技术领域technical field
本发明属于皮肤精原细胞保存的技术领域，特别涉及一种皮肤精原细胞保护液。The invention belongs to the technical field of skin spermatogonia preservation, and in particular relates to a skin spermatogonia protective solution.
背景技术Background technique
目前我国的临床和细胞研究实验中，细胞的采集、长期保存和使用是时空分离的，采集的细胞离体后对外部环境的要求非常苛刻，特别是细胞采集与长期保存之间的细胞转运时，由于细胞还没有经过长期保存处理，外部环境不好会降低细胞活力，因此细胞采集后立即加入保护液来保持细胞的活力成了一个很重要的课题。因为采集的细胞无论是培养增殖或者保存最终都会进入人体血液，所以添加的保护液一方面要求可以保持细胞离体后在体外的活力，另一方面，还要求保护液中不能有不适宜静脉注射的成分，特别是致敏成分。At present, in my country's clinical and cell research experiments, the collection, long-term storage and use of cells are separated in time and space, and the collected cells have very strict requirements on the external environment after isolation, especially when cells are transported between cell collection and long-term storage. , because the cells have not been preserved for a long time, the external environment will reduce the viability of the cells, so adding the protective solution immediately after the cells are collected to maintain the viability of the cells has become a very important topic. Because the collected cells will eventually enter the human blood whether they are cultured, proliferated or preserved, the added protective solution must on the one hand maintain the viability of the cells in vitro, on the other hand, it is also required that the protective solution should not contain any substances that are not suitable for intravenous injection. ingredients, especially allergenic ingredients.
常规的细胞保护液是生理盐水，方便安全，缺点是保护效果不好，专利文献CN101919380A报道，用生理盐水保护间充质干细胞，2小时后间充质干细胞活力就会降低10％左右，因此生理盐水只适用于间充质干细胞的即时保存。The conventional cell protection solution is physiological saline, which is convenient and safe, but the disadvantage is that the protection effect is not good. According to the patent document CN101919380A, when using normal saline to protect mesenchymal stem cells, the viability of mesenchymal stem cells will decrease by about 10% after 2 hours, so physiological Saline is only suitable for immediate preservation of MSCs.
对于离体皮肤精原细胞的保存可以分为，几天以上几个月甚至几年、几十年的长时间的保存，一般采用液氮超低温保存，细胞需要预处理，加入甘油或者DMSO等防结晶的保护剂；另一种是几小时到几天之内的短时间的临时保存，目前研究的比较少，细胞临时保存的保护液，一般是生理盐水或者培养基中引入生长因子等。发明人在采集皮肤精原细胞采集盒转运过程中，还发现一般采集过程都是把25ml的细胞或者更多体积的细胞与保护液混合装在试管中静置保存。一个容易忽视的问题，就是皮肤精原细胞在一个细长的试管中细胞的供氧问题。皮肤精原细胞需要一个低氧的环境，氧气缺乏和供应过量都会对皮肤精原细胞活力产生影响。The preservation of isolated skin spermatogonia can be divided into long-term preservation of more than a few days, months, even years, and decades. Generally, liquid nitrogen is used for ultra-low temperature preservation. The protective agent for crystallization; the other is short-term temporary storage within a few hours to a few days. At present, there are relatively few studies. The protective solution for temporary storage of cells is generally physiological saline or growth factors introduced into the culture medium. The inventor also found that during the transfer process of the skin spermatogonial cell collection box, 25ml of cells or more cells were mixed with the protective solution and stored in a test tube in a general collection process. An easily overlooked problem is the oxygen supply of skin spermatogonia in a long and thin test tube. Skin spermatogonia need a hypoxic environment, and oxygen deficiency and oversupply will have an impact on the vitality of skin spermatogonia.
