CN108815189A - A kind of preparation containing self endometrium mescenchymal stem cell excretion body - Google Patents
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Abstract
本发明提供了一种含有自体子宫内膜间充质干细胞外泌体的制剂，涉及生物干细胞技术领域，按照体积份数比，包括5份间充质干细胞外泌体以及95份基质溶液，所述基质溶液包括95‑99份复方电解质注射液以及1‑5份药用人血白蛋白，所述间充质干细胞外泌体通过自体子宫内膜间充质干细胞饥饿培养所得，饥饿培养时间为12‑24小时，所述自体子宫内膜间充质干细胞提取自人子宫内膜的间充质干细胞，按照正常培养的方法进行培养得到，培养时间为48‑72小时，本发明实现了为治疗子宫内膜损伤等疾病提供了安全、有效、使用方便的治疗药物。
The invention provides a preparation containing exosomes of autologous endometrial mesenchymal stem cells, which relates to the technical field of biological stem cells, and comprises 5 parts of mesenchymal stem cell exosomes and 95 parts of matrix solution according to the ratio of parts by volume. The matrix solution includes 95-99 parts of compound electrolyte injection and 1-5 parts of medicinal human serum albumin, and the mesenchymal stem cell exosomes are obtained by starvation culture of autologous endometrial mesenchymal stem cells, and the starvation culture time is 12-24 hours, the autologous endometrial mesenchymal stem cells are extracted from human endometrial mesenchymal stem cells, cultured according to the normal culture method, the culture time is 48-72 hours, the present invention realizes the Diseases such as endometrial injury provide safe, effective, and easy-to-use therapeutic drugs.
Description
技术领域technical field
本发明涉及生物干细胞技术领域，尤其涉及一种含有自体子宫内膜间充质干细胞外泌体的制剂。The invention relates to the technical field of biological stem cells, in particular to a preparation containing autologous endometrial mesenchymal stem cell exosomes.
背景技术Background technique
子宫对于女性的一生至关重要，但是一旦子宫内膜损伤，则会造成一系列后果，现有的子宫内膜修复难度大，治疗时间较长，给很多女性带来了极大的困扰。The uterus is very important to a woman's life, but once the endometrium is damaged, it will cause a series of consequences. The existing endometrium is difficult to repair and the treatment time is long, which has brought great troubles to many women.
间充质干细胞(Mesenchymal Stem Cells,MSC)间充质细胞属于多能干细胞，具有多向分化、造血支持和促进干细胞植入、免疫调控等多种功能，主要存在于子宫内膜、脂肪和骨髓中。Mesenchymal stem cells (Mesenchymal Stem Cells, MSC) are pluripotent stem cells, which have multiple functions such as multidirectional differentiation, hematopoietic support, promotion of stem cell implantation, and immune regulation. They mainly exist in the endometrium, fat, and bone marrow. middle.
因此如何使得间充质干细胞能够用于治疗子宫内膜损伤成为了本领域技术人员亟待解决的问题。Therefore, how to use mesenchymal stem cells to treat endometrial injury has become an urgent problem to be solved by those skilled in the art.
发明内容Contents of the invention
本发明所要解决的技术问题是克服现有技术中存在的不足，提供一种含有自体子宫内膜间充质干细胞外泌体的制剂，该注射制剂利用干细胞分泌的外泌体为治疗子宫内膜损伤等疾病提供了安全、有效、使用方便的治疗药物。The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art and provide a preparation containing exosomes from autologous endometrial mesenchymal stem cells. The injection preparation utilizes exosomes secreted by stem cells to treat endometrial It provides safe, effective and easy-to-use therapeutic drugs for diseases such as injuries.
