Flt-1在小细胞肺癌细胞穿过人脑微血管内皮细胞单层中的作用及机制研究
摘要
目的
脑部肿瘤严重危害人类的健康,以继发性脑瘤为主。肺癌为癌症的首要杀手,小细胞肺癌(small cell lung cancer,SCLC)为临床常见的恶性肿瘤,生长快,侵袭力强,易发生胸内或远端转移,近一半的SCLC患者会发生脑转移,SCLC发生脑转移是导致这类疾病死亡的主要原因。脑转移的关键步骤是小细胞肺癌从血液中游离出跨血脑屏障(Blood brain barrier,BBB)进入脑组织。血脑屏障是位于脑组织与血液之间的一个复杂细胞系统,血脑屏障的物质基础是脑部的微血管,脑微血管内皮细胞间的紧密连接(tight junction,TJ)使得血管内皮细胞间相互“焊接”得十分紧密,不象其他组织微血管壁那样有较大的缝隙,对于保护脑组织周围稳定的生理环境和防止血液中有害物质侵入脑内具有重要的生理意义。
紧密连接由许多蛋白构成,完整的紧密连接结构蛋白主要由跨膜蛋白、胞质附着蛋白和细胞骨架(微丝)三种组分构成。跨膜蛋白如occludin、claudins和连接粘附分子(junctional adhesive molecule,JAM)等,为跨膜糖蛋白,参与封锁相邻细胞间的空隙;胞质附着蛋白如ZO-1、ZO-2和ZO-3等,ZO为紧密连接处的膜外周蛋白,将occludin与其下面的actin骨架相连。这些蛋白附着到细胞骨架上,彼此相互作用,参与维持和调节紧密连接的屏障功能。
多种机制参与调节细胞间TJ的功能,occludin蛋白的高度磷酸化在TJ结构的组装和功能的调节上起到重要的作用。膜周边蛋白ZO-1将跨膜蛋白occludin和actin骨架纤维相连,actin既是细胞间TJ结构组分,又是TJ渗透性的调节者。一些调控actin骨架纤维组装的信号分子也与TJ渗透性功能的调节相关。小GTP结合蛋白RhoA为公认的actin骨架纤维的调节者,我们实验室最近的研究表明:小细胞肺癌细胞(NCI-H209细胞)能够通过内皮细胞的Rho/ROCK信号通路指导其跨过人脑微血管内皮细胞(HBMEC)单层迁移,但有关其穿过HBMEC单层的详细机制尚未清楚。
血管内皮生长因子(Vascular endothelial growth factor,VEGF)又称为血管渗透因子,其蛋白家族包括VEGF-A,B,C,D和胎盘生长因子(Placental growthfactor,PlGF)。PlGF为分泌的糖蛋白,与VEGF在氨基酸序列残基上有53%的同源性,且其生物学功能与VEGF也有相似之处,如刺激内皮细胞生长等。血管内皮生长因子受体1(Vascular endothelial growth factor receper,VEGFR-1,又称为Fms-1ike tyrosine kinase-1,Flt-1),为PlGF在细胞膜上的专一受体,属于受体酪氨酸激酶家族成员,由七个免疫球蛋白样结构的胞外区、跨膜区、近膜区、激酶插入区和C-末端构成。PlGF与Flt-1的胞外区结合后,可活化受体的酪氨酸激酶,活化的酪氨酸激酶在信号转导的传递和生物学功能的发挥中起到重要的作用。
本实验室通过免疫组化技术检测到小细胞肺癌细胞高表达PlGF,并证实PlGF在小细胞肺癌细胞跨人脑微血管内皮细胞单层迁移过程中发挥作用,但有关PlGF的受体Flt-1在这一过程中所发挥的作用及机制尚不清楚,本研究试图回答该问题。
方法
一、探讨Flt-1受体在小细胞肺癌细胞跨人脑微血管内皮细胞单层迁移中的作用
1、细胞培养
(1)HBMEC细胞培养于含10%胎牛血清和10%Nu血清的RPMI1640培养液
(2)NCI-H209细胞培养于含10%新生牛血清的RPMI1640培养液
2、免疫荧光技术
观察NCI-H209细胞与HBMEC单层共培养后内皮细胞间TJ结构改变
3、蛋白抽提和Western blot方法
分析NCI-H209细胞与HBMEC单层共培养后TJ蛋白occludin的可溶性片段和不可溶性片段的分布情况
4、跨内皮迁移实验
分析NCI-H209细胞与HBMEC单层共培养后其跨HBMEC单层迁移能力
5、HRP flux检测
分析NCI-H209细胞与HBMEC单层共培养后HBMEC单层渗透性改变
6、Western blot方法
检测NCI-H209细胞与HBMEC单层共培养后Flt-1蛋白表达情况
7、中和抗体及抑制剂封闭实验
利用Flt-1中和抗体和VEGFR-1、2受体抑制剂,分析Flt-1受体及其磷酸化形式在NCI-H209细胞的跨内皮单层迁移中的作用
8、建立和培养稳定干扰Flt-1的HBMECs并检测其与NCI-H209细胞共培养后相关生物学特性的变化
二、探讨Rho/ROCK信号通路通过Flt-1受体启动小细胞肺癌细胞跨人脑微血管内皮单层迁移中的作用
1、细胞培养
2、免疫荧光技术
细胞骨架蛋白的形态观察
3、中和抗体实验
利用Flt-1中和抗体能够封闭Flt-1受体结合区的特性,观察受体封闭后,NCI-H209细胞致HBMEC单层细胞骨架形态变化的改变
4、Rho活性检测
利用Flt-1中和抗体和稳定干扰Flt-1沉默的HBMECs,检测经NCI-H209细胞作用后,HBMEC单层细胞内活化形式的GTP-RhoA表达情况;利用不同浓度的PlGF纯品,检测其对HBMEC单层细胞内活化形式的GTP-RhoA表达情况的影响
5、Western blot方法
利用稳定干扰Flt-1的HBMECs,检测NCI-H209细胞与其单层共培养后,内皮细胞内的p-cofilin和cofilin的表达情况;利用PlGF纯品,检测HBMEC单层在PlGF作用下内皮细胞内的p-cofilin和cofilin的表达情况
6、蛋白抽提和Western blot方法
分析PlGF纯品对TJ结构蛋白occludin可溶性片段和不可溶性片段分布的影响
7、跨内皮迁移实验和HRP flux检测
利用高表达ROCK的HBMECs(ROCK-WT)、Rho结合突变的HBMEs(ROCK-KDIA)和ROCK特异性抑制剂(Y27632),分析内皮细胞内的Rho/ROCK信号通路在NCI-H209细胞的跨内皮单层迁移及致内皮单层渗透性改变中的作用三、探讨Sre分子在参与Flt-1受体介导的小细胞肺癌细胞跨人脑微血管内皮细胞单层迁移过程中的作用
1、细胞培养
2、Western blot方法
检测NCI-H209细胞及PlGF纯品作用后的HBMEC单层细胞内的p-Src和c-Src的表达情况;利用稳定干扰Flt-1的HBMECs,检测其与NCI-H209细胞共培养后内皮细胞内的p-Src和c-Src的表达情况;利用Src分子的特异性抑制剂(pp1),分析NCI-H209细胞与HBMEC单层共培养后内皮细胞内的p-cofilin和cofilin表达情况
3、免疫共沉淀
检测Flt-1受体与c-Src分子间的相互作用
4、Rho活性检测