发明内容Contents of the invention
针对上述问题，本发明提出了一种皮肤精原细胞保护液，除了对保护液中一般培养基组分和抗生素的筛选、优化，该保护液还可以为皮肤细胞或精原细胞提供低氧环境，同时引入抗氧化剂可起到保持细胞活率的效果。因为保护液所保护的皮肤细胞或精原细胞最终是要用于人的，因此除非必要，尽量选择人体血液中已经存在的组分，尽量避免外源物质的引入。In view of the above problems, the present invention proposes a skin spermatogonia protection solution, in addition to screening and optimizing the general medium components and antibiotics in the protection solution, the protection solution can also provide a hypoxic environment for skin cells or spermatogonia , At the same time, the introduction of antioxidants can maintain the effect of cell viability. Because the skin cells or spermatogonia protected by the protective solution are ultimately used in humans, unless necessary, try to select components that already exist in human blood, and try to avoid the introduction of foreign substances.
本发明公开的皮肤精原细胞保护液，具体技术方案如下：The skin spermatogonia protective solution disclosed by the invention has a specific technical scheme as follows:
一种皮肤精原细胞保护液，所述保护液主要是将氨基糖苷类抗生素用DMEM培养基水溶液溶解而成，每毫升所述DMEM培养基水溶液溶解所述氨基糖苷类抗生素50-200单位，所述DMEM培养基水溶液的浓度为10-25g/L。A skin spermatogonial cell protection solution, the protection solution is mainly formed by dissolving aminoglycoside antibiotics with DMEM medium aqueous solution, and dissolving 50-200 units of the aminoglycoside antibiotics per milliliter of the DMEM medium aqueous solution, the The concentration of the DMEM medium aqueous solution is 10-25g/L.
优选的，所述氨基糖苷类抗生素选自庆大霉素、卡那霉素、阿米卡星或妥布霉素中的一种或多种。Preferably, the aminoglycoside antibiotic is selected from one or more of gentamicin, kanamycin, amikacin or tobramycin.
优选的，所述氨基糖苷类抗生素为重量份数比为10:2:2的庆大霉素、卡那霉素和妥布霉素的混合物，所述DMEM培养基水溶液的浓度为16.9g/L。Preferably, the aminoglycoside antibiotic is a mixture of gentamycin, kanamycin and tobramycin with a weight and number ratio of 10:2:2, and the concentration of the DMEM medium aqueous solution is 16.9g/ L.
优选的，所述保护液还包括氧合血红蛋白，每毫升所述DMEM培养基水溶液溶解所述氧合血红蛋白400-1000μg。进一步优选的，每毫升所述DMEM培养基水溶液溶解所述氧合血红蛋白600μg。Preferably, the protective solution further includes oxyhemoglobin, and 400-1000 μg of oxyhemoglobin is dissolved per milliliter of the DMEM medium aqueous solution. Further preferably, 600 μg of the oxyhemoglobin is dissolved per milliliter of the DMEM medium aqueous solution.
优选的，所述保护液还包括抗氧化剂，每毫升所述DMEM培养基水溶液溶解所述抗氧化剂0.1-1.0μmol。Preferably, the protection solution also includes antioxidants, and 0.1-1.0 μmol of the antioxidants are dissolved per milliliter of the DMEM medium aqueous solution.
优选的，所述抗氧化剂为维生素C、维生素E或谷胱甘肽中的一种或多种。进一步优选的，所述抗氧化剂为重量份数比为1：1的维生素E和谷胱甘肽的混合物。Preferably, the antioxidant is one or more of vitamin C, vitamin E or glutathione. Further preferably, the antioxidant is a mixture of vitamin E and glutathione with a ratio of parts by weight of 1:1.
优选的，所述保护液还包括吐温80、棕榈酰胺、海藻酸钾和月桂酰肌氨酸，每毫升所述DMEM培养基水溶液分别溶解所述吐温80 0.1-1.0μg、棕榈酰胺20-80μg、海藻酸钾10-50μg、月桂酰肌氨酸100-200μg。Preferably, the protective solution also includes Tween 80, palmitamide, potassium alginate and lauroyl sarcosine, and each milliliter of the DMEM medium aqueous solution dissolves 0.1-1.0 μg of Tween 80, palmitamide 20- 80μg, potassium alginate 10-50μg, lauroyl sarcosine 100-200μg.
