神经调节蛋白1对冠状动脉平滑肌细胞表达血管生成因子的影响*

2016-12-26 06:29庞毅恒陈力铨
中国病理生理杂志 2016年11期
关键词:平滑肌磷酸化受体

庞毅恒, 桂 春, 陈力铨

(广西医科大学第一附属医院西院心血管内科, 广西 南宁 530021)



神经调节蛋白1对冠状动脉平滑肌细胞表达血管生成因子的影响*

庞毅恒, 桂 春△, 陈力铨

(广西医科大学第一附属医院西院心血管内科, 广西 南宁 530021)

目的: 探讨神经调节蛋白1(neuregulin-1,NRG-1)对人冠状动脉平滑肌细胞(HCASMCs)表达血管生成因子的影响。方法:培养HCASMCs,实验使用第3代细胞。用Western blot法检测细胞ErbB的表达和磷酸化。在正常、缺氧缺血清或NRG-1(100 μg/L)处理条件下,用Western blot法检测血管内皮生长因子(VEGF)、血管生成素1(Ang-1)和血管生成素2(Ang-2)表达的改变。结果:ErbB2、ErbB3和ErbB4均能在HCASMCs中表达,加入NRG-1后,这3种ErbB的磷酸化水平均增加。与对照组比较,在缺氧缺血清组HCASMCs中VEGF和Ang-1的表达明显增加(P<0.05),而Ang-2的表达差异无统计学显著性。与缺氧缺血清组比较,NRG-1处理组的HCASMCs表达VEGF和Ang-1进一步显著增加(P<0.05),而Ang-2的表达差异无统计学显著性。结论:HCASMCs能表达ErbB2、ErbB3和ErbB4,加入NRG-1增强ErbB2、ErbB3 和ErbB4 的磷酸化。缺氧缺血清和NRG-1处理均能增加VEGF和Ang-1在HCASMCs中的表达。

神经调节蛋白1; 人冠脉平滑肌细胞; 血管内皮生长因子; 血管生成素

神经调节蛋白(neuregulins,NRGs)是一个与表皮生长因子(epidermal growth factor,EGF)相关的生长和分化因子家族,目前研究较多的为NRG-1,其对胚胎心脏的发育和成年心脏功能的维护起着非常重要的作用[1]。NRG-1对血管生成的作用也有些研究报道。Hedhli等[2]研究发现在内皮细胞NRG-1敲除的小鼠中结扎股动脉,内皮NRG-1缺乏显著减慢腿部血流的恢复、降低毛细血管密度和减少动脉发生,外源性给予NRG-1能加速血流的恢复,提示内皮产生的NRG-1是缺血诱导的血管生成和动脉发生所必需的。Xiao等[3]研究显示,在心肌梗死中高表达NRG-1能增加缺血心肌的微血管数量。Nakaoka等[4]研究发现,NRG-1处理小鼠后,小鼠心脏中血管生成素1(angiopoietin-1,Ang-1)表达明显增加。我们前期研究显示,给予NRG-1治疗可显著提高糖尿病大鼠心肌组织的血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达、增加Flk和Tie-2受体磷酸化。我们研究还发现,在糖尿病和不稳定型心绞痛患者中,血清NRG-1浓度与VEGF和Ang-1浓度呈正相关[5]。以上研究提示NRG-1与其它血管生成因子可能存在某些相互作用和调控,在心肌血管生成中起着一定的作用。

所以本研究通过体外培养的人冠状动脉平滑肌细胞(human coronary artery smooth muscle cells,HCASMCs),检测HCASMCs是否表达NRG-1及其受体,以及NRG-1处理后,HCASMCs VEGF、Ang-1和Ang-2表达的变化,为探索NRG-1在心肌血管生成中的作用提供基础。

材 料 和 方 法

1 试剂与材料

HCASMCs原代细胞FC-0031、平滑肌细胞专用培养基、抗坏血酸、L-谷氨酰胺(L-glutamine)和胎牛血清(fetal bovine serum,FBS)购自Lifeline;抗NRG-1抗体为RD产品;抗VEGF、Ang-1和Ang-2抗体购自Santa Cruz;NRG-1、酪氨酸磷酸化抑制剂AG1478、兔抗ErbB2、ErbB3、ErbB4 多克隆抗体以及兔抗磷酸化的ErbB2、ErbB3、ErbB4 多克隆抗体购自Cell Signaling Technology;内参照GAPDH抗体为中国中杉金桥公司产品。

2 HCASMCs的复苏和培养

从液氮中取出原代人冠脉平滑肌细胞冻存管,37 ℃水浴完全溶解,经过清洗,转移至25 cm2透气培养瓶内于37 ℃、5% CO2培养箱进行培养。当细胞生长成单层达80%~90%的培养面积,用0.25%胰酶消化,然后传代,实验选用第3次传代的HCASMCs进行。