如图所示，本发明是通过以下技术方案予以实现：一种含有自体子宫内膜间充质干细胞外泌体的制剂，按照体积份数比，包括5份间充质干细胞外泌体以及95份基质溶液，按照体积份数比，基质溶液包括95-99份复方电解质注射液以及1-5份药用人血白蛋白，间充质干细胞外泌体通过自体子宫内膜间充质干细胞饥饿培养所得，饥饿培养时间为12-24小时，自体子宫内膜间充质干细胞提取自人子宫内膜的间充质干细胞，按照正常培养的方法进行培养得到，培养时间为48-72小时。As shown in the figure, the present invention is realized through the following technical scheme: a preparation containing autologous endometrial mesenchymal stem cell exosomes, including 5 parts of mesenchymal stem cell exosomes and 95 parts by volume. Parts of matrix solution, according to the ratio of parts by volume, the matrix solution includes 95-99 parts of compound electrolyte injection and 1-5 parts of medicinal human serum albumin, mesenchymal stem cell exosomes are starved by autologous endometrial mesenchymal stem cells For the cultured product, the starvation culture time is 12-24 hours, and the autologous endometrial mesenchymal stem cells are extracted from human endometrial mesenchymal stem cells, which are obtained by culturing according to the normal culture method, and the culture time is 48-72 hours.
根据上述技术方案，优选地，基质溶液的pH值为6.0-8.0。According to the above technical solution, preferably, the pH value of the matrix solution is 6.0-8.0.
根据上述技术方案，优选地，复方电解质注射液为复方电解质葡萄糖MG3注射液。According to the above technical solution, preferably, the compound electrolyte injection is the compound electrolyte glucose MG3 injection.
根据上述技术方案，优选地，药用人血白蛋白为奥地利百特公司所生产。According to the above technical solution, preferably, the pharmaceutical human albumin is produced by Austria Baxter Company.
根据上述技术方案，优选地，其用于治疗子宫内膜损伤等用途。According to the above technical solution, preferably, it is used for the treatment of endometrial injury and other purposes.
一种含有自体子宫内膜间充质干细胞外泌体的制剂的制备方法，包括以下步骤：A method for preparing a preparation containing autologous endometrial mesenchymal stem cell exosomes, comprising the following steps:
(1)自体子宫内膜间充质干细胞的培养：从细胞库中选取自人子宫内膜的间充质干细胞进行复苏，用无酚红DMEM/DF12和体积比浓度为10％胎牛血清做完全培养基，放入T75细胞培养瓶在37℃、5％CO2条件下进行培养；(1) Cultivation of autologous endometrial mesenchymal stem cells: The mesenchymal stem cells from human endometrium were selected from the cell bank for resuscitation, and prepared with phenol red-free DMEM/DF12 and 10% fetal bovine serum by volume. Complete culture medium, put into T75 cell culture flask and culture at 37°C, 5% CO2 ;
(2)间充质干细胞外泌体的制备：待细胞生长程度达到70％-80％，使用生理盐水清洗细胞2-4次，用复方电解质注射液进行饥饿培养12-24小时，待细胞生长程度达到90％-95％后，收取细胞培养上清液，并采用外泌体提取试剂盒对间充质干细胞外泌体进行提取，按照质量体积比2-4％加入人血清白蛋白，混合均匀后待用；(2) Preparation of exosomes from mesenchymal stem cells: when the cell growth reaches 70%-80%, wash the cells 2-4 times with normal saline, starve and culture them with compound electrolyte injection for 12-24 hours, wait for the cells to grow After the degree reaches 90%-95%, collect the cell culture supernatant, and use the exosome extraction kit to extract the exosomes of mesenchymal stem cells, add human serum albumin according to the mass volume ratio of 2-4%, and mix Uniform and ready for use;
(3)基质溶液的制备：按配比，将药用人血白蛋白，按照质量体积比2-4％溶于复方电解质注射液中，配制成均一的基质溶液待用；(3) Preparation of matrix solution: according to the ratio, the medicinal human serum albumin is dissolved in the compound electrolyte injection according to the mass volume ratio of 2-4%, and prepared into a uniform matrix solution for use;
(4)制备腔内注射制剂：按照体积份数比，选取95份基质溶液以及5份间充质干细胞外泌体，进行充分混合搅拌，待混合均匀后，分装到无菌保存管中，放入-80℃冰箱中冷冻待用。(4) Preparation of intracavitary injection preparations: according to the ratio of parts by volume, 95 parts of matrix solution and 5 parts of mesenchymal stem cell exosomes were selected, mixed and stirred thoroughly, and after being mixed evenly, they were distributed into sterile storage tubes. Store in a -80°C freezer for later use.