优选的，所述保护液还包括柠檬酸、丙酮酸、延胡索酸和乙酰辅酶A，每毫升所述DMEM培养基水溶液分别溶解柠檬酸1-10μg、丙酮酸50-80μg、延胡索酸10-20μg和乙酰辅酶A80-150μg。Preferably, the protection solution also includes citric acid, pyruvic acid, fumaric acid and acetyl coenzyme A, and 1-10 μg of citric acid, 50-80 μg of pyruvic acid, 10-20 μg of fumaric acid and acetyl coenzyme A are respectively dissolved in each milliliter of the DMEM medium aqueous solution. A80-150μg.
DMEM培养基中含有皮肤细胞或精原细胞生长代谢所需的糖类、氨基酸、无机盐和维生素等物质，对于维持细胞渗透压及活力有较好的作用。本发明优选了抗菌效果好、性质稳定、适于保护需要注入人体的皮肤细胞或精原细胞的氨基糖苷类抗生素，并优选了四种抗生素庆大霉素、卡那霉素、阿米卡星或妥布霉素。抗生素的选择不但具有抗菌效果，更重要的是不会对保存的细胞活力产生影响。DMEM medium contains carbohydrates, amino acids, inorganic salts, vitamins and other substances required for the growth and metabolism of skin cells or spermatogonia, which has a good effect on maintaining cell osmotic pressure and vitality. The present invention preferably has good antibacterial effect, stable properties, and is suitable for protecting skin cells or spermatogonia that need to be injected into the human body. Aminoglycoside antibiotics, and four antibiotics, gentamicin, kanamycin, and amikacin or tobramycin. The choice of antibiotics not only has antibacterial effect, more importantly, it will not affect the preserved cell viability.
在保护液内加入氧合血红蛋白可以为保护液提供低氧氧气来源，进而提高皮肤细胞或精原细胞的保存活率。Adding oxyhemoglobin in the protection solution can provide a hypoxic oxygen source for the protection solution, thereby improving the survival rate of skin cells or spermatogonia.
在保护液内加入抗氧化剂可以为保护液提供抗氧化保护。Adding antioxidants to the protective solution can provide antioxidant protection for the protective solution.
在保护液中加入吐温80、棕榈酰胺、海藻酸钾和月桂酰肌氨酸的混合物可以改善保护液内的氧气传递和物质交换，提高皮肤细胞或精原细胞的活率。Adding a mixture of Tween 80, palmitamide, potassium alginate and lauroyl sarcosine to the protective solution can improve the oxygen transfer and material exchange in the protective solution, and increase the vitality of skin cells or spermatogonia.
在保护液中加入柠檬酸、丙酮酸、延胡索酸和乙酰辅酶A可以为保存的细胞进一步提供能量，可以延长对皮肤细胞或精原细胞的保存时间。Adding citric acid, pyruvic acid, fumaric acid and acetyl coenzyme A to the protection solution can further provide energy for the preserved cells and prolong the preservation time of skin cells or spermatogonia.
不同于一般培养基中添加的血红蛋白作为营养物质，本发明的氧合血红蛋白主要作用是作为低浓度的氧气供应物质，皮肤细胞或精原细胞起初消耗的是溶液中物理溶解的氧气，随着氧分压下降和二氧化氮浓度的提高，逐步可以从氧合血红蛋白获得氧气。添加的人血液中天然存在的抗氧化剂不同于一般意义上的保护细胞抗氧化，而是作为一个氧气供应过量的平衡保护。Different from the hemoglobin added in the general culture medium as a nutrient substance, the main function of the oxyhemoglobin of the present invention is as a low-concentration oxygen supply substance. What the skin cells or spermatogonia initially consume is the physically dissolved oxygen in the solution. Oxygen can gradually be obtained from oxyhemoglobin as the partial pressure decreases and the concentration of nitrogen dioxide increases. The addition of antioxidants naturally present in human blood is not intended to protect cells against oxidation in the general sense, but instead serves as a balanced protection against excess oxygen supply.
