技术领域technical field
本发明属于干细胞技术领域，具体涉及一种人脐带间充质干细胞外泌体冻干粉及其制备的方法。The invention belongs to the technical field of stem cells, and in particular relates to a human umbilical cord mesenchymal stem cell exosome freeze-dried powder and a preparation method thereof.
背景技术Background technique
外泌体是由细胞经过“内吞-融合-外排”等一系列调控过程而形成，并主动分泌至胞外的大小均一，分子直径为40～100nm的亚细胞双层膜囊泡，其内含有与细胞来源相关的蛋白质、miRNA及mRNA等物质。外泌体既可以通过质膜受体直接激活受体细胞，也可以转运蛋白质、miRNA及mRNA甚至细胞器进入受体细胞内，同时也可以携带处于不同病例状态下的细胞所含有的特异性物质，从而在生理学和病理学上都发挥着重要的作用。外泌体的应用主要集中在临床诊断、抗肿瘤和美容抗衰方面。Exosomes are subcellular double-layer membrane vesicles with a uniform size and a molecular diameter of 40-100 nm, which are formed by cells through a series of regulatory processes such as "endocytosis-fusion-efflux", and are actively secreted to the outside of the cell. Contains proteins, miRNA and mRNA related to cell origin. Exosomes can not only directly activate recipient cells through plasma membrane receptors, but also transport proteins, miRNA, mRNA and even organelles into recipient cells, and can also carry specific substances contained in cells in different case states. It plays an important role in both physiology and pathology. The application of exosomes mainly focuses on clinical diagnosis, anti-tumor and cosmetic anti-aging.
间充质干细胞(human mesenchymal stem cells,hMSC）是具有自我更新和多向分化能力的细胞，产生并释放营养性物质，通过旁分泌途径促进细胞修复及血管生成，在再生医学领域有重要作用。许多的研究表明MSCs外泌体在治疗组织损伤性疾病中具有重要的作用。人脐带组织中含有丰富的间充质干细胞，所获得的人脐带间充质干细胞(UC MSCs)可以在体外条件下稳定培养，细胞活性强，分泌旺盛，其细胞培养上清液中含有大量的外泌体。同时，所分泌的外泌体来源单一稳定。Mesenchymal stem cells (human mesenchymal stem cells, hMSC) are cells with self-renewal and multi-directional differentiation capabilities, produce and release nutrients, promote cell repair and angiogenesis through paracrine pathways, and play an important role in the field of regenerative medicine. Many studies have shown that MSCs exosomes play an important role in the treatment of tissue damage diseases. Human umbilical cord tissue is rich in mesenchymal stem cells. The obtained human umbilical cord mesenchymal stem cells (UC MSCs) can be stably cultured in vitro, with strong cell activity and strong secretion. The cell culture supernatant contains a large amount of exosomes. At the same time, the source of secreted exosomes is single and stable.
目前所用的提取外泌体的方法主要包括超高速离心法，免疫磁珠法，液相色谱法，密度梯度离心法等。超高速离心机由于造价昂贵，普及率低并且耗时较长，免疫磁珠法和液相色谱法同样存在效率低，难以广泛普及的问题。基于外泌体的大小，我们利用旋转超滤技术能够快速地、大批量地从人脐带间充质干细胞细胞的培养上清液中分离出大量的外泌体。由于所提纯的外泌体悬液需要冷冻保存(-20℃到-80℃)，这使其在储存和运输以及应用方面带来了许多不便。The methods currently used to extract exosomes mainly include ultrahigh-speed centrifugation, immunomagnetic bead method, liquid chromatography, density gradient centrifugation and so on. Due to the high cost of ultra-high-speed centrifuges, low penetration rate and long time-consuming, immunomagnetic bead method and liquid chromatography also have low efficiency and are difficult to be widely used. Based on the size of exosomes, we can quickly and in large quantities isolate a large number of exosomes from the culture supernatant of human umbilical cord mesenchymal stem cells by using rotary ultrafiltration technology. Since the purified exosome suspension needs to be frozen (-20°C to -80°C), it brings a lot of inconvenience in storage, transportation and application.
