小鼠KyoT家族的新异构发现及其对血管内皮细胞的影响
发布时间：2018-09-11 17:26
【摘要】：血管生成是多数情况下血管新生的重要方式——从创伤愈合到特定性生理周期（女性月经周期），从多种人类疾病的发生到肿瘤的生长和转移，血管生成参与众多重要生理性和病理性过程。 血管生成是由多步骤组成的复杂过程：首先细胞外基质（Extracellularmatrix, ECM）发生降解，接着血管内皮细胞（Endothelial cells, ECs）从已有血管中出芽，随后ECs经历增殖、迁移、分化而形成血管，最后平滑肌细胞被募集至新生的血管上。ECs不仅是构成血管的重要组成部分，更是血管生成过程重要的“执行者”。 近年来随着体外血管生成体系的应用、体内基因剔除/敲入小鼠模型的建立，对血管生成机制的研究也不断深入，发现多条信号途径在细胞、分子水平调控血管生成和ECs功能。Notch信号途径因参与调控ECs的多种功能，决定ECs的命运，而成为备受关注的调控信号。 Notch信号途径是进化中高度保守的、通过细胞与细胞之间直接接触而激活的信号途径，参与调控包括细胞增殖、分化、凋亡、命运决定等多种重要过程。当相邻细胞间Notch配体与其受体接触时，Notch受体的胞内段（Notch intracellular domain, NIC）就会释放进入细胞核，与核内DNA结合蛋白——重组信号结合蛋白-J（Recombination signal binding protein-Jκ,RBP-J）结合，从而激活下游基因的转录。在缺少NIC的情况下，RBP-J则通过募集多种转录共抑制分子而发挥抑制转录的作用。本室前期研究发现：RBP-J可通过与LIM蛋白KyoT2结合而募集多种转录共抑制分子。但是，KyoT是由选择性拼接形成的分子亚家族，与RBP-J结合的只有KyoT2吗？是否还有其它KyoT分子能和RBP-J结合？这种结合对Notch信号途径有什么样的调控作用？对ECs的功能又有怎样的影响？ 基于上述设想，本课题发现了KyoT另一个剪接变异体KyoT3，深入研究了与RBP-J结合的LIM蛋白家族KyoT3的组织分布与细胞定位，寻找其与RBP-J结合的直接证据，阐明KyoT3对Notch信号途径的调控作用，探究KyoT家族成员对ECs的功能的影响。主要研究结果如下： 1.发现了KyoT家族的新异构体。 首先，通过以小鼠胚胎cDNA文库为模板，经聚合酶链反应（Polymerasechain reaction, PCR）获得KyoT家族另一剪接变异体，KyoT3。KyoT3，其全长为969bp，编码包含323个氨基酸的大小为36.26KD的蛋白质。KyoT3的N-端是与KyoT1相同的3个半LIM结构域，在LIM结构域之后为KyoT1所不具备的3个核定位信号（Nuclear localization signals, NLS）和1个出核序列（Nuclear export sequence, NES）。与KyoT2相同的是：KyoT3也拥有由C-端27个氨基酸构成的RBP-J结合基序，这提示KyoT3可能也具有与KyoT2相似的功能。利用位于KyoT3序列上的酶切位点XhoⅠ，将KyoT3分为前后2段，通过对2段分别扩增后再拼接的方式，在不改变KyoT3序列、不引入新碱基的情况下，成功构建pMD18-T-KyoT3，为进一步研究KyoT3的功能打下基础。 其次，探明了KyoT3在组织中的分布和细胞内的定位，并确认其在细胞核内的定位依赖于其NLS。提取小鼠不同组织器官的RNA，经过反转录为cDNA之后，采用KyoT3特异性引物，通过PCR的方法研究KyoT3在组织内的分布，发现KyoT3的mRNA在小鼠脾脏、胸腺、睾丸、卵巢、小肠、结肠、心脏、大脑、胎盘、肺脏、肝脏、骨骼肌、肾脏和胰腺都有表达，说明其组织分布十分广泛。为明确KyoT3在细胞内的定位，构建了含KyoT3全长的pEGFP-C2-KyoT3和pEGFP-N1-KyoT3；仅含NLS的pEGFP-C2-NLS和pEGFP-N1-NLS；不含有NLS的pEGFP-C2-KyoT3N和pEGFP-N1-KyoT3N质粒。Western blot的方法发现其中C2-KyoT3N的表达量很低后，通过其它5种质粒分别转染HeLa细胞，而确定了KyoT3主要分布于细胞核内，并且KyoT3在细胞核内的定位是依赖于其3个串联的NLS实现的。 最后，通过免疫共沉淀验证了KyoT3与RBP-J之间的相互作用，并进一步明确了KyoT3具有抑制RBP-J依赖的转录的作用。