WO2019233032A1 - 谷氨酰胺转氨酶介导的细胞膜表面修饰方法 - Google Patents

谷氨酰胺转氨酶介导的细胞膜表面修饰方法 Download PDF

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WO2019233032A1
WO2019233032A1 PCT/CN2018/114035 CN2018114035W WO2019233032A1 WO 2019233032 A1 WO2019233032 A1 WO 2019233032A1 CN 2018114035 W CN2018114035 W CN 2018114035W WO 2019233032 A1 WO2019233032 A1 WO 2019233032A1
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cell
modification
cells
modified
cell membrane
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杜亚楠
齐春晓
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清华大学
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0006—Modification of the membrane of cells, e.g. cell decoration
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells

Definitions

  • the invention relates to the field of biomedical engineering, in particular to a method for modifying the surface of a cell membrane mediated by glutamine aminotransferase.
  • Conditional stimulation mainly refers to stimulating cells through physical or chemical factors, so that cells respond to specific stimuli.
  • Such as hypoxic culture, drug intervention, growth factor induction, etc. help cells to upregulate the expression of certain targeting molecules to achieve the purpose of changing cell characteristics.
  • stimuli can cause a series of uncontrollable changes in the overall genetic and epigenetic aspects of the cell, so there are potential safety hazards.
  • the pre-stimulation of cell differentiation will greatly increase its immunogenicity. If used for cell therapy, it may lead to poor treatment after cell implantation.
  • the realization of special physical conditions generally requires special equipment to complete. Differences in accuracy result in errors, which will result in poor repeatability between different operators, which will limit widespread use.
  • chemical stimuli are mainly drugs and growth factors.
  • heterologous proteins will make cells immune. Primarily, if it enters the human body, it will cause serious side effects, such as interleukin 6 residues, which are the main causes of symptoms such as fever, vomiting, and endocrine dysfunction after cell implantation. And drug residues will cause certain toxic effects on the body.
  • Genetic modification is the introduction of certain foreign molecules with specific targeting molecules into the cell, so that the surface of the cell membrane can be recognized by specific molecules.
  • CAR-T chimeric antigen receptor T cells
  • the genetic modification has successfully modified the cell epitope, and the cell targeting has been improved to a certain extent, the problems faced by genetic modification are low transfection rate, high off-target rate, and genotoxicity. What is more important is that there are differences in the loading capacity, effectiveness, manipulability, and compatibility of newly introduced heterologous genes among different vectors. According to the existing experimental data, no matter whether it is viral or non-viral transfection, it can not only guarantee cell viability and differentiation ability, but also ensure efficient and stable transfection.
  • Cell membrane engineering is an emerging field of research. Its purpose is to modify the cell membrane by using biological, chemical, or physical methods to change the target molecule's surface characteristics.
  • One of its main purposes It is to achieve the targeted homing problem in cell therapy.
  • the most conventional methods for modifying the surface of cell membranes are mainly chemical methods, but the process of chemically modifying hair is very complicated. Multi-step operations are required to attach peptides to the cell membrane, which will affect the production of cell activity to varying degrees.
  • the disadvantage is the randomness of the substrates.
  • the physical method is currently based on the principle of similar compatibility between the cell membrane and the modifier, so that the modifier is chimeric on the cell membrane.
  • the treatment of the modified target is more demanding, and after modification, it exists on the cell membrane for a long time, and the half-life is longer. long.
  • the existing biological modification is mainly performed on a specific enzyme molecule of a specific cell, and is not universal.
  • the current cell modification methods mainly take the above measures, which can solve some of the basic research and clinical treatment needs, but there are still a series of problems, especially the pursuit of personalized precision medicine requires a universal
  • the modification method can make modification adjustments for different patients. Therefore, a cell modification method that is fast and simple in operation, easy to control in quality, non-damaging to cells, and non-toxic to metabolites needs to be urgently addressed.
  • the purpose of the present invention is to provide a universal method for modifying the surface of cell membranes to compensate for certain aspects or aspects of targeted therapy, adhesion, migration, proliferation, differentiation, and drug resistance of cells in basic research and cell therapy.
  • the function is insufficient.
  • the present invention claims a method for surface modification of a cell membrane mediated by glutamine aminotransferase.
  • the glutamine transaminase-mediated cell membrane surface modification method provided by the present invention is a cell membrane engineering method for changing the characteristics of the cell membrane surface.
  • the modification method is based on the glutamine aminotransferase-mediated polypeptide / protein polymerization reaction, which is a naturally occurring enzymatic reaction in the cell.
  • the target substance on the cell membrane surface is connected without changing the physiological characteristics of the modified cell to achieve Specific purpose.
  • Glutamine transaminase is a protein macromolecule widely present in many mammalian cells. It is a naturally occurring transaminase that plays an indispensable role in cell reproductive metabolism.
  • glutamine transaminase can catalyze the deamination of glutamine and lysine-containing polypeptides / proteins, thereby covalently linking two peptides / proteins, that is, peptides containing glutamine residues in vitro Linked to proteins that contain lysine residues in the body.