发明内容Contents of the invention
本发明的目的是为了克服现有技术提取外泌体的方法存在的不足。The purpose of the present invention is to overcome the shortcomings of the prior art methods for extracting exosomes.
为此，本发明提供了一种人脐带间充质干细胞外泌体冻干粉，包括人间充质干细胞外泌体以及冻干剂。To this end, the present invention provides a human umbilical cord mesenchymal stem cell exosome freeze-dried powder, including human mesenchymal stem cell exosomes and a freeze-dried agent.
所述冻干剂的成份包括氯化钠、海藻糖、甘露醇、右旋糖酐、葡萄糖、乳酸钠、氯化钾、氯化钙。The ingredients of the freeze-dried preparation include sodium chloride, trehalose, mannitol, dextran, glucose, sodium lactate, potassium chloride and calcium chloride.
所述冻干剂的各成份浓度比为氯化钠0.7%-1.1%，葡萄糖0.1%-5%，甘露醇5%-20%，右旋糖酐0.1%-2%，海藻糖0.2%-3%，乳酸钠0.2%-0.4%，氯化钾0.01%-0.05%，氯化钙0.01%-0.04%。The concentration ratio of each component of the freeze-dried agent is sodium chloride 0.7%-1.1%, glucose 0.1%-5%, mannitol 5%-20%, dextran 0.1%-2%, trehalose 0.2%-3%, Sodium lactate 0.2%-0.4%, potassium chloride 0.01%-0.05%, calcium chloride 0.01%-0.04%.
一种人脐带间充质干细胞外泌体冻干粉的制备方法，包括如下步骤：A preparation method for human umbilical cord mesenchymal stem cell exosome freeze-dried powder, comprising the steps of:
（1）人脐带间充质干细胞的培养；(1) Culture of human umbilical cord mesenchymal stem cells;
（2）进行外泌体的提取；(2) Extraction of exosomes;
（3）制备人脐带间充质干细胞外泌体冻干粉。(3) Prepare human umbilical cord mesenchymal stem cell exosome freeze-dried powder.
所述（1）人脐带间充质干细胞的培养，具体操作是：取生长良好的人脐带间充质干细胞，用α-MEM 培养基培养，待其生长至融合度80％以上，收集培养液，用PBS洗三遍，0.25％胰酶消化传代，继续传代培养，每次收集培养上清液，并更换新鲜的培养基，连续培养过程中收集培养上清液。(1) The cultivation of human umbilical cord mesenchymal stem cells, the specific operation is: take well-grown human umbilical cord mesenchymal stem cells, culture them in α-MEM medium, wait until they grow to a confluence of more than 80%, and collect the culture medium , washed three times with PBS, digested and subcultured with 0.25% trypsin, continued to subculture, collected the culture supernatant each time, and replaced with fresh medium, and collected the culture supernatant during continuous culture.
所述（2）进行外泌体的提取，具体操作步骤是：In (2) extracting exosomes, the specific operation steps are:
a、取细胞培养上清液在300g～400g离心10min~20min，收集上清液，弃沉淀；a. Take the cell culture supernatant and centrifuge at 300g-400g for 10min-20min, collect the supernatant and discard the precipitate;
b、将步骤a收集的上清液取在2000g～3000g离心20min～30min，继续收集上清液，弃沉淀；b. Take the supernatant collected in step a and centrifuge at 2000g-3000g for 20min-30min, continue to collect the supernatant, and discard the precipitate;
c、将步骤b收集的上清液用0.22μm孔径过滤器进行过滤，收集过滤液；c. Filter the supernatant collected in step b with a 0.22 μm pore size filter, and collect the filtrate;
d、在Stirred Cell 8050型超滤装置上超滤上清液，滤膜的孔径为30~60nm，超滤完毕后，加入PBS并再次超滤，重复3次。d. Ultrafilter the supernatant on a Stirred Cell 8050 ultrafiltration device. The pore size of the filter membrane is 30-60 nm. After the ultrafiltration is completed, add PBS and perform ultrafiltration again, repeating 3 times.
e、PBS洗涤滤膜上的外泌体，制得浓缩培养液，用BCA试剂盒测蛋白浓度，并进行冷藏保存。e. Wash the exosomes on the filter membrane with PBS to prepare a concentrated culture solution, measure the protein concentration with a BCA kit, and store it in cold storage.
