【作者】 苏踊跃；

【导师】 罗向东；

【作者基本信息】 第三军医大学 ， 外科学， 2004， 博士

【摘要】 血管内皮细胞不仅仅是血液和组织间的一道机械屏障，而且是一个动态的、异质的、播散分布的器官，具有重要的分泌、合成、代谢和免疫功能。血管内皮细胞是创伤、感染、休克、心血管疾病、肿瘤等多种疾病中极易受损的细胞，同时也是参与机体应激反应、创伤组织修复的重要成员，其增殖和分化的相互协调是烧伤、创伤后创面愈合和修复关键因素之一。 本室前期研究发现：人钙／钙调蛋白依赖的丝氨酸蛋白激酶(CASK)可以抑制血管内皮细胞株ECV304细胞的增殖，而其调节血管内皮细胞增殖的作用机制尚不清楚，对此深入探讨将有助于我们对创伤修复等涉及血管发生、重建的病理生理现象的理解。 CASK的主要功能是作为一个支架蛋白质参与细胞膜蛋白骨架的构建，细胞连接的形成，以及调节功能蛋白质的分布，参与细胞的信号传导和基因调控等。我们发现，人血管内皮细胞株ECV304中CASK能够与分化抑制因子1(Id1)相互作用。Id家族属于HLH蛋白，其结构中不含有与DNA特异性结合的碱性区，与很多bHLH转录因子结合形成无功能活性的异二聚体，是bHLH家族的负调控因子，在细胞增殖和分化的调节中具有重要作用。CASK与Id1在物理结构上存在相互作用提示两者可能在生物学功能上有所关联。本室对此做了一些探索性工作，我们发现，过表达CASK的ECV304细胞的生长率显著降低，细胞周期依赖性激酶抑制因子p16、p21表达上调，我们推测CASK过表达后所观察到的细胞增殖抑制现象可能与Id1有关，为了进一步验证以上推测和深入了解CASK抑制细胞增殖的信号机制，我们从几个方面进行了初步探索。 本室前期的研究已经成功构建了CASK过表达的ECV304细胞，本课题主要承担CASK缺陷表达ECV304细胞模型的建立以及CASK影响细胞增殖的细胞内信号机制研究。采用近年发展起来的RNA干扰技术(RNAi)，应用质粒载体介导的细胞内小干扰RNA(siRNA)表达策略，共设计和成功构建7个siRNA表达质粒，分别对应于靶基因CASK、Id1，报告基因萤火虫荧光素酶、EGFP和GFP，全部经测序验证。 首先通过重组siRNA表达质粒对外源报告基因EGFP和萤火虫荧光素酶的抑制效果，确认了载体介导的RNAi策略在ECV304细胞中应用的可行性。根据蛋白和mRNA第三军医大学博士学位论文水平的阻断效率，筛选出对内源性靶基因CASK和Idl抑制效率较高的重组质粒。应用RNAi方法抑制CASK表达，ECV3O4细胞生长加快，而CASK过表达，ECV304细胞生长受到一定的抑制，实验从正调节和负调节两个方向证明，CASK能够参与ECv304细胞增殖的抑制信号。流式细胞术分析表明，应用RNAi方法抑制CASK表达，ECV304细胞的增殖指数(PI)明显高于对照组，细胞较多地进入有丝分裂期和DNA合成期，这从另一个侧面说明，CASK是ECV3O4增殖的负调控分子。 本研究结果显示，CASK过表达时，Idl与其拼接型Idl’的mRNA组成比例发生显著的改变，Idl逐渐减少，而Idl’逐渐增高。我们推测CASK调节Idl和Idl’的组成比例可能是CASK抑制细胞增殖的原因之一。同时，细胞计数实验结果表明，抑制Idl表达削弱了CASK对细胞增殖的影响，提示Idl参与了CASK抑制细胞增殖的信号。 Idl分子通过与bHLH类转录因子形成无功能活性的异二聚体而抑制bHLH家族转录因子的活性，有研究证实，周期素依赖的蛋白激酶抑制因子pl6和pZI在哺乳动物细胞中受bHLH转录因子和Idl分子的共同调节，因此我们观察了CASK下调表达情况下P16和/或pZI的表达。实验结果表明，以RNAi方法抑制cAsK的表达，P16和P21的表达也受到了明显的抑制，这提示cAsK抑制细胞增殖的生物学效应可能是通过pl6和/或pZI影响细胞周期转换而实现的。 为深入探讨CAsK影响pl6和pZI表达的机制，我们研究了cAsK表达抑制或者增强的情况下，ldl与bHLH转录因子家族成员EZA形成二聚化的定量改变，结果表明，当CASK表达增加时，与EZA分子免疫共沉淀的Idl减少，相反当CASK表达抑制时，与EZA分子免疫共沉淀的Idl明显增多。提示同样作为Idl分子的结合伙伴，CASK与EZA分子可能存在类似竞争的关系。 通过上述研究，我们提出一个尚未见报道的哺乳动物细胞增殖抑制信号:细胞外(细胞间质)信号传递到支架蛋白CASK，CASK与细胞内分子Idl结合，减少了Idl与bHLH类转录因子EZA的结合，由于Idl是EZA负调控因子，EZA转录因子与Idl结合减少，相应地对某些下游靶基因的转录活性增强，通过结合p21/P16基因调控序列的E一box(CANNTG)区，促进pl6、pZI表达增加，抑制细胞周期转换，抑制细胞增殖。更多还原