  • the specific reaction process is divided into two steps: First, in the presence of calcium ions, the enzymatic functional region Cys277-his335-Asp358 of glutamine transaminase is activated. The activated glutamine transaminase recognizes glutamine residues and catalyzes itself Lysine reacts with the recognized glutamine to form a dimerization intermediate.
  • the glutamine transaminase-mediated cell membrane surface modification method provided by the present invention may include the following steps: using glutamine transaminase to connect a modification to a substrate protein on the cell membrane surface of a target cell; the modification contains glutamine Glycine residues; the substrate protein contains lysine residues.
  • the aforementioned substrate protein may be a polypeptide molecule containing a glutamine residue, a protein molecule, or even a DNA, RNA, lipid, organic macromolecule, etc. linked to a polypeptide containing a glutamine residue.
  • glutamine transaminase is the glutamine transaminase expressed by the target cell or the target cell is induced to express (either glutamine transaminase may be self-introduced or exogenously introduced).
  • the modifier includes a modified region and a functional region.
  • the modified region contains a glutamine residue to meet the needs of being recognized and catalyzed by a glutamine transaminase on the cell membrane surface (that is, the modified region is used to connect with a lysine residue on the substrate protein ).
  • the functional region is a custom molecule, which is used to confer new properties on the target cell, and can be appropriately changed according to different purposes after modification.
  • the modified region may be any molecule that can be linked to an amino acid, such as a protein, a polypeptide sequence, a DNA sequence, a sugar chain, a lipid, an organic chemical molecule, and the like.
  • the modified region mainly has two functions, one is covalent connection with the cell membrane, and this part of the sequence is required to contain glutamine residues; the other is to control the overall characteristics of the modifier, such as length, molecular weight, electrical properties, hydrophilicity, etc. This part of the sequence can be adjusted according to the different purposes of the modification.
  • the target cell may be a cell capable of expressing or induced expression of glutamine transaminase.
  • the target cells are mesenchymal cells, endothelial cells or immune cells.
  • the mesenchymal cells may be mesenchymal stem cells; the endothelial cells may be vascular endothelial cells; and the immune cells may be white blood cells, B lymphocytes or T lymphocytes.
  • the target cell may be any one of the following: stem cells (adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, placental mesenchymal stem cells, umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, amniotic fluid) Mesenchymal stem cells, uterine blood mesenchymal stem cells, neural stem cells), immune cells (oligodendrocytes, dendritic cells, macrophages, neutrophils, T lymphocytes, B lymphocytes), somatic cells (osteochondral Protocells, chondrocytes, osteoblasts, vascular smooth muscle cells, vascular endothelial cells, epithelial cells, fibroblasts, stem cell differentiation-derived cells, liver cells, neural cells, cancer cells, etc. cell.
  • stem cells asdipose mesenchymal stem cells, bone marrow mesenchymal stem cells, placental mesenchymal stem cells, umbil
  • the above method may include the following steps: adding the modifier to a cell basal medium (ie, a serum-free cell culture medium), and the resulting mixture is referred to as a working solution; placing the target cell in the working solution, and incubating, Get the cells of interest.
  • a cell basal medium ie, a serum-free cell culture medium
  • the glutamine transaminase-mediated cell membrane surface modification method provided by the present invention is a "one-step cell membrane modification method" and may include the following steps:
  • the function of the functional area is mainly divided into enhancing certain aspects of the cell (such as the addition of targeting sequences in cell therapy) and reducing certain aspects of the cell (such as overcoming the interaction between cell surface molecules and the surrounding microenvironment).
  • the functional region may be selected from a protein, a polypeptide sequence, a DNA sequence, a sugar chain, a lipid, an organic chemical molecule, and the like.
  • the modified region mainly has two functions, one is covalent connection with the cell membrane, and this part of the sequence is required to contain glutamine residues; the other is to control the overall characteristics of the modifier, such as length, molecular weight, electrical properties, hydrophilicity, etc. This part of the sequence can be adjusted according to the different purposes of the modification.
  • the target cells were suspended in the modification of the working fluid, wherein the content of said target cell is 105 to 106 / mL (e.g., 10 5 / mL).
  • the method may further include the steps of: centrifuging the incubation solution (for example, 1000 g for 5 minutes) to remove the modified working solution; and then washing the modified target cells with HBSS; Centrifuge at 1000 g for 5 minutes) and remove the washing solution.
  • the HBSS rinse-centrifugation step can be repeated.
  • the quality is evaluated according to the purpose of the modification, and then subsequent experiments are performed.
  • the target cell is specifically a fat-derived human mesenchymal stem cell;
  • the modification is a FITC-labeled polypeptide GQLKHLEQQEG (sequence 1), wherein the modification region is GQLKH; the function The district is LEQQEG.
  • the present invention claims a modified cell prepared by the method described above.
  • Figure 1 is the basic principle of cell membrane modification: under the catalysis of glutamine transaminase on the cell membrane, the modification is covalently linked to the cell membrane through glutamine.
  • Figure 2 shows the surface of the cell membrane modified with different concentrations of the target (reaction time is 25 minutes).
  • iMSC refers to human mesenchymal stem cells whose glutamine transaminase is inhibited, that is, the cells no longer contain enzyme molecules that catalyze cell modification, and are induced by adding 0.5 ⁇ g / ML cystamine to the culture medium for 24 hours.