利用src活性缺失突变的HBMECs(Src-DN),检测经NCI-H209细胞作用后的内皮细胞单层细胞内活化形式的GTP-RhoA表达情况;利用不同浓度的PlGF纯品,检测其对HBMEC单层细胞内活化形式的GTP-RhoA表达情况的影响
5、跨内皮迁移实验和HRP flux检测
利用Src分子的特异性抑制剂(PP1),分析抑制Src分子的活性对NCI-H209细胞跨内皮单层迁移能力及致HBMEC单层渗透性改变的影响
结果
一、Flt-1受体与小细胞肺癌细胞穿人脑微血管内皮细胞单层有关
1、NCI-H209细胞能够穿过人脑微血管内皮细胞单层,并引起内皮单层细胞间紧密连接“开放”
(1)NCI-H209细胞能够穿过HBMEC单层
(2)HRP flux实验分析显示:NCI-H209细胞能够引起HBMEC单层渗透性增高
(3)NCI-H209细胞与HBMEC单层共培养,能够引起内皮细胞间TJ结构改变
(4)Occludin蛋白抽提实验显示:NCI-H209细胞与HBMEC单层共培养能够引起TJ构成蛋白occludin片段分布改变
2、Flt-1受体参与NCI-H209细胞跨人脑微血管内皮细胞单层迁移
(1)NCI-H209细胞与HBMEC共培养,Flt-1表达略有升高
(2)Flt-I磷酸化激酶抑制剂能够降低NCI-H209细胞跨HBMEC单层迁移
(3)Flt-1中和抗体能够降低NCI-H209细胞跨HBMEC单层迁移
(4)Flt-1中和抗体能够消除NCI-H209细胞引起的HBMEC单层TJ结构的改变
3、干扰Flt-1的HBMECs能够下调NCI-H209细胞的跨内皮迁移及消除NCI-H209细胞引起的内皮细胞间TJ的改变
(1)成功建立稳定干扰Flt-1的HBMECs
(2)干扰Flt-1的HBMECs能够下调NCI-H209细胞的跨内皮迁移
(3)干扰Flt-1的HBMECs能够消除NCI-H209细胞引起的内皮细胞间TJ的“开放”
二、Flt-1通过Rho/ROCK信号通路启动小细胞肺癌细胞穿过人脑微血管内皮单层
1、Flt-1受体阻断能够消除NCI-H209细胞引起的HBMEC单层细胞内actin骨架纤维的改变
(1)NCI-H209细胞能够引起HBMEC单层细胞内actin骨架纤维发生变化
(2)Flt-1受体阻断及干扰沉默能够消除NCI-H209细胞引起的HBMEC单层细胞actin骨架纤维的变化
2、Flt-1受体参与NCI-H209细胞引起的HBMEC单层细胞内的Rho/ROCK信号通路的活化及引起的骨架纤维的重排
(1)Flt-1中和抗体和干扰Flt-1的HBMECs均能够消除NCI-H209细胞引起的Rho/ROCK信号途径的活化
(2)干扰Flt-1的HBMECs能消除NCI-H209细胞引起的内皮细胞骨架相关蛋白cofilin磷酸化水平增高
3、PlGF纯品能够活化HBMEC单层细胞内的Rho/ROCK信号通路,并引起TJ相关蛋白分布改变
(1)PlGF能够活化HBMEC单层细胞内的RhoA蛋白,具有浓度依赖性
(2)PlGF能够引起HBNEC单层细胞内骨架相关蛋白cofilin磷酸化水平增高,具有浓度依赖性
(3)PlGF能够引起HBMEC单层TJ相关蛋白Occludin片段分布改变
4、抑制或活化Rho/ROCK信号通路可下调或增加NCI-H209细胞的跨内皮迁移
(1)ROCK抑制剂Y27632可降低NCI-H209细胞跨HBMEC单层迁移
(2)过表达ROCK及Rho结合突变的(ROCK DN)的HBMECs分别能够增强和下调NCI-H209细胞跨HBMEC单层迁移
三、Src参与Flt-1介导的小细胞肺癌细胞穿过人脑微血管内皮细胞单层
1、抑制Src信号通路能够下调NCI-H209细胞的跨内皮单层迁移
(1)Src抑制剂PP1可下调NCI-H209细胞跨HBMEC单层的迁移
(2)Src活性缺失突变的HBMECs(Src-DN)能够降低NCI-H209细胞的跨内皮单层的迁移
(3)Src抑制剂PP1能够降低PlGF引起的内皮单层渗透性的增加
2、NCI-H209细胞及PlGF均能够活化HBMEC内的Src分子
(1)NCI-H209细胞能够引起HBMEC单层细胞内的Src信号分子的磷酸化水平表达增高
(2)PlGF能够引起HBMEC单层细胞内的Src信号分子的磷酸化水平表达增高
3、Flt-1受体与Src信号分子相互作用,参与NCI-H209跨内皮迁移过程中Src信号分子的活化
(1)干扰Flt-1的HBMECs能够消除NCI-H209细胞引起的Src分子的活化
(2)Flt-1磷酸化激酶抑制剂能够消除NCI-H209细胞引起的Src分子的活化
(3)Flt-1受体能够与HBMEC内的Src信号分子结合,形成复合物
4、Src分子与RhoA分子相关,共同调节RhoA下游效应蛋白cofilin的活性
(1)Src活性缺失突变的HBMECs(Src-DN)能够消除NCI-H209细胞引起的RhoA蛋白的活化
(2)Src信号分子抑制剂PP1能够消除NCI-H209细胞引起的Rho/ROCK下游效应蛋白cofilin磷酸化
结论
1、小细胞肺癌细胞通过Flt-1受体开通人脑微血管内皮细胞单层细胞间的紧密连接,引起内皮细胞单层渗透性增加,实现其跨内皮迁移。
2、NCI-H209细胞在跨内皮单层迁移过程中,通过Flt-1受体活化内皮细胞Rho/ROCK信号,调节actin骨架纤维重排和TJ完整性,使其穿过内皮细胞单层。
3、Src分子及其磷酸化形式参与了Flt-1受体介导的NCI-H209细胞穿人脑微血管内皮细胞单层。
Src与RhoA在Flt-1受体介导NCI-H209细胞穿过人脑微血管内皮细胞单层中的相互关系有待进一步研究。
Brain tumors remain one of the leading cause of death.The Lung cancer is the largest cancer killer.Brain metastasis,which is an important cause of cancer morbidity and mortality,occurs in at least 50%of patients with small cell lung cancer(SCLC). A key event of brain metastasis is the migration of cancer cells through the blood-brain barrier(BBB),which constitutes the endothelium,basement membrane and foot processes of astrocytes.The BBB can protect the brain from the intrusion of harmful substances.BBB is a physical barrier comprised of tight junctions(TJs),which form a tight seal to intercellular diffusion.
