CN115094090B - Cell modified by specific heparan sulfate modified enzyme gene, construction method and application thereof - Google Patents
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Abstract
本发明提出了特定硫酸肝素修饰酶基因修饰的细胞及其构建方法和应用。更进一步地，本发明提出了所述HS3ST5基因在诱导细胞分化成血管中的用途。所述HS3ST5基因可以有效促进细胞，尤其是内皮细胞向成血管方向分化，增强细胞的黏附和增殖、提高血管内皮化效率、增强成血管活性和血管生成相关基因的表达，有助于实现组织工程组织的血管化、解码内皮细胞所构筑的微环境对于实质细胞的影响，在组织器官发育与损伤修复再生、肿瘤发生发展等过程中发挥关键作用，为进一步寻找促进组织再生和疾病(如恶性肿瘤)治疗的策略提供新的模型与思路，具有广阔的应用前景。The present invention proposes cells modified by specific heparin sulfate modifying enzyme genes and their construction methods and applications. Furthermore, the present invention proposes the use of the HS3ST5 gene in inducing cell differentiation into blood vessels. The HS3ST5 gene can effectively promote the differentiation of cells, especially endothelial cells, in the direction of angiogenesis, enhance cell adhesion and proliferation, improve the efficiency of vascular endothelialization, enhance angiogenic activity and the expression of angiogenesis-related genes, and help realize tissue engineering. The vascularization of tissues and the influence of the microenvironment constructed by endothelial cells on parenchymal cells play a key role in the development of tissues and organs, damage repair and regeneration, and tumor development. ) treatment strategies provide new models and ideas and have broad application prospects.
Description
技术领域Technical field
本发明涉及生物技术领域，具体地，本发明涉及特定硫酸肝素修饰酶基因修饰的细胞及其构建方法和应用。The present invention relates to the field of biotechnology. Specifically, the present invention relates to cells modified by specific heparin sulfate modifying enzyme genes and their construction methods and applications.
背景技术Background technique
重要生命器官的复杂结构决定其功能的完整性，而其血供分布特点与其功能的独特性有着必然联系。正常的血管系统是维持内环境稳定和细胞生长代谢的基础。The complex structure of important vital organs determines the integrity of their functions, and their blood supply distribution characteristics are inevitably related to the uniqueness of their functions. The normal vascular system is the basis for maintaining homeostasis and cell growth and metabolism.
细胞微环境由基质细胞及其分泌的可溶性细胞因子及不溶性ECM构成。ECM主要包括胶原、糖蛋白、蛋白聚糖(proteoglycan,PG)及其调控蛋白，PGs主要由核心蛋白和以糖链形式存在的糖氨聚糖构成，为组织器官和机体的完整性提供重要的力学和生物学支持。硫酸肝素蛋白聚糖(heparan sulfate proteoglycan，HSPG)因其作为细胞外基质的主要成分和抗凝活性而被熟知。系列酶对HS糖链复杂合成过程的精细调控，使得不同类型和修饰的HSPG具有高度发育阶段和组织细胞特异性。且3-OST2,5磺基化酶修饰的糖链3ST5-HS(NS3ST5)明显促进内皮细胞在体外增殖和成血管活性。The cellular microenvironment consists of stromal cells and their secreted soluble cytokines and insoluble ECM. ECM mainly includes collagen, glycoproteins, proteoglycans (PG) and their regulatory proteins. PGs are mainly composed of core proteins and glycosaminoglycans in the form of sugar chains. They provide important components for the integrity of tissues, organs and the body. Mechanical and biological support. Heparan sulfate proteoglycan (HSPG) is well known for its main component of the extracellular matrix and its anticoagulant activity. The fine regulation of the complex synthesis process of HS sugar chains by a series of enzymes makes different types and modified HSPGs highly specific to developmental stages and tissue cells. And the sugar chain 3ST5-HS (NS3ST5) modified by 3-OST2,5 sulfonylase significantly promoted the proliferation and angiogenic activity of endothelial cells in vitro.
肝脏是损伤后能够快速再生的实质性器官，但是这种再生能力并不是无限的。脱细胞技术最大限度保持了器官原有结构包括完整脉管系统及组分，为细胞生长及功能发挥提供天然再生环境，在未来器官替代治疗中将有广阔应用前景。然而，由于血管系统再内皮化不完整将导致胶原暴露，激活凝血系统引发血栓形成，进而器官堵塞缺氧和失功能。因此，血管化是制约肝脏等复杂脏器组织工程构建成功和功能发挥的瓶颈。The liver is a solid organ that can rapidly regenerate after injury, but this regeneration capacity is not unlimited. Decellularization technology maintains the original structure of the organ to the greatest extent, including the complete vasculature and components, and provides a natural regenerative environment for cell growth and function. It will have broad application prospects in organ replacement therapy in the future. However, incomplete re-endothelialization of the vascular system will lead to collagen exposure, activation of the coagulation system and triggering thrombosis, resulting in organ blockage, hypoxia and dysfunction. Therefore, vascularization is a bottleneck that restricts the successful construction and functional performance of complex organ tissue engineering such as the liver.
目前，HS3ST5在疾病如肝损伤后再生以及肿瘤发生发展特别是在血管发生及生长中的分子机制仍有待研究。Currently, the molecular mechanisms of HS3ST5 in regeneration after diseases such as liver injury and tumor development, especially in angiogenesis and growth, remain to be studied.
发明内容Contents of the invention
本申请是基于发明人对下列问题和事实的发现而提出的：This application is filed based on the inventor's discovery of the following issues and facts:
血管化是制约肝脏等复杂脏器组织工程构建成功和功能发挥的瓶颈，本申请的发明人经过大量实验验证，发现HS3ST5基因可以促进内皮细胞、SK-Hep-1或HUVEC向成血管方向分化，增强内皮细胞的黏附和增殖、提高内皮化效率、增强成血管活性和血管生成相关基因的表达，有助于基于脱细胞肝脏生物支架构建再血管化再细胞化组织工程肝脏等模型，可应用于脂肪肝、药物性肝损伤和肝脏肿瘤等的机制研究以及临床前靶向药物的研发和肝移植中仿生肝组织构建等领域，具有广阔的应用前景。Vascularization is a bottleneck that restricts the successful construction and functional performance of complex organ tissue engineering such as the liver. The inventor of this application has verified through a large number of experiments and found that the HS3ST5 gene can promote the differentiation of endothelial cells, SK-Hep-1 or HUVEC in the direction of vasculogenesis. Enhance the adhesion and proliferation of endothelial cells, improve endothelialization efficiency, enhance angiogenic activity and expression of angiogenesis-related genes, which is helpful for constructing revascularized and recellularized tissue engineering liver models based on decellularized liver bioscaffolds, which can be applied to Research on the mechanisms of fatty liver, drug-induced liver injury and liver tumors, as well as the development of preclinical targeted drugs and the construction of bionic liver tissue for liver transplantation, have broad application prospects.
因此，在本发明的的第一方面，本发明提出了HS3ST5基因在诱导细胞分化成血管中的用途。根据本发明的实施例，高表达HS3ST5基因的细胞的增殖速度、成血管活性及血管生成相关基因的表达量均得到显著提升，因此，HS3ST5基因可以有效促进内皮细胞向成血管方向分化。Therefore, in a first aspect of the invention, the invention proposes the use of the HS3ST5 gene in inducing cell differentiation into blood vessels. According to embodiments of the present invention, the proliferation rate, angiogenic activity, and expression of angiogenesis-related genes of cells that highly express the HS3ST5 gene are significantly increased. Therefore, the HS3ST5 gene can effectively promote the differentiation of endothelial cells in the direction of angiogenesis.
根据本发明的实施例，上述HS3ST5基因在诱导内皮细胞分化成血管中的用途还可以包括下列附加技术特征中的至少之一：According to embodiments of the present invention, the use of the above-mentioned HS3ST5 gene in inducing endothelial cells to differentiate into blood vessels may also include at least one of the following additional technical features:
根据本发明的实施例，所述HS3ST5基因具有SEQ ID NO:2所示的核苷酸序列。According to an embodiment of the present invention, the HS3ST5 gene has the nucleotide sequence shown in SEQ ID NO:2.
ATGCTATTCAAACAGCAGGCGTGGCTGAGACAGAAGCTCCTGGTGCTGGGAAGCCTTGCCGTTGGGAGTCTCCTGTATCTAGTCGCCAGAGTTGGGAGCTTGGATAGGCTACAACCCATTTGCCCCATTGAAGGTCGACTGGGTGGAGCCCGCACTCAGGCTGAATTCCCACTTCGCGCCCTGCAGTTTAAGCGTGGCCTGCTGCACGAGTTCCGGAAGGGCAACGCTTCCAAGGAGCAGGTTCGCCTCCATGACCTGGTCCAGCAGCTCCCCAAGGCCATTATCATTGGGGTGAGGAAAGGAGGCACAAGGGCCCTGCTTGAAATGCTGAACCTACATCCGGCAGTAGTCAAAGCCTCTCAAGAAATCCACTTTTTTGATAATGATGAGAATTATGGTAAGGGCATTGAGTGGTATAGGAAAAAGATGCCTTTTTCCTACCCTCAGCAAATCACAATTGAAAAGAGCCCAGCATATTTTATCACAGAGGAGGTTCCAGAAAGGATTTACAAAATGAACTCATCCATCAAGTTGTTGATCATTGTCAGGGAGCCAACCACAAGAGCTATTTCTGATTATACTCAGGTGCTAGAGGGGAAGGAGAGGAAGAACAAAACTTATTACAAGTTTGAGAAGCTGGCCATAGACCCTAATACATGCGAAGTGAACACAAAATACAAAGCAGTAAGAACCAGCATCTACACCAAACATCTGGAAAGGTGGTTGAAATACTTTCCAATTGAGCAATTTCATGTCGTCGATGGAGATCGCCTCATCACGGAACCTCTGCCAGAACTTCAGCTCGTGGAGAAGTTCCTAAATCTGCCTCCAAGGATAAGTCAATACAATTTATACTTCAATGCTACCAGAGGGTTTTACTGCTTGCGGTTTAATATTATCTTTAATAAGTGCCTGGCGGGCAGCAAGGGGCGCATTCATCCAGAGGTGGACCCCTCTGTCATTACTAAATTGCGCAAATTCTTTCATCCTTTTAATCAAAAATTTTACCAGATCACTGGGAGGACATTGAACTGGCCCTAA(SEQ ID NO:2)。ATGCTATTCAAACAGCAGGCGTGGCTGAGACAGAAGCTCTGGTGCTGGGAAGCCTTGCCGTTGGGAGTCTCCTGTATCTAGTCGCCAGAGTTGGGAGCTTGGATAGGCTACAACCCATTTGCCCCATTGAAGGTCGACTGGGTGGAGCCCGCACTCAGGCTGAATTCCCACTTCGCGCCCTGCAGTTTAAGCGTGGCCTGCTGCACGAGTTCCGGAAGGGCAACGCTTCCAAGGAGCAGGTTCGCCTCCATGACCTGGTCCA GCAGCTCCCCAAGGCCATTATCATTGGGGTGAGGAAAGGAGGCACAAGGGCCCTGCTTGAAATGCTGAACCTACATCCGGCAGTAGTCAAAGCCTCTCAAGAAATCCACTTTTTTGATAATGATGAGAATTATGGTAAGGGCATTGAGTGGTATAGGAAAAAGATGCCTTTTTCCTACCCTCAGCAAATCACAATTGAAAAGAGCCCAGCATATTTTATCACAGAGGAGGTTCCAGAAAGGATTTACAAAATGAACTCATCCATCAAGTTGTTGATC ATTGTCAGGGAGCCAACCACAAGAGCTATTTCTGATTATACTCAGGTGCTAGAGGGGAAGGAGAGGAAGAACAAAACTTATTACAAGTTTGAGAAGCTGGCCATAGACCCTAATACATGCGAAGTGAACACAAAATACAAAGCAGTAAGAACCAGCATCTACACCAAACATCTGGAAAGGTGGTTGAAATACTTCCAATTGAGCAATTTCATGTCGTCGATGGAGATCGCCTCATCACGGAACCTCTGCCAGAACTTCAGCTCGTGGAGAAG TTCCTAAATCTGCCTCCAAGGATAAGTCAATACAATTTATACTTCAATGCTACCAGAGGGTTTTACTGCTTGCGGTTTAATATTATCTTAATAAGTGCCTGGCGGGCAGCAAGGGGCGCATTCATCCAGAGGTGGACCCCTCTGTCATTACTAAATTGCGCAAATTCTTTCATCCTTTTAATCAAAAATTTTACCAGATCACTGGGAGGACATTGAACTGGCCCTAA (SEQ ID NO: 2).