所述（3）人脐带间充质干细胞外泌体冻干粉，具体操作是：取制得的人脐带间充质干细胞外泌体，加入成份包括氯化钠、海藻糖、甘露醇、右旋糖酐、葡萄糖、乳酸钠、氯化钾、氯化钙的冻干剂，然后用0.22μm滤膜过滤，分装后低温冷冻24h，然后在超低温冷冻干燥机冷冻干燥24h，冷冻干燥结束后，得到外泌体冻干粉，室温，阴凉通风处储存。(3) Human umbilical cord mesenchymal stem cell exosome freeze-dried powder, the specific operation is: take the obtained human umbilical cord mesenchymal stem cell exosome, add ingredients including sodium chloride, trehalose, mannitol, dextran , glucose, sodium lactate, potassium chloride, calcium chloride, and then filtered with a 0.22 μm filter membrane, subpackaged and frozen for 24 hours, and then freeze-dried in an ultra-low temperature freeze dryer for 24 hours. After the freeze-drying, exocrine Freeze-dried powder, stored at room temperature in a cool and ventilated place.
本发明的有益效果：本发明提供的这种人脐带间充质干细胞外泌体冻干粉及其制备的方法，所制备的外泌体冻干粉既能在室温条件下长期储存，又能很好保持外泌体的活性；冻干前和冻干后外泌体的活性不存在显著性差异；而且该人脐带间充质干细胞外泌体冻干粉，制备过程简单，便于进行保存和运输。Beneficial effects of the present invention: the human umbilical cord mesenchymal stem cell exosome freeze-dried powder and its preparation method provided by the present invention, the prepared exosome freeze-dried powder can be stored at room temperature for a long time, and can The activity of exosomes is well maintained; there is no significant difference in the activity of exosomes before and after freeze-drying; and the human umbilical cord mesenchymal stem cell exosome freeze-dried powder has a simple preparation process and is convenient for storage and storage. transportation.
以下将结合附图对本发明做进一步详细说明。The present invention will be described in further detail below in conjunction with the accompanying drawings.
附图说明Description of drawings
图1a为人脐带间充质干细胞收集培养上清的细胞放大40 倍状态图。Figure 1a is a 40-fold magnified cell state diagram of the collected culture supernatant of human umbilical cord mesenchymal stem cells.
图1b为人脐带间充质干细胞收集培养上清的细胞放大100倍状态图。Figure 1b is a 100-fold magnified cell state diagram of the collected culture supernatant of human umbilical cord mesenchymal stem cells.
图2人脐带间充质干细胞的表面标志物的流式检测图。Figure 2 Flow cytometric detection of surface markers of human umbilical cord mesenchymal stem cells.
图3a为制备的人脐带间充质干细胞外泌体的形态图。Figure 3a is a morphological diagram of the prepared human umbilical cord mesenchymal stem cell exosomes.
图3b添加冻干保护剂冻干后的人脐带间充质干细胞外泌体的形态图。Fig. 3b is a morphological diagram of human umbilical cord mesenchymal stem cell exosomes after adding lyoprotectant and freeze-drying.
图4a为人脐带间充质干细胞提取的外泌体在冻干前后对人成纤维细胞生长促进作用的比较结果示意图。Figure 4a is a schematic diagram of the comparison results of exosomes extracted from human umbilical cord mesenchymal stem cells before and after freeze-drying on the growth promotion of human fibroblasts.
图4b为人脐带间充质干细胞提取的外泌体在冻干前后对人成纤维细胞生长促进作用的比较结果条形图。Figure 4b is a bar graph of the comparison results of exosomes extracted from human umbilical cord mesenchymal stem cells before and after freeze-drying on the growth promotion of human fibroblasts.
图5为人脐带间充质干细胞提取的外泌体在冻干前后对人成纤维细胞抗氧化效果比较的结果条形图。Figure 5 is a bar graph showing the comparison of the antioxidative effects of exosomes extracted from human umbilical cord mesenchymal stem cells on human fibroblasts before and after freeze-drying.