本发明的有益效果是：将间充质干细胞外泌体混合到基质溶液中，使得外泌体的活性及功能得到了保存和利用，而且存活的时间可以更长，可达到长时间促进表皮细胞增长，消除炎症，快速修复内膜损伤重建，从而实现子宫内膜结构和功能的恢复。The beneficial effect of the present invention is: the exosomes of mesenchymal stem cells are mixed into the matrix solution, so that the activity and function of the exosomes are preserved and utilized, and the survival time can be longer, which can achieve long-term promotion of epidermal cells. Growth, elimination of inflammation, rapid repair of endometrial damage and reconstruction, so as to achieve the recovery of endometrial structure and function.
附图说明Description of drawings
图1示出了根据本发明的实施例的不同培养方法提取的外泌体数量图。Figure 1 shows a graph of the number of exosomes extracted by different culture methods according to an embodiment of the present invention.
图2示出了根据本发明的实施例的不同实验组子宫内膜三种细胞因子的基因表达差异图。Fig. 2 shows the gene expression differences of three cytokines in the endometrium of different experimental groups according to an embodiment of the present invention.
图3示出了根据本发明的实施例的动物子宫内膜损伤面积统计图。Fig. 3 shows a statistical chart of the damage area of endometrium of animals according to the embodiment of the present invention.
具体实施方式Detailed ways
为了使本技术领域的技术人员更好地理解本发明的技术方案，下面结合附图和最佳实施例对本发明作进一步的详细说明。In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below in conjunction with the accompanying drawings and preferred embodiments.
本发明提供了一种含有自体子宫内膜间充质干细胞外泌体的制剂，按照体积份数比，包括5份间充质干细胞外泌体以及95份基质溶液，按照体积份数比，基质溶液包括95-99份复方电解质注射液以及1-5份药用人血白蛋白，间充质干细胞外泌体通过自体子宫内膜间充质干细胞饥饿培养所得，饥饿培养时间为12-24小时，自体子宫内膜间充质干细胞提取自人子宫内膜的间充质干细胞，按照正常培养的方法进行培养得到，培养时间为48-72小时。The invention provides a preparation containing autologous endometrial mesenchymal stem cell exosomes, which comprises 5 parts of mesenchymal stem cell exosomes and 95 parts of matrix solution according to the volume ratio, and the matrix The solution includes 95-99 parts of compound electrolyte injection and 1-5 parts of medicinal human serum albumin. Mesenchymal stem cell exosomes are obtained by starvation culture of autologous endometrial mesenchymal stem cells. The starvation culture time is 12-24 hours , Autologous endometrial mesenchymal stem cells are extracted from human endometrial mesenchymal stem cells, cultured according to the normal culture method, and the culture time is 48-72 hours.
根据上述实施例，优选地，基质溶液的pH值为6.0-8.0。According to the above embodiment, preferably, the pH value of the matrix solution is 6.0-8.0.
根据上述实施例，优选地，复方电解质注射液为复方电解质葡萄糖MG3注射液。According to the above embodiment, preferably, the compound electrolyte injection is compound electrolyte glucose MG3 injection.
根据上述实施例，优选地，药用人血白蛋白为奥地利百特公司所生产。According to the above embodiments, preferably, the pharmaceutical human albumin is produced by Baxter, Austria.