构建了带Myc标签的pCMV-Myc-KyoT3真核表达质粒，，通过与带Flag标签的pCMV-RBP-J-Flag共转染HeLa细胞的方式，采用免疫共沉淀实验，利用不同的抗体检测，确认了KyoT3和RBP-J之间存在物理相互作用。随即使用双荧光报告基因系统，在HeLa细胞和HEK293细胞内证实KyoT3具有抑制RBP-J依赖的转录的作用，并且这种作用具有剂量依赖效应。此外，通过将KyoT3与NIC质粒共转染HeLa细胞，24h后用实时定量PCR的方法检测下游基因Hes-1的mRNA水平的方法，结果也表明：共转染KyoT3和NIC时，KyoT3能显著抑制NIC激活的Hes-1的转录。 2.阐明KyoT家族成员对血管内皮细胞的影响。 首先，成功分离和培养人脐静脉内皮细胞（Human umbilical veinendothelial cells, HUVECs），在其中检测到KyoT家族成员KyoT2的表达。为研究在血管生成中KyoT家族成员的作用，在本室建立了HUVECs的分离、培养的方法，并通过其形态表现为典型的铺路石样、表面CD31分子表达平均约为99%、具有形成管腔的能力，对分离、培养的细胞进行了确认。通过PCR的方法检测HUVECs中KyoT家族成员的表达。结果发现：在HUVECs中仅KyoT2表达，于是将研究聚焦于KyoT2。 其次，发现转染KyoT2能使HUVECs细胞系（HUVEC Cell line,HUVEC-CL）中管腔形成增多，tip细胞的数目增加，细胞增殖减少。在后续的实验中，使用脂质体LTXPLUS瞬时转染EGFP-KyoT2于HUVECs和HUVEC-CL，通过计数绿色细胞总数的方法，发现：无论在HUVECs或是HUVEC-CL中，与转染EGFP的对照组相比，转染KyoT2后细胞增殖减弱，然而转染KyoT2却能使HUVEC-CL中管腔形成增多，tip细胞的数目增加。 通过本课题的研究证实：KyoT另一剪接变异体KyoT3的存在，并明确了其在组织中的分布和细胞内的定位，并且KyoT3在细胞核内的定位是依赖于其3个串联的NLS的。KyoT3能够与RBP-J发生物理上的相互作用，也因此参与了RBP-J介导的Notch信号途径的调控。KyoT3能够抑制RBP-J介导的转录激活，并且这种抑制作用具有剂量依赖效应。为研究KyoT家族成员在血管生成中的作用，在本室建立了HUVECs的分离与培养方法，通过该方法能够获得纯度高、功能好的HUVECs，作为研究ECs的模型。使用PCR的方法，检测到仅KyoT2在HUVECs内的表达，因此也将研究重心转移到KyoT2对ECs的功能影响。进一步研究发现瞬时转染KyoT2能够抑制ECs增殖，促使管腔形成增多，tip细胞的数目增加。为更深入地研究KyoT2在血管生成过程中的作用奠定基础。
[Abstract]:Angiogenesis is an important way of angiogenesis in most cases - from wound healing to specific physiological cycles (female menstrual cycles), from the occurrence of a variety of human diseases to the growth and metastasis of tumors, angiogenesis is involved in many important physiological and pathological processes.
Angiogenesis is a complex process consisting of multiple steps: first, the extracellular matrix (ECM) is degraded, then the endothelial cells (ECs) sprout from the existing vessels, then the ECs undergo proliferation, migration, differentiation and angiogenesis, and finally the smooth muscle cells are recruited to the new blood vessels. An important component of blood vessels is also an important "executor" in the angiogenesis process.