图6a为人脐带间充质干细胞提取的外泌体在冻干前后对人成纤维细胞活性氧清除能力比较的示意图。Fig. 6a is a schematic diagram showing the comparison of the ability of exosomes extracted from human umbilical cord mesenchymal stem cells to scavenge reactive oxygen species in human fibroblasts before and after freeze-drying.
图6b为人脐带间充质干细胞提取的外泌体在冻干前后对人成纤维细胞活性氧清除能力比较的条形图。Figure 6b is a bar graph comparing the ability of exosomes extracted from human umbilical cord mesenchymal stem cells to scavenge reactive oxygen species in human fibroblasts before and after freeze-drying.
具体实施方式Detailed ways
为进一步阐述本发明达成预定目的所采取的技术手段及功效，以下结合附图及实施例对本发明的具体实施方式、结构特征及其功效，详细说明如下。In order to further illustrate the technical means and effects adopted by the present invention to achieve the intended purpose, the specific implementation, structural features and effects of the present invention will be described in detail below in conjunction with the accompanying drawings and examples.
实施例1Example 1
为了克服现有技术提取外泌体的方法存在的不足；本发明提供一种人脐带间充质干细胞外泌体冻干粉，包括人间充质干细胞外泌体以及冻干剂。In order to overcome the shortcomings of existing methods for extracting exosomes in the prior art, the present invention provides a freeze-dried powder of exosomes from human umbilical cord mesenchymal stem cells, including exosomes from human mesenchymal stem cells and a freeze-dried agent.
上述冻干剂的成份包括氯化钠、海藻糖、甘露醇、右旋糖酐、葡萄糖、乳酸钠、氯化钾、氯化钙。The ingredients of the freeze-dried preparation include sodium chloride, trehalose, mannitol, dextran, glucose, sodium lactate, potassium chloride and calcium chloride.
进一步，冻干剂各成份的浓度可以为：冻干剂的各成份浓度比为氯化钠0.7%-1.1%，葡萄糖0.1%-5%，甘露醇5%-20%，右旋糖酐0.1%-2%，海藻糖0.2%-3%，乳酸钠0.2%-0.4%，氯化钾0.01%-0.05%，氯化钙0.01%-0.04%。Further, the concentration of each component of the freeze-dried agent can be: the concentration ratio of each component of the freeze-dried agent is 0.7%-1.1% of sodium chloride, 0.1%-5% of glucose, 5%-20% of mannitol, and 0.1%-2% of dextran. %, trehalose 0.2%-3%, sodium lactate 0.2%-0.4%, potassium chloride 0.01%-0.05%, calcium chloride 0.01%-0.04%.
实施例2Example 2
一种人脐带间充质干细胞外泌体冻干粉的制备方法，包括如下步骤：A preparation method for human umbilical cord mesenchymal stem cell exosome freeze-dried powder, comprising the steps of:
一、人脐带间充质干细胞的培养；1. Culture of human umbilical cord mesenchymal stem cells;
取生长良好的人脐带间充质干细胞，用α-MEM培养基培养，待其生长至融合度80％以上，收集培养液，用PBS洗三遍，0.25％胰酶消化传代，继续传代培养，每次收集培养上清液，并更换新鲜的培养基，连续培养过程中收集培养上清液。其中，用于收集培养上清的细胞状态如图1所示；细胞的表面标志物检测如图2所示。Well-grown human umbilical cord mesenchymal stem cells were taken and cultured in α-MEM medium. After they grew to a confluence of more than 80%, the culture medium was collected, washed three times with PBS, digested and subcultured with 0.25% trypsin, and continued to be subcultured. The culture supernatant was collected each time and replaced with fresh medium, and the culture supernatant was collected during continuous culture. Among them, the state of cells used to collect the culture supernatant is shown in Figure 1; the detection of cell surface markers is shown in Figure 2.