3 不同干预与分组

取第3次传代的HCASMCs进行培养,当长至铺满瓶底90%时,用2瓶按1∶3的比例传代6瓶,待长至铺满瓶底90%左右,将细胞原有培养基吸弃,按分组要求加入含不同成分的培养基并置于不同要求条件下培养24 h后进行Western blot法检测。实验分为4组:空白对照组:培养基含血清不含NRG-1,该组细胞在37 ℃、5% CO2培养箱进行培养;缺氧缺血清组:培养基不含血清、不含NRG-1,在95%氮气、5% CO2厌氧培养箱,37 ℃进行培养;NRG-1组:培养基不含血清,含100 μg/L NRG-1,在95%氮气、5% CO2厌氧培养箱,37 ℃进行培养;NRG-1+抑制剂组:培养基不含血清,含NRG-1(100 μg/L)和抑制剂AG1478(10 μmol/L),在95%氮气、5% CO2厌氧培养箱,37 ℃进行培养。

4 Western blot检测

将各组细胞收集到1.5 mL EP管,加入裂解液和PMSF提取细胞总蛋白,Bradford法检测蛋白浓度,根据蛋白分子量,选取适宜浓度的分离胶进行聚丙烯酰胺凝胶电泳。电泳结束后根据marker和目的条带分子量大小进行切胶。因为ErbB2、ErbB3和ErbB4这3个蛋白表达于心肌组织,所以用大鼠心肌作为阳性对照。然后将蛋白转至硝酸纤维素膜上,分步与适当浓度的 I 抗和辣根过氧化物酶标记的 II 抗结合。显影后,计算机扫描胶片,用ImageJ成像系统对扫描胶片上对应的蛋白条带的灰度进行分析,以目的条带与内参照GAPDH 条带的灰度比值来代表目标蛋白的相对表达量。

5 统计学处理

用SPSS 16.0统计软件分析,数据用均数±标准差(mean±SD)表示,组间多重比较采用单因素方差分析,组间两两比较采用SNK-q检验,以P<0.05为差异有统计学意义。

结 果

1 NRG-1及ErbB在HCASMCs中的表达

HCASMCs经体外培养后,进行Western blot检测,发现有NRG-1的受体ErbB2、ErbB3和ErbB4的表达,但HCASMCs不表达NRG-1。将受体的表达量与大鼠心肌组织比较,发现ErbB2和ErbB3表达丰度比心肌组织强,ErbB4表达丰度比心肌组织弱,见图1。

2 NRG-1处理增加HCASMCs的ErbB磷酸化

经NRG-1干预后, HCASMCs的ErbB2、ErbB3和ErbB4磷酸化较空白对照组明显增强(P<0.05);

Figure 1. The expression of NRG-1 and ErbBs in the HCASMCs. A: the HCASMCs under microscope (×100); B: the expression of ErbB2, ErbB3 and ErbB4 in the HCASMCs and cardiac muscle (CM) detected by Western blot; C: the expression of NRG-1 and GAPDH in HCASMCs and CM detected by Western blot.

图1 NRG-1及ErbBs在HCASMCs中的表达

然而在同时含有NRG-1和酪氨酸磷酸化抑制剂AG1478的HCASMCs中,ErbB2、ErbB3和ErbB4的磷酸化水平较仅经NRG-1处理的HCASMCs明显降低,见图2。

Figure 2. The phosphorylation levels of ErbB2, ErbB3 and ErbB4 in the HCASMCs with NRG-1 treatment. Mean±SD.n=3.*P<0.05vscontrol group;#P<0.05vsNRG-1 group.

图2 NRG-1作用下HCASMCs ErbB2/3/4磷酸化水平

3 NRG-1处理对HCASMCs血管生成因子表达的影响

与对照组相比,缺氧缺血清条件下HCASMCs的VEGF和Ang-1表达水平明显增加(P<0.05);NRG-1干预下,VEGF和Ang-1的表达量与仅缺氧缺血清时相比较进一步增强(P<0.05)。但加入酪氨酸磷酸化抑制剂AG1478后,酪氨酸磷酸化受到抑制,VEGF和Ang-1的表达明显减少。而Ang-2的表达量不受缺氧缺血清和NRG-1处理的影响,见图3。

讨 论

本研究首次发现HCASMCs能表达ErbB2、ErbB3和ErbB4,但不能表达NRG-1;NRG-1处理能增加ErbB2、ErbB3和ErbB4的磷酸化,提示NRG-1可能通过激活受体而产生生理作用;缺氧缺血清及NRG-1处理能增强VEGF和Ang-1的表达,但Ang-2因子的表达量未受影响。

Figure 3. The protein expression of VEGF, Ang-1 and Ang-2 in the HCASMCs with NRG-1 treatment. Mean±SD.n=3.*P<0.05vscontrol group;#P<0.05vshypoxia and serum deprivation group;△P<0.05vsNRG-1 group.