根据本发明的实施例，所述HS3ST5蛋白具有SEQ ID NO:4所述的氨基酸序列。According to an embodiment of the present invention, the HS3ST5 protein has the amino acid sequence described in SEQ ID NO: 4.
MLFKQQAWLRQKLLVLGSLAVGSLLYLVARVGSLDRLQPICPIEGRLGGARTQAEFPLRALQFKRGLLHEFRKGNASKEQVRLHDLVQQLPKAIIIGVRKGGTRALLEMLNLHPAVVKASQEIHFFDNDENYGKGIEWYRKKMPFSYPQQITIEKSPAYFITEEVPERIYKMNSSIKLLIIVREPTTRAISDYTQVLEGKERKNKTYYKFEKLAIDPNTCEVNTKYKAVRTSIYTKHLERWLKYFPIEQFHVVDGDRLITEPLPELQLVEKFLNLPPRISQYNLYFNATRGFYCLRFNIIFNKCLAGSKGRIHPEVDPSVITKLRKFFHPFNQKFYQITGRTLNWP(SEQ ID NO:4)。MLFKQQAWLRQKLLVLGSLAVGSLLYLVARVGSLDRLQPICPIEGRLGGARTQAEFPLRALQFKRGLLHEFRKGNASKEQVRLHDLVQQLPKAIIIGVRKGGTRALLEMLNLHPAVVKASQEIHFFDNDENYGKGIEWYRKKMPFSYPQQITIEKSPAYFITEEVPERIYKMNSSIKLLIIVREPTTRAISDYTQVLEGKERKNKTY YKFEKLAIDPNTCEVNTKYKAVRTSIYTKHLERWLKYFPIEQFHVVDGDRLITEPLPELQLVEKFLNLPPRISQYNLYFNATRGFYCLRFNIIFNKCLAGSKGRIHPEVDPSVITKLRKFFHPFNQKFYQITGRTLNWP (SEQ ID NO: 4).
根据本发明的实施例，所述细胞选自下列中的至少之一：内皮细胞、SK-Hep-1和HUVEC。According to an embodiment of the present invention, the cells are selected from at least one of the following: endothelial cells, SK-Hep-1 and HUVEC.
根据本发明的实施例，所述细胞包括选自下列中的至少之一：动脉血管内皮细胞、静脉血管内皮细胞、毛细血管内皮细胞、肝脏内皮细胞、肺内皮细胞、心室内皮细胞、脑内皮细胞、骨骼肌内皮细胞、胃内皮细胞、视网膜内皮细胞、皮肤内皮细胞、小肠内皮细胞、肾内皮细胞、关节滑膜内皮细胞、食道内皮细胞、胰岛内皮细胞、骨髓内皮细胞、胎盘内皮细胞、脉络膜内皮细胞、SK-Hep-1、HUVEC和原代内皮细胞。According to an embodiment of the present invention, the cells include at least one selected from the group consisting of arterial endothelial cells, venous endothelial cells, capillary endothelial cells, liver endothelial cells, lung endothelial cells, ventricular endothelial cells, and brain endothelial cells. cells, skeletal muscle endothelial cells, gastric endothelial cells, retinal endothelial cells, skin endothelial cells, small intestinal endothelial cells, renal endothelial cells, joint synovial endothelial cells, esophageal endothelial cells, pancreatic islet endothelial cells, bone marrow endothelial cells, placental endothelial cells, choroid Endothelial cells, SK-Hep-1, HUVEC and primary endothelial cells.
根据本发明的实施例，所述细胞包括SK-Hep-1、HUVEC、HepaRG或原代内皮细胞。According to embodiments of the present invention, the cells include SK-Hep-1, HUVEC, HepaRG or primary endothelial cells.
在本发明的第二方面，本发明提出了试剂在制备试剂盒中的用途。根据本发明的实施例，所述试剂盒用于诱导细胞分化成血管，所述试剂包括HS3ST5蛋白或HS3ST5功能域肽段，或用于激活HS3ST5基因或HS3ST5蛋白或过表达HS3ST5基因。如前所述，HS3ST5基因可以有效促进内皮细胞、SK-Hep-1或HUVEC细胞向成血管方向分化，因此，包含HS3ST5蛋白或HS3ST5功能域肽段，或用于激活HS3ST5基因或HS3ST5蛋白或过表达HS3ST5基因的试剂可以有效诱导上述细胞向成血管方向分化。In a second aspect of the invention, the invention provides the use of reagents in the preparation of kits. According to an embodiment of the present invention, the kit is used to induce cells to differentiate into blood vessels, and the reagent includes HS3ST5 protein or HS3ST5 functional domain peptide, or is used to activate the HS3ST5 gene or HS3ST5 protein or overexpress the HS3ST5 gene. As mentioned before, the HS3ST5 gene can effectively promote the differentiation of endothelial cells, SK-Hep-1 or HUVEC cells in the direction of vasculogenesis. Therefore, the HS3ST5 protein or HS3ST5 functional domain peptide can be used to activate the HS3ST5 gene or HS3ST5 protein or process. Reagents expressing the HS3ST5 gene can effectively induce the differentiation of the above cells in the direction of vasculogenesis.
根据本发明的实施例，上述试剂在制备试剂盒中的用途还可以包括下列附加技术特征中的至少之一：According to embodiments of the present invention, the use of the above reagents in preparing kits may also include at least one of the following additional technical features:
根据本发明的实施例，所述试剂包括携带表达所述HS3ST5基因功能区的载体。According to an embodiment of the present invention, the reagent includes a vector carrying a functional region expressing the HS3ST5 gene.
根据本发明的实施例，所述HS3ST5基因功能区具有SEQ ID NO:1所示的核苷酸序列。According to an embodiment of the present invention, the HS3ST5 gene functional region has the nucleotide sequence shown in SEQ ID NO: 1.
ATGCTATTCAAACAGCAGGCGTGGCTGAGACAGAAGCTCCTGGTGCTGGGAAGCCTTGCCGTTGGGAGTCTCCTGTATCTAGTCGCCAGAGTTGGGAGCTTGGATAGGCTACAACCCATTTGCCCCATTGAAGGTCGACTGGGTGGAGCCCGCACTCAGGCTGAATTCCCACTTCGCGCCCTGCAGTTTAAGCGTGGCCTGCTGCACGAGTTCCGGAAGGGCAACGCTTCCAAGGAGCAGGTTCGCCTCCATGACCTGGTCCAGCAGCTCCCCAAGGCCATTATCATTGGGGTGAGGAAAGGAGGCACAAGGGCCCTGCTTGAAATGCTGAACCTACATCCGGCAGTAGTCAAAGCCTCTCAAGAAATCCACTTTTTTGATAATGATGAGAATTATGGTAAGGGCATTGAGTGGTATAGGAAAAAGATGCCTTTTTCCTACCCTCAGCAAATCACAATTGAAAAGAGCCCAGCATATTTTATCACAGAGGAGGTTCCAGAAAGGATTTACAAAATGAACTCATCCATCAAGTTGTTGATCATTGTCAGGGAGCCAACCACAAGAGCTATTTCTGATTATACTCAGGTGCTAGAGGGGAAGGAGAGGAAGAACAAAACTTATTACAAGTTTGAGAAGCTGGCCATAGACCCTAATACATGCGAAGTGAACACAAAATACAAAGCAGTAAGAACCAGCATCTACACCAAACATCTGGAAAGGTGGTTGAAATACTTTCCAATTGAGCAATTTCATGTCGTCGATGGAGATCGCCTCATCACGGAACCTCTGCCAGAACTTCAGCTCGTGGAGAAGTTCCTAAATCTGCCTCCAAGGATAAGTCAATACAATTTATACTTCAATGCTACCAGAGGGTTTTACTGCTTGCGGTTTAATATTATCTTTAATAAGTGCCTGGCGGGCAGCAAGGGGCGCATTCATCCAGAGGTGGACCCCTCTGTCATTACTAAATTGCGCAAATTCTTTCATCCTTTTAATCAAAAATTTTACCAGATCACTGGGAGGACATTGAACTGGCCCTAA(SEQ ID NO:1)。ATGCTATTCAAACAGCAGGCGTGGCTGAGACAGAAGCTCTGGTGCTGGGAAGCCTTGCCGTTGGGAGTCTCCTGTATCTAGTCGCCAGAGTTGGGAGCTTGGATAGGCTACAACCCATTTGCCCCATTGAAGGTCGACTGGGTGGAGCCCGCACTCAGGCTGAATTCCCACTTCGCGCCCTGCAGTTTAAGCGTGGCCTGCTGCACGAGTTCCGGAAGGGCAACGCTTCCAAGGAGCAGGTTCGCCTCCATGACCTGGTCCA GCAGCTCCCCAAGGCCATTATCATTGGGGTGAGGAAAGGAGGCACAAGGGCCCTGCTTGAAATGCTGAACCTACATCCGGCAGTAGTCAAAGCCTCTCAAGAAATCCACTTTTTTGATAATGATGAGAATTATGGTAAGGGCATTGAGTGGTATAGGAAAAAGATGCCTTTTTCCTACCCTCAGCAAATCACAATTGAAAAGAGCCCAGCATATTTTATCACAGAGGAGGTTCCAGAAAGGATTTACAAAATGAACTCATCCATCAAGTTGTTGATC ATTGTCAGGGAGCCAACCACAAGAGCTATTTCTGATTATACTCAGGTGCTAGAGGGGAAGGAGAGGAAGAACAAAACTTATTACAAGTTTGAGAAGCTGGCCATAGACCCTAATACATGCGAAGTGAACACAAAATACAAAGCAGTAAGAACCAGCATCTACACCAAACATCTGGAAAGGTGGTTGAAATACTTCCAATTGAGCAATTTCATGTCGTCGATGGAGATCGCCTCATCACGGAACCTCTGCCAGAACTTCAGCTCGTGGAGAAG TTCCTAAATCTGCCTCCAAGGATAAGTCAATACAATTTATACTTCAATGCTACCAGAGGGTTTTACTGCTTGCGGTTTAATATTATCTTAATAAGTGCCTGGCGGGCAGCAAGGGGCGCATTCATCCAGAGGTGGACCCCTCTGTCATTACTAAATTGCGCAAATTCTTTCATCCTTTTAATCAAAAATTTTACCAGATCACTGGGAGGACATTGAACTGGCCCTAA (SEQ ID NO: 1).