  • Figure 3 shows the modification of the target substance on the cell membrane surface at different times (the concentration of the modifier is 1 mM).
  • iMSC refers to human mesenchymal stem cells whose glutamine transaminase is inhibited, that is, the cells no longer contain enzyme molecules that catalyze cell modification, and are induced by adding 0.5 ⁇ g / ML cystamine to the culture medium for 24 hours.
  • Figure 4 shows the results of measuring the residual amount of the target substance on the cell membrane at different times after modification.
  • (a) is the result of measuring the residual amount of the target substance on the cell membrane at different time points after the modification;
  • (b) is the change law of the polypeptide residue on the cell over time by flow cytometry.
  • Figure 5 is the effect of modification on cell proliferation.
  • Figure 6 shows the effect of modification on cell function.
  • MSC refers to adipose-derived human mesenchymal stem cells
  • fMSC refers to functional polypeptide modified MSC
  • cMSC refers to control polypeptide modified MSC.
  • control group (Neg) involved in the above figures are all unmodified cells, that is, adipose-derived human mesenchymal stem cells.
  • Primer synthesis services are completed by China Biotech Engineering Co., Ltd. (Table 1).
  • the basic principle of the cell membrane modification of the present invention under the catalysis of glutamine transaminase on the cell membrane, the modified substance is covalently linked to the cell membrane through glutamine ( Figure 1).
  • the target cells and modifiers used in this example are as follows:
  • Target cells adipose-derived human mesenchymal stem cells.
  • Modifier FITC-labeled polypeptide GQLKHLEQQEG (sequence 1, ie, functional polypeptide), in which the modified region is GQLKH; the functional region is LEQQEG.
  • the control polypeptide is GNLRHLENNEG, in which the amino acid residues of the substrate of glutamine aminotransferase are replaced with N and R, so that the polypeptide no longer has the ability to be recognized by glutamine aminotransferase.
  • the control polypeptide is used to exclude the effect of the characteristics of the polypeptide on the modification effect .
  • Modification purpose to attach the modification to the surface of the cell membrane.
  • (1) cell modification FITC labeled polypeptide functions at a series of concentrations (see FIG. 2) was dissolved in a mesenchymal stem cell basal medium (pH 7.4-7.6), and digested at a final concentration of 105 cells / mL or after Mesenchymal stem cells react at 37 ° C for a corresponding time (see Figure 3). The reaction process ensures that the cells do not aggregate (can be achieved by shaking, vortexing, etc.). Remove the modified working solution by centrifugation (centrifugation at 1000 g for 5 minutes), wash with HBSS three times, remove the cleaning solution by centrifugation (centrifugation at 1000 g for 5 minutes), and resuspend the cells in HBSS to complete the cell modification.
  • the modified cells are directly subjected to flow cytometry to characterize the amount of peptides on the cells, as shown in Figure 2 (b) and Figure 3 (b); the total of 10,000 cells can also be read with a microplate reader.
  • the fluorescence value is then divided by the number of cells to obtain the average fluorescence intensity of each cell, as shown in Figure 2 (c) and Figure 3 (c).
  • the modified cells are fixed with tissue fixation solution, and the cell membrane and nucleus are stained according to the cell membrane dye Cell Plasma Membrane Staining Kit product manual. After staining, imaging is performed using a confocal microscope, as shown in Figure 2 (a) and Figure 3 (a ).
  • step (2) The modified cells are seeded back into the cell culture dish, and the cells are re-digested at 0h, 6h, 12h, 24h, and 48h after modification to obtain a cell suspension.
  • step (2) The same operation as in step (2) is performed for flow analysis. Analysis with a microplate reader, a quantitative analysis of the residue of the peptide on the cell membrane is obtained, as shown in (a) and (b) of FIG. 4.
  • the modified cells extract cellular RNA and perform reverse transcription to obtain cDNA, and then perform RT-PCR to characterize the expression of related genes such as dryness maintenance, growth factor secretion, and anti-inflammatory factor secretion function (target genes for detection And the primers used are shown in Table 1, GAPDH is an internal reference), as shown in Figure 6 (b).
  • results shown in (b) of FIG. 6 indicate that the modification does not affect the mesenchymal stem cell's stem maintenance, growth factor secretion, and anti-inflammatory factor secretion function.
  • the one-step modification method of the cell membrane surface of the present invention can effectively modify the modifier on the surface of the cell membrane, and can adjust the concentration and modification time of the modifier, and at the same time, can modify the modifier according to the modification purpose.
  • this modification method has no significant effect on the cell's proliferation ability, activity, and gene expression, which is conducive to the later use of the cell, and provides an improvement or remedy for cell function deficiency for regenerative medicine and basic cell research Technology platform.
  • the "one-step cell membrane modification method” provided by the present invention has the following significant advantages:
  • the method has short steps, convenient operation and high repeatability.
  • the operation cycle is short, which is convenient for continuous progress with other experiments or operations without affecting the state of cell survival.
  • the required reagents and instruments are conventional cell culture equipment, and no special equipment is required.