配置保护液时，先配置DMEM培养基水溶液，然后加入氨基糖甙类抗生素，还可以加入氧合血红蛋白、人血液中天然存在的抗氧化剂、吐温80、棕榈酰胺、海藻酸钾、月桂酰肌氨酸、柠檬酸、丙酮酸、延胡索酸和乙酰辅酶A，过滤除菌，存储于4℃冰箱。使用时与刚采集的皮肤细胞或精原细胞按1:1体积比例混合。When configuring the protective solution, first configure the DMEM medium aqueous solution, and then add aminoglycoside antibiotics, and also add oxyhemoglobin, antioxidants naturally present in human blood, Tween 80, palmitamide, potassium alginate, and lauroyl inosine amino acid, citric acid, pyruvic acid, fumaric acid and acetyl-CoA, filter-sterilized, and stored in a 4°C refrigerator. When used, it is mixed with freshly collected skin cells or spermatogonia in a volume ratio of 1:1.
皮肤精原细胞保护液与皮肤细胞或精原细胞的混合液可以临时存储于4℃冰箱或者转运过程中存储于含有冰盒的、并且保障温度低于4℃的密闭容器内。The mixture of skin spermatogonia protection solution and skin cells or spermatogonia can be temporarily stored in a refrigerator at 4°C or stored in an airtight container with an ice box and a temperature lower than 4°C during transportation.
本发明的提供的皮肤精原细胞保护液为皮肤细胞或精原细胞提供了适宜和稳定的保存环境，其更接近体内环境，可以显著提高皮肤细胞或精原细胞保存后的细胞活率。The skin spermatogonia protective solution provided by the present invention provides a suitable and stable preservation environment for skin cells or spermatogonia, which is closer to the environment in the body, and can significantly improve the cell viability of skin cells or spermatogonia after preservation.
具体实施方式detailed description
实施例1Example 1
一种皮肤精原干细胞保护液，该保护液主要是将庆大霉素用DMEM培养基水溶液溶解而成，每毫升所述DMEM培养基水溶液溶解庆大霉素100单位，所述DMEM培养基水溶液的浓度为16.9g/L。A skin spermatogonial stem cell protection solution, the protection solution is mainly formed by dissolving gentamicin with DMEM medium aqueous solution, 100 units of gentamicin are dissolved in each milliliter of the DMEM medium aqueous solution, and the DMEM medium aqueous solution The concentration is 16.9g/L.
实施例2Example 2
一种皮肤精原干细胞保护液，该保护液主要是将卡那霉素用DMEM培养基水溶液溶解而成，每毫升所述DMEM培养基水溶液溶解卡那霉素100单位，所述DMEM培养基水溶液的浓度为16.9g/L。A skin spermatogonial stem cell protection solution, the protection solution is mainly formed by dissolving kanamycin with DMEM medium aqueous solution, and dissolving 100 units of kanamycin per milliliter of the DMEM medium aqueous solution, and the DMEM medium aqueous solution The concentration is 16.9g/L.
实施例3Example 3
一种皮肤精原干细胞保护液，该保护液主要是将阿米卡星用DMEM培养基水溶液溶解而成，每毫升所述DMEM培养基水溶液溶解阿米卡星100单位，所述DMEM培养基水溶液的浓度为16.9g/L。A skin spermatogonial stem cell protection solution, the protection solution is mainly formed by dissolving amikacin with DMEM medium aqueous solution, dissolving 100 units of amikacin per milliliter of the DMEM medium aqueous solution, and the DMEM medium aqueous solution The concentration is 16.9g/L.