二、进行外泌体的提取；2. Extraction of exosomes;
a、取细胞培养上清液在300g～400g离心10min~20min，收集上清液，弃沉淀；a. Take the cell culture supernatant and centrifuge at 300g-400g for 10min-20min, collect the supernatant and discard the precipitate;
b、将步骤a收集的上清液取在2000g～3000g离心20min～30min，继续收集上清液，弃沉淀；b. Take the supernatant collected in step a and centrifuge at 2000g-3000g for 20min-30min, continue to collect the supernatant, and discard the precipitate;
c、将步骤b收集的上清液用0.22μm孔径过滤器进行过滤，收集过滤液；c. Filter the supernatant collected in step b with a 0.22 μm pore size filter, and collect the filtrate;
d、在Stirred Cell 8050型超滤装置上超滤上清液，滤膜的孔径为30~60nm，超滤完毕后，加入PBS并再次超滤，重复3次。d. Ultrafilter the supernatant on a Stirred Cell 8050 ultrafiltration device. The pore size of the filter membrane is 30-60 nm. After the ultrafiltration is completed, add PBS and perform ultrafiltration again, repeating 3 times.
e、PBS洗涤滤膜上的外泌体，制得浓缩培养液，用BCA试剂盒测蛋白浓度，并进行冷藏保存。e. Wash the exosomes on the filter membrane with PBS to prepare a concentrated culture solution, measure the protein concentration with a BCA kit, and store it in cold storage.
三、制备人脐带间充质干细胞外泌体冻干粉；3. Preparation of human umbilical cord mesenchymal stem cell exosome freeze-dried powder;
取制得的人脐带间充质干细胞外泌体，加入成份包括氯化钠、海藻糖、甘露醇、右旋糖酐、葡萄糖、乳酸钠、氯化钾、氯化钙的冻干剂，然后用0.22μm滤膜过滤，分装后低温冷冻24h，然后在超低温冷冻干燥机冷冻干燥24h，冷冻干燥结束后，得到外泌体冻干粉，室温，阴凉通风处储存。Take the obtained human umbilical cord mesenchymal stem cell exosomes, add ingredients including sodium chloride, trehalose, mannitol, dextran, glucose, sodium lactate, potassium chloride, calcium chloride freeze-dried agent, and then filter with 0.22 μm Membrane filtration, subpackaging, low-temperature freezing for 24 hours, and then freeze-drying in an ultra-low temperature freeze dryer for 24 hours. After the freeze-drying, exosome freeze-dried powder was obtained, and stored at room temperature in a cool and ventilated place.
实施例3Example 3
除了上述实施例的外泌体提取方法外，所述人脐带间充质干细胞外泌体的提取方法为在4℃下，人脐带间充质干细胞培养上清在300g～10000g的离心力下离心去除培养上清中的细胞及细胞碎片，收集上清，利用旋转超滤仪得到培养基中的外泌体。In addition to the method for extracting exosomes in the above examples, the method for extracting exosomes from human umbilical cord mesenchymal stem cells is to centrifuge and remove the culture supernatant of human umbilical cord mesenchymal stem cells under a centrifugal force of 300g to 10000g at 4°C. The cells and cell debris in the supernatant were cultured, the supernatant was collected, and the exosomes in the medium were obtained by using a rotary ultrafiltration apparatus.
实施例4Example 4
外泌体的形态检测：Morphological detection of exosomes:
第一组即为a为实施例2制备得到的人脐带间充质干细胞外泌体在冻干前的液体状态；第二组即为b为冻干后的外泌体状态，如图3所示。The first group is a, which is the liquid state of human umbilical cord mesenchymal stem cell exosomes prepared in Example 2 before lyophilization; the second group is b, which is the state of exosomes after lyophilization, as shown in Figure 3 Show.
实施例5Example 5
外泌体对人成纤维细胞增殖的影响：Effects of exosomes on the proliferation of human fibroblasts:
对于冻干前超低温保存和冻干后室温保存的已知浓度的外泌体，分别取0.1、1、10ug/ml的浓度与人成纤维细胞共培养72h后检测其对人成纤维细胞增殖的影响，对照组即不添加外泌体的培养基培养，结果如图4所示；For the known concentrations of exosomes stored at cryopreservation before freeze-drying and at room temperature after freeze-drying, the concentrations of 0.1, 1, and 10 μg/ml were respectively cultured with human fibroblasts for 72 hours to detect their effects on the proliferation of human fibroblasts. Influence, the control group is culture medium without adding exosomes, the results are shown in Figure 4;
从图4结果可以看出，实施例2制备的人脐带间充质干细胞外泌体在冻干前和复溶后对细胞的增殖均有促进作用，且效果没有显著性差异。It can be seen from the results in Figure 4 that the human umbilical cord mesenchymal stem cell exosomes prepared in Example 2 can promote cell proliferation before freeze-drying and after reconstitution, and there is no significant difference in effect.