根据上述实施例，优选地，其用于治疗子宫内膜损伤等用途。According to the above-mentioned embodiments, preferably, it is used for treating endometrial injury and the like.
一种含有自体子宫内膜间充质干细胞外泌体的制剂的制备方法，包括以下步骤：A method for preparing a preparation containing autologous endometrial mesenchymal stem cell exosomes, comprising the following steps:
实施例1：Example 1:
(1)自体子宫内膜间充质干细胞的培养：从细胞库中选取自人子宫内膜的间充质干细胞进行复苏，用无酚红DMEM/DF12和体积比浓度为10％胎牛血清做完全培养基，放入T75细胞培养瓶在37℃、5％CO2条件下进行培养；(1) Cultivation of autologous endometrial mesenchymal stem cells: The mesenchymal stem cells from human endometrium were selected from the cell bank for resuscitation, and prepared with phenol red-free DMEM/DF12 and 10% fetal bovine serum by volume. Complete culture medium, put into T75 cell culture flask and culture at 37°C, 5% CO2 ;
(2)间充质干细胞外泌体的制备：待细胞生长程度达到70％-80％，使用生理盐水清洗细胞2-4次，用复方电解质注射液进行饥饿培养12小时，待细胞生长程度达到90％后，收取细胞培养上清液，并采用外泌体提取试剂盒对间充质干细胞外泌体进行提取，按照质量体积比2％加入人血清白蛋白，混合均匀后待用；(2) Preparation of exosomes from mesenchymal stem cells: when the growth degree of the cells reaches 70%-80%, the cells are washed 2-4 times with normal saline, starvation culture is carried out with compound electrolyte injection for 12 hours, and the growth degree of the cells reaches After 90%, collect the cell culture supernatant, and use the exosome extraction kit to extract the exosomes of mesenchymal stem cells, add human serum albumin according to the mass volume ratio of 2%, mix well and set aside;
(3)基质溶液的制备：按配比，将药用人血白蛋白，按照质量体积比2％溶于复方电解质注射液中，配制成均一的基质溶液待用；(3) Preparation of matrix solution: according to the ratio, the medicinal human serum albumin was dissolved in the compound electrolyte injection according to the mass volume ratio of 2%, and prepared into a uniform matrix solution for use;
(4)制备腔内注射制剂：按照体积份数比，选取95份基质溶液以及5份间充质干细胞外泌体，进行充分混合搅拌，待混合均匀后，分装到无菌保存管中，放入-80℃冰箱中冷冻待用。(4) Preparation of intracavitary injection preparations: according to the ratio of parts by volume, 95 parts of matrix solution and 5 parts of mesenchymal stem cell exosomes were selected, mixed and stirred thoroughly, and after being mixed evenly, they were distributed into sterile storage tubes. Store in a -80°C freezer for later use.