实施例4Example 4
一种皮肤精原干细胞保护液，该保护液主要是将庆大霉素、卡那霉素和妥布霉素用DMEM培养基水溶液溶解而成，每毫升所述DMEM培养基水溶液分别溶解庆大霉素100单位、卡那霉素20单位、妥布霉素20单位，所述DMEM培养基水溶液的浓度为16.9g/L。A skin spermatogonial stem cell protection solution, the protection solution is mainly formed by dissolving gentamicin, kanamycin and tobramycin with DMEM medium aqueous solution, and each milliliter of the DMEM medium aqueous solution dissolves gentamicin respectively Mycin 100 units, kanamycin 20 units, tobramycin 20 units, the concentration of the DMEM medium aqueous solution is 16.9g/L.
实施例5Example 5
一种皮肤精原干细胞保护液，该保护液主要是将妥布霉素用DMEM培养基水溶液溶解而成，每毫升所述DMEM培养基水溶液溶解妥布霉素100单位，所述DMEM培养基水溶液的浓度为10.0g/L。A skin spermatogonial stem cell protection solution, the protection solution is mainly formed by dissolving tobramycin in DMEM medium aqueous solution, 100 units of tobramycin are dissolved in each milliliter of the DMEM medium aqueous solution, and the DMEM medium aqueous solution The concentration is 10.0g/L.
实施例6Example 6
一种皮肤精原干细胞保护液，该保护液主要是将庆大霉素用DMEM培养基水溶液溶解而成，每毫升所述DMEM培养基水溶液溶解庆大霉素100单位，所述DMEM培养基水溶液的浓度为25.0g/L。A skin spermatogonial stem cell protection solution, the protection solution is mainly formed by dissolving gentamicin with DMEM medium aqueous solution, 100 units of gentamicin are dissolved in each milliliter of the DMEM medium aqueous solution, and the DMEM medium aqueous solution The concentration is 25.0g/L.
实施例7Example 7
一种皮肤精原干细胞保护液，该保护液主要是将庆大霉素和氧合血红蛋白用DMEM培养基水溶液溶解而成，每毫升所述DMEM培养基水溶液分别溶解庆大霉素100单位、氧合血红蛋白400μg，所述DMEM培养基水溶液的浓度为16.9g/L。A skin spermatogonial stem cell protection solution, the protection solution is mainly formed by dissolving gentamicin and oxyhemoglobin with DMEM medium aqueous solution, and each milliliter of the DMEM medium aqueous solution dissolves 100 units of gentamycin and oxygenated hemoglobin respectively. Synthesized hemoglobin 400 μ g, the concentration of described DMEM medium aqueous solution is 16.9g/L.
实施例8Example 8
一种皮肤精原干细胞保护液，该保护液主要是将庆大霉素和氧合血红蛋白用DMEM培养基水溶液溶解而成，每毫升所述DMEM培养基水溶液分别溶解庆大霉素100单位、氧合血红蛋白600μg，所述DMEM培养基水溶液的浓度为16.9g/L。A skin spermatogonial stem cell protection solution, the protection solution is mainly formed by dissolving gentamicin and oxyhemoglobin with DMEM medium aqueous solution, and each milliliter of the DMEM medium aqueous solution dissolves 100 units of gentamycin and oxygenated hemoglobin respectively. Hemoglobin 600 μg, the concentration of described DMEM medium aqueous solution is 16.9g/L.
实施例9Example 9
一种皮肤精原干细胞保护液，该保护液主要是将庆大霉素和氧合血红蛋白用DMEM培养基水溶液溶解而成，每毫升所述DMEM培养基水溶液分别溶解庆大霉素100单位、氧合血红蛋白1000μg，所述DMEM培养基水溶液的浓度为16.9g/L。A skin spermatogonial stem cell protection solution, the protection solution is mainly formed by dissolving gentamicin and oxyhemoglobin with DMEM medium aqueous solution, and each milliliter of the DMEM medium aqueous solution dissolves 100 units of gentamycin and oxygenated hemoglobin respectively. Hemoglobin 1000 μg, the concentration of described DMEM medium aqueous solution is 16.9g/L.