  • the modified target can be adjusted and planned in a wide range according to the purpose, which can meet various experimental purposes and application requirements (that is, the universality of the modified target, such as protein, polypeptide sequences, DNA sequences, sugar chains, etc. Lipids, organic chemical molecules, and any molecule that can be linked to an amino acid).
  • Glutamine transaminase is a protein molecule that is widely present in a variety of cells in humans and animals. This method can modify a variety of cell types. As long as the cell expresses or induces the expression of glutamine transaminase, it is applicable to this one-step cell membrane. Modification (that is, to modify the diversity of cells, such as mesenchymal stem cells, vascular endothelial cells, leukocytes, B lymphocytes, T lymphocytes and any other cells that express or induce the expression of glutamine transaminase).
  • Modification that is, to modify the diversity of cells, such as mesenchymal stem cells, vascular endothelial cells, leukocytes, B lymphocytes, T lymphocytes and any other cells that express or induce the expression of glutamine transaminase).
  • the method uses a naturally occurring enzymatic reaction to connect the target object without causing damage to the characteristics of the cell itself.
  • this modification method can precisely control the modification time, concentration and other parameters, so that the functional fragments form a proper coverage on the surface of the cell membrane, so as to fully reduce the inefficiency of the modifiers while ensuring the function.
  • the present invention has developed a precise and controllable method for modifying the surface of cell membranes based on natural enzymatic reactions.
  • the platform is guided by the improvement of cell function, and does not change the physiological characteristics of cells. It is based on cell basic research and cell therapy.
  • the exit is based on the various variability of functional fragments, and is characterized by simple operation, fast response and high repeatability, providing a new idea for regenerative medicine such as drug development, cell therapy and basic research.