根据本发明的实施例，所述HS3ST5功能域肽段具有SEQ ID NO:3所述的氨基酸序列。According to an embodiment of the present invention, the HS3ST5 functional domain peptide has the amino acid sequence described in SEQ ID NO: 3.
MLFKQQAWLRQKLLVLGSLAVGSLLYLVARVGSLDRLQPICPIEGRLGGARTQAEFPLRALQFKRGLLHEFRKGNASKEQVRLHDLVQQLPKAIIIGVRKGGTRALLEMLNLHPAVVKASQEIHFFDNDENYGKGIEWYRKKMPFSYPQQITIEKSPAYFITEEVPERIYKMNSSIKLLIIVREPTTRAISDYTQVLEGKERKNKTYYKFEKLAIDPNTCEVNTKYKAVRTSIYTKHLERWLKYFPIEQFHVVDGDRLITEPLPELQLVEKFLNLPPRISQYNLYFNATRGFYCLRFNIIFNKCLAGSKGRIHPEVDPSVITKLRKFFHPFNQKFYQITGRTLNWP(SEQ ID NO:3)。MLFKQQAWLRQKLLVLGSLAVGSLLYLVARVGSLDRLQPICPIEGRLGGARTQAEFPLRALQFKRGLLHEFRKGNASKEQVRLHDLVQQLPKAIIIGVRKGGTRALLEMLNLHPAVVKASQEIHFFDNDENYGKGIEWYRKKMPFSYPQQITIEKSPAYFITEEVPERIYKMNSSIKLLIIVREPTTRAISDYTQVLEGKERKNKTY YKFEKLAIDPNTCEVNTKYKAVRTSIYTKHLERWLKYFPIEQFHVVDGDRLITEPLPELQLVEKFLNLPPRISQYNLYFNATRGFYCLRFNIIFNKCLAGSKGRIHPEVDPSVITKLRKFFHPFNQKFYQITGRTLNWP (SEQ ID NO: 3).
根据本发明的实施例，所述载体为病毒载体。According to an embodiment of the invention, the vector is a viral vector.
根据本发明的实施例，所述病毒包括慢病毒。According to an embodiment of the invention, the virus includes a lentivirus.
根据本发明的实施例，所述HS3ST5基因具有SEQ ID NO:2所示的核苷酸序列。According to an embodiment of the present invention, the HS3ST5 gene has the nucleotide sequence shown in SEQ ID NO:2.
根据本发明的实施例，所述HS3ST5蛋白具有SEQ ID NO:4所述的氨基酸序列。According to an embodiment of the present invention, the HS3ST5 protein has the amino acid sequence described in SEQ ID NO: 4.
根据本发明的实施例，所述细胞包括选自下列中的至少之一：内皮细胞、SK-Hep-1、HUVEC。According to an embodiment of the present invention, the cells include at least one selected from the following: endothelial cells, SK-Hep-1, and HUVEC.
根据本发明的实施例，所述细胞包括选自下列中的至少之一：动脉血管内皮细胞、静脉血管内皮细胞、毛细血管内皮细胞、肝脏内皮细胞、肺内皮细胞、心室内皮细胞、脑内皮细胞、骨骼肌内皮细胞、胃内皮细胞、视网膜内皮细胞、皮肤内皮细胞、小肠内皮细胞、肾内皮细胞、关节滑膜内皮细胞、食道内皮细胞、胰岛内皮细胞、骨髓内皮细胞、胎盘内皮细胞、脉络膜内皮细胞、SK-Hep-1、HUVEC和原代内皮细胞。According to an embodiment of the present invention, the cells include at least one selected from the group consisting of arterial endothelial cells, venous endothelial cells, capillary endothelial cells, liver endothelial cells, lung endothelial cells, ventricular endothelial cells, and brain endothelial cells. cells, skeletal muscle endothelial cells, gastric endothelial cells, retinal endothelial cells, skin endothelial cells, small intestinal endothelial cells, renal endothelial cells, joint synovial endothelial cells, esophageal endothelial cells, pancreatic islet endothelial cells, bone marrow endothelial cells, placental endothelial cells, choroid Endothelial cells, SK-Hep-1, HUVEC and primary endothelial cells.
根据本发明的实施例，所述细胞包括选自下列中的至少之一：SK-Hep-1、HUVEC和原代内皮细胞。According to an embodiment of the present invention, the cells include at least one selected from: SK-Hep-1, HUVEC and primary endothelial cells.
在本发明的第三方面，本发明提出了试剂在制备试剂盒中的用途。根据本发明的实施例，所述试剂盒用于抑制细胞分化成血管，所述试剂用于抑制HS3ST5基因或HS3ST5蛋白。研究表明，当血管为肿瘤或是其他病变组织提供营养，或是血管过度生长侵入到周围组织时，它便对身体形成危害；如前所述，HS3ST5基因可以促进内皮细胞、SK-Hep-1和HUVEC向成血管方向分化，因此，当所述试剂可以抑制HS3ST5基因或HS3ST5蛋白时，可以有效抑制上述细胞向成血管方向分化。In a third aspect of the invention, the invention provides the use of reagents in the preparation of kits. According to an embodiment of the present invention, the kit is used to inhibit cell differentiation into blood vessels, and the reagent is used to inhibit HS3ST5 gene or HS3ST5 protein. Studies have shown that when blood vessels provide nutrients for tumors or other diseased tissues, or when blood vessels overgrow and invade surrounding tissues, it will cause harm to the body; as mentioned above, the HS3ST5 gene can promote endothelial cells, SK-Hep-1 and HUVEC differentiate in the direction of angiogenesis. Therefore, when the reagent can inhibit the HS3ST5 gene or HS3ST5 protein, it can effectively inhibit the differentiation of the above cells in the direction of angiogenesis.
根据本发明的实施例，上述试剂在制备试剂盒中的用途进一步包括下列附加技术特征中的至少之一：According to an embodiment of the present invention, the use of the above-mentioned reagents in preparing a kit further includes at least one of the following additional technical features:
根据本发明的实施例，所述抑制HS3ST5基因是通过沉默HS3ST5基因实现的。According to an embodiment of the present invention, the suppression of the HS3ST5 gene is achieved by silencing the HS3ST5 gene.
根据本发明的实施例，所述HS3ST5基因具有SEQ ID NO:2所示的核苷酸序列。According to an embodiment of the present invention, the HS3ST5 gene has the nucleotide sequence shown in SEQ ID NO:2.
根据本发明的实施例，所述HS3ST5蛋白具有SEQ ID NO:4所述的氨基酸序列。According to an embodiment of the present invention, the HS3ST5 protein has the amino acid sequence described in SEQ ID NO: 4.
根据本发明的实施例，所述细胞选自下列中的至少之一：内皮细胞、SK-Hep-1和HUVEC。According to an embodiment of the present invention, the cells are selected from at least one of the following: endothelial cells, SK-Hep-1 and HUVEC.
根据本发明的实施例，所述细胞包括选自下列中的至少之一：SK-Hep-1、动脉血管内皮细胞、静脉血管内皮细胞、毛细血管内皮细胞、肝脏内皮细胞、肺内皮细胞、心室内皮细胞、脑内皮细胞、骨骼肌内皮细胞、胃内皮细胞、视网膜内皮细胞、皮肤内皮细胞、小肠内皮细胞、肾内皮细胞、关节滑膜内皮细胞、食道内皮细胞、胰岛内皮细胞、骨髓内皮细胞、胎盘内皮细胞、脉络膜内皮细胞、SK-Hep-1、HUVEC和原代内皮细胞；According to an embodiment of the present invention, the cells include at least one selected from the following: SK-Hep-1, arterial vascular endothelial cells, venous vascular endothelial cells, capillary endothelial cells, liver endothelial cells, pulmonary endothelial cells, heart Indoor endothelial cells, brain endothelial cells, skeletal muscle endothelial cells, gastric endothelial cells, retinal endothelial cells, skin endothelial cells, small intestinal endothelial cells, renal endothelial cells, joint synovial endothelial cells, esophageal endothelial cells, pancreatic islet endothelial cells, bone marrow endothelial cells , placental endothelial cells, choroidal endothelial cells, SK-Hep-1, HUVEC and primary endothelial cells;
根据本发明的实施例，所述细胞包括选自下列中的至少之一：SK-Hep-1、HUVEC和原代内皮细胞。According to an embodiment of the present invention, the cells include at least one selected from: SK-Hep-1, HUVEC and primary endothelial cells.