图3 NRG-1影响HCASMCs表达VEGF、Ang-1、Ang-2蛋白

不同的血管平滑肌细胞ErbBs的表达种类不尽相同。有研究报道,胸主动脉血管平滑肌细胞表达3个ErbB[6],但还有研究结果相反,人主动脉平滑肌细胞仅表达ErbB1和ErbB2,不表达ErbB3和ErbB4[7]。近期研究人员表明大鼠主动脉平滑肌细胞能表达ErbB2/3/4的mRNA和ErbBs蛋白[8]。本研究结果表明,HCASMCs体外培养可表达NRG-1的3种特异受体——ErbB2、ErbB3和ErbB4。虽然我们的实验结果表明HCASMCs其本身在体外培养时检测不到NRG-1的表达,但这并不意味着HCASMCs不受NRG-1因子的调控,因为无论是在靠近心肌细胞的心肌微血管内皮细胞[9-10],还是冠脉粥样斑块内的巨噬泡沫细胞都有高表达的NRG-1[11]。因此研究NRG-1对HCASMCs的影响具有现实意义。

前期研究发现,在多种组织和细胞发现存在一种缺氧诱导调控,适度的缺氧缺血清会使得缺氧诱导因子1表达升高,进而调控下游诸如VEGF、Ang-1、内皮素等多种血管生成因子的增加[12-13]。在本研究中,我们证实HCASMCs也会受缺氧缺血清诱导调控,上调血管生成因子VEGF和Ang-1的表达,但对Ang-2没有显著影响。在缺氧缺血清条件下,经NRG-1处理诱导,HCASMCs表达VEGF和Ang-1的水平还会进一步升高,提示NRG-1与缺氧缺血清协同对HCASMCs表达VEGF和Ang-1起正调控增强作用。NRG-1能促进HCASMCs VEGF和Ang-1的表达,推测与NRG-1/ErbB信号传导有关。先前在心脏发育、成熟心脏[14-15]及癌细胞的血管生成[16]研究中发现,NRG-1可通过激活受体酪氨酸激酶的磷酸化进行系列的信号转导发挥其生物学效应,使得VEGF因子表达增强。我们的研究发现,虽然HCASMCs不表达NRG-1,但受体ErbB2、ErbB3和ErbB4得到表达。在外源的NRG-1处理下,ErbB2、ErbB3和ErbB4磷酸化增强,说明NRG-1触发了NRG-1/ErbB信号传导,进而使得血管生成因子VEGF和Ang-1的表达量也随之增加。这一结论可以由加入NRG-1的竞争性抑制剂AG1478获得反证。当加入AG1478后,AG1478和ErbB结合,阻碍了NRG-1/ErbB传导,从实验检测结果可以看到HCASMCs的ErbB2、ErbB3和ErbB4磷酸化受到抑制,血管生成因子VEGF和Ang-1的表达量也随之回落。

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(责任编辑: 陈妙玲, 罗 森)

Neuregulin-1 induces expression of angiogenic factors in coronary artery smooth muscle cells

PANG Yi-heng, GUI Chun, CHEN Li-quan

(DepartmentofCardiology,TheFirstAffiliatedHospitalofGuangxiMedicalUniversity,Nanning530021,China.E-mail:gui_chun@126.com)

AIM: To investigate the effects of neuregulin-1 (NRG-1) on the expression of angiogenic factors in human coronary artery smooth muscle cells (HCASMCs). METHODS: HCASMCs were culturedinvitro, and the cells at the 3rd passage were collected to assess the expression and phosphorylation of ErbB by Western blot. After HCASMCs were cultured under normal condition, with hypoxia and serum deprivation, or with NRG-1 treatment, the expression of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) was determined by Western blot. RESULTS: The expression of ErbB2, ErbB3 and ErbB4 was observed in the HCASMCs, and the phosphorylation of these receptors was increased by NRG-1 treatment. Compared with control group, the expression of VEGF and Ang-1 in the HCASMCs was significantly increased in hypoxia and serum deprivation group (P<0.05), while no difference in the expression of Ang-2 between the 2 groups was found. Compared with hypoxia and serum deprivation group, the expression of VEGF and Ang-1 in the HCASMCs treated with NRG-1 was further increased (P<0.05), and no difference in the expression of Ang-2 between the 2 groups was observed. CONCLUSION: HCASMCs express ErbB2, ErbB3 and ErbB4, and the phosphorylation of the receptors is increased by NRG-1. Hypoxia, serum deprivation and NRG-1 treatment induce the increased expression of VEGF and Ang-1 significantly.

Neuregulin-1; Human coronary artery smooth muscle cells; Vascular endothelial growth factor; Angiopoietin

1000- 4718(2016)11- 1945- 04

2016- 03- 24

2016- 09- 09

国家自然科学基金资助项目(No. 81160021;No. 81460063);广西自然科学基金重点课题 (No. 2014GXNSFDA118024)

R543.3; R363

A

10.3969/j.issn.1000- 4718.2016.11.005

杂志网址: http://www.cjpp.net

△通讯作者 Tel: 0771-3278737; E-mail: gui_chun@126.com

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