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Abstract

提供了一种谷氨酰胺转氨酶介导的细胞膜表面修饰方法。所述的谷氨酰胺转氨酶介导的细胞膜表面修饰方法，包括如下步骤：利用谷氨酰胺转氨酶将修饰物连接到靶细胞的细胞膜表面的底物蛋白上；所述修饰物上含有谷氨酰胺残基；所述底物蛋白上含有赖氨酸残基。

Description

谷氨酰胺转氨酶介导的细胞膜表面修饰方法 技术领域
本发明涉及生物医学工程领域，具体涉及一种谷氨酰胺转氨酶介导的细胞膜表面修饰方法。
背景技术
目前已报道的细胞修饰有：1、条件刺激；2、基因改造；3、细胞膜修饰三种方法。
条件刺激主要是指通过物理或者化学因素对细胞实施刺激，使细胞对特定刺激做出反馈。如低氧培养、药物干预、生长因子诱导等，有利于细胞上调某些靶向分子的表达，以实现改变细胞特性的目的。但是这类刺激会导致细胞在整体基因和表观遗传方面都会出现一系列不可控的变化，因此存在安全隐患。例如预刺激细胞分化的同时会使其免疫原性大幅度提高，如果用于细胞治疗可能导致细胞植入后治疗效果不佳；其次，特殊物理条件的实现一般都需要特殊设备才能完成，设备的不同导致精确性存在误差，因此会造成不同的操作者间可重复性差，对广泛应用造成限制；第三，化学刺激物，主要以药物、生长因子为主，异种蛋白的引入会使细胞获得免疫原性，如果进入人体会引起严重的副反应，如白介素6残留是造成细胞植入后发热，呕吐，内分泌功能紊乱等症状的主要原因。而药物残留更是会对机体造成一定的毒害作用。
基因改造是通过给细胞导入某些具有特定靶向性的分子的外源基因从而使细胞膜表面获得被特异性分子识别的能力。如目前临床在使用的通过基因编辑方法获得的嵌合抗原受体T细胞(chimeric antigen receptor t cells，CAR-T)。虽然基因修饰成功对细胞表位进行了改造，而且对细胞的靶向性有了一定程度的提高，但是基因改造所面临的问题是转染率低，脱靶率高，及基因毒性等。而更为重要的是不同载体的装载能力，有效性，可操控性，新导入异种基因的相融性等都存在差异。就现有的实验数据表明，无论是病毒性或非病毒性转染，都不能既保证细胞活性和分化能力，又保证比较高效稳定的转染。
细胞膜工程是一种新兴的研究领域，其目的在于利用生物的、化学的、 或物理的方法对细胞膜进行目的分子的修饰，使被修饰细胞的细胞膜表面特性发生变化，其最主要的目的之一就是为了实现细胞治疗中的靶向归巢问题。现有的最常规的细胞膜表面修饰方法主要是化学法，但是化学修饰发过程非常复杂，需要多步操作才能将多肽等连接在细胞膜上，会不同程度的影响细胞活性产生，而且其最明显的缺点就是连接底物的随机性。物理方法目前主要是依据细胞膜与修饰物的相似相容原理完成，使修饰物嵌合在细胞膜上，此种方法中修饰目的物的处理要求较高，而且修饰之后长期存在于细胞膜上，半衰期较长。而现有的生物法修饰主要针对某种特定细胞的特定酶分子进行，不具有普适性。
总体而言，目前细胞修饰的方法主要采取以上措施，能够解决一部分基础研究和临床治疗需求，但是仍然存在一系列的问题，特别是对于个体化精准医疗的追求更是要求一种普适性的修饰方法能够针对不同患者做出修饰调整。因此一种操作过程快速简单，质量容易控制，对细胞无损伤，代谢产物无毒害的细胞修饰方法亟需解决。
发明公开
本发明的目的是提供一种普适性的细胞膜表面修饰方法，以弥补基础研究和细胞治疗中细胞在靶向治疗、粘附、迁移、增殖、分化、抗药性等某一方面或某几方面的功能不足。
第一方面，本发明要求保护一种谷氨酰胺转氨酶介导的细胞膜表面修饰方法。
本发明所提供的谷氨酰胺转氨酶介导的细胞膜表面修饰方法，系一种细胞膜工程手段，用于改变细胞膜表面特性。该修饰方法以细胞天然存在的酶促反应——谷氨酰胺转氨酶介导的多肽/蛋白聚合反应为依据，在不改变被修饰细胞生理特性的基础上，进行细胞膜表面目的物的连接，以实现特定目的。谷氨酰胺转氨酶是一种广泛存在于多种哺乳动物细胞的蛋白大分子，是一种天然存在的转氨酶，在细胞的生殖代谢中起到不可或缺的作用。其催化原理是谷氨酰胺转氨酶能够催化含有谷氨酰胺和赖氨酸的多肽/蛋白发生脱氨基作用，从而使两段多肽/蛋白发生共价连接，即体外的含谷氨酰胺残基的多肽链接在了体内含有赖氨酸残基的蛋白上。其具体反应过程分为两步：一、在钙离子存在下，谷氨酰胺转氨酶酶促功能区 Cys277-his335-Asp358处于激活状态，激活的谷氨酰胺转氨酶识别谷氨酰胺残基，并催化自身赖氨酸与识别的谷氨酰胺发生脱氨基反应而形成二聚中间体；二、中间体在遇到其他含有赖氨酸残基的底物时，发生转氨基作用，将谷氨酰胺转移至赖氨酸底物上，并游离出谷氨酰胺转氨酶，从而完成多肽/蛋白分子间的化学连接。
本发明所提供的谷氨酰胺转氨酶介导的细胞膜表面修饰方法，可包括如下步骤：利用谷氨酰胺转氨酶将修饰物连接到靶细胞的细胞膜表面的底物蛋白上；所述修饰物上含有谷氨酰胺残基；所述底物蛋白上含有赖氨酸残基。
上述底物蛋白可以为含有谷氨酰胺残基的多肽分子，蛋白分子，甚至是含有谷氨酰胺残基的多肽连接的DNA，RNA，脂质，有机大分子等。
其中，谷氨酰胺转氨酶为靶细胞表达或靶细胞经过诱导表达的谷氨酰胺转氨酶(谷氨酰胺转氨酶为自身的或外源导入的均可以)。