在本发明的第四方面，本发明提出了试剂在制备药物中的用途。根据本发明的实施例，所述药物用于治疗或预防血管生成障碍相关疾病，所述试剂包括HS3ST5蛋白或HS3ST5功能域肽段，或用于激活或过表达HS3ST5基因。如前所述，HS3ST5基因可以有效促进细胞向成血管方向分化，因此，当所述试剂包括HS3ST5基因表达的HS3ST5蛋白或HS3ST5功能域肽段，或用于激活或过表达HS3ST5基因时，包含所述试剂的药物可以有效促进细胞向成血管方向分化，用于治疗或预防血管生成障碍相关疾病。In a fourth aspect of the invention, the invention provides the use of the reagent in the preparation of a medicament. According to embodiments of the present invention, the drug is used to treat or prevent diseases related to angiogenesis disorders, and the reagent includes HS3ST5 protein or HS3ST5 functional domain peptide, or is used to activate or overexpress the HS3ST5 gene. As mentioned above, the HS3ST5 gene can effectively promote the differentiation of cells in the direction of angiogenesis. Therefore, when the reagent includes the HS3ST5 protein or HS3ST5 functional domain peptide expressed by the HS3ST5 gene, or is used to activate or overexpress the HS3ST5 gene, it contains all The above-mentioned reagents can effectively promote the differentiation of cells in the direction of angiogenesis and are used to treat or prevent diseases related to angiogenesis disorders.
根据本发明的实施例，上述试剂在制备药物中的用途还可以包括下列附加技术特征中的至少之一：According to embodiments of the present invention, the use of the above-mentioned reagents in preparing medicines may also include at least one of the following additional technical features:
根据本发明的实施例，所述试剂包括携带表达所述HS3ST5基因功能区的载体。According to an embodiment of the present invention, the reagent includes a vector carrying a functional region expressing the HS3ST5 gene.
根据本发明的实施例，所述HS3ST5基因功能区具有SEQ ID NO:1所示的核苷酸序列。According to an embodiment of the present invention, the HS3ST5 gene functional region has the nucleotide sequence shown in SEQ ID NO: 1.
根据本发明的实施例，所述HS3ST5功能域肽段具有SEQ ID NO:3所述的氨基酸序列。According to an embodiment of the present invention, the HS3ST5 functional domain peptide has the amino acid sequence described in SEQ ID NO: 3.
根据本发明的实施例，所述载体为病毒载体。According to an embodiment of the invention, the vector is a viral vector.
根据本发明的实施例，所述病毒包括慢病毒。According to an embodiment of the invention, the virus includes a lentivirus.
根据本发明的实施例，所述HS3ST5基因具有SEQ ID NO:2所示的核苷酸序列。According to an embodiment of the present invention, the HS3ST5 gene has the nucleotide sequence shown in SEQ ID NO:2.
根据本发明的实施例，所述HS3ST5蛋白具有SEQ ID NO:4所述的氨基酸序列。According to an embodiment of the present invention, the HS3ST5 protein has the amino acid sequence described in SEQ ID NO: 4.
根据本发明的实施例，所述血管生成障碍相关疾病包括选自下列中的至少之一：缺血性脑栓塞、冠心病、心肌梗死、血管闭塞性血栓性脉管炎等。According to an embodiment of the present invention, the angiogenesis disorder-related diseases include at least one selected from the following: ischemic cerebral embolism, coronary heart disease, myocardial infarction, vaso-occlusive thromboangiitis, and the like.
在本发明的第五方面，本发明提出了试剂在制备药物中的用途。根据本发明的实施例，所述药物用于治疗或预防血管过度增生相关疾病，所述试剂用于抑制HS3ST5基因或HS3ST5蛋白。研究表明，当血管为肿瘤或是其他病变组织提供营养，或是血管过度生长侵入到周围组织时，它便对身体形成危害，如前所述，HS3ST5基因可以有效促进细胞向成血管方向分化，当所述试剂用于抑制HS3ST5基因或HS3ST5基因表达获得的HS3ST5蛋白时，包含所述试剂的药物同样可以抑制HS3ST5基因或HS3ST5基因表达获得的HS3ST5蛋白，用于治疗或预防血管过度增生相关疾病。In a fifth aspect of the invention, the invention provides the use of the reagent in the preparation of a medicament. According to an embodiment of the present invention, the drug is used to treat or prevent diseases related to excessive vascular proliferation, and the agent is used to inhibit the HS3ST5 gene or HS3ST5 protein. Studies have shown that when blood vessels provide nutrients for tumors or other diseased tissues, or when blood vessels overgrow and invade surrounding tissues, it will cause harm to the body. As mentioned above, the HS3ST5 gene can effectively promote the differentiation of cells into the direction of vasculogenesis. When the reagent is used to inhibit the HS3ST5 gene or the HS3ST5 protein obtained from the expression of the HS3ST5 gene, the medicine containing the reagent can also inhibit the HS3ST5 gene or the HS3ST5 protein obtained from the expression of the HS3ST5 gene, and is used to treat or prevent diseases related to excessive vascular proliferation.
根据本发明的实施例，上述试剂在制备药物中的用途进一步包括下列附加技术特征中的至少之一：According to embodiments of the present invention, the use of the above-mentioned reagents in preparing medicines further includes at least one of the following additional technical features:
根据本发明的实施例，所述抑制HS3ST5基因是通过沉默HS3ST5基因实现的。According to an embodiment of the present invention, the suppression of the HS3ST5 gene is achieved by silencing the HS3ST5 gene.
根据本发明的实施例，所述HS3ST5基因具有SEQ ID NO:2所示的核苷酸序列。According to an embodiment of the present invention, the HS3ST5 gene has the nucleotide sequence shown in SEQ ID NO:2.
根据本发明的实施例，所述HS3ST5蛋白具有SEQ ID NO:4所述的氨基酸序列。According to an embodiment of the present invention, the HS3ST5 protein has the amino acid sequence described in SEQ ID NO: 4.
根据本发明的实施例，血管过度增生相关疾病包括老年性黄斑变性、肿瘤、糖尿病、视网膜病、血管瘤、类风湿性关节炎、肥胖症等。According to embodiments of the present invention, diseases related to excessive vascular proliferation include age-related macular degeneration, tumors, diabetes, retinopathy, hemangiomas, rheumatoid arthritis, obesity, and the like.
在本发明的第六方面，本发明提出了一种诱导细胞分化成血管的方法。根据本发明的实施例，将试剂与所述细胞进行接触，所述试剂包括HS3ST5蛋白或HS3ST5功能域肽段或用于激活或过表达HS3ST5基因。如前所述，HS3ST5基因可以有效促进细胞向成血管方向分化，当所述试剂包括HS3ST5蛋白或HS3ST5功能域肽段或用于激活或过表达HS3ST5基因并与所述细胞进行接触时可以有效诱导细胞往成血管方向分化。根据本发明的具体实施例的方法可以有效诱导细胞往成血管方向分化。In a sixth aspect of the present invention, the present invention provides a method for inducing cell differentiation into blood vessels. According to an embodiment of the present invention, a reagent is brought into contact with the cell, and the reagent includes HS3ST5 protein or HS3ST5 functional domain peptide or is used to activate or overexpress the HS3ST5 gene. As mentioned above, the HS3ST5 gene can effectively promote the differentiation of cells into the angiogenic direction, and can be effectively induced when the reagent includes the HS3ST5 protein or HS3ST5 functional domain peptide or is used to activate or overexpress the HS3ST5 gene and come into contact with the cells. Cells differentiate into blood vessels. Methods according to specific embodiments of the present invention can effectively induce cells to differentiate in the direction of vasculogenesis.
根据本发明的实施例，上述诱导细胞分化成血管的方法还可以包括下列附加技术特征中的至少之一：According to embodiments of the present invention, the above-mentioned method of inducing cell differentiation into blood vessels may further include at least one of the following additional technical features:
根据本发明的实施例，所述试剂包括携带表达所述HS3ST5基因功能区的载体。According to an embodiment of the present invention, the reagent includes a vector carrying a functional region expressing the HS3ST5 gene.
根据本发明的实施例，所述HS3ST5基因功能区具有SEQ ID NO:1所示的核苷酸序列。According to an embodiment of the present invention, the HS3ST5 gene functional region has the nucleotide sequence shown in SEQ ID NO: 1.
根据本发明的实施例，所述HS3ST5功能域肽段具有SEQ ID NO:3所述的氨基酸序列。According to an embodiment of the present invention, the HS3ST5 functional domain peptide has the amino acid sequence described in SEQ ID NO: 3.
根据本发明的实施例，所述载体为病毒载体。According to an embodiment of the invention, the vector is a viral vector.
根据本发明的实施例，所述病毒包括慢病毒。According to an embodiment of the invention, the virus includes a lentivirus.
根据本发明的实施例，所述HS3ST5基因具有SEQ ID NO:2所示的核苷酸序列。According to an embodiment of the present invention, the HS3ST5 gene has the nucleotide sequence shown in SEQ ID NO:2.
根据本发明的实施例，所述HS3ST5蛋白具有SEQ ID NO:4所述的氨基酸序列。According to an embodiment of the present invention, the HS3ST5 protein has the amino acid sequence described in SEQ ID NO: 4.
根据本发明的实施例，所述细胞包括选自下列中的至少之一：内皮细胞、SK-Hep-1和HUVEC。According to an embodiment of the present invention, the cells include at least one selected from the following: endothelial cells, SK-Hep-1 and HUVEC.
根据本发明的实施例，所述内皮细胞包括选自下列中的至少之一：动脉血管内皮细胞、静脉血管内皮细胞、毛细血管内皮细胞、肝脏内皮细胞、肺内皮细胞、心室内皮细胞、脑内皮细胞、骨骼肌内皮细胞、胃内皮细胞、视网膜内皮细胞、皮肤内皮细胞、小肠内皮细胞、肾内皮细胞、关节滑膜内皮细胞、食道内皮细胞、胰岛内皮细胞、骨髓内皮细胞、胎盘内皮细胞、脉络膜内皮细胞、SK-Hep-1、HUVEC和原代内皮细胞；According to an embodiment of the present invention, the endothelial cells include at least one selected from the following: arterial endothelial cells, venous endothelial cells, capillary endothelial cells, liver endothelial cells, lung endothelial cells, ventricular endothelial cells, brain Endothelial cells, skeletal muscle endothelial cells, gastric endothelial cells, retinal endothelial cells, skin endothelial cells, small intestinal endothelial cells, renal endothelial cells, joint synovial endothelial cells, esophageal endothelial cells, pancreatic islet endothelial cells, bone marrow endothelial cells, placental endothelial cells, Choroidal endothelial cells, SK-Hep-1, HUVEC and primary endothelial cells;
根据本发明的实施例，所述细胞包括选自下列中的至少之一：SK-Hep-1、HUVEC和原代内皮细胞。According to an embodiment of the present invention, the cells include at least one selected from: SK-Hep-1, HUVEC and primary endothelial cells.