Numerous studies on TJ's molecular constituents have provided ample evidence that the complex junctional structure consists of transmembrane proteins,cytoplasmic proteins,and cytoskeleton components,all together forming a delicate multifunctional cytoarchitecture.The major interlocking transmembrane proteins is occludin-a 65 kDa phosphoprotein,claudins-a family of 22 kDa proteins and JAM-1,a 32 kDa protein,are association with other several cytoplasmic proteins,cytoplasmic proteins,including ZO-1 and ZO-2,interact either directly or indirectly with occludin.These proteins may act as a protein scaffold to organize occludin at the TJ.In addition to these TJ structural proteins,actin fibers are one of the major cytoskeletal structures in TJ and are linked to the plasma membrane.
Permeability across the TJ is tightly regulated by numerous mechanisms.Studies on TJ regulation have revealed that the hyperphosphorylated form of occludin appears to play an important role in the functional assembly of TJs.ZO-1,a peripheral membrane protein of TJs,links occludin to the underlying actin cytoskeleton. cytoskeletal component-actin,both structural support and functional influence on the organization of TJ complex,and subsequently modulating its junctional permeability. Signaling molecules that directly control actin cytoskeleton organization are particularly intriguing with regard to TJ function regulation.The family of Ras-related small GTP-binding proteins RhoA is regulators of the actin cytoskeleton. Our recent studies have implicated that Rho/ROCK signalling in HBMECs was required for NCI-H209 cells transendothelial.But the precise molecular mechanism of tumor cells penetrating the BBB is poorly defined.
Vascular endothelial growth factor(VEGF) induces permeabilization of blood vessels.VEGF belongs to a protein family,within which Placental growth factor(P1GF) is a member(other members include VEGF-A,B,CandD).P1GF is a secreted, disulfide-linked dimeric glycoprotein.P1GF shares 53%of similarity in its overall amino acid(aa) residues with VEGF.The biological functions of VEGF and P1GF are similar,including stimulation of the growth of vascular endothelial cell.We have used immunohistochemistry to assess the P1GF protein expressing in SCLC.P1GF was positively stained mainly in cytoplasm of lung cancer cells.High level staining of P1GF was found in SCLC patients.P1GF expression is significantly more in SCLC tumour tissues than in matched normal tissues.This finding suggests that P1GF may be active in tumor metastasis.P1GF may have a pivotal role in brain metastasis of SCLC.
Fit-1,the known P1GF receptor,is a member of the tyrosine kinase family of receptor.They consist of seven immunoglobulin-like loops in the extracellular part,a transmembrane domain,a juxtamembrane domain,a kinase domain interrupted by a 69-amino acid residue long insert,and a C-terminal tail.Receptor tyrosine phosphorylation is crucial for signal transduction by the activation of The Flt-1 extracellular domain interacts with P1GF.
Based on these results,the aim of this study is to test whether activation of Flt-1 affects the permeabilization of endothelialcells of blood vessels.Therefore,this study focused on investigation the role of Flt-1 and further analysis about molecular mechanism of signaling during SCLC cells migration across the HBMECs.The study will be useful for brain metastases of SCLC therapy in the futher.
Methods
1.To study the role of Flt-1 in NCI-H209 cells transendothelial migration.
(1)Cell culture
①HBMEC is cultured in RPMI1640,supplemented with 10%Nu-serum etc
②NCI-H209 cell is cultured in RPMI1640,10%FBS
(2)Immunofluorescence assay
To analyzed tight junction protein ZO-1 expression.
The HBMEC monolayers grown on glass coverslips were fixed with paraformaldehyde and permeabilized with Triton X-100.After blocking with BSA in PBS,the cells were incubated with anti-ZO-1 Ab,The glass slides were analyzed using immunofluorescence microscopy.
(3)Transendothelial migration assay
NCI-H209 cells are added to the upper chamber of Transwell(the BBB model in vitro) for 8h,the cells that had transmigrated into the lower chamber were harvestedand counted in a hemocytometer.To analyzed the ability of NCI-H209 cells transendothelial migration.
(4)HRP flux measurement
The analysis of permeability of HBMEC monolayers.
The HBMEC monolayer in Transwell inserts were cocultured with NCI-H209 cells.HRP in serumfree RPMI1640 medium was added to the upper compartment of the Transwell system.After 1 h,the media from the lower chamber were collected and the HRP content of the samples were assayed colorimetrically.
(5)The western blot assay
Analysis of Flt-1 expression on HBMEC
(6)Neutralization Ab and inhibitor assay
The analysis of effects of neutralization Ab and inhibitor
Neutralization Ab and inhibitor at different dose added to the upper compartment of Transwell system,after a certain time,the cells that had transmigrated into the lower chamber were harvestedand counted in a hemocytometer.
(7)The establish of HBMEC(siRNA Flt-1) cell line by stable transfection.
2.To investigated the role of Flt-1 on HBMEC in the process of NCI-H209 cell transendothelial migration induced by Rho/ROCK
(1)Cell culture
(2)Immunofluorescence assay
To analyzed the expression of F-actin
The cell incubated with rhodamine-labeled phalloidin for the actin filaments.
(3)Effects of neutralization Ab
To analyzed the effects of neutralization Ab
(4)The western blot assay
To analyzed the p-cofilin/cofilin expression on HBMEC
(6)Cell fractionation and Western blot assay
Confluent HBMECs were washed with D-PBS,extracted in TritonX-100 lysis buffer.The supernatant was collected as the soluble fraction(S).The pellets were dissolved in SDS lysis buffer.The supernatant was collected as the insoluble material (IS).Equal amounts of proteins were separated by SDS-PAGE and Western blot analysis.