实施例10Example 10
一种皮肤精原干细胞保护液，该保护液主要是将庆大霉素和维生素E用DMEM培养基水溶液溶解而成，每毫升所述DMEM培养基水溶液分别溶解庆大霉素100单位、维生素E0.1μmol，所述DMEM培养基水溶液的浓度为16.9g/L。A skin spermatogonial stem cell protective solution, the protective solution is mainly formed by dissolving gentamicin and vitamin E with DMEM medium aqueous solution, and 100 units of gentamicin and vitamin E are dissolved in each milliliter of the DMEM medium aqueous solution respectively. .1 μmol, the concentration of the DMEM medium aqueous solution is 16.9g/L.
实施例11Example 11
一种皮肤精原干细胞保护液，该保护液主要是将庆大霉素、维生素E和谷胱甘肽用DMEM培养基水溶液溶解而成，每毫升所述DMEM培养基水溶液分别溶解庆大霉素100单位、维生素E0.3μmol、谷胱甘肽0.3μmol，所述DMEM培养基水溶液的浓度为16.9g/L。A skin spermatogonial stem cell protection solution, the protection solution is mainly formed by dissolving gentamicin, vitamin E and glutathione with DMEM medium aqueous solution, and each milliliter of the DMEM medium aqueous solution dissolves gentamicin respectively 100 units, vitamin E 0.3 μmol, glutathione 0.3 μmol, the concentration of the DMEM medium aqueous solution is 16.9 g/L.
实施例12Example 12
一种皮肤精原干细胞保护液，该保护液主要是将庆大霉素、氧合血红蛋白、维生素E和谷胱甘肽用DMEM培养基水溶液溶解而成，每毫升所述DMEM培养基水溶液分别溶解庆大霉素100单位、氧合血红蛋白600μg、维生素E0.25μmol、谷胱甘肽0.25μmol，所述DMEM培养基水溶液的浓度为16.9g/L。A skin spermatogonial stem cell protection solution, the protection solution is mainly formed by dissolving gentamicin, oxyhemoglobin, vitamin E and glutathione with DMEM medium aqueous solution, and each milliliter of the DMEM medium aqueous solution dissolves respectively Gentamicin 100 units, oxyhemoglobin 600 μg, vitamin E 0.25 μmol, glutathione 0.25 μmol, the concentration of the DMEM medium aqueous solution is 16.9 g/L.
实施例13Example 13
一种皮肤精原干细胞保护液，该保护液主要是将庆大霉素、氧合血红蛋白、维生素C和维生素E用DMEM培养基水溶液溶解而成，每毫升所述DMEM培养基水溶液分别溶解庆大霉素100单位、氧合血红蛋白600μg、维生素C0.5μmol、维生素E0.5μmol，所述DMEM培养基水溶液的浓度为16.9g/L。A skin spermatogonial stem cell protection solution, the protection solution is mainly formed by dissolving gentamicin, oxyhemoglobin, vitamin C and vitamin E with DMEM medium aqueous solution, and each milliliter of the DMEM medium aqueous solution dissolves gentamicin respectively. Mycin 100 units, oxyhemoglobin 600 μg, vitamin C 0.5 μmol, vitamin E 0.5 μmol, the concentration of the DMEM medium aqueous solution is 16.9 g/L.