进一步地，所述修饰物包含修饰区和功能区。所述修饰区上含有谷氨酰胺残基，以满足被细胞膜表面上谷氨酰胺转氨酶识别并催化聚合反应的需求(即所述修饰区用于与所述底物蛋白上的赖氨酸残基连接)。所述功能区为自定义分子，用于赋予所述靶细胞新的性能，可根据修饰后不同的目的进行合适的改变。所述修饰区可以是蛋白、多肽序列，DNA序列，糖链，脂质，有机化学分子等任何可以与氨基酸相连的分子。
所述修饰区主要有两方面功能，一是与细胞膜发生共价连接，这一部分序列要求含有谷氨酰胺残基；二是控制修饰物整体特性，如长度，分子量大小，电性，亲疏水性等，这一部分序列可以根据修饰的不同目的进行调整。
进一步地，所述靶细胞可为能够表达或者经过诱导表达谷氨酰胺转氨酶的细胞。
如，所述靶细胞为***、内皮细胞或免疫细胞。
更进一步地，所述***可为间充质干细胞；所述内皮细胞可为血管内皮细胞；所述免疫细胞可为白细胞、B淋巴细胞或T淋巴细胞。
更加具体的，所述靶细胞可为以下任意一种：干细胞(脂肪间充质干细胞，骨髓间充质干细胞，胎盘间充质干细胞，脐带间充质干细胞，脐带 血间充质干细胞，羊水间充质干细胞，宫血间充质干细胞，神经干细胞)，，免疫细胞(少突细胞，树突细胞，巨噬细胞，中性粒细胞，T淋巴细胞，B淋巴细胞)，体细胞(骨软骨原细胞，软骨细胞，成骨细胞，血管平滑肌细胞，血管内皮细胞，上皮细胞，纤维细胞，干细胞分化来源细胞，肝脏细胞，神经细胞，)，癌细胞等以及经过诱导可表达谷氨酰胺转氨酶的细胞。
上述方法可以包括如下步骤：将修饰物加入细胞基础培养基(即不含血清的细胞培养基)中，得到的混合物记为工作液；将所述靶细胞置于所述工作液中，孵育，得到目的细胞。
本领域技术人员可以根据修饰物不同，选择合适的修饰时间和修饰浓度。
具体而言，本发明所提供的谷氨酰胺转氨酶介导的细胞膜表面修饰方法，为“一步细胞膜修饰法”，可包括如下步骤：
(a1)根据修饰目的确定所述修饰物的所述功能区和所述修饰区，并合成所述修饰物。
其中，所述功能区的作用主要分为增强细胞某方面功能(如细胞治疗中靶向序列的加入)和降低细胞某方面功能(如克服细胞表面分子与周围微环境的相互作用)。根据修饰后的目的，所述功能区可以选择蛋白、多肽序列，DNA序列，糖链，脂质，有机化学分子等任何一种。
所述修饰区主要有两方面功能，一是与细胞膜发生共价连接，这一部分序列要求含有谷氨酰胺残基；二是控制修饰物整体特性，如长度，分子量大小，电性，亲疏水性等，这一部分序列可以根据修饰的不同目的进行调整。
(a2)用基础细胞培养基对所述修饰物进行稀释，使所述修饰物在稀释液中的浓度为0.1mM-100mM(如0.1mM到10mM，进一步如1-5mM)，并调整pH到7.4-7.6，得到修饰工作液。
(a3)将所述靶细胞悬浮于所述修饰工作液中，37℃条件下，孵育5-40分钟(如5-35分钟，进一步如15-25分钟)。在此期间保证细胞悬浮状态，可以通过摇床，涡旋仪等实现。
进一步地，步骤(a3)中，将所述靶细胞悬浮于所述修饰工作液中，其中所述靶细胞的含量为10 5-10 6个/mL(如10 5个/mL)。
另外，在步骤(a3)之后，还可包括如下步骤：将孵育液离心(如1000g离心5分钟)，去掉所述修饰工作液；然后用HBSS冲洗修饰后的所述靶细胞；再离心(如1000g离心5分钟)，去掉清洗液。其中HBSS冲洗-离心的步骤可以重复进行。
在得到修饰完成的细胞后，根据修饰目的进行质量评价，然后进行后续实验。
在本发明的具体实施方式中，所述靶细胞具体为脂肪来源的人类间充质干细胞；所述修饰物为FITC标记的多肽GQLKHLEQQEG(序列1)，其中所述修饰区为GQLKH；所述功能区为LEQQEG。
第二方面，本发明要求保护利用前文所述方法制备得到的修饰后细胞。
第三方面，本发明要求保护如下任一应用：
(A1)前文所述方法在改变细胞膜表面特性中的应用；
(A2)前文所述方法在调控(如提高)细胞靶向性、细胞粘附性、细胞迁移能力、细胞增殖能力、细胞分化能力和/或细胞抗药性等方面中的应用；
(A3)前文所述修饰后细胞在制备用于细胞治疗的药物中的应用。
附图说明
图1为细胞膜修饰的基本原理：在细胞膜上谷氨酰胺转氨酶的催化作用下，修饰物通过谷氨酰胺与细胞膜发生共价连接。
图2为不同浓度目的物修饰在细胞膜表面(反应时间为25分钟)。(a)为不同多肽浓度修饰后的荧光图片；(b)为不同浓度修饰后细胞进行流式分析；(c)为对荧光强度进行定量(其中，“X”即表示mM)。其中，iMSC是指谷氨酰胺转氨酶被抑制的人间充质干细胞，即细胞中不再含有催化细胞修饰的酶分子，通过在培养基中加入0.5μg/ML胱胺诱导24小时实现。
图3为不同时间目的物修饰在细胞膜表面(修饰物浓度为1mM)。(a)为不同修饰时间修饰后的荧光图片；(b)为不同时间修饰后细胞进行流式分析；(c)为对荧光强度进行定量。其中，iMSC是指谷氨酰胺转氨酶被抑制的人间充质干细胞，即细胞中不再含有催化细胞修饰的酶分子，通过在培养基中加入0.5μg/ML胱胺诱导24小时实现。
图4为修饰后不同时间目的物在细胞膜上的残留量测定结果。(a)为修饰后不同时间点目的物在细胞膜上的残留量测定结果；(b)为流式分析多肽在细胞上残留情况随时间的变化规律。