【Abstract】 CASK(calcium/calmodulin-dependent serine protein kinase)is a member of the membrane associated guanylate kinase(MAGUK) family, a group of conserved cytoskeletal proteins. CASK form scaffolds for protein networks at cell membranes, and play important role in construction of cytoskelecton, formation of cell junctions, signal transduction and regulation on gene expression.Our previous studies have shown that guanylate kinase (GUK) domain of hCASK interact with Id1 by yeast two-hybrid method. CASK and Id1 were coprecipitated in western blotting detection and distributed co-localized at cytoplasmThe Id family of helix-loop-helix (HLH) proteins are thought to affect the balance between cell growth and differentiation by negatively regulating the function of basic helix-loop-helix (bHLH) transcription factors. Id family has a constellation of cellular functions including proliferation and cell cycle progression, migration and invasiveness, cell fate determination, but it is not well elucidated on the upstream signal of Id protein.A large body of evidence has shown that bHLH transcription factors are involved in the transcription of pl6 and p21.We deduced that CASK might regulate these genes by impacting Idl/bHLH transcription factor binding.To identify this hypothesis, we established a CASK knockdown model of ECV304 cell line with RNA interference technique.Methods1. Cell culture: Human cell line ECV304 were cultured at 37C /5% CO2in Ml99 medium supplemented with 100IU/ml penicillin, 10ug/ml streptomycin, and 10% heat-inactivated fetal calf serum(FCS).2. Construction of recombinant siRNA expression plasmids: (1) Choose appropriate 19bp sequence on the transcript of target gene and blast to eliminate any target sequences with significant homology to other coding sequences. The oligonucleotides were synthesized by Shanghai Bioasia corporation. (2) Annealing of each pair of oligonucieotides (3) Double digestion of vector by endonuclease (4)Ligation of annealed oligonucleotide inserts to linearized vector.(5) Transformation of DH5 a (6) Identificationof recombinant plasmids by sequencing.3. Cell Transfection: (1) Optimizing the transfection condition according to manufacture’s introductions, choosing the best ratio of FuGene6 to plasmid DNA. (2) Plate the ECV304 cells one day before the transfection experiment.When cells were 60-80% confluent, transfected cells according to the optimal condition.4. Dual-Luciferase Reporter 1000 Assay :ECV304 cells were transfected by siLuciferase(target to firefly luciferase) plasmid and pGL-3promoter plasmid/pRL plasmid. Collected the cell lysate and measure the firefly luciferase and renilla luciferase activity.5. Western blotting and immuno-coprecipitation: Two or three days after transfection, cells were washed with PBS and collected by scraping. They were lysed in ice-cold RIPA buffer and centrifuged. The supernatant was used for protein concentration determination by BCA-200 method. The proteins were resolved on 10% SDS-polyacrylamide gels, transferred onto nitrocellulose membranes, and incubated with the appropriate antibodies. The peroxidase-based detection was performed with Chemiluminescence Reagent (Pierce) according to the manufacturer’s instructions.6. RT-PCR: Total RNA was prepared from ECV304 cells 48h after transfection using the Tripure reagent according to the manufacturer’s protocol. RT-PCR amplification products were resolved by 1 % agarose gel.7. Growth rate and cell cycle analysis:The cells were seeded in 24 well plates and transfected with siCASK or pBS/U6 vector by using FuGene 6. On different time points after transfection, cells were trypsinized and counted with a hemocytometer. All samples were done in tripicate and every sample counted was repeated at least 6 times. ECV304 cells were seeded in 6.0cm dishes with M199 medium free of serum. The cells were synchronized in cell cycle 24h later. Then the cells were transfected with siCASK or pBS/U6 vector with FuGene6 reag更多还原