在本发明的第七方面，本发明提出了生物模型在筛选药物中的用途。根据本发明的实施例，所述生物模型高表达或低表达HS3ST5蛋白。如前所述，HS3ST5基因可以有效促进细胞向成血管方向分化，因此，当所述HS3ST5基因表达的HS3ST5蛋白水平发生变化时，可以构建不同的生物模型，例如，当所述生物模型高表达HS3ST5蛋白时，所述生物模型能够用来筛选降低HS3ST5蛋白表达量的药物，根据本发明具体实施例的生物模型可以有效筛选到目标药物。In the seventh aspect of the present invention, the present invention proposes the use of biological models in screening drugs. According to embodiments of the present invention, the biological model expresses high or low expression of HS3ST5 protein. As mentioned before, the HS3ST5 gene can effectively promote the differentiation of cells in the direction of vasculogenesis. Therefore, when the HS3ST5 protein level expressed by the HS3ST5 gene changes, different biological models can be constructed. For example, when the biological model highly expresses HS3ST5 protein, the biological model can be used to screen for drugs that reduce the expression of HS3ST5 protein. The biological model according to specific embodiments of the present invention can effectively screen for target drugs.
根据本发明的实施例，上述生物模型在筛选药物中的用途还可以包括下列附加技术特征中的至少之一：According to embodiments of the present invention, the use of the above-mentioned biological model in screening drugs may also include at least one of the following additional technical features:
根据本发明的实施例，所述药物用于治疗或预防血管生长过度相关疾病或血管生成障碍相关疾病。According to an embodiment of the present invention, the medicament is used to treat or prevent diseases related to excessive blood vessel growth or disorders of angiogenesis.
根据本发明的实施例，所述血管生成过度相关疾病包括选自下列中的至少之一：老年性黄斑变性、肿瘤、糖尿病和视网膜病、血管瘤、类风湿性关节炎、肥胖症等。According to an embodiment of the present invention, the hyperangiogenesis-related disease includes at least one selected from the following: age-related macular degeneration, tumors, diabetes and retinopathy, hemangiomas, rheumatoid arthritis, obesity, and the like.
根据本发明的实施例，所述血管生成障碍相关疾病包括选自下列中的至少之一：缺血性脑栓塞、冠心病、心肌梗死、血管闭塞性血栓性脉管炎等。According to an embodiment of the present invention, the angiogenesis disorder-related diseases include at least one selected from the following: ischemic cerebral embolism, coronary heart disease, myocardial infarction, vaso-occlusive thromboangiitis, and the like.
根据本发明的实施例，所述生物模型为细胞模型或者动物模型。According to an embodiment of the present invention, the biological model is a cell model or an animal model.
根据本发明的实施例，所述生物模型为脱细胞生物支架。According to an embodiment of the present invention, the biological model is an acellular biological scaffold.
根据本发明的实施例，所述生物模型为脱细胞肝脏生物支架。According to an embodiment of the present invention, the biological model is an acellular liver biological scaffold.
根据本发明的实施例，所述生物模型为脱细胞肝脏基质。According to an embodiment of the invention, the biological model is an acellular liver matrix.
在本发明的第八方面，本发明提出了一种筛选药物的方法，所述药物用于治疗或预防血管过度增生相关疾病。根据本发明的实施例，将待筛选药物与生物模型进行接触处理；基于所述生物模型中HS3ST5蛋白的表达量，确定待筛选药物是否为目标药物；其中，待筛选药物与所述生物模型接触后相比于待筛选药物与生物模型接触前，所述生物模型中HS3ST5蛋白的表达量下降，是待筛选药物为目标药物的指示。如前所述，HS3ST5基因可以有效促进细胞向成血管方向分化，同样地，所述HS3ST5基因表达的蛋白可以有效促进细胞向成血管方向分化，当待筛选药物与所述生物模型进行接触处理后，所述生物模型中HS3ST5蛋白的表达量低于待筛选药物与所述生物模型接触前地表达量，则待筛选药物能够降低所述HS3ST5蛋白的表达量，根据本发明具体实施例地方法可以有效获得目标药物。In an eighth aspect of the present invention, the present invention provides a method for screening drugs used to treat or prevent diseases related to vascular hyperplasia. According to an embodiment of the present invention, the drug to be screened is brought into contact with a biological model; based on the expression level of HS3ST5 protein in the biological model, it is determined whether the drug to be screened is a target drug; wherein the drug to be screened is in contact with the biological model Afterwards, compared with before the drug to be screened comes into contact with the biological model, the expression of HS3ST5 protein in the biological model decreases, which is an indication that the drug to be screened is the target drug. As mentioned before, the HS3ST5 gene can effectively promote the differentiation of cells in the direction of angiogenesis. Similarly, the protein expressed by the HS3ST5 gene can effectively promote the differentiation of cells in the direction of angiogenesis. When the drug to be screened is contacted with the biological model, , the expression level of HS3ST5 protein in the biological model is lower than the expression level before the drug to be screened comes into contact with the biological model, then the drug to be screened can reduce the expression level of the HS3ST5 protein, and the method according to specific embodiments of the present invention can Effectively obtain target drugs.
根据本发明的实施例，上述筛选药物的方法还可以包括下列附加技术特征中的至少之一：According to embodiments of the present invention, the above-mentioned method for screening drugs may further include at least one of the following additional technical features:
根据本发明的实施例，所述生物模型中正常表达或过表达HS3ST5蛋白。According to embodiments of the present invention, the HS3ST5 protein is normally expressed or overexpressed in the biological model.
在本发明的第九方面，本发明提出了一种筛选药物的方法，所述药物用于治疗或预防血管生成障碍相关疾病。根据本发明的实施例，将待筛选药物与生物模型进行接触处理；基于生物模型中HS3ST5蛋白的表达量，确定待筛选药物是否为目标药物；其中，待筛选药物与生物模型接触后相比于待筛选药物与生物模型接触前，所述生物模型中HS3ST5蛋白的表达量上升，是待筛选药物为目标药物的指示。如前所述，HS3ST5基因可以有效促进细胞向成血管方向分化，同样地，所述HS3ST5基因表达的蛋白可以有效促进细胞向成血管方向分化，当待筛选药物与所述生物模型进行接触处理后，所述生物模型中HS3ST5蛋白的表达量高于待筛选药物与所述生物模型接触前的表达量，则待筛选药物能够升高所述HS3ST5蛋白的表达量，根据本发明具体实施例的方法可以有效获得目标药物。In a ninth aspect of the present invention, the present invention provides a method for screening drugs for treating or preventing diseases related to angiogenesis disorders. According to embodiments of the present invention, the drug to be screened is brought into contact with the biological model; based on the expression level of HS3ST5 protein in the biological model, it is determined whether the drug to be screened is the target drug; wherein, after the drug to be screened is in contact with the biological model, compared with Before the drug to be screened comes into contact with the biological model, the expression of HS3ST5 protein in the biological model increases, which is an indication that the drug to be screened is the target drug. As mentioned before, the HS3ST5 gene can effectively promote the differentiation of cells in the direction of angiogenesis. Similarly, the protein expressed by the HS3ST5 gene can effectively promote the differentiation of cells in the direction of angiogenesis. When the drug to be screened is contacted with the biological model, , the expression level of HS3ST5 protein in the biological model is higher than the expression level of the drug to be screened before contact with the biological model, then the drug to be screened can increase the expression level of the HS3ST5 protein, according to the method of specific embodiments of the present invention Target drugs can be obtained effectively.
根据本发明的实施例，上述筛选药物的方法还可以包括下列附加技术特征中的至少之一：According to embodiments of the present invention, the above-mentioned method for screening drugs may further include at least one of the following additional technical features:
根据本发明的实施例，所述生物模型中正常表达或低表达HS3ST5蛋白。According to embodiments of the present invention, the HS3ST5 protein is expressed normally or at low levels in the biological model.
在本发明的第十方面，本发明提出了一种促进脱细胞生物支架再血管化的方法。根据本发明的实施例，包括：将所述脱细胞生物支架与试剂进行接触，所述试剂包括HS3ST5蛋白或HS3ST5功能域肽段，或用于激活HS3ST5基因或HS3ST5蛋白或过表达HS3ST5基因。如前所述，HS3ST5基因可以有效促进细胞向成血管方向分化，同样地，所述HS3ST5基因表达的蛋白或蛋白的功能域可以有效促进细胞向成血管方向分化，根据本发明具体实施例的方法可以有效促进细胞生物支架再血管化。In a tenth aspect of the present invention, the present invention proposes a method for promoting revascularization of an acellular biological scaffold. According to an embodiment of the present invention, the method includes: contacting the decellularized biological scaffold with a reagent, the reagent including HS3ST5 protein or HS3ST5 functional domain peptide, or used to activate the HS3ST5 gene or HS3ST5 protein or overexpress the HS3ST5 gene. As mentioned above, the HS3ST5 gene can effectively promote the differentiation of cells in the direction of angiogenesis. Similarly, the protein or functional domain of the protein expressed by the HS3ST5 gene can effectively promote the differentiation of cells in the direction of angiogenesis. According to the method of specific embodiments of the present invention It can effectively promote the revascularization of cell biological scaffolds.
根据本发明的实施例，上述促进脱细胞生物支架再血管化的方法还可以包括下列附加技术特征中的至少之一：According to embodiments of the present invention, the above-mentioned method for promoting revascularization of acellular biological scaffolds may further include at least one of the following additional technical features:
根据本发明的实施例，所述试剂包括携带表达所述HS3ST5基因功能区的载体。According to an embodiment of the present invention, the reagent includes a vector carrying a functional region expressing the HS3ST5 gene.
根据本发明的实施例，所述HS3ST5蛋白具有SEQ ID NO:4所示的氨基酸序列。According to an embodiment of the present invention, the HS3ST5 protein has the amino acid sequence shown in SEQ ID NO: 4.
根据本发明的实施例，所述HS3ST5功能域肽段具有SEQ ID NO:3所述的氨基酸序列。According to an embodiment of the present invention, the HS3ST5 functional domain peptide has the amino acid sequence described in SEQ ID NO: 3.
根据本发明的实施例，所述HS3ST5基因具有SEQ ID NO:2所示的核苷酸序列。According to an embodiment of the present invention, the HS3ST5 gene has the nucleotide sequence shown in SEQ ID NO:2.
根据本发明的实施例，所述载体为病毒载体。According to an embodiment of the invention, the vector is a viral vector.
根据本发明的实施例，所述病毒包括慢病毒。According to an embodiment of the invention, the virus includes a lentivirus.