(7)Rho activation assay
Rho activation assay kit was used to determine the activation of Rho proteins in HBMECs cocultured with NCI-H209 cells.Briefly,cell lysates were incubated with Rho-binding peptide immobilized on agarose beads for 45 min,and activated GTP-Rho bound to agarose was detected by Western blot using anti-RhoA antibody
3.To study the role of Src in regulation the progress of NCI-H209 cells transendothelial migration by the activated RhoA
(1)Cell culture
(2)The western blot assay
The western blot analysis of p-cofilin/cofilin,p-Src/c-Src expression on HBMEC
(3)Immunoprecipitation assay
The cells were then scraped in 1 mL of lysis buffer.Cell extract samples for immunoprecipitation were precleared using packed Protein A/G(1:1) and mixing on a rotator at 4℃30 min.Centrifugation at 14 000 rpm in a cold microcentrifuge for 2 min was then conducted and the precleared cell extract supernatant was collected. Immunoprecipitations were performed using c-Src and packed Protein A/G agarose. The immunoprecipitations were mixed on a rotator at 4℃for 2 h.The immune complexes were washed 3 times with lysis buffer.Samples for Westerns blot were boiled for 5 min in Laemmli's sample buffer,and resolved on SDS-PAGE gels.
(4)Rho activation assay
(5)Transendothelial migration assay
To investigated the ability of NCI-H209 cells transendothelial migration
Results
1.Flt-1 plays an important role in NCI-H209 cells transendothelial migration.
(1)The transendothelial migration of NCI-H209 cells induced by coculturing with HBMEC is associated with the permeability of HBMEC monolayers and TJ
①NCI-H209 can migrate through the HBMEC monolayer
②HRP flux measurement indicated that the disruption of tight junction is induced by HBMECs coculturing with NCI-H209 cells
③NCI-H209 cells,coculturing with HBMECs at different time,showed dramatic cell morphology changes
④The biological character analysis of The different detergent solubility of endothelial occludin during the colocalization between NCI-H209 cells with HBMECs analyzed by Western blot
(2)Flt-1 participated the progression of the transendothelial migration of NCI-H209 cells
①The expression of Flt-1 on HBNECs induced by NCI-H209 cells
②The ability of NCI-H209's transendothelial migration decreased by VEGFR (flt-1,VEGFR2) tyrosine kinase inhibitor
③The ability of NCI-H209's transendothelial migration decreased by Flt-1 neutralization antibody
④Flt-1 neutralization antibody can maitaine tight junction integrity followed by NCI-H209 cells are treated
(3)The ability of NCI-H209's transendothelial migration decreased by Flt-1 siRNA and changes of TJ induced by NCI-H209 cells
①A single-clone cell strain of Flt-1 siRNA is obtained
②The ability of NCI-H209's transendothelial migration decreased by Flt-1 siRNA
③NCI-H209 cells cocultured with Flt-1 siRNA HBMECs can maitaine tight junction integrity
2.Rho/ROCK signalling can induced the process of NCI-H209 cells transendothelial migration by the role of Flt-1 on HBMEC
(1)The recruitment of F-actin' changes induced by NCI-H209 cells is obtained from the blocking of Flt-1
①The alteration of actin cytoskeleton in HBMECs during NCI-H209 cells transendothelial migration.
②The recruitment of F-actin' changes induced by NCI-H209 cells is obtained from Flt-1 neutralization antibody and Flt-1 siRNA
(2)NCI-H209 cells-induced changes of endothelial actin cytoskeleton were mediated by Rho/ROCK
①Overexpression of Rho-GTP in HBMECs followed by cocultured with NCI-H209 cells is down-regulated by Flt-1 siRNA HBMECs and Flt-1 neutralization antibody
②The levels of phospho-cofilin in HBMECs cocultured with NCI-H209 cells decreased by Flt-1 neutralization antibody.
(3)P1GF can promote the Rho/ROCK signalling activation,up-regulated levels of phospho-cofilin,the distribution changes of occludin
①The distribution change of tight junction protein,occluding,induced by P1GF at different time.
②The activation of endothelial RhoA protein followed by HBMECs are treated by P1GF at different dose
③The up-regulated levels of phospho-cofilin proteins is detected followed by HBMECs are treated by P1GF at different dose
(4)NCI-H209 cells transendothelial migration was associated with Rho/ROCK signalling activation
①ROCK inhibitor,Y27632,could block NCI-H209 cells transendothelial migration
②Overexpression of ROCK and its dominant-negative mutant in HBMECs significantly affected NCI-H209 cells transendothelial migration
3.The important role of Src on HBMECs in the process of NCI-H209 cells transendothelial migration by Flt-1 receptor
(1)The ability of NCI-H209 cell transendothelial migration is abolished by the functional deletion of Src
①The ability of NCI-H209 cell transendothelial migration is abolished by the inhibitor of Src
②The ability of NCI-H209 cell transendothelial migration is abolished by the r of Src-DN
③HRP flux measurement indicated that the disruption of tight junction is induced by P1GF is abolished by the inhibitor of Src
(2)The activation of Src in HBMECs can be induced by NCI-H209 cells or P1GF
①The up-regulated levels of c-Src protein and phospho-Src is detected followed by NCI-H209 cells at different time
②The up-regulated levels of c-Src protein and phospho-Src is detected followed by P1GF
(3)The interaction between Flt-1 and Src can promote the activation of Src during the NCI-H209 cells cocultivated with HBMECs
①Flt-1 siRNA in HBMECs abolished the activative effect of c-Src that induced by NCI-H209 cells
②The activation of Src during the NCI-H209 cells cocultivated with HBMECs can be abolished by the VEGFR(flt-1,VEGFR2) tyrosine kinase inhibitor
③Flt-1 can interacted with c-Src
(4)The role of Src in regulation the progress of NCI-H209 cells transendothelial migration by the activated RhoA
①The important role of Src in the activation of endothelial RhoA protein, GTP-RhoA,induced by the NCI-H209 cells
②Cofilin phosphorylation in HBMECs were increasing after NCI-H209 cells treatment compared with HBMECs alone,While,the levels of phospho-cofilin is down-regulated by the inhibitor of Src
Conclusion
flt-1 receptor participate in NCI-H209 cells migration through the HBMEC in vitro.
Rho/ROCK was required for SCLC cells transendothelial migration by the activation of Flt-1 receptor.
The important role of Src on HBMEC in the process of NCI-H209 cells transendothelial migration by Flt-1 receptor.The study will analysis about the interaction between Src and Rho in the further.
