase-3、Caspase-8及Caspase-9的表达,分光光度法检测Caspase-8的相对活性,荧光定量PCR检测凋亡相关基因p53、Bcl-2、Bax及Bcl-xL的表达。结果与对照组相比,低剂量普萘洛尔(50μM/L)对HemECs的活性无明显影响,而100μM/L普萘洛尔作用24小时能显著降低细胞活性至48%左右。100μM/L普萘洛尔作用24小时后,HemECs的增殖受到明显抑制,细胞早期凋亡显著增高,出现典型的凋亡细胞核形态学变化。普萘洛尔显著抑制细胞内VEGF的表达,激活凋亡蛋白Caspase-3与Caspase-9,但对Caspase-8活性无明显影响。荧光定量PCR检测发现普萘洛尔干预后,凋亡相关基因p53与Bax表达量显著升高、Bcl-xl表达量显著下降,而Bcl-2基因表达量无显著变化。结论普萘洛尔对体外培养的HemECs具有明确的抗增殖、促凋亡作用,其机制可能为普萘洛尔能抑制HemECs中VEGF的表达,激活内源性凋亡通路,上调促凋亡基因p53与Bax及下调凋亡抑制基因Bcl-xL。第二部分β受体信号通路对血管瘤内皮细胞增殖的调控作用目的探讨β受体信号通路对血管瘤内皮细胞(HemECs)增殖的调控作用及其可能的机制。方法采用real time-PCR与western blot检测β受体在HemECs中的表达情况。选用β受体激动剂异丙肾上腺素(ISO)、选择性β1受体阻断剂美托洛尔(MET)及选择性β2受体阻断剂ICI118551(ICI)作用于体外HemECs。通过细胞增殖与活性实验、流式细胞仪细胞周期分析,观察ISO、MET及ICI对HemECs增殖的影响；采用酶联免疫分析观察细胞中cAMP水平变化；通过western blot检测相关蛋白与磷酸化位点的变化。结果β1与β2受体在HemECs中均有表达。与对照组相比,生理浓度(10nM/L)的ISO即能刺激HemEC增殖,在浓度达到1μM/L时作用最为明显。同时,ISO能显著促进cAMP的生成,但这种作用可被MET或ICI阻断,其中以ICI作用更为明显。Western blot检测发现,ISO能显著促进细胞周期蛋白D1、细胞周期蛋白依赖激酶-4(CDK-4)与CDK-6表达及Rb蛋白磷酸化。给予血管内皮生长因子受体-2(VEGFR-2)抑制剂或细胞外信号调节酶(ERK)抑制剂能显著抑制ISO诱导HemEC增殖的作用。进一步研究发现,β受体阻断剂与VEGFR-2阻断剂能显著抑制ISO导致的ERK磷酸化。提前给予ICI能显著抑制ISO诱导升高的VEGF表达与VEGFR-2活性,而MET无此作用。结论激活p受体可经ERK通路促进HemEC的增殖；在p受体激活的同时,可反式激活VEGFR-2信号通路,从而进一步促进HemEC的增殖。第三部分VEGF-VEGFR-2自分泌环对血管瘤内皮细胞凋亡影响的分子作用机制目的VEGF-VEGFR-2信号通路在多数肿瘤中处于异常激活状态,其对维持肿瘤的生物学特征中起着重要的作用。异常的VEGF-VEGFR-2信号通路可显著促进血管瘤内皮细胞(HemECs)的增殖与迁移,但对HemEC凋亡的影响尚无报道。故本研究拟对比HemEC与普通的脐静脉内皮细胞(HUVEC)中VEGF-VEGFR-2活化状态,探讨其对细胞凋亡的影响与机制。方法无血清培养环境下诱导血管内皮细胞凋亡。应用Annexin V-FITC/PI双染流式细胞仪术、Hoechst33342与TUNEL荧光染色检测观察VEGF中和抗体与VEGFR-2阻断剂对细胞凋亡的影响。采用western blot技术检测VEGF、VEGFR-2及PTEN在HemEC和HUVEC中的表达或活化状态。应用P13K与ERK抑制剂处理血管内皮细胞,通过上述各种方法检测VEGF-VEGFR-2信号通路抑制细胞凋亡的下游靶点。在获得初步论证的基础上,建立PTEN高表达的HemEC系,检测细胞凋亡状态变化及其对Akt磷酸化的影响。结果无血清培养12h后,HUVEC可见凋亡率明显较对照组增高,而HemEC凋亡率增高不明显。无血清培养24h或48h, HemEC中出现增多的凋亡细胞,但显著低于HUVEC。Western blot检测发现HemEC中VEGF表达水平与VEGFR-2磷酸化均显著高于HUVEC,而PTEN表达水平显著低于HUVEC.中和VEGF或阻断VEGFR-2磷酸化后,HemEC凋亡率与PTEN蛋白水平均明显增高。与利用P13K阻断剂作用于HemEC时情况相同,高表达PTEN的HemEC细胞,其Akt活性明显降低,细胞更易发生凋亡。而ERK阻断剂对无血清培养环境下的HemEC并无促凋亡的作用。结论HemEC中的VEGF自分泌环,能显著地抑制HemEC发生凋亡,其作用是通过VEGFR-2受体及下游的PTEN/PI3K/Akt通路来实现的。
[Abstract]:Infant hemangioma ( IH ) is a very common benign tumor with a high incidence rate of up to 10 % in the newborn . It is the first choice for the treatment of infantile hemangioma ( IHs ) . In the first few weeks after birth , it is not sensitive to the treatment of steroid drugs . In addition , many IH children , even the newborn IH children , are not sensitive to the treatment of steroid drugs .