附图说明Description of the drawings
图1是根据本发明实施例1的HS3st5基因表达质粒图谱；Figure 1 is a map of the HS3st5 gene expression plasmid according to Embodiment 1 of the present invention;
图2是根据本发明实施例3的HS3ST5过表达内皮细胞系qPCR过表达鉴定结果；Figure 2 is the qPCR overexpression identification result of the HS3ST5 overexpressing endothelial cell line according to Example 3 of the present invention;
图3是根据本发明实施例3的HS3ST5过表达内皮细胞系WB过表达鉴定结果；Figure 3 is the identification result of WB overexpression of the HS3ST5 overexpressing endothelial cell line according to Example 3 of the present invention;
图4是根据本发明实施例4的SK-HEP-1-HS3ST5细胞成管实验结果及定量图；其中，Figure 4 is a tube-forming experimental result and quantitative chart of SK-HEP-1-HS3ST5 cells according to Embodiment 4 of the present invention; wherein,
4-A是所述SK-HEP-1-HS3ST5细胞成管实验结果图，4-A is a picture of the results of the tube formation experiment of the SK-HEP-1-HS3ST5 cells,
4-B是所述SK-HEP-1-HS3ST5细胞成管实验定量分析结果图，横坐标表示不同处理组的细胞，纵坐标(Nb Junctions和Tot.Branching lenght)分别表示分支点的数量和分枝长度；4-B is a diagram of the quantitative analysis results of the tube formation experiment of the SK-HEP-1-HS3ST5 cells. The abscissa represents cells in different treatment groups, and the ordinate (Nb Junctions and Tot.Branching lenght) represents the number and branching points respectively. branch length;
图5是根据本发明实施例4的SK-HEP-1-HS3ST5细胞粘附曲线；Figure 5 is an SK-HEP-1-HS3ST5 cell adhesion curve according to Example 4 of the present invention;
图6是根据本发明实施例5的SK-HEP-1-HS3ST5细胞与正常肝类器官共培养10天后肝功能指glutamine synthetase表达量变化的荧光定量图。Figure 6 is a fluorescence quantitative graph showing changes in expression of glutamine synthetase, a liver function indicator, after co-culture of SK-HEP-1-HS3ST5 cells and normal liver organoids for 10 days according to Example 5 of the present invention.
具体实施方式Detailed ways
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements with the same or similar functions. The embodiments described below with reference to the drawings are exemplary and are intended to explain the present invention and are not to be construed as limiting the present invention.
在对本发明描述的过程中，对于本文中有关的术语进行了解释和说明，这些解释和说明仅仅是为了方便对于方案的理解，并不能看做是对本发明保护方案的限制。In the process of describing the present invention, the relevant terms in this article have been explained and explained. These explanations and explanations are only for the convenience of understanding of the scheme and cannot be regarded as limiting the protection scheme of the present invention.
本申请中，所述“脱细胞支架”是指由经化学和物理的方法去除异体或异种组织中的细胞，形成无免疫原性或低免疫原性的材料构建的组织工程支架。In this application, the "decellularized scaffold" refers to a tissue engineering scaffold constructed by removing cells from allogeneic or xenogeneic tissues through chemical and physical methods to form non-immunogenic or low-immunogenic materials.
在本申请中，所述“正常表达HS3ST5蛋白”是指正常健康的机体未经过任何实验处理，如基因表达干预等，健康机体中表达HS3ST5蛋白的水平。In this application, the "normal expression of HS3ST5 protein" refers to the level of expression of HS3ST5 protein in a normal healthy body without any experimental treatment, such as gene expression intervention, etc.
在本申请中，所述“高表达HS3ST5蛋白”是指正常健康的机体经过HS3ST5蛋白表达量干预，如HS3ST5基因过表达等，经过所述干预后，所述HS3ST5蛋白的含量高于，或，显著或极显著(常规统计学定义P<0.05、P<0.01)高于所述正常健康的机体的HS3ST5蛋白含量。In this application, the "highly expressed HS3ST5 protein" refers to a normal and healthy body that has undergone intervention in HS3ST5 protein expression, such as HS3ST5 gene overexpression, etc. After the intervention, the content of the HS3ST5 protein is higher than, or, Significantly or extremely significantly (conventional statistics define P<0.05, P<0.01) higher than the HS3ST5 protein content of the normal healthy body.
在本申请中，所述“低表达HS3ST5蛋白”是指正常健康的机体经过HS3ST5蛋白表达量干预，如HS3ST5基因沉默等，经过所述干预后，所述HS3ST5蛋白的含量低于，或，显著或极显著(常规统计学定义P<0.05、P<0.01)低于所述正常健康的机体的HS3ST5蛋白含量。In this application, the "low expression of HS3ST5 protein" refers to a normal and healthy body that has undergone intervention in the expression of HS3ST5 protein, such as HS3ST5 gene silencing, etc. After the intervention, the content of the HS3ST5 protein is lower than, or significantly or extremely significantly (conventional statistical definitions of P<0.05, P<0.01) lower than the HS3ST5 protein content of the normal healthy body.
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。The solutions of the present invention will be explained below with reference to examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If specific techniques or conditions are not specified in the examples, the techniques or conditions described in literature in the field or product instructions will be followed. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.
实施例1质粒的扩增Example 1 Amplification of plasmid
本试验用于HS3ST5表达质粒(GV492)和包装质粒(Helper 1.0和Helper 2.0)的扩增，其中，表达质粒GV492(购于上海吉凯基因科技有限公司)，用于在哺乳动物细胞中对HS3ST5进行真核表达，其质粒图谱如图1所示，具体实验操作步骤如下：This test is used for the amplification of HS3ST5 expression plasmid (GV492) and packaging plasmid (Helper 1.0 and Helper 2.0). Among them, expression plasmid GV492 (purchased from Shanghai Jikai Gene Technology Co., Ltd.) is used to amplify HS3ST5 in mammalian cells. For eukaryotic expression, the plasmid map is shown in Figure 1. The specific experimental steps are as follows:
1)质粒的构建：1) Construction of plasmid:
化学合成编码HS3ST5的核苷酸序列，并将其插入表达质粒上述GV492中，Chemically synthesize the nucleotide sequence encoding HS3ST5 and insert it into the expression plasmid GV492 mentioned above,
2)转化：按全式金Trans5αChemicallyCompetentCell(货号CD201)说明书进行。取1μL溶解的质粒加入50μL刚刚溶解的大肠杆菌DH5α感受态细胞(购于北京全式金生物技术有限公司)中，冰上放置30min，42℃金属浴45秒后立即转到冰上放置2min，加入500μL无抗生素的液体LB培养基，置于37℃摇床200转速1小时使感受态细胞复苏。1000rpm/min离心1min，去掉400μL上清，重悬剩余感受态细胞，将其均匀涂在含有氨苄抗性的固体LB培养板上，将平板在37℃倒置培养12-14小时。挑取LB平板内的单个克隆菌落到含有氨苄青霉素的LB液体培养基中，并对之进行编号，37℃、200r/min摇菌培养过夜。2) Transformation: Carry out according to the instruction manual of Trans5αChemically CompetentCell (Cat. No. CD201). Add 1 μL of the dissolved plasmid to 50 μL of freshly dissolved E. coli DH5α competent cells (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), place on ice for 30 min, and then transfer to the metal bath at 42°C for 45 seconds and immediately transfer to ice for 2 min. Add 500 μL of antibiotic-free liquid LB medium and place on a 37°C shaker at 200 rpm for 1 hour to revive the competent cells. Centrifuge at 1000 rpm/min for 1 min, remove 400 μL of supernatant, resuspend the remaining competent cells, spread them evenly on a solid LB culture plate containing ampicillin resistance, and incubate the plate upside down at 37°C for 12-14 hours. Pick single clones from the LB plate into LB liquid culture medium containing ampicillin, number them, and culture overnight at 37°C and 200r/min shaking.
3)质粒测序鉴定：取过夜培养的菌液各1mL进行测序，用Snapgene软件分析测序结果，获得测序正确的阳性克隆，其核苷酸序列如下所示：3) Plasmid sequencing identification: Take 1 mL of each bacterial liquid cultured overnight for sequencing, use Snapgene software to analyze the sequencing results, and obtain a positive clone with correct sequencing. Its nucleotide sequence is as follows:
ATGCTATTCAAACAGCAGGCGTGGCTGAGACAGAAGCTCCTGGTGCTGGGAAGCCTTGCCGTTGGGAGTCTCCTGTATCTAGTCGCCAGAGTTGGGAGCTTGGATAGGCTACAACCCATTTGCCCCATTGAAGGTCGACTGGGTGGAGCCCGCACTCAGGCTGAATTCCCACTTCGCGCCCTGCAGTTTAAGCGTGGCCTGCTGCACGAGTTCCGGAAGGGCAACGCTTCCAAGGAGCAGGTTCGCCTCCATGACCTGGTCCAGCAGCTCCCCAAGGCCATTATCATTGGGGTGAGGAAAGGAGGCACAAGGGCCCTGCTTGAAATGCTGAACCTACATCCGGCAGTAGTCAAAGCCTCTCAAGAAATCCACTTTTTTGATAATGATGAGAATTATGGTAAGGGCATTGAGTGGTATAGGAAAAAGATGCCTTTTTCCTACCCTCAGCAAATCACAATTGAAAAGAGCCCAGCATATTTTATCACAGAGGAGGTTCCAGAAAGGATTTACAAAATGAACTCATCCATCAAGTTGTTGATCATTGTCAGGGAGCCAACCACAAGAGCTATTTCTGATTATACTCAGGTGCTAGAGGGGAAGGAGAGGAAGAACAAAACTTATTACAAGTTTGAGAAGCTGGCCATAGACCCTAATACATGCGAAGTGAACACAAAATACAAAGCAGTAAGAACCAGCATCTACACCAAACATCTGGAAAGGTGGTTGAAATACTTTCCAATTGAGCAATTTCATGTCGTCGATGGAGATCGCCTCATCACGGAACCTCTGCCAGAACTTCAGCTCGTGGAGAAGTTCCTAAATCTGCCTCCAAGGATAAGTCAATACAATTTATACTTCAATGCTACCAGAGGGTTTTACTGCTTGCGGTTTAATATTATCTTTAATAAGTGCCTGGCGGGCAGCAAGGGGCGCATTCATCCAGAGGTGGACCCCTCTGTCATTACTAAATTGCGCAAATTCTTTCATCCTTTTAATCAAAAATTTTACCAGATCACTGGGAGGACATTGAACTGGCCCTAA(SEQ ID NO:1)。ATGCTATTCAAACAGCAGGCGTGGCTGAGACAGAAGCTCTGGTGCTGGGAAGCCTTGCCGTTGGGAGTCTCCTGTATCTAGTCGCCAGAGTTGGGAGCTTGGATAGGCTACAACCCATTTGCCCCATTGAAGGTCGACTGGGTGGAGCCCGCACTCAGGCTGAATTCCCACTTCGCGCCCTGCAGTTTAAGCGTGGCCTGCTGCACGAGTTCCGGAAGGGCAACGCTTCCAAGGAGCAGGTTCGCCTCCATGACCTGGTCCA GCAGCTCCCCAAGGCCATTATCATTGGGGTGAGGAAAGGAGGCACAAGGGCCCTGCTTGAAATGCTGAACCTACATCCGGCAGTAGTCAAAGCCTCTCAAGAAATCCACTTTTTTGATAATGATGAGAATTATGGTAAGGGCATTGAGTGGTATAGGAAAAAGATGCCTTTTTCCTACCCTCAGCAAATCACAATTGAAAAGAGCCCAGCATATTTTATCACAGAGGAGGTTCCAGAAAGGATTTACAAAATGAACTCATCCATCAAGTTGTTGATC ATTGTCAGGGAGCCAACCACAAGAGCTATTTCTGATTATACTCAGGTGCTAGAGGGGAAGGAGAGGAAGAACAAAACTTATTACAAGTTTGAGAAGCTGGCCATAGACCCTAATACATGCGAAGTGAACACAAAATACAAAGCAGTAAGAACCAGCATCTACACCAAACATCTGGAAAGGTGGTTGAAATACTTCCAATTGAGCAATTTCATGTCGTCGATGGAGATCGCCTCATCACGGAACCTCTGCCAGAACTTCAGCTCGTGGAGAAG TTCCTAAATCTGCCTCCAAGGATAAGTCAATACAATTTATACTTCAATGCTACCAGAGGGTTTTACTGCTTGCGGTTTAATATTATTCTTTAATAAGTGCCTGGCGGGCAGCAAGGGGCGCATTCATCCAGAGGTGGACCCCTCTGTCATTACTAAATTGCGCAAATTCTTTCATCCTTTTAATCAAAAATTTTACCAGATCACTGGGAGGACATTGAACTGGCCCTAA (SEQ ID NO: 1).
