小鼠KyoT家族的新異構(gòu)發(fā)現(xiàn)及其對血管內(nèi)皮細(xì)胞的影響
發(fā)布時(shí)間：2018-09-11 17:26
【摘要】：血管生成是多數(shù)情況下血管新生的重要方式——從創(chuàng)傷愈合到特定性生理周期（女性月經(jīng)周期），從多種人類疾病的發(fā)生到腫瘤的生長和轉(zhuǎn)移，血管生成參與眾多重要生理性和病理性過程。 血管生成是由多步驟組成的復(fù)雜過程：首先細(xì)胞外基質(zhì)（Extracellularmatrix, ECM）發(fā)生降解，接著血管內(nèi)皮細(xì)胞（Endothelial cells, ECs）從已有血管中出芽，隨后ECs經(jīng)歷增殖、遷移、分化而形成血管，最后平滑肌細(xì)胞被募集至新生的血管上。ECs不僅是構(gòu)成血管的重要組成部分，更是血管生成過程重要的“執(zhí)行者”。 近年來隨著體外血管生成體系的應(yīng)用、體內(nèi)基因剔除/敲入小鼠模型的建立，對血管生成機(jī)制的研究也不斷深入，發(fā)現(xiàn)多條信號(hào)途徑在細(xì)胞、分子水平調(diào)控血管生成和ECs功能。Notch信號(hào)途徑因參與調(diào)控ECs的多種功能，決定ECs的命運(yùn)，而成為備受關(guān)注的調(diào)控信號(hào)。 Notch信號(hào)途徑是進(jìn)化中高度保守的、通過細(xì)胞與細(xì)胞之間直接接觸而激活的信號(hào)途徑，參與調(diào)控包括細(xì)胞增殖、分化、凋亡、命運(yùn)決定等多種重要過程。當(dāng)相鄰細(xì)胞間Notch配體與其受體接觸時(shí)，Notch受體的胞內(nèi)段（Notch intracellular domain, NIC）就會(huì)釋放進(jìn)入細(xì)胞核，與核內(nèi)DNA結(jié)合蛋白——重組信號(hào)結(jié)合蛋白-J（Recombination signal binding protein-Jκ,RBP-J）結(jié)合，從而激活下游基因的轉(zhuǎn)錄。在缺少NIC的情況下，RBP-J則通過募集多種轉(zhuǎn)錄共抑制分子而發(fā)揮抑制轉(zhuǎn)錄的作用。本室前期研究發(fā)現(xiàn)：RBP-J可通過與LIM蛋白KyoT2結(jié)合而募集多種轉(zhuǎn)錄共抑制分子。但是，KyoT是由選擇性拼接形成的分子亞家族，與RBP-J結(jié)合的只有KyoT2嗎？是否還有其它KyoT分子能和RBP-J結(jié)合？這種結(jié)合對Notch信號(hào)途徑有什么樣的調(diào)控作用？對ECs的功能又有怎樣的影響？ 基于上述設(shè)想，本課題發(fā)現(xiàn)了KyoT另一個(gè)剪接變異體KyoT3，深入研究了與RBP-J結(jié)合的LIM蛋白家族KyoT3的組織分布與細(xì)胞定位，尋找其與RBP-J結(jié)合的直接證據(jù)，闡明KyoT3對Notch信號(hào)途徑的調(diào)控作用，探究KyoT家族成員對ECs的功能的影響。主要研究結(jié)果如下： 1.發(fā)現(xiàn)了KyoT家族的新異構(gòu)體。 首先，通過以小鼠胚胎cDNA文庫為模板，經(jīng)聚合酶鏈反應(yīng)（Polymerasechain reaction, PCR）獲得KyoT家族另一剪接變異體，KyoT3。KyoT3，其全長為969bp，編碼包含323個(gè)氨基酸的大小為36.26KD的蛋白質(zhì)。KyoT3的N-端是與KyoT1相同的3個(gè)半LIM結(jié)構(gòu)域，在LIM結(jié)構(gòu)域之后為KyoT1所不具備的3個(gè)核定位信號(hào)（Nuclear localization signals, NLS）和1個(gè)出核序列（Nuclear export sequence, NES）。與KyoT2相同的是：KyoT3也擁有由C-端27個(gè)氨基酸構(gòu)成的RBP-J結(jié)合基序，這提示KyoT3可能也具有與KyoT2相似的功能。利用位于KyoT3序列上的酶切位點(diǎn)XhoⅠ，將KyoT3分為前后2段，通過對2段分別擴(kuò)增后再拼接的方式，在不改變KyoT3序列、不引入新堿基的情況下，成功構(gòu)建pMD18-T-KyoT3，為進(jìn)一步研究KyoT3的功能打下基礎(chǔ)。 其次，探明了KyoT3在組織中的分布和細(xì)胞內(nèi)的定位，并確認(rèn)其在細(xì)胞核內(nèi)的定位依賴于其NLS。提取小鼠不同組織器官的RNA，經(jīng)過反轉(zhuǎn)錄為cDNA之后，采用KyoT3特異性引物，通過PCR的方法研究KyoT3在組織內(nèi)的分布，發(fā)現(xiàn)KyoT3的mRNA在小鼠脾臟、胸腺、睪丸、卵巢、小腸、結(jié)腸、心臟、大腦、胎盤、肺臟、肝臟、骨骼肌、腎臟和胰腺都有表達(dá)，說明其組織分布十分廣泛。為明確KyoT3在細(xì)胞內(nèi)的定位，構(gòu)建了含KyoT3全長的pEGFP-C2-KyoT3和pEGFP-N1-KyoT3；僅含NLS的pEGFP-C2-NLS和pEGFP-N1-NLS；不含有NLS的pEGFP-C2-KyoT3N和pEGFP-N1-KyoT3N質(zhì)粒。