In recent years, with the application of angiogenesis system in vitro, the establishment of gene knock-out/knock-in mice model in vivo and the study of angiogenesis mechanism have been deepened. It has been found that many signal pathways regulate angiogenesis and ECs function at the cellular and molecular level. Notch signaling pathway has become the fate of ECs because it participates in the regulation of various functions of ECs. Signals of concern.
Notch signaling pathways are highly conserved in evolution. They are activated by direct cell-to-cell contact and participate in many important processes including cell proliferation, differentiation, apoptosis, and fate determination. In the absence of NIC, RBP-J can inhibit transcription by recruiting multiple co-inhibitors of transcription. RBP-J can recruit multiple transcriptional co-inhibitors by binding to LIM protein KyoT2. However, KyoT is a molecular subfamily formed by selective splicing, and only KyoT2 binds to RBP-J. Are there any other KyoT molecules that bind to RBP-J? What are the regulatory effects of this binding on Notch signaling pathway? What are the functions of ECs? The impact?
Based on the above assumption, another splicing variant of KyoT, KyoT3, was discovered. The tissue distribution and cellular localization of the LIM protein family KyoT3 binding to RBP-J were studied in depth, and the direct evidence of its binding to RBP-J was found. The regulatory effect of KyoT3 on Notch signaling pathway was clarified, and the effects of KyoT family members on ECs were explored. The results are as follows:
1. new isomers of the KyoT family were discovered.
Firstly, another splicing variant of KyoT family, KyoT3. KyoT3, was obtained by polymerase chain reaction (PCR) using mouse embryo cDNA library as template. Its full length was 969 bp, encoding a protein of 36.26 KD containing 323 amino acids. The N-terminal of KyoT3 was the same three semi-LIM domains as KyoT1 in the LIM domain. Like KyoT2, KyoT3 also has a RBP-J binding motif composed of 27 amino acids at the C-terminal, suggesting that KyoT3 may have similar functions to KyoT2. The enzyme digestion site Xho I was used to divide KyoT3 into two segments. By amplifying and splicing the two segments respectively, pMD18-T-KyoT3 was successfully constructed without changing the sequence of KyoT3 and introducing new bases, which laid a foundation for further study on the function of KyoT3.
Secondly, the distribution of KyoT3 in tissues and its localization in cells were investigated, and it was confirmed that its localization in the nucleus was dependent on its NLS. RNA from different tissues and organs of mice was extracted. After reverse transcription into cDNA, KyoT3 specific primers were used to study the distribution of KyoT3 in tissues by PCR. It was found that the expression of KyoT3 mRNA in spleen and chest of mice. The expression of KyoT3 in gland, testis, ovary, small intestine, colon, heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas showed that the tissue was widely distributed. Two-KyoT3N and pEGFP-N1-KyoT3N plasmids were transfected into HeLa cells with low expression of C2-KyoT3N by Western blot. The results showed that KyoT3 was mainly distributed in the nucleus of HeLa cells, and the localization of KyoT3 in the nucleus was dependent on its three tandem NLS.
Finally, the interaction between KyoT3 and RBP-J was verified by immunoprecipitation, and the inhibition of RBP-J dependent transcription by KyoT3 was further clarified. The eukaryotic expression plasmid of pCMV-Myc-KyoT3 with Myc tag was constructed, and the co-transfection of pCMV-RBP-J-Flag with pCMV-RBP-J-Flag tag was carried out. Physical interactions between KyoT3 and RBP-J were confirmed by different antibody assays. With the use of a dual fluorescent reporter gene system, KyoT3 inhibited RBP-J-dependent transcription in HeLa cells and HEK293 cells, and this effect was dose-dependent. In addition, KyoT3 was co-transfected with NIC plasmid into HeLa cells, 2. Four hours later, real-time quantitative PCR was used to detect the mRNA level of downstream gene Hes-1. The results also showed that KyoT3 significantly inhibited the transcription of NIC-activated Hes-1 when co-transfected with KyoT3 and NIC.