In this study , the effect of 尾 - blocker on the proliferation and apoptosis of vascular endothelial cells ( HemECs ) in infants and children has been confirmed .

Effect of first part of prodinol on proliferation and apoptosis of vascular endothelial cells and its mechanism

Objective To study the effect and mechanism of prodinol on proliferation and apoptosis of vascular endothelial cells ( HemECs ) in vitro .

Methods The cell activity was detected by the cell culture Kit ( CCK - 8 ) method , and the cell proliferation status was detected by means of the cell culture Kit ( CCK - 8 ) method . The expression of VEGF , Caspase - 3 , Caspase - 8 and Caspase - 9 was detected by annexin V - FITC / PI double staining flow cytometry . The relative activity of Caspase - 8 was detected by Western blot , and the expression of p53 , Bcl - 2 , Bax and Bcl - xL was detected by fluorescence quantitative PCR .

Results Compared with the control group , low - dose prodinol ( 50 渭M / L ) had no significant effect on the activity of hemECs . After 24 hours of 100 渭M / L naptalol , the proliferation of apoptotic cells was significantly inhibited . Apoptosis of the cells was significantly inhibited . Apoptosis - related gene p53 and Caspase - 9 were significantly inhibited by fluorescent quantitative PCR . The expression of Bcl - xl decreased significantly , while Bcl - 2 gene expression was not significantly altered .

Conclusion The mechanism may inhibit the expression of VEGF in HemECs , activate endogenous apoptosis pathway , up - regulate p53 and Bax and down - regulate Bcl - xL .

Regulation of second partial 尾 - receptor signaling pathway on proliferation of vascular endothelial cells

Objective To investigate the effect of 尾 - receptor signaling pathway on proliferation of vascular endothelial cells ( HemECs ) and its possible mechanism .

Methods The expression of 尾 - receptor in HemECs was detected by real time - PCR and western blot . The effects of ISO , MET and ICI on the proliferation of HemECs were observed by means of cell proliferation and activity experiment and flow cytometry .
The level of cAMP in the cells was observed by enzyme - linked immunoassay .
The changes of related protein and phosphorylation sites were detected by western blot .

Results 尾 - adrenergic receptor - 2 ( VEGFR - 2 ) inhibitor or extracellular signal - regulating enzyme ( ERK ) inhibitor can significantly inhibit the proliferation of cell cycle protein D1 , cyclin dependent kinase - 4 ( CDKs - 4 ) and CDKs - 6 .

Conclusion The activation of p - receptor can promote the proliferation of HemEC via ERK pathway .
At the same time as the p - receptor is activated , the VEGFR - 2 signal pathway may be activated trans , thereby further promoting the proliferation of HemEC .

Molecular Mechanism of the Third Part of VEGF - VEGFR - 2 Self - Secreting Ring on the Apoptosis of Vascular Endothelial Cells

Objective VEGF - VEGFR - 2 signaling pathway plays an important role in the biological characteristics of tumor . The abnormal VEGF - VEGFR - 2 signaling pathway can significantly promote the proliferation and migration of vascular endothelial cells ( HemECs ) . However , the effect of VEGF - VEGFR - 2 signal pathway on apoptosis of vascular endothelial cells ( HUVEC ) has not been reported .

Methods The expression or activation status of VEGF , VEGFR - 2 and PTEN in HemEC and HUVEC was observed by the method of western blot . The expression or activation status of VEGF , VEGFR - 2 and PTEN in HemEC and HUVEC was detected by western blot .

Results Compared with the control group , the apoptosis rate of HUVEC was significantly higher than that in control group , but the apoptosis rate was significantly lower than that in HUVEC . The expression level of VEGF was significantly higher than that in HUVEC . The expression of PTEN was significantly lower than that in HUVEC .

Conclusion The VEGF self - secreting ring in HemEC can significantly inhibit the apoptosis of HemEC , and its function is achieved by means of the VEGFR - 2 receptor and the downstream PTEN / PI3 pathway .
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R739.5

【共引文献】