图5为修饰对细胞增殖的影响。(a)为不同多肽修饰对细胞增殖能力的影响(修饰物浓度为1mM，修饰时间为25分钟)；(b)为不同时间，浓度为1mM的功能多肽修饰对细胞活性的影响。
图6为修饰对细胞功能的影响。(a)为修饰对细胞活性的影响；其中，MSC即是脂肪来源的人类间充质干细胞，fMSC是指功能多肽修饰的MSC，cMSC是指对照多肽修饰的MSC。(b)为修饰对细胞基因的影响。
以上各图中的所涉及的对照组(Neg)均为未做任何修饰的细胞，即脂肪来源的人类间充质干细胞。
实施发明的最佳方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中实验所用试剂和材料：
(1)培养基及相关试剂：磷酸盐缓冲液(PBS)，胎牛血清(FBS)，青霉素/链霉素，HBSS，0.25％胰蛋白酶均为加拿大Wisent公司产品；人间充质干细胞培养基为中国武汉维诺赛生物技术有限公司产品；二甲基亚砜(Dimethyl sulfoxide，DMSO)为美国Biomol公司产品；谷氨酰胺转氨酶抑制剂cystamine(胱胺)为sigma公司产品。
(2)生物实验试剂：TriZol，Hoechst 33342，过硫酸铵为美国Sigma公司产品；Calcein-AM和Propidiumiodide(PI)为日本Wako公司产品，细胞膜染料Cell Plasma Membrane Staining Kit为abcom公司产品，4％组织细胞固定液为北京索莱宝科技有限公司产品。
(3)分子实验试剂：First-strand cDNA synthesis kit，SYBR Green II，RNase抑制剂、Oligo(dT)和10mM dNTP均为中国Takara公司产品；提取RNA所用的无霉无菌离心管和枪头为Life Technologies公司产品。
(4)引物合成与测序服务：由中国生工生物工程有限公司完成引物合成服务(表1)。
(5)多肽合成：由上海淘普生物科技有限公司完成。
表1 下述实施例中所用到引物序列
Figure PCTCN2018114035-appb-000001
实施例1、谷氨酰胺转氨酶介导的细胞膜表面修饰
本发明细胞膜修饰的基本原理：在细胞膜上谷氨酰胺转氨酶的催化作用下，修饰物通过谷氨酰胺与细胞膜发生共价连接(图1)。本实施例中所采用的靶细胞和修饰物具体如下：
靶细胞：脂肪来源的人类间充质干细胞。
修饰物：FITC标记的多肽GQLKHLEQQEG(序列1，即功能多肽)，其中修饰区为GQLKH；功能区为LEQQEG。
对照多肽为GNLRHLENNEG，其中谷氨酰胺转氨酶的底物氨基酸残基被替换为N和R，使多肽不再具备被谷氨酰胺转氨酶识别的能力，对照多肽用以排除多肽自身特性对修饰效果的影响。
修饰目的：将修饰物连接在细胞膜表面。
(1)细胞修饰：FITC标记的功能多肽以系列浓度(参见图2)溶解在间充质干细胞基础培养基中(pH 7.4-7.6)，与以细胞终浓度10 5个/mL的消化后的间充质干细胞在37℃条件下反应相应时间(参见图3)，反应过程保证细胞不会聚集(可以通过摇床，涡旋仪等实现)。离心(1000g离心5分钟)去掉修饰工作液，用HBSS清洗3次，离心(1000g离心5分钟)去掉清洗液，将细胞重悬于HBSS中，完成细胞修饰。
(2)修饰完成的细胞直接进行流式分析，用于表征细胞上的多肽量，如图2中(b)和图3中(b)；也可以用酶标仪读取10000个细胞的总荧光值，然后除以细胞数目得到平均每个细胞的荧光强度，如图2中(c)和图3中(c)。
(3)修饰完成的细胞用组织固定液固定，按照细胞膜染料Cell Plasma Membrane Staining Kit产品说明书染细胞膜和细胞核，染色后用共聚焦显微镜进行成像，如图2中(a)和图3中(a)。
图2和图3所示结果表明修饰物在细胞膜上的修饰量呈现时间和浓度依赖性，即修饰时间越长，或者修饰物浓度越高越有利于修饰物与细胞膜表面的连接，但是当浓度过高(10mM)或时间过长(35分钟)时修饰物被内吞进细胞内部，反而造成细胞膜上残存量不足，因此根据修饰物不同，选择合适的修饰时间和修饰浓度。
(4)修饰完成的细胞重新种回细胞培养皿中，分别在修饰后的0h，6h，12h，24h，48h重新消化细胞得到细胞悬液，同步骤(2)操作相同，分别进行流式分析和酶标仪分析，得到多肽在细胞膜上的残留定量分析，如图4中(a)和(b)。
图4所示结果表明修饰物在6小时之内迅速降解，不影响细胞长期功 能和表型的表现。
(5)细胞增殖能力的测定：分别将不同浓度的功能多肽和对照多肽修饰后的间充质干细胞重新种回细胞培养皿中，分别在修饰后4h(0d)、1d、2d进行细胞计数，确定不同多肽修饰对细胞增殖的影响，如图5中(a)；以及不同时间功能多肽修饰对细胞增殖能力的影响，如图5中(b)。
图5所示结果表明修饰物的种类和修饰时间对细胞增殖能力均没有显著影响，说明该修饰方法不会对细胞增殖造成不良影响。
(6)修饰后的细胞重新种回细胞培养皿中6h后，按照产品说明书进行细胞活/死染色，后计算活细胞与死细胞数目比值，确定细胞修饰对细胞活性的影响，如图6中(a)。
图6中(a)所示结果表明修饰物修饰后不会对细胞活性造成影响。