脑部肿瘤严重危害人类的健康,以继发性脑瘤为主。肺癌为癌症的首要杀手,小细胞肺癌(small cell lung cancer,SCLC)为临床常见的恶性肿瘤,生长快,侵袭力强,易发生胸内或远端转移,近一半的SCLC患者会发生脑转移,SCLC发生脑转移是导致这类疾病死亡的主要原因。脑转移的关键步骤是小细胞肺癌从血液中游离出跨血脑屏障(Blood brain barrier,BBB)进入脑组织。血脑屏障是位于脑组织与血液之间的一个复杂细胞系统,血脑屏障的物质基础是脑部的微血管,脑微血管内皮细胞间的紧密连接(tight junction,TJ)使得血管内皮细胞间相互“焊接”得十分紧密,不象其他组织微血管壁那样有较大的缝隙,对于保护脑组织周围稳定的生理环境和防止血液中有害物质侵入脑内具有重要的生理意义。
紧密连接由许多蛋白构成,完整的紧密连接结构蛋白主要由跨膜蛋白、胞质附着蛋白和细胞骨架(微丝)三种组分构成。跨膜蛋白如occludin、claudins和连接粘附分子(junctional adhesive molecule,JAM)等,为跨膜糖蛋白,参与封锁相邻细胞间的空隙;胞质附着蛋白如ZO-1、ZO-2和ZO-3等,ZO为紧密连接处的膜外周蛋白,将occludin与其下面的actin骨架相连。这些蛋白附着到细胞骨架上,彼此相互作用,参与维持和调节紧密连接的屏障功能。
多种机制参与调节细胞间TJ的功能,occludin蛋白的高度磷酸化在TJ结构的组装和功能的调节上起到重要的作用。膜周边蛋白ZO-1将跨膜蛋白occludin和actin骨架纤维相连,actin既是细胞间TJ结构组分,又是TJ渗透性的调节者。一些调控actin骨架纤维组装的信号分子也与TJ渗透性功能的调节相关。小GTP结合蛋白RhoA为公认的actin骨架纤维的调节者,我们实验室最近的研究表明:小细胞肺癌细胞(NCI-H209细胞)能够通过内皮细胞的Rho/ROCK信号通路指导其跨过人脑微血管内皮细胞(HBMEC)单层迁移,但有关其穿过HBMEC单层的详细机制尚未清楚。
血管内皮生长因子(Vascular endothelial growth factor,VEGF)又称为血管渗透因子,其蛋白家族包括VEGF-A,B,C,D和胎盘生长因子(Placental growthfactor,PlGF)。PlGF为分泌的糖蛋白,与VEGF在氨基酸序列残基上有53%的同源性,且其生物学功能与VEGF也有相似之处,如刺激内皮细胞生长等。血管内皮生长因子受体1(Vascular endothelial growth factor receper,VEGFR-1,又称为Fms-1ike tyrosine kinase-1,Flt-1),为PlGF在细胞膜上的专一受体,属于受体酪氨酸激酶家族成员,由七个免疫球蛋白样结构的胞外区、跨膜区、近膜区、激酶插入区和C-末端构成。PlGF与Flt-1的胞外区结合后,可活化受体的酪氨酸激酶,活化的酪氨酸激酶在信号转导的传递和生物学功能的发挥中起到重要的作用。
本实验室通过免疫组化技术检测到小细胞肺癌细胞高表达PlGF,并证实PlGF在小细胞肺癌细胞跨人脑微血管内皮细胞单层迁移过程中发挥作用,但有关PlGF的受体Flt-1在这一过程中所发挥的作用及机制尚不清楚,本研究试图回答该问题。
方法
一、探讨Flt-1受体在小细胞肺癌细胞跨人脑微血管内皮细胞单层迁移中的作用
1、细胞培养
(1)HBMEC细胞培养于含10%胎牛血清和10%Nu血清的RPMI1640培养液
(2)NCI-H209细胞培养于含10%新生牛血清的RPMI1640培养液
2、免疫荧光技术
观察NCI-H209细胞与HBMEC单层共培养后内皮细胞间TJ结构改变
3、蛋白抽提和Western blot方法
分析NCI-H209细胞与HBMEC单层共培养后TJ蛋白occludin的可溶性片段和不可溶性片段的分布情况
4、跨内皮迁移实验
分析NCI-H209细胞与HBMEC单层共培养后其跨HBMEC单层迁移能力
5、HRP flux检测
分析NCI-H209细胞与HBMEC单层共培养后HBMEC单层渗透性改变
6、Western blot方法
检测NCI-H209细胞与HBMEC单层共培养后Flt-1蛋白表达情况
7、中和抗体及抑制剂封闭实验
利用Flt-1中和抗体和VEGFR-1、2受体抑制剂,分析Flt-1受体及其磷酸化形式在NCI-H209细胞的跨内皮单层迁移中的作用
8、建立和培养稳定干扰Flt-1的HBMECs并检测其与NCI-H209细胞共培养后相关生物学特性的变化
二、探讨Rho/ROCK信号通路通过Flt-1受体启动小细胞肺癌细胞跨人脑微血管内皮单层迁移中的作用
1、细胞培养
2、免疫荧光技术
细胞骨架蛋白的形态观察
3、中和抗体实验
利用Flt-1中和抗体能够封闭Flt-1受体结合区的特性,观察受体封闭后,NCI-H209细胞致HBMEC单层细胞骨架形态变化的改变
4、Rho活性检测
利用Flt-1中和抗体和稳定干扰Flt-1沉默的HBMECs,检测经NCI-H209细胞作用后,HBMEC单层细胞内活化形式的GTP-RhoA表达情况;利用不同浓度的PlGF纯品,检测其对HBMEC单层细胞内活化形式的GTP-RhoA表达情况的影响
5、Western blot方法
利用稳定干扰Flt-1的HBMECs,检测NCI-H209细胞与其单层共培养后,内皮细胞内的p-cofilin和cofilin的表达情况;利用PlGF纯品,检测HBMEC单层在PlGF作用下内皮细胞内的p-cofilin和cofilin的表达情况
6、蛋白抽提和Western blot方法
分析PlGF纯品对TJ结构蛋白occludin可溶性片段和不可溶性片段分布的影响
7、跨内皮迁移实验和HRP flux检测
利用高表达ROCK的HBMECs(ROCK-WT)、Rho结合突变的HBMEs(ROCK-KDIA)和ROCK特异性抑制剂(Y27632),分析内皮细胞内的Rho/ROCK信号通路在NCI-H209细胞的跨内皮单层迁移及致内皮单层渗透性改变中的作用三、探讨Sre分子在参与Flt-1受体介导的小细胞肺癌细胞跨人脑微血管内皮细胞单层迁移过程中的作用
1、细胞培养
2、Western blot方法
检测NCI-H209细胞及PlGF纯品作用后的HBMEC单层细胞内的p-Src和c-Src的表达情况;利用稳定干扰Flt-1的HBMECs,检测其与NCI-H209细胞共培养后内皮细胞内的p-Src和c-Src的表达情况;利用Src分子的特异性抑制剂(pp1),分析NCI-H209细胞与HBMEC单层共培养后内皮细胞内的p-cofilin和cofilin表达情况
3、免疫共沉淀
检测Flt-1受体与c-Src分子间的相互作用
4、Rho活性检测
利用src活性缺失突变的HBMECs(Src-DN),检测经NCI-H209细胞作用后的内皮细胞单层细胞内活化形式的GTP-RhoA表达情况;利用不同浓度的PlGF纯品,检测其对HBMEC单层细胞内活化形式的GTP-RhoA表达情况的影响