实施例6Example 6
外泌体抗氧化功效的检测：Exosome Antioxidant Efficacy Detection:
对于冻干前超低温保存和冻干后室温保存的已知浓度的外泌体，分别取1、10、20ug/ml的浓度与人成纤维细胞共培养72h后检测细胞内Mn-SOD的相对表达量，对照组即不添加外泌体的培养基培养，结果如图5所示；For the known concentrations of exosomes stored at cryopreservation before freeze-drying and at room temperature after freeze-drying, the concentrations of 1, 10, and 20 ug/ml were respectively co-cultured with human fibroblasts for 72 hours to detect the relative expression of Mn-SOD in the cells amount, the control group was cultured in medium without adding exosomes, the results are shown in Figure 5;
从图5结果可以看出，实施例2制备的人脐带间充质干细胞外泌体在冻干前和复溶后的抗氧化功效没有显著性差异。It can be seen from the results in Figure 5 that there is no significant difference in the antioxidant efficacy of the human umbilical cord mesenchymal stem cell exosomes prepared in Example 2 before freeze-drying and after reconstitution.
实施例7Example 7
外泌体对活性氧的清除能力检测：Detection of the scavenging ability of exosomes on active oxygen:
对于冻干前超低温保存和冻干后室温保存的已知浓度的外泌体，分别取1、10、20ug/ml的浓度与人成纤维细胞共培养72h后，利用荧光探针DCFH-DA进行活性氧的检测，验证外泌体对胞内活性氧清除能力的影响，对照组即不添加外泌体的培养基培养，结果如图6所示；For the known concentrations of exosomes stored at cryopreservation before freeze-drying and at room temperature after freeze-drying, the concentrations of 1, 10, and 20 ug/ml were respectively co-cultured with human fibroblasts for 72 hours, and the fluorescent probe DCFH-DA was used to detect exosomes. Detection of active oxygen to verify the effect of exosomes on the ability to scavenge intracellular active oxygen. The control group was cultured in a medium without exosomes. The results are shown in Figure 6;
从图6结果可见，实施例2制备的人脐带间充质干细胞外泌体在冻干后对活性氧清除能力有轻微下降，但在冻干前和复溶后的活性氧清除能力抗氧化能力不存在显著性差异。From the results in Figure 6, it can be seen that the exosomes from human umbilical cord mesenchymal stem cells prepared in Example 2 had a slight decrease in the ability to scavenge active oxygen after lyophilization, but the ability to scavenge active oxygen before and after reconstitution and the antioxidant capacity There is no significant difference.
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be assumed that the specific implementation of the present invention is limited to these descriptions. For those of ordinary skill in the technical field of the present invention, without departing from the concept of the present invention, some simple deduction or replacement can be made, which should be regarded as belonging to the protection scope of the present invention.
| Application Number | Priority Date | Filing Date | Title |
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| CN201810473929.4ACN108633877A (en) | 2018-05-17 | 2018-05-17 | A kind of human umbilical cord mesenchymal stem cells excretion body freeze-dried powder and its method of preparation |
| Application Number | Priority Date | Filing Date | Title |
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| CN201810473929.4ACN108633877A (en) | 2018-05-17 | 2018-05-17 | A kind of human umbilical cord mesenchymal stem cells excretion body freeze-dried powder and its method of preparation |
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| CN108633877Atrue CN108633877A (en) | 2018-10-12 |
| Application Number | Title | Priority Date | Filing Date |
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| CN201810473929.4AWithdrawnCN108633877A (en) | 2018-05-17 | 2018-05-17 | A kind of human umbilical cord mesenchymal stem cells excretion body freeze-dried powder and its method of preparation |
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