(7)修饰后的细胞提取细胞RNA后进行逆转录得到cDNA，然后进行RT-PCR对干性维持，生长因子分泌，抗炎因子分泌功能等相关基因表达量进行表征(用于检测的目标基因以及所用引物如表1所示，GAPDH为内参)，如图6中(b)。
图6中(b)所示结果表明修饰物修饰后不会对间充质干细胞的干性维持，生长因子分泌，抗炎因子分泌功能造成影响。
综上所述，以上结果表明本发明的一步细胞膜表面修饰方法能够有效的将修饰物修饰在细胞膜表面，并且能够进行修饰物浓度和修饰时间的调整，同时能过根据修饰目的的不同对修饰物进行“量身定制”，该修饰方法对细胞的增殖能力、活性、基因表达均不产生显著性影响，有利于细胞的后期利用，为再生医学和细胞基础研究提供一种改善或弥补细胞功能不足的技术平台。
以上只是本发明的一个具体实施方式，但本发明并不局限于上述具体实施例，任何在本发明思想下的任何变形、替换方式均属于本发明的保护范围。
工业应用
本发明所提供的“一步细胞膜修饰法”具有如下显著优势：
1、方法步骤简短，操作方便，可重复性高。
2、操作周期短，便于与其他实验或操作连续进展而不影响细胞存活状 态。
3、所需试剂和仪器设备都是常规细胞培养设备，不需特殊设备。
4、修饰的目的物可根据目的不同进行大范围调整与规划，能够满足各种不同的实验目的和应用需求(即修饰目的物的普适性，例如蛋白、多肽序列，DNA序列，糖链，脂质，有机化学分子等任何可以与氨基酸相连的分子)。
5、谷氨酰胺转氨酶是一种广泛存在于人和动物体多种细胞的蛋白质分子，该方法可以修饰的细胞类型繁多，只要细胞表达或者经过诱导表达谷氨酰胺转氨酶即适用于此一步法细胞膜修饰(即修饰细胞的多样性，例如以间充质干细胞、血管内皮细胞、白细胞、B淋巴细胞、T淋巴细胞等任何表达或者诱导表达谷氨酰胺转氨酶的细胞)。
6、该方法利用天然存在的酶促反应进行目的物的连接，不会对细胞本身特性造成伤害。
7、对修饰细胞没有特殊要求，可以针对特定病患提取其自身细胞进行修饰后回输，能够满足精准医疗的需求。
8、同时这种修饰方法可以精确控制修饰时间、浓度等参数，使功能片段在细胞膜表面形成合适的覆盖率，从而在保证功能的前提下充分降低修饰物的无效率。
因此，本发明开发了一种依据天然酶促反应的精确可控的细胞膜表面修饰方法，该平台以细胞功能改善为导向，以不改变细胞自身生理特性为前提，以细胞基础研究和细胞治疗为出口，以功能片段的多样可变性为媒介，以操作简单、反应快速和可重复性高为特点，为药物研发、细胞治疗和基础研究等再生医学提供一种新思路。

Claims (14)

  1. 一种谷氨酰胺转氨酶介导的细胞膜表面修饰方法，包括如下步骤：利用谷氨酰胺转氨酶将修饰物连接到靶细胞的细胞膜表面的底物蛋白上；所述修饰物上含有谷氨酰胺残基；所述底物蛋白上含有赖氨酸残基。
  2. 根据权利要求1所述的方法，其特征在于：所述修饰物包含修饰区和功能区；所述修饰区上含有谷氨酰胺残基。
  3. 根据权利要求1或2所述的方法，其特征在于：所述功能区为蛋白、多肽、DNA、糖链、脂质或有机化学分子。
  4. 根据权利要求1-3中任一所述的方法，其特征在于：所述靶细胞为能够表达或经过诱导表达谷氨酰胺转氨酶的细胞。
  5. 根据权利要求4所述的方法，其特征在于：所述靶细胞为***、内皮细胞或免疫细胞。
  6. 根据权利要求1-5中任一所述的方法，其特征在于：
    所述方法包括如下步骤：将修饰物加入基础细胞培养基中，得到的混合物记为工作液，将所述靶细胞置于所述工作液中，孵育，得到目的细胞。
  7. 根据权利要求1-6中任一所述的方法，其特征在于：
    所述方法包括如下步骤：
    (a1)根据修饰目的确定所述修饰物的所述功能区和所述修饰区，并合成所述修饰物；
    (a2)用基础细胞培养基对所述修饰物进行稀释，使所述修饰物在稀释液中的浓度为0.1mM-100mM，并调整pH到7.4-7.6，得到修饰工作液；
    (a3)将所述靶细胞悬浮于所述修饰工作液中，37℃条件下，孵育5-40分钟。
  8. 根据权利要求7所述的方法，其特征在于：步骤(a3)中，将所述靶细胞悬浮于所述修饰工作液中，其中所述靶细胞的含量为10 5-10 6个/mL。
  9. 根据权利要求7或8所述的方法，其特征在于：在步骤(a3)之后，还包括如下步骤：将孵育液离心，去掉所述修饰工作液；然后用HBSS冲洗修饰后的所述靶细胞；再离心，去掉清洗液。
  10. 利用权利要求1-9所述方法制备得到的修饰后细胞。
  11. 权利要求1-9中任一所述方法在改变细胞膜表面特性中的应用。
  12. 权利要求1-9中任一所述方法在调控细胞靶向性、细胞粘附性、细胞迁移能力、细胞增殖能力、细胞分化能力和/或细胞抗药性中的应用；
  13. 权利要求1-9中任一所述方法在用于细胞治疗中的应用。
  14. 权利要求10所述修饰后细胞在制备用于细胞治疗的药物中的应用。
PCT/CN2018/114035 2018-06-04 2018-11-06 谷氨酰胺转氨酶介导的细胞膜表面修饰方法 WO2019233032A1 (zh)

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