2. elucidate the effects of KyoT family members on vascular endothelial cells.
Firstly, human umbilical vein endothelial cells (HUVECs) were successfully isolated and cultured, in which the expression of KyoT2 was detected. In order to study the role of KyoT family members in angiogenesis, a method of isolation and culture of HUVECs was established in our laboratory, and the morphology of HUVECs was typical paving stone. The results showed that only KyoT2 was expressed in HUVECs, so the study focused on KyoT2.
Secondly, it was found that transfection of KyoT2 could increase lumen formation, increase the number of tip cells and decrease cell proliferation in HUVECs cell line (HUVEC-CL). In-CL, compared with EGFP-transfected control group, KyoT2-transfected cells decreased proliferation, but KyoT2-transfected cells increased lumen formation and tip cell number in HUVEC-CL.
The present study confirmed that another splicing variant of KyoT, KyoT3, was present, and its distribution in tissues and intracellular localization were clarified. The localization of KyoT3 in the nucleus was dependent on its three series of NLS. KyoT3 could interact physically with RBP-J and therefore participate in the Notch signaling pathway mediated by RBP-J. KyoT3 can inhibit RBP-J-mediated transcriptional activation, and this inhibition has a dose-dependent effect. To study the role of KyoT family members in angiogenesis, a method of isolation and culture of HUVECs was established in our laboratory. High purity and good function HUVECs could be obtained by this method as a model for studying ECs. Methods: The expression of KyoT2 in HUVECs was detected only, so the focus of the study was shifted to the effect of KyoT2 on the function of ECs. Further studies showed that transient transfection of KyoT2 could inhibit the proliferation of ECs, promote the formation of lumen and increase the number of tip cells.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R331.32
本文编号：2237361
[Abstract]:Angiogenesis is an important way of angiogenesis in most cases - from wound healing to specific physiological cycles (female menstrual cycles), from the occurrence of a variety of human diseases to the growth and metastasis of tumors, angiogenesis is involved in many important physiological and pathological processes.
Angiogenesis is a complex process consisting of multiple steps: first, the extracellular matrix (ECM) is degraded, then the endothelial cells (ECs) sprout from the existing vessels, then the ECs undergo proliferation, migration, differentiation and angiogenesis, and finally the smooth muscle cells are recruited to the new blood vessels. An important component of blood vessels is also an important "executor" in the angiogenesis process.
In recent years, with the application of angiogenesis system in vitro, the establishment of gene knock-out/knock-in mice model in vivo and the study of angiogenesis mechanism have been deepened. It has been found that many signal pathways regulate angiogenesis and ECs function at the cellular and molecular level. Notch signaling pathway has become the fate of ECs because it participates in the regulation of various functions of ECs. Signals of concern.
Notch signaling pathways are highly conserved in evolution. They are activated by direct cell-to-cell contact and participate in many important processes including cell proliferation, differentiation, apoptosis, and fate determination. In the absence of NIC, RBP-J can inhibit transcription by recruiting multiple co-inhibitors of transcription. RBP-J can recruit multiple transcriptional co-inhibitors by binding to LIM protein KyoT2. However, KyoT is a molecular subfamily formed by selective splicing, and only KyoT2 binds to RBP-J. Are there any other KyoT molecules that bind to RBP-J? What are the regulatory effects of this binding on Notch signaling pathway? What are the functions of ECs? The impact?
Based on the above assumption, another splicing variant of KyoT, KyoT3, was discovered. The tissue distribution and cellular localization of the LIM protein family KyoT3 binding to RBP-J were studied in depth, and the direct evidence of its binding to RBP-J was found. The regulatory effect of KyoT3 on Notch signaling pathway was clarified, and the effects of KyoT family members on ECs were explored. The results are as follows:
1. new isomers of the KyoT family were discovered.