4)质粒抽提：根据以上获得的阳性克隆菌落将相应菌液1mL加入50mL含氨苄抗性的液体LB培养基中培养过夜，按天根生化科技(北京)有限公司DP108-无内毒素质粒中量提取试剂盒说明书进行质粒提取。4) Plasmid extraction: According to the positive clone colonies obtained above, add 1 mL of the corresponding bacterial solution to 50 mL of liquid LB medium containing ampicillin resistance and culture overnight. Perform plasmid extraction according to the instructions of the quantitative extraction kit.
实施例2慢病毒包装Example 2 Lentivirus packaging
本实施例采用上述实施例1获得测序正确的质粒进行慢病毒包装和纯化，以获得携带有目的基因HS3ST5的慢病毒浓缩液，具体的包装和纯化步骤为：This example uses the above-mentioned Example 1 to obtain the correctly sequenced plasmid for lentivirus packaging and purification to obtain a lentivirus concentrate carrying the target gene HS3ST5. The specific packaging and purification steps are:
1)转染前一天，将293FT慢病毒包装细胞接种于10cm培养皿中，使第二天细胞密度达到70％左右可进行转染。1) One day before transfection, inoculate 293FT lentivirus packaging cells into a 10cm culture dish so that the cell density reaches about 70% the next day for transfection.
2)转染体系如表1所示。2) The transfection system is shown in Table 1.
表1：Table 1:
3)用枪头以吹泡泡的形式反复吹打至少100次，可见液体中出现絮状沉淀，室温放置20分钟，以“Z”字形加入HEK-293FT细胞中。37℃培养箱培养6-8h后更换新的培养基，并加入丁酸钠使其终浓度为5mM。在培养过程中通过荧光显微镜对HEK-293FT细胞进行GFP表达效果观察，如图2所示，转染第三天HEK-293FT细胞绿色荧光强度高，产毒效果好，可见表达质粒可以成功转染HEK-293FT细胞。3) Use a pipette tip to blow bubbles repeatedly at least 100 times. A flocculent precipitate can be seen in the liquid. Leave it at room temperature for 20 minutes and add it to the HEK-293FT cells in a "Z" shape. After culturing for 6-8 hours in a 37°C incubator, replace the culture medium with new one, and add sodium butyrate to a final concentration of 5mM. During the culture process, the GFP expression effect of HEK-293FT cells was observed through a fluorescence microscope. As shown in Figure 2, on the third day of transfection, the green fluorescence intensity of HEK-293FT cells was high and the toxin production effect was good. It can be seen that the expression plasmid can be successfully transfected. HEK-293FT cells.
5)转染72h后收取培养上清作为病毒原液，并将收集的病毒原液用0.45μm滤膜过滤到50mL离心管中，4℃、12000×g离心15分钟去除细胞碎片，获得病毒粗提液。5) 72 hours after transfection, collect the culture supernatant as the virus stock solution, filter the collected virus stock solution with a 0.45 μm filter membrane into a 50 mL centrifuge tube, and centrifuge at 4°C and 12,000×g for 15 minutes to remove cell debris to obtain a crude virus extract. .
6)按全式金TransLv lentivirus precipitation solution(5X)说明书进行病毒液浓缩，获得HS3ST5慢病毒浓缩液。6) Concentrate the virus liquid according to the instructions of the full gold TransLv lentivirus precipitation solution (5X) to obtain the HS3ST5 lentivirus concentrated solution.
实施例3构建HS3ST5过表达SK-HEP-1内皮细胞系(SK-HEP-1-HS3ST5)Example 3 Construction of HS3ST5 overexpression SK-HEP-1 endothelial cell line (SK-HEP-1-HS3ST5)
该实施例利用实施例2获得的HS3ST5慢病毒浓缩液感染SK-HEP-1细胞(购自中国医学科学院细胞中心)，并检测目的质粒对SK-HEP-1细胞的感染效果，具体包括以下步骤：This example uses the HS3ST5 lentivirus concentrate obtained in Example 2 to infect SK-HEP-1 cells (purchased from the Cell Center of the Chinese Academy of Medical Sciences), and detects the infection effect of the target plasmid on SK-HEP-1 cells, which specifically includes the following steps :
1)所述病毒包装第三天，可接种目的细胞SK-HEP-1于6cm皿中，以含10％胎牛血清(fetal bovine serum,FBS)的MEM培养基进行贴壁培养，使第二天细胞密度达到30％-50％即可感染。1) On the third day after the virus is packaged, the target cells SK-HEP-1 can be inoculated into a 6cm dish and cultured adherently in MEM medium containing 10% fetal bovine serum (FBS). Infection can occur as soon as the cell density reaches 30%-50%.
2)将刚收集的或者从-80℃取出化冻的病毒浓缩液200μL加入SK-HEP-1培养基中，同时加入4μg/mL的polybrene以增加感染效率。2) Add 200 μL of freshly collected or thawed virus concentrate taken out from -80°C to the SK-HEP-1 culture medium, and add 4 μg/mL polybrene at the same time to increase the infection efficiency.
3)48小时后，对SK-HEP-1细胞的病毒感染情况进行荧光观察，期间可重复以上步骤2)增强感染效率。病毒感染SK-HEP-1细胞第十天荧光显微镜观察结果如图3所示，可见绿色荧光蛋白稳定表达，感染效果良好，证实SK-HEP-1细胞可被目的质粒pLV-HS3ST5-GFPSpark成功感染。将上述确认经慢病毒感染的SK-HEP-1细胞命名为pSK-HEP-1-HS3ST5细胞。3) After 48 hours, fluorescently observe the viral infection of SK-HEP-1 cells. During this period, the above steps 2) can be repeated to enhance the infection efficiency. The results of fluorescence microscopy observation on the tenth day after virus infection of SK-HEP-1 cells are shown in Figure 3. It can be seen that green fluorescent protein is stably expressed and the infection effect is good, confirming that SK-HEP-1 cells can be successfully infected by the target plasmid pLV-HS3ST5-GFPSpark. . The SK-HEP-1 cells confirmed to be infected with lentivirus were named pSK-HEP-1-HS3ST5 cells.
4)流式分选：待pSK-HEP-1-HS3ST5细胞在荧光显微镜下GFP阳性细胞达到80-90％时，用1mL MEM(2xFBS+2xPS+10μM Y-27)重悬细胞，准备1*107个SK-HEP-1和pSK-HEP-1-HS3ST5细胞，70μm滤器过滤细胞到无菌流式管中，放于冰上，准备上机。接收管内提前加入1mL MEM(2xFBS+2xPS+10μM Y-27)收集GFP高表达的pSK-HEP-1-HS3ST5细胞，对应HS3ST5高表达的pSK-HEP-1-HS3ST5细胞，命名为SK-HEP-1-HS3ST5细胞(于2022年01月21日保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO：45039)。4) Flow sorting: When the GFP-positive cells of pSK-HEP-1-HS3ST5 cells reach 80-90% under a fluorescence microscope, resuspend the cells in 1mL MEM (2xFBS+2xPS+10μM Y-27) and prepare 1* 10 7 SK-HEP-1 and pSK-HEP-1-HS3ST5 cells, filter the cells with a 70μm filter into a sterile flow tube, place on ice, and prepare for use. Add 1mL MEM (2xFBS+2xPS+10μM Y-27) to the receiving tube in advance to collect pSK-HEP-1-HS3ST5 cells with high GFP expression, which correspond to pSK-HEP-1-HS3ST5 cells with high HS3ST5 expression, named SK-HEP- 1-HS3ST5 cells (deposited in the General Microbiology Center of China Microbial Culture Collection Committee on January 21, 2022, with the deposit number of CGMCC NO: 45039).