5、跨内皮迁移实验和HRP flux检测
利用Src分子的特异性抑制剂(PP1),分析抑制Src分子的活性对NCI-H209细胞跨内皮单层迁移能力及致HBMEC单层渗透性改变的影响
结果
一、Flt-1受体与小细胞肺癌细胞穿人脑微血管内皮细胞单层有关
1、NCI-H209细胞能够穿过人脑微血管内皮细胞单层,并引起内皮单层细胞间紧密连接“开放”
(1)NCI-H209细胞能够穿过HBMEC单层
(2)HRP flux实验分析显示:NCI-H209细胞能够引起HBMEC单层渗透性增高
(3)NCI-H209细胞与HBMEC单层共培养,能够引起内皮细胞间TJ结构改变
(4)Occludin蛋白抽提实验显示:NCI-H209细胞与HBMEC单层共培养能够引起TJ构成蛋白occludin片段分布改变
2、Flt-1受体参与NCI-H209细胞跨人脑微血管内皮细胞单层迁移
(1)NCI-H209细胞与HBMEC共培养,Flt-1表达略有升高
(2)Flt-I磷酸化激酶抑制剂能够降低NCI-H209细胞跨HBMEC单层迁移
(3)Flt-1中和抗体能够降低NCI-H209细胞跨HBMEC单层迁移
(4)Flt-1中和抗体能够消除NCI-H209细胞引起的HBMEC单层TJ结构的改变
3、干扰Flt-1的HBMECs能够下调NCI-H209细胞的跨内皮迁移及消除NCI-H209细胞引起的内皮细胞间TJ的改变
(1)成功建立稳定干扰Flt-1的HBMECs
(2)干扰Flt-1的HBMECs能够下调NCI-H209细胞的跨内皮迁移
(3)干扰Flt-1的HBMECs能够消除NCI-H209细胞引起的内皮细胞间TJ的“开放”
二、Flt-1通过Rho/ROCK信号通路启动小细胞肺癌细胞穿过人脑微血管内皮单层
1、Flt-1受体阻断能够消除NCI-H209细胞引起的HBMEC单层细胞内actin骨架纤维的改变
(1)NCI-H209细胞能够引起HBMEC单层细胞内actin骨架纤维发生变化
(2)Flt-1受体阻断及干扰沉默能够消除NCI-H209细胞引起的HBMEC单层细胞actin骨架纤维的变化
2、Flt-1受体参与NCI-H209细胞引起的HBMEC单层细胞内的Rho/ROCK信号通路的活化及引起的骨架纤维的重排
(1)Flt-1中和抗体和干扰Flt-1的HBMECs均能够消除NCI-H209细胞引起的Rho/ROCK信号途径的活化
(2)干扰Flt-1的HBMECs能消除NCI-H209细胞引起的内皮细胞骨架相关蛋白cofilin磷酸化水平增高
3、PlGF纯品能够活化HBMEC单层细胞内的Rho/ROCK信号通路,并引起TJ相关蛋白分布改变
(1)PlGF能够活化HBMEC单层细胞内的RhoA蛋白,具有浓度依赖性
(2)PlGF能够引起HBNEC单层细胞内骨架相关蛋白cofilin磷酸化水平增高,具有浓度依赖性
(3)PlGF能够引起HBMEC单层TJ相关蛋白Occludin片段分布改变
4、抑制或活化Rho/ROCK信号通路可下调或增加NCI-H209细胞的跨内皮迁移
(1)ROCK抑制剂Y27632可降低NCI-H209细胞跨HBMEC单层迁移
(2)过表达ROCK及Rho结合突变的(ROCK DN)的HBMECs分别能够增强和下调NCI-H209细胞跨HBMEC单层迁移
三、Src参与Flt-1介导的小细胞肺癌细胞穿过人脑微血管内皮细胞单层
1、抑制Src信号通路能够下调NCI-H209细胞的跨内皮单层迁移
(1)Src抑制剂PP1可下调NCI-H209细胞跨HBMEC单层的迁移
(2)Src活性缺失突变的HBMECs(Src-DN)能够降低NCI-H209细胞的跨内皮单层的迁移
(3)Src抑制剂PP1能够降低PlGF引起的内皮单层渗透性的增加
2、NCI-H209细胞及PlGF均能够活化HBMEC内的Src分子
(1)NCI-H209细胞能够引起HBMEC单层细胞内的Src信号分子的磷酸化水平表达增高
(2)PlGF能够引起HBMEC单层细胞内的Src信号分子的磷酸化水平表达增高
3、Flt-1受体与Src信号分子相互作用,参与NCI-H209跨内皮迁移过程中Src信号分子的活化
(1)干扰Flt-1的HBMECs能够消除NCI-H209细胞引起的Src分子的活化
(2)Flt-1磷酸化激酶抑制剂能够消除NCI-H209细胞引起的Src分子的活化
(3)Flt-1受体能够与HBMEC内的Src信号分子结合,形成复合物
4、Src分子与RhoA分子相关,共同调节RhoA下游效应蛋白cofilin的活性
(1)Src活性缺失突变的HBMECs(Src-DN)能够消除NCI-H209细胞引起的RhoA蛋白的活化
(2)Src信号分子抑制剂PP1能够消除NCI-H209细胞引起的Rho/ROCK下游效应蛋白cofilin磷酸化
结论
1、小细胞肺癌细胞通过Flt-1受体开通人脑微血管内皮细胞单层细胞间的紧密连接,引起内皮细胞单层渗透性增加,实现其跨内皮迁移。
2、NCI-H209细胞在跨内皮单层迁移过程中,通过Flt-1受体活化内皮细胞Rho/ROCK信号,调节actin骨架纤维重排和TJ完整性,使其穿过内皮细胞单层。
3、Src分子及其磷酸化形式参与了Flt-1受体介导的NCI-H209细胞穿人脑微血管内皮细胞单层。
Src与RhoA在Flt-1受体介导NCI-H209细胞穿过人脑微血管内皮细胞单层中的相互关系有待进一步研究。
Brain tumors remain one of the leading cause of death.The Lung cancer is the largest cancer killer.Brain metastasis,which is an important cause of cancer morbidity and mortality,occurs in at least 50%of patients with small cell lung cancer(SCLC). A key event of brain metastasis is the migration of cancer cells through the blood-brain barrier(BBB),which constitutes the endothelium,basement membrane and foot processes of astrocytes.The BBB can protect the brain from the intrusion of harmful substances.BBB is a physical barrier comprised of tight junctions(TJs),which form a tight seal to intercellular diffusion.