实施例2：Example 2:
(1)自体子宫内膜间充质干细胞的培养：从细胞库中选取自人子宫内膜的间充质干细胞进行复苏，用无酚红DMEM/DF12和体积比浓度为10％胎牛血清做完全培养基，放入T75细胞培养瓶在37℃、5％CO2条件下进行培养；(1) Cultivation of autologous endometrial mesenchymal stem cells: The mesenchymal stem cells from human endometrium were selected from the cell bank for resuscitation, and prepared with phenol red-free DMEM/DF12 and 10% fetal bovine serum by volume. Complete culture medium, put into T75 cell culture flask and culture at 37°C, 5% CO2 ;
(2)间充质干细胞外泌体的制备：待细胞生长程度达到70％-80％，使用生理盐水清洗细胞2-4次，用复方电解质注射液进行饥饿培养16小时，待细胞生长程度达到90％后，收取细胞培养上清液，并采用外泌体提取试剂盒对间充质干细胞外泌体进行提取，按照质量体积比3％加入人血清白蛋白，混合均匀后待用；(2) Preparation of exosomes from mesenchymal stem cells: when the growth degree of the cells reaches 70%-80%, the cells are washed 2-4 times with normal saline, starvation culture is carried out with compound electrolyte injection for 16 hours, and the growth degree of the cells reaches After 90%, collect the cell culture supernatant, and use the exosome extraction kit to extract the exosomes of mesenchymal stem cells, add human serum albumin according to the mass volume ratio of 3%, mix well and set aside;
(3)基质溶液的制备：按配比，将药用人血白蛋白，按照质量体积比3％溶于复方电解质注射液中，配制成均一的基质溶液待用；(3) Preparation of matrix solution: according to the ratio, the medicinal human serum albumin was dissolved in the compound electrolyte injection according to the mass volume ratio of 3%, and prepared into a uniform matrix solution for use;
(4)制备腔内注射制剂：按照体积份数比，选取95份基质溶液以及5份间充质干细胞外泌体，进行充分混合搅拌，待混合均匀后，分装到无菌保存管中，放入-80℃冰箱中冷冻待用。(4) Preparation of intracavitary injection preparations: according to the ratio of parts by volume, 95 parts of matrix solution and 5 parts of mesenchymal stem cell exosomes were selected, mixed and stirred thoroughly, and after being mixed evenly, they were distributed into sterile storage tubes. Store in a -80°C freezer for later use.
实施例3：Example 3:
(1)自体子宫内膜间充质干细胞的培养：从细胞库中选取自人子宫内膜的间充质干细胞进行复苏，用无酚红DMEM/DF12和体积比浓度为10％胎牛血清做完全培养基，放入T75细胞培养瓶在37℃、5％CO2条件下进行培养；(1) Cultivation of autologous endometrial mesenchymal stem cells: The mesenchymal stem cells from human endometrium were selected from the cell bank for resuscitation, and prepared with phenol red-free DMEM/DF12 and 10% fetal bovine serum by volume. Complete culture medium, put into T75 cell culture flask and culture at 37°C, 5% CO2 ;
(2)间充质干细胞外泌体的制备：待细胞生长程度达到70％-80％，使用生理盐水清洗细胞2-4次，用复方电解质注射液进行饥饿培养24小时，待细胞生长程度达到90％后，收取细胞培养上清液，并采用外泌体提取试剂盒对间充质干细胞外泌体进行提取，按照质量体积比4％加入人血清白蛋白，混合均匀后待用；(2) Preparation of exosomes from mesenchymal stem cells: when the growth degree of the cells reaches 70%-80%, the cells are washed 2-4 times with normal saline, starvation culture is carried out with compound electrolyte injection for 24 hours, and the growth degree of the cells reaches After 90%, collect the cell culture supernatant, and use the exosome extraction kit to extract the exosomes of mesenchymal stem cells, add human serum albumin according to the mass volume ratio of 4%, mix well and set aside;
(3)基质溶液的制备：按配比，将药用人血白蛋白，按照质量体积比4％溶于复方电解质注射液中，配制成均一的基质溶液待用；(3) Preparation of matrix solution: according to the ratio, the medicinal human serum albumin was dissolved in the compound electrolyte injection according to the mass volume ratio of 4%, and prepared into a uniform matrix solution for use;
(4)制备腔内注射制剂：按照体积份数比，选取95份基质溶液以及5份间充质干细胞外泌体，进行充分混合搅拌，待混合均匀后，分装到无菌保存管中，放入-80℃冰箱中冷冻待用。(4) Preparation of intracavitary injection preparations: according to the ratio of parts by volume, 95 parts of matrix solution and 5 parts of mesenchymal stem cell exosomes were selected, mixed and stirred thoroughly, and after being mixed evenly, they were distributed into sterile storage tubes. Store in a -80°C freezer for later use.