Firstly, another splicing variant of KyoT family, KyoT3. KyoT3, was obtained by polymerase chain reaction (PCR) using mouse embryo cDNA library as template. Its full length was 969 bp, encoding a protein of 36.26 KD containing 323 amino acids. The N-terminal of KyoT3 was the same three semi-LIM domains as KyoT1 in the LIM domain. Like KyoT2, KyoT3 also has a RBP-J binding motif composed of 27 amino acids at the C-terminal, suggesting that KyoT3 may have similar functions to KyoT2. The enzyme digestion site Xho I was used to divide KyoT3 into two segments. By amplifying and splicing the two segments respectively, pMD18-T-KyoT3 was successfully constructed without changing the sequence of KyoT3 and introducing new bases, which laid a foundation for further study on the function of KyoT3.
Secondly, the distribution of KyoT3 in tissues and its localization in cells were investigated, and it was confirmed that its localization in the nucleus was dependent on its NLS. RNA from different tissues and organs of mice was extracted. After reverse transcription into cDNA, KyoT3 specific primers were used to study the distribution of KyoT3 in tissues by PCR. It was found that the expression of KyoT3 mRNA in spleen and chest of mice. The expression of KyoT3 in gland, testis, ovary, small intestine, colon, heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas showed that the tissue was widely distributed. Two-KyoT3N and pEGFP-N1-KyoT3N plasmids were transfected into HeLa cells with low expression of C2-KyoT3N by Western blot. The results showed that KyoT3 was mainly distributed in the nucleus of HeLa cells, and the localization of KyoT3 in the nucleus was dependent on its three tandem NLS.
Finally, the interaction between KyoT3 and RBP-J was verified by immunoprecipitation, and the inhibition of RBP-J dependent transcription by KyoT3 was further clarified. The eukaryotic expression plasmid of pCMV-Myc-KyoT3 with Myc tag was constructed, and the co-transfection of pCMV-RBP-J-Flag with pCMV-RBP-J-Flag tag was carried out. Physical interactions between KyoT3 and RBP-J were confirmed by different antibody assays. With the use of a dual fluorescent reporter gene system, KyoT3 inhibited RBP-J-dependent transcription in HeLa cells and HEK293 cells, and this effect was dose-dependent. In addition, KyoT3 was co-transfected with NIC plasmid into HeLa cells, 2. Four hours later, real-time quantitative PCR was used to detect the mRNA level of downstream gene Hes-1. The results also showed that KyoT3 significantly inhibited the transcription of NIC-activated Hes-1 when co-transfected with KyoT3 and NIC.
2. elucidate the effects of KyoT family members on vascular endothelial cells.
Firstly, human umbilical vein endothelial cells (HUVECs) were successfully isolated and cultured, in which the expression of KyoT2 was detected. In order to study the role of KyoT family members in angiogenesis, a method of isolation and culture of HUVECs was established in our laboratory, and the morphology of HUVECs was typical paving stone. The results showed that only KyoT2 was expressed in HUVECs, so the study focused on KyoT2.
Secondly, it was found that transfection of KyoT2 could increase lumen formation, increase the number of tip cells and decrease cell proliferation in HUVECs cell line (HUVEC-CL). In-CL, compared with EGFP-transfected control group, KyoT2-transfected cells decreased proliferation, but KyoT2-transfected cells increased lumen formation and tip cell number in HUVEC-CL.
The present study confirmed that another splicing variant of KyoT, KyoT3, was present, and its distribution in tissues and intracellular localization were clarified. The localization of KyoT3 in the nucleus was dependent on its three series of NLS. KyoT3 could interact physically with RBP-J and therefore participate in the Notch signaling pathway mediated by RBP-J. KyoT3 can inhibit RBP-J-mediated transcriptional activation, and this inhibition has a dose-dependent effect. To study the role of KyoT family members in angiogenesis, a method of isolation and culture of HUVECs was established in our laboratory. High purity and good function HUVECs could be obtained by this method as a model for studying ECs. Methods: The expression of KyoT2 in HUVECs was detected only, so the focus of the study was shifted to the effect of KyoT2 on the function of ECs. Further studies showed that transient transfection of KyoT2 could inhibit the proliferation of ECs, promote the formation of lumen and increase the number of tip cells.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R331.32
【参考文献】
相关期刊论文 前1条
1 黄红艳,李荣,孙强,王健,周鹏,韩骅,张万会;LIM蛋白KyoT2与人类紧密连接蛋白2的相互作用[J];遗传学报;2002年11期
本文编号：2237361
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