Numerous studies on TJ's molecular constituents have provided ample evidence that the complex junctional structure consists of transmembrane proteins,cytoplasmic proteins,and cytoskeleton components,all together forming a delicate multifunctional cytoarchitecture.The major interlocking transmembrane proteins is occludin-a 65 kDa phosphoprotein,claudins-a family of 22 kDa proteins and JAM-1,a 32 kDa protein,are association with other several cytoplasmic proteins,cytoplasmic proteins,including ZO-1 and ZO-2,interact either directly or indirectly with occludin.These proteins may act as a protein scaffold to organize occludin at the TJ.In addition to these TJ structural proteins,actin fibers are one of the major cytoskeletal structures in TJ and are linked to the plasma membrane.
Permeability across the TJ is tightly regulated by numerous mechanisms.Studies on TJ regulation have revealed that the hyperphosphorylated form of occludin appears to play an important role in the functional assembly of TJs.ZO-1,a peripheral membrane protein of TJs,links occludin to the underlying actin cytoskeleton. cytoskeletal component-actin,both structural support and functional influence on the organization of TJ complex,and subsequently modulating its junctional permeability. Signaling molecules that directly control actin cytoskeleton organization are particularly intriguing with regard to TJ function regulation.The family of Ras-related small GTP-binding proteins RhoA is regulators of the actin cytoskeleton. Our recent studies have implicated that Rho/ROCK signalling in HBMECs was required for NCI-H209 cells transendothelial.But the precise molecular mechanism of tumor cells penetrating the BBB is poorly defined.
Vascular endothelial growth factor(VEGF) induces permeabilization of blood vessels.VEGF belongs to a protein family,within which Placental growth factor(P1GF) is a member(other members include VEGF-A,B,CandD).P1GF is a secreted, disulfide-linked dimeric glycoprotein.P1GF shares 53%of similarity in its overall amino acid(aa) residues with VEGF.The biological functions of VEGF and P1GF are similar,including stimulation of the growth of vascular endothelial cell.We have used immunohistochemistry to assess the P1GF protein expressing in SCLC.P1GF was positively stained mainly in cytoplasm of lung cancer cells.High level staining of P1GF was found in SCLC patients.P1GF expression is significantly more in SCLC tumour tissues than in matched normal tissues.This finding suggests that P1GF may be active in tumor metastasis.P1GF may have a pivotal role in brain metastasis of SCLC.
Fit-1,the known P1GF receptor,is a member of the tyrosine kinase family of receptor.They consist of seven immunoglobulin-like loops in the extracellular part,a transmembrane domain,a juxtamembrane domain,a kinase domain interrupted by a 69-amino acid residue long insert,and a C-terminal tail.Receptor tyrosine phosphorylation is crucial for signal transduction by the activation of The Flt-1 extracellular domain interacts with P1GF.
Based on these results,the aim of this study is to test whether activation of Flt-1 affects the permeabilization of endothelialcells of blood vessels.Therefore,this study focused on investigation the role of Flt-1 and further analysis about molecular mechanism of signaling during SCLC cells migration across the HBMECs.The study will be useful for brain metastases of SCLC therapy in the futher.
Methods
1.To study the role of Flt-1 in NCI-H209 cells transendothelial migration.
(1)Cell culture
①HBMEC is cultured in RPMI1640,supplemented with 10%Nu-serum etc
②NCI-H209 cell is cultured in RPMI1640,10%FBS
(2)Immunofluorescence assay
To analyzed tight junction protein ZO-1 expression.
The HBMEC monolayers grown on glass coverslips were fixed with paraformaldehyde and permeabilized with Triton X-100.After blocking with BSA in PBS,the cells were incubated with anti-ZO-1 Ab,The glass slides were analyzed using immunofluorescence microscopy.
(3)Transendothelial migration assay
NCI-H209 cells are added to the upper chamber of Transwell(the BBB model in vitro) for 8h,the cells that had transmigrated into the lower chamber were harvestedand counted in a hemocytometer.To analyzed the ability of NCI-H209 cells transendothelial migration.
(4)HRP flux measurement
The analysis of permeability of HBMEC monolayers.
The HBMEC monolayer in Transwell inserts were cocultured with NCI-H209 cells.HRP in serumfree RPMI1640 medium was added to the upper compartment of the Transwell system.After 1 h,the media from the lower chamber were collected and the HRP content of the samples were assayed colorimetrically.
(5)The western blot assay
Analysis of Flt-1 expression on HBMEC
(6)Neutralization Ab and inhibitor assay
The analysis of effects of neutralization Ab and inhibitor
Neutralization Ab and inhibitor at different dose added to the upper compartment of Transwell system,after a certain time,the cells that had transmigrated into the lower chamber were harvestedand counted in a hemocytometer.
(7)The establish of HBMEC(siRNA Flt-1) cell line by stable transfection.
2.To investigated the role of Flt-1 on HBMEC in the process of NCI-H209 cell transendothelial migration induced by Rho/ROCK
(1)Cell culture
(2)Immunofluorescence assay
To analyzed the expression of F-actin
The cell incubated with rhodamine-labeled phalloidin for the actin filaments.
(3)Effects of neutralization Ab
To analyzed the effects of neutralization Ab
(4)The western blot assay
To analyzed the p-cofilin/cofilin expression on HBMEC
(6)Cell fractionation and Western blot assay
Confluent HBMECs were washed with D-PBS,extracted in TritonX-100 lysis buffer.The supernatant was collected as the soluble fraction(S).The pellets were dissolved in SDS lysis buffer.The supernatant was collected as the insoluble material (IS).Equal amounts of proteins were separated by SDS-PAGE and Western blot analysis.
