炎性介质IL-1β和IL-3调节内皮细胞表达淋巴管表型的作用

发布时间：2018-06-22 22:15

  本文选题：人脐静脉内皮细胞 + 淋巴管内皮表型　； 参考：《山东大学》2012年硕士论文

【摘要】：研究背景：淋巴管是心血管循环系统的重要辅助系统,在调节体内渗透压、维持内环境稳定以及免疫、炎症反应中发挥重要作用。淋巴管新生(lymphangiogenesis)与机体组织许多病理生理过程密切相关,尤其是在炎症反应、创伤修复以及肿瘤转移等方面发挥重要作用。因此,探寻淋巴管新生机制对于上述疾病的治疗具有重要意义。 以往的研究认为,淋巴管新生的途径主要有两个方面：原有的淋巴管在血管内皮生长因子C (VEGF-C)的作用下以出芽方式形成新的淋巴管；外周血内皮祖细胞迁移到局部组织,在VEGF-C等的诱导下转分化为淋巴管内皮细胞(lymphatic endothelial cells, LECs)。近期的研究发现,许多炎性介质与淋巴管标志物(Prox-1、Podoplanin、VEGFR-3、LYVE-1等)的表达和淋巴管新生有密切联系,例如：LPS、TNF-α、IFN-γ、IL-1、IL-3、IL-6等。然而,炎性介质调节淋巴管内皮表型的作用机制尚不十分清楚。 文献报道,炎性介质LPS、IL-3等能刺激内皮细胞的NF-κB的P50同源二聚体途径诱导内皮细胞淋巴管标志物的表达；另有文献认为,血管内皮细胞(blood endothelial cells, BECs)在炎性介质IFN-γ、TNF-α的作用下表达IL-3,间接通过IL-3刺激淋巴管标志物的表达。IL-1是许多病理过程中重要的炎性因子,能否通过NF-κB途径诱导内皮细胞表达淋巴管表型即向淋巴管内皮细胞转分化?尚未见报道。本文重点研究了IL-1β、IL-3诱导血管内皮细胞转分化为淋巴管内皮细胞的调节作用。 目的：本实验采用炎性介质IL-1β和IL-3分别处理分离培养的人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs)和人脐静脉内皮细胞株CRL-1730,研究诱导或维持内皮细胞的淋巴管内皮表型特性的关键因素。 方法：①取脐带,按常规分离培养原代HUVECs,细胞融合后观察并记录其形态,用vWF相关抗原的免疫细胞化学染色验证是否为BECs,传代进行后续实验；取人脐静脉内皮细胞株CRL-1730,按同样条件培养。②采用RT-PCR和免疫细胞化学法检测HUVECs和CRL-1730细胞株对淋巴管标志物Prox-1、VEGFR-3、 Podoplanin、LYVE-1的表达情况。③分别用IL-1p或IL-3刺激CRL-1730细胞株一定时间,观察刺激前后细胞形态的改变；采用RT-PCR和免疫细胞化学法检测细胞对淋巴管标志物的表达情况。④采用Real-time PCR法检测CRL-1730细胞株经IL-1β刺激前、后对淋巴管标志物mRNA表达量的差异。⑤将NF-κB阻断剂PDTC加入到培养的CRL-1730细胞株中,1h后再加入IL-1β刺激一定时间后,采用免疫细胞化学法检测细胞NF-κB活性改变,并用RT-PCR法检测CRL-1730细胞株对淋巴管内皮标志物的表达情况。 结果：①RT-PCR和免疫细胞化学法发现：分离培养的HUVECs表达淋巴管标志物,而CRL-1730细胞株则不表达淋巴管标志物。②CRL-1730细胞株经IL-1β或IL-3刺激24h后,生长汇合的内皮细胞形态由鹅卵石状变为长梭形；RT-PCR和免疫细胞化学法证实表达淋巴管标志物。③Real-time PCR检测结果显示：与未经刺激的分离培养的HUVECs相比,未经刺激的内皮细胞株CRL-1730对淋巴管内皮标志物Prox-1、Podoplanin、VEGFR-3、LYVE-1的mRNA表达量极少；经IL-1β刺激1.5h后表达量显著升高(表1,图10),P0.05。进一步验证了之前刺激实验做出的RT-PCR结果。④CRL-1730细胞株在加入NF-κB阻断剂PDTC之后再用炎性因子IL-1β刺激,免疫细胞化学和RT-PCR法检测其呈阴性表达。 结论：IL-1p和IL-3均能诱导血管内皮细胞表达淋巴管内皮表型；IL-1p能通过NF-κB途径诱导BECs表达淋巴管内皮表型。 意义：研究炎性介质诱导血管内皮转分化为淋巴管内皮细胞作用,对于解释炎症环境下淋巴管形成机制具有重要意义,并为治疗淋巴管生成或生成障碍相关疾病奠定基础。
[Abstract]:Background : lymphatic vessel is an important auxiliary system of cardiovascular circulatory system , plays an important role in regulating osmotic pressure in vivo , maintaining homeostasis of internal environment and immune and inflammatory response .

Previous studies suggest that there are two main pathways for lymphatic angiogenesis : the original lymphatic vessels form new lymphatic vessels in budding manner under the action of vascular endothelial growth factor C ( VEGF - C ) ;
Peripheral vascular endothelial progenitor cells migrate to local tissues and differentiate into lymphatic endothelial cells ( LECs ) under the induction of VEGF - C and the like . Recent studies have found that many inflammatory mediators are closely related to the expression of lymphatic markers ( Prox - 1 , Podopathy , VEGFR - 3 , LYVE - 1 , etc . ) , such as LPS , TNF - 伪 , IFN - 纬 , IL - 1 , IL - 3 , IL - 6 , etc . However , the mechanism of inflammatory mediators for modulating lymphatic endothelial phenotype is not very clear .

It is reported that inflammatory mediators LPS , IL - 3 and the like can stimulate the expression of endothelial cell lymphoma markers by stimulating the P50 homologous dimer pathway of NF - 魏B in endothelial cells ;
IL - 1 is an important inflammatory factor in many pathological processes . It has not been reported that IL - 1 is an important inflammatory factor in many pathological processes . It has not been reported that IL - 1尾 and IL - 3 can induce endothelial cells to differentiate into lymphatic endothelial cells .

Objective : To investigate the key factors in the induction or maintenance of endothelial cell endothelial phenotype in cultured human umbilical vein endothelial cells ( HUVEC ) and human umbilical vein endothelial cells ( CRL - 1730 ) using inflammatory mediators IL - 1尾 and IL - 3 respectively .

Methods : ( 1 ) The umbilical cord was isolated and cultured in vitro . The morphology was observed and recorded after cell fusion .
The expression of Prox - 1 , VEGFR - 3 , Podopathy and LYVE - 1 was detected by RT - PCR and immunohistochemical method .
The expression of lymphocyte markers was detected by RT - PCR and immune cytochemical method . The mRNA expression of lymphocytes was detected by Real - time PCR .

Results : 鈶，

本文编号：2054403