实施例4Example 4
(1)选取7周龄的SD大鼠12只，平均分为3组，①空白组；②阴性对照组；③实验组制备大鼠子宫内膜损伤模型。(1) Select 12 7-week-old SD rats and divide them into 3 groups on average, ① blank group; ② negative control group; ③ experimental group to prepare rat endometrial injury models.
(2)造模成功后，按照①空白组在子宫内膜腔内注射生理盐水；②阴性对照组在子宫内膜腔内注射基质溶液；③实验组在子宫内膜腔内注射间充质干细胞外泌体注射液进行治疗，分别在给药后的第1、3、5、7、9、天进行数据测量，并统计。(2) After successful modeling, inject physiological saline into the endometrial cavity according to ① the blank group; ② inject matrix solution into the endometrial cavity in the negative control group; ③ inject mesenchymal stem cells into the endometrial cavity in the experimental group The exosome injection was used for treatment, and the data were measured and counted on the 1st, 3rd, 5th, 7th, 9th, and day after administration.
(3)数据分析，分析各实验组伤口愈合的时间差异。(3) Data analysis, analyzing the difference in wound healing time of each experimental group.
外泌体是由细胞分泌的直径为30～150nm的小囊泡，内含来源于细胞相关的蛋白质与核苷酸等生物分子，在细胞交流间起重要作用。Exosomes are small vesicles secreted by cells with a diameter of 30-150 nm, which contain biomolecules such as cell-related proteins and nucleotides, and play an important role in cell communication.
外泌体是细胞分泌的小生物膜囊泡。与间充质干细胞相比，外泌体体积更小、复杂性更低，易于生产和存储，质干细胞外泌体富含生物活性分子，如蛋白质和RNA；存在于外泌体中的许多蛋白质都属于酶类，具有催化活性，间充质干细胞外泌体没有细胞的活性，并且不存在形成肿瘤的风险；此外，由于膜结合蛋白含量较低，外泌体的免疫原性低于间充质干细胞；间充质干细胞外泌体对维持细胞内稳态以及对外界刺激作出反应起到了重要的作用；当组织微环境稳态因疾病或损伤而遭到破坏时，这种作用就显得更加重要了；目前间充质干细胞分泌的外泌体的治疗效果越来越得到认可，且外泌体的使用比干细胞更方便且成本更低，相对于细胞的使用外泌体的接受度也更大。Exosomes are small biofilm vesicles secreted by cells. Compared with mesenchymal stem cells, exosomes are smaller, less complex, and easy to produce and store. Mesenchymal stem cell exosomes are rich in bioactive molecules, such as proteins and RNA; many proteins present in exosomes They all belong to enzymes and have catalytic activity. Mesenchymal stem cell exosomes have no cell activity, and there is no risk of tumor formation; in addition, due to the lower content of membrane-bound proteins, exosomes are less immunogenic than mesenchymal stem cells. Mesenchymal stem cells; Mesenchymal stem cell exosomes play an important role in maintaining cell homeostasis and responding to external stimuli; when the tissue microenvironment homeostasis is disrupted due to disease or injury, this role is even more important It is important; at present, the therapeutic effect of exosomes secreted by mesenchymal stem cells is more and more recognized, and the use of exosomes is more convenient and lower cost than stem cells, and the acceptance of exosomes is also higher than that of cells. big.
本发明的有益效果是：将间充质干细胞外泌体混合到基质溶液中，使得外泌体的活性及功能得到了保存和利用，而且存活的时间可以更长，可达到长时间促进表皮细胞增长，消除炎症，快速修复内膜损伤重建，从而实现子宫内膜结构和功能的恢复。The beneficial effect of the present invention is: the exosomes of mesenchymal stem cells are mixed into the matrix solution, so that the activity and function of the exosomes are preserved and utilized, and the survival time can be longer, which can achieve long-term promotion of epidermal cells. Growth, elimination of inflammation, rapid repair of endometrial damage and reconstruction, so as to achieve the recovery of endometrial structure and function.
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.
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