Western blot的方法發(fā)現(xiàn)其中C2-KyoT3N的表達(dá)量很低后，通過其它5種質(zhì)粒分別轉(zhuǎn)染HeLa細(xì)胞，而確定了KyoT3主要分布于細(xì)胞核內(nèi)，并且KyoT3在細(xì)胞核內(nèi)的定位是依賴于其3個(gè)串聯(lián)的NLS實(shí)現(xiàn)的。 最后，通過免疫共沉淀驗(yàn)證了KyoT3與RBP-J之間的相互作用，并進(jìn)一步明確了KyoT3具有抑制RBP-J依賴的轉(zhuǎn)錄的作用。構(gòu)建了帶Myc標(biāo)簽的pCMV-Myc-KyoT3真核表達(dá)質(zhì)粒，，通過與帶Flag標(biāo)簽的pCMV-RBP-J-Flag共轉(zhuǎn)染HeLa細(xì)胞的方式，采用免疫共沉淀實(shí)驗(yàn)，利用不同的抗體檢測，確認(rèn)了KyoT3和RBP-J之間存在物理相互作用。隨即使用雙熒光報(bào)告基因系統(tǒng)，在HeLa細(xì)胞和HEK293細(xì)胞內(nèi)證實(shí)KyoT3具有抑制RBP-J依賴的轉(zhuǎn)錄的作用，并且這種作用具有劑量依賴效應(yīng)。此外，通過將KyoT3與NIC質(zhì)粒共轉(zhuǎn)染HeLa細(xì)胞，24h后用實(shí)時(shí)定量PCR的方法檢測下游基因Hes-1的mRNA水平的方法，結(jié)果也表明：共轉(zhuǎn)染KyoT3和NIC時(shí)，KyoT3能顯著抑制NIC激活的Hes-1的轉(zhuǎn)錄。 2.闡明KyoT家族成員對血管內(nèi)皮細(xì)胞的影響。 首先，成功分離和培養(yǎng)人臍靜脈內(nèi)皮細(xì)胞（Human umbilical veinendothelial cells, HUVECs），在其中檢測到KyoT家族成員KyoT2的表達(dá)。為研究在血管生成中KyoT家族成員的作用，在本室建立了HUVECs的分離、培養(yǎng)的方法，并通過其形態(tài)表現(xiàn)為典型的鋪路石樣、表面CD31分子表達(dá)平均約為99%、具有形成管腔的能力，對分離、培養(yǎng)的細(xì)胞進(jìn)行了確認(rèn)。通過PCR的方法檢測HUVECs中KyoT家族成員的表達(dá)。結(jié)果發(fā)現(xiàn)：在HUVECs中僅KyoT2表達(dá)，于是將研究聚焦于KyoT2。 其次，發(fā)現(xiàn)轉(zhuǎn)染KyoT2能使HUVECs細(xì)胞系（HUVEC Cell line,HUVEC-CL）中管腔形成增多，tip細(xì)胞的數(shù)目增加，細(xì)胞增殖減少。在后續(xù)的實(shí)驗(yàn)中，使用脂質(zhì)體LTXPLUS瞬時(shí)轉(zhuǎn)染EGFP-KyoT2于HUVECs和HUVEC-CL，通過計(jì)數(shù)綠色細(xì)胞總數(shù)的方法，發(fā)現(xiàn)：無論在HUVECs或是HUVEC-CL中，與轉(zhuǎn)染EGFP的對照組相比，轉(zhuǎn)染KyoT2后細(xì)胞增殖減弱，然而轉(zhuǎn)染KyoT2卻能使HUVEC-CL中管腔形成增多，tip細(xì)胞的數(shù)目增加。 通過本課題的研究證實(shí)：KyoT另一剪接變異體KyoT3的存在，并明確了其在組織中的分布和細(xì)胞內(nèi)的定位，并且KyoT3在細(xì)胞核內(nèi)的定位是依賴于其3個(gè)串聯(lián)的NLS的。KyoT3能夠與RBP-J發(fā)生物理上的相互作用，也因此參與了RBP-J介導(dǎo)的Notch信號(hào)途徑的調(diào)控。KyoT3能夠抑制RBP-J介導(dǎo)的轉(zhuǎn)錄激活，并且這種抑制作用具有劑量依賴效應(yīng)。為研究KyoT家族成員在血管生成中的作用，在本室建立了HUVECs的分離與培養(yǎng)方法，通過該方法能夠獲得純度高、功能好的HUVECs，作為研究ECs的模型。使用PCR的方法，檢測到僅KyoT2在HUVECs內(nèi)的表達(dá)，因此也將研究重心轉(zhuǎn)移到KyoT2對ECs的功能影響。進(jìn)一步研究發(fā)現(xiàn)瞬時(shí)轉(zhuǎn)染KyoT2能夠抑制ECs增殖，促使管腔形成增多，tip細(xì)胞的數(shù)目增加。為更深入地研究KyoT2在血管生成過程中的作用奠定基礎(chǔ)。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R331.32
本文編號(hào)：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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R331.32
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
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