5)HS3ST5过表达验证：5) HS3ST5 overexpression verification:
过表达验证主要从mRNA转录水平和蛋白翻译水平进行验证，分别通过qPCR和WB实现，具体步骤如下：Overexpression verification is mainly verified from the mRNA transcription level and protein translation level, which are achieved through qPCR and WB respectively. The specific steps are as follows:
a)qPCR:a)qPCR:
利用Trizol法提取RNA，按东洋纺逆转录试剂盒说明书逆转录为cDNA，然后进行qPCR检测：Extract RNA using the Trizol method, reverse transcribe into cDNA according to the instructions of the Toyobo reverse transcription kit, and then perform qPCR detection:
i.cDNA的稀释：向逆转录后的20μL cDNA中加入180μL ddH2O，混匀离心，10倍稀释后的cDNA用于实时定量PCR反应。i. Dilution of cDNA: Add 180 μL ddH 2 O to 20 μL cDNA after reverse transcription, mix and centrifuge, and use the 10-fold diluted cDNA for real-time quantitative PCR reaction.
ii.引物的准备：先将引物用ddH2O稀释至100μM，然后按10μL forward+10μLreverse+380μL ddH2O配成引物混合物400μL，混匀离心后用于实时定量PCR反应。ii. Primer preparation: First dilute the primer to 100 μM with ddH 2 O, then mix 10 μL forward+10 μL reverse+380 μL ddH 2 O to prepare 400 μL of primer mixture, mix and centrifuge for real-time quantitative PCR reaction.
iii.实时定量PCR反应体系按表2进行配制：iii. The real-time quantitative PCR reaction system is prepared according to Table 2:
表2：Table 2:
iv.qPCR反应程序如表3所示iv.qPCR reaction program is shown in Table 3
表3：table 3:
v.引物特异性检测：追加溶解曲线反应检测引物特异性，从退火温度开始每10sec上升0.5℃直到90℃，观察峰形图从而检测引物特异性。v. Primer specificity detection: Add a melting curve reaction to detect primer specificity. Starting from the annealing temperature, increase 0.5°C every 10 seconds until 90°C. Observe the peak shape to detect primer specificity.
vi.结果分析：由仪器配套专用软件进行结果分析，检测不同细胞中目的基因mRNA相对内参基因的表达情况。vi. Result analysis: The instrument is equipped with special software to perform result analysis and detect the expression of the target gene mRNA relative to the internal reference gene in different cells.
qPCR结果如图2所示，可见HS3ST5基因在SK-HEP-1-HS3ST5细胞中mRNA水平的表达较对照质粒组细胞SK-HEP-1明显增高，说明经过目的质粒感染改造的SK-HEP-1-HS3ST5细胞在转录水平成功过表达HS3ST5。The qPCR results are shown in Figure 2. It can be seen that the expression of HS3ST5 gene in SK-HEP-1-HS3ST5 cells at the mRNA level is significantly higher than that in SK-HEP-1 cells in the control plasmid group, indicating that SK-HEP-1 has been transformed by infection with the target plasmid. -HS3ST5 cells successfully overexpress HS3ST5 at the transcriptional level.
b)Wstern blot:b)Western blot:
1.蛋白裂解：裂解液配方如表4所示1. Protein lysis: The lysis solution formula is shown in Table 4
表4：Table 4:
吸尽6cm培养皿中的培养基，用预冷的l×PBS洗两遍，加入400μL细胞裂解液(含溴酚蓝)，搅动细胞收集于1.5mL的EP管中，98℃金属浴l0分钟使蛋白变性，4℃，12000rpm/min离心5min，去除细胞碎片，上清用于电泳或储存于-20℃备用。Aspirate the culture medium in the 6cm culture dish, wash it twice with pre-cooled l×PBS, add 400μL cell lysis solution (containing bromophenol blue), stir the cells and collect them in a 1.5mL EP tube, and place in a metal bath at 98°C for 10 minutes. Denature the protein, centrifuge at 12,000 rpm/min for 5 min at 4°C, remove cell debris, and use the supernatant for electrophoresis or store at -20°C for later use.
2.SDS聚丙烯酰胺凝胶的配方如表5所示：2. The formula of SDS polyacrylamide gel is shown in Table 5:
表5：table 5:
3.经过电泳、转膜、5％脱脂牛奶PBST溶液封闭，一抗4℃孵育过夜，洗涤后，以与一抗对应的辣根过氧化物酶标记的IgG抗体进行二抗孵育，最后在化学发光仪上显色。3. After electrophoresis, transfer, and blocking with 5% skim milk PBST solution, the primary antibody was incubated overnight at 4°C. After washing, the secondary antibody was incubated with the horseradish peroxidase-labeled IgG antibody corresponding to the primary antibody. Finally, in the chemical Color development on a luminometer.
WB检测结果如图3所示，可见HS3ST5在SK-HEP-1-HS3ST5细胞中蛋白水平的表达较SK-HEP-l细胞明显增高，说明经过目的质粒感染改造的SK-HEP-1-HS3ST5细胞在蛋白水平成功过表达HS3ST5。The WB detection results are shown in Figure 3. It can be seen that the protein level expression of HS3ST5 in SK-HEP-1-HS3ST5 cells is significantly higher than that in SK-HEP-l cells, indicating that the SK-HEP-1-HS3ST5 cells have been transformed by the target plasmid infection. Successfully overexpressed HS3ST5 at the protein level.
实施例4：HS3ST5基因过表达对内皮细胞系SK-HEP-1的影响Example 4: Effect of HS3ST5 gene overexpression on endothelial cell line SK-HEP-1
1.对细胞成管的影响：1. Effect on cell tube formation:
将SK-HEP-1和SK-HEP-1-HS3ST5细胞提前一天用基础培养基饥饿24h，BME(一种来源于Engelbreth-Holm-Swarm肿瘤的减生长因子基质成分)原液50μL预先包被96孔板，37℃成膜后，胰酶消化细胞，完全培养基重悬，以1*104/孔细胞数接种细胞于BME包被的96孔板中，从接种开始分别于2、8、24小时显微镜下观察成管情况并拍照记录。SK-HEP-1 and SK-HEP-1-HS3ST5 cells were starved for 24 hours with basal medium one day in advance, and 96 wells were pre-coated with 50 μL of BME (a growth factor-reducing matrix component derived from Engelbreth-Holm-Swarm tumors). plate, after film formation at 37°C, trypsinize the cells, resuspend them in complete culture medium, and inoculate the cells into a BME-coated 96-well plate at a cell number of 1*10 4 /well, starting at 2, 8, and 24 Observe the tube formation under a microscope and take photos for recording.
成管结果如图4所示，可见SK-HEP-1-HS3ST5细胞成管较SK-HEP-1细胞快，成管能力较强，说明HS3ST5过表达提高了SK-HEP-1细胞的成管能力。The tube formation results are shown in Figure 4. It can be seen that SK-HEP-1-HS3ST5 cells form tubes faster than SK-HEP-1 cells and have stronger tube formation ability, indicating that HS3ST5 overexpression improves the tube formation of SK-HEP-1 cells. ability.
2.对细胞粘附的影响：2. Effect on cell adhesion:
将SK-HEP-1和SK-HEP-1-HS3ST5细胞提前用含5％血清的完全培养基预处理2h，0.5％BME(一种来源于Engelbreth-Holm-Swarm肿瘤的减生长因子基质成分)原液50μl预先包被96孔板中一半的孔，37℃成膜后，胰酶消化细胞，完全培养基重悬，以3*104/孔细胞数接种细胞于96孔板中，分别于0，5，10，15，20，30，40，50，60，75，90，105，120min用PBS清洗未贴壁的细胞，用4％PFA固定15min，用DAPI孵育15min，上机检测。SK-HEP-1 and SK-HEP-1-HS3ST5 cells were pretreated for 2 h with complete medium containing 5% serum and 0.5% BME (a growth factor-reducing matrix component derived from Engelbreth-Holm-Swarm tumors). 50 μl of the original solution was pre-coated to half of the wells in the 96-well plate. After film formation at 37°C, the cells were digested with trypsin and resuspended in complete culture medium. The cells were seeded in the 96-well plate at 3*10 4 /well cells, and the cells were inoculated at 0 , 5, 10, 15, 20, 30, 40, 50, 60, 75, 90, 105, 120 minutes, wash the non-adherent cells with PBS, fix them with 4% PFA for 15 minutes, incubate with DAPI for 15 minutes, and detect on the machine.
细胞粘附结果如图5所示，可见SK-HEP-1-HS3ST5较SK-HEP-1细胞贴壁速率明显增加，说明HS3ST5过表达促进了SK-HEP-1细胞的粘附能力。The cell adhesion results are shown in Figure 5. It can be seen that SK-HEP-1-HS3ST5 has a significantly higher adhesion rate than SK-HEP-1 cells, indicating that HS3ST5 overexpression promotes the adhesion ability of SK-HEP-1 cells.
实施例5：HS3ST5过表达的内皮细胞对肝实质细胞的影响Example 5: Effects of HS3ST5 overexpressed endothelial cells on liver parenchymal cells
将SK-HEP-1和SK-HEP-1-HS3ST5细胞分别与正常肝类器官进行三维共培养，通过共聚焦成像和高内涵成像观察共培养后glutamine synthetase(GS)的表达情况，包括以下步骤：SK-HEP-1 and SK-HEP-1-HS3ST5 cells were co-cultured with normal liver organoids in three dimensions, and the expression of glutamine synthetase (GS) after co-culture was observed through confocal imaging and high-content imaging, including the following steps :
1)收集细类器官与1.5ml管中，4％多聚甲醛室温固定30min。1) Collect fine organoids in a 1.5ml tube and fix with 4% paraformaldehyde at room temperature for 30 minutes.
2)PBS洗三遍，0.25％Triton X-100细胞破膜15min。2) Wash three times with PBS and rupture the cell membrane with 0.25% Triton X-100 for 15 minutes.
3)PBS洗三遍，1％BSA封闭1小时。3) Wash three times with PBS and block with 1% BSA for 1 hour.
4)一抗4℃孵育过夜。4) Incubate the primary antibody at 4°C overnight.
5)PBS洗三遍，二抗室温避光孵育1小时。5) Wash three times with PBS and incubate with secondary antibody for 1 hour at room temperature in the dark.
6)PBS洗三遍，0.1-1μg/ml DAPI孵育细胞30min.6) Wash three times with PBS and incubate cells with 0.1-1μg/ml DAPI for 30 minutes.
7)PBS洗三遍，上机拍照成像。7) Wash three times with PBS and take pictures on the machine.
免疫荧光的检测结果如图6所示，可见SK-HEP-1-HS3ST5共培养的正常肝类器官中GS表达明显较对照组增高，说明过表达HS3ST5的内皮细胞SK-HEP-1-HS3ST5增强了肝实质细胞的肝功能。The immunofluorescence detection results are shown in Figure 6. It can be seen that the expression of GS in normal liver organoids co-cultured with SK-HEP-1-HS3ST5 is significantly higher than that in the control group, indicating that SK-HEP-1-HS3ST5 enhances endothelial cells overexpressing HS3ST5. The liver function of liver parenchymal cells.
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。In the description of this specification, reference to the terms "one embodiment," "some embodiments," "an example," "specific examples," or "some examples" or the like means that specific features are described in connection with the embodiment or example. , structures, materials or features are included in at least one embodiment or example of the invention. In this specification, the schematic expressions of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art may combine and combine different embodiments or examples and features of different embodiments or examples described in this specification unless they are inconsistent with each other.
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。Although the embodiments of the present invention have been shown and described above, it can be understood that the above-mentioned embodiments are illustrative and should not be construed as limitations of the present invention. Those of ordinary skill in the art can make modifications to the above-mentioned embodiments within the scope of the present invention. The embodiments are subject to changes, modifications, substitutions and variations.
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