(7)Rho activation assay
Rho activation assay kit was used to determine the activation of Rho proteins in HBMECs cocultured with NCI-H209 cells.Briefly,cell lysates were incubated with Rho-binding peptide immobilized on agarose beads for 45 min,and activated GTP-Rho bound to agarose was detected by Western blot using anti-RhoA antibody
3.To study the role of Src in regulation the progress of NCI-H209 cells transendothelial migration by the activated RhoA
(1)Cell culture
(2)The western blot assay
The western blot analysis of p-cofilin/cofilin,p-Src/c-Src expression on HBMEC
(3)Immunoprecipitation assay
The cells were then scraped in 1 mL of lysis buffer.Cell extract samples for immunoprecipitation were precleared using packed Protein A/G(1:1) and mixing on a rotator at 4℃30 min.Centrifugation at 14 000 rpm in a cold microcentrifuge for 2 min was then conducted and the precleared cell extract supernatant was collected. Immunoprecipitations were performed using c-Src and packed Protein A/G agarose. The immunoprecipitations were mixed on a rotator at 4℃for 2 h.The immune complexes were washed 3 times with lysis buffer.Samples for Westerns blot were boiled for 5 min in Laemmli's sample buffer,and resolved on SDS-PAGE gels.
(4)Rho activation assay
(5)Transendothelial migration assay
To investigated the ability of NCI-H209 cells transendothelial migration
Results
1.Flt-1 plays an important role in NCI-H209 cells transendothelial migration.
(1)The transendothelial migration of NCI-H209 cells induced by coculturing with HBMEC is associated with the permeability of HBMEC monolayers and TJ
①NCI-H209 can migrate through the HBMEC monolayer
②HRP flux measurement indicated that the disruption of tight junction is induced by HBMECs coculturing with NCI-H209 cells
③NCI-H209 cells,coculturing with HBMECs at different time,showed dramatic cell morphology changes
④The biological character analysis of The different detergent solubility of endothelial occludin during the colocalization between NCI-H209 cells with HBMECs analyzed by Western blot
(2)Flt-1 participated the progression of the transendothelial migration of NCI-H209 cells
①The expression of Flt-1 on HBNECs induced by NCI-H209 cells
②The ability of NCI-H209's transendothelial migration decreased by VEGFR (flt-1,VEGFR2) tyrosine kinase inhibitor
③The ability of NCI-H209's transendothelial migration decreased by Flt-1 neutralization antibody
④Flt-1 neutralization antibody can maitaine tight junction integrity followed by NCI-H209 cells are treated
(3)The ability of NCI-H209's transendothelial migration decreased by Flt-1 siRNA and changes of TJ induced by NCI-H209 cells
①A single-clone cell strain of Flt-1 siRNA is obtained
②The ability of NCI-H209's transendothelial migration decreased by Flt-1 siRNA
③NCI-H209 cells cocultured with Flt-1 siRNA HBMECs can maitaine tight junction integrity
2.Rho/ROCK signalling can induced the process of NCI-H209 cells transendothelial migration by the role of Flt-1 on HBMEC
(1)The recruitment of F-actin' changes induced by NCI-H209 cells is obtained from the blocking of Flt-1
①The alteration of actin cytoskeleton in HBMECs during NCI-H209 cells transendothelial migration.
②The recruitment of F-actin' changes induced by NCI-H209 cells is obtained from Flt-1 neutralization antibody and Flt-1 siRNA
(2)NCI-H209 cells-induced changes of endothelial actin cytoskeleton were mediated by Rho/ROCK
①Overexpression of Rho-GTP in HBMECs followed by cocultured with NCI-H209 cells is down-regulated by Flt-1 siRNA HBMECs and Flt-1 neutralization antibody
②The levels of phospho-cofilin in HBMECs cocultured with NCI-H209 cells decreased by Flt-1 neutralization antibody.
(3)P1GF can promote the Rho/ROCK signalling activation,up-regulated levels of phospho-cofilin,the distribution changes of occludin
①The distribution change of tight junction protein,occluding,induced by P1GF at different time.
②The activation of endothelial RhoA protein followed by HBMECs are treated by P1GF at different dose
③The up-regulated levels of phospho-cofilin proteins is detected followed by HBMECs are treated by P1GF at different dose
(4)NCI-H209 cells transendothelial migration was associated with Rho/ROCK signalling activation
①ROCK inhibitor,Y27632,could block NCI-H209 cells transendothelial migration
②Overexpression of ROCK and its dominant-negative mutant in HBMECs significantly affected NCI-H209 cells transendothelial migration
3.The important role of Src on HBMECs in the process of NCI-H209 cells transendothelial migration by Flt-1 receptor
(1)The ability of NCI-H209 cell transendothelial migration is abolished by the functional deletion of Src
①The ability of NCI-H209 cell transendothelial migration is abolished by the inhibitor of Src
②The ability of NCI-H209 cell transendothelial migration is abolished by the r of Src-DN
③HRP flux measurement indicated that the disruption of tight junction is induced by P1GF is abolished by the inhibitor of Src
(2)The activation of Src in HBMECs can be induced by NCI-H209 cells or P1GF
①The up-regulated levels of c-Src protein and phospho-Src is detected followed by NCI-H209 cells at different time
②The up-regulated levels of c-Src protein and phospho-Src is detected followed by P1GF
(3)The interaction between Flt-1 and Src can promote the activation of Src during the NCI-H209 cells cocultivated with HBMECs
①Flt-1 siRNA in HBMECs abolished the activative effect of c-Src that induced by NCI-H209 cells
②The activation of Src during the NCI-H209 cells cocultivated with HBMECs can be abolished by the VEGFR(flt-1,VEGFR2) tyrosine kinase inhibitor
③Flt-1 can interacted with c-Src
(4)The role of Src in regulation the progress of NCI-H209 cells transendothelial migration by the activated RhoA
①The important role of Src in the activation of endothelial RhoA protein, GTP-RhoA,induced by the NCI-H209 cells
②Cofilin phosphorylation in HBMECs were increasing after NCI-H209 cells treatment compared with HBMECs alone,While,the levels of phospho-cofilin is down-regulated by the inhibitor of Src
Conclusion
flt-1 receptor participate in NCI-H209 cells migration through the HBMEC in vitro.
Rho/ROCK was required for SCLC cells transendothelial migration by the activation of Flt-1 receptor.
The important role of Src on HBMEC in the process of NCI-H209 cells transendothelial migration by Flt-1 receptor.The study will analysis about the interaction between Src and Rho in the further.
引文
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