实施例14Example 14
一种皮肤精原干细胞保护液，该保护液主要是将庆大霉素、吐温80、棕榈酰胺、海藻酸钾和月桂酰肌氨酸用DMEM培养基水溶液溶解而成，每毫升所述DMEM培养基水溶液分别溶解庆大霉素100单位、吐温80 0.5μg、棕榈酰胺80μg、海藻酸钾50μg、月桂酰肌氨酸100μg，所述DMEM培养基水溶液的浓度为16.9g/L。A skin spermatogonial stem cell protective solution, the protective solution is mainly formed by dissolving gentamicin, Tween 80, palmitamide, potassium alginate and lauroyl sarcosine with DMEM medium aqueous solution, and each milliliter of the DMEM 100 units of gentamicin, 0.5 μg of Tween 80, 80 μg of palmitamide, 50 μg of potassium alginate, and 100 μg of lauroyl sarcosine were respectively dissolved in the aqueous medium solution, and the concentration of the aqueous DMEM medium solution was 16.9 g/L.
实施例15Example 15
一种皮肤精原干细胞保护液，该保护液主要是将庆大霉素、氧合血红蛋白、吐温80、棕榈酰胺、海藻酸钾和月桂酰肌氨酸用DMEM培养基水溶液溶解而成，每毫升所述DMEM培养基水溶液分别溶解庆大霉素100单位、氧合血红蛋白600μg、吐温80 1.0μg、棕榈酰胺20μg、海藻酸钾10μg、月桂酰肌氨酸200μg，所述DMEM培养基水溶液的浓度为16.9g/L。A skin spermatogonial stem cell protection solution, the protection solution is mainly formed by dissolving gentamicin, oxyhemoglobin, Tween 80, palmitoamide, potassium alginate and lauroyl sarcosine with DMEM medium aqueous solution, each Dissolve 100 units of gentamicin, 600 μg of oxyhemoglobin, 1.0 μg of Tween 80, 20 μg of palmitamide, 10 μg of potassium alginate, and 200 μg of lauroyl sarcosine in milliliters of the DMEM medium aqueous solution. The concentration is 16.9g/L.
实施例16Example 16
一种皮肤精原干细胞保护液，该保护液主要是将庆大霉素、氧合血红蛋白、维生素E、谷胱甘肽、吐温80、棕榈酰胺、海藻酸钾和月桂酰肌氨酸用DMEM培养基水溶液溶解而成，每毫升所述DMEM培养基水溶液分别溶解庆大霉素100单位、氧合血红蛋白600mg、维生素E0.25μmol、谷胱甘肽0.25μmol、吐温80 0.5μg、棕榈酰胺60μg、海藻酸钾30μg、月桂酰肌氨酸150μg，所述DMEM培养基水溶液的浓度为16.9g/L。A protective solution for skin spermatogonial stem cells, the protective solution mainly uses DMEM with gentamicin, oxyhemoglobin, vitamin E, glutathione, Tween 80, palmitamide, potassium alginate and lauroyl sarcosine It is formed by dissolving an aqueous medium solution, and each milliliter of the DMEM aqueous medium solution dissolves 100 units of gentamicin, 600 mg of oxyhemoglobin, 0.25 μmol of vitamin E, 0.25 μmol of glutathione, 0.5 μg of Tween 80, and 60 μg of palmitamide , potassium alginate 30 μg, lauroyl sarcosine 150 μg, the concentration of the DMEM medium aqueous solution is 16.9 g/L.
实施例17Example 17
一种皮肤精原干细胞保护液，该保护液主要是将庆大霉素、柠檬酸、丙酮酸、延胡索酸和乙酰辅酶A用DMEM培养基水溶液溶解而成，每毫升所述DMEM培养基水溶液溶解分别庆大霉素100单位、柠檬酸10μg、丙酮酸80μg、延胡索酸10μg和乙酰辅酶A 80μg，所述DMEM培养基水溶液的浓度为16.9g/L。A skin spermatogonial stem cell protection solution, the protection solution is mainly formed by dissolving gentamicin, citric acid, pyruvic acid, fumaric acid and acetyl-CoA with DMEM medium aqueous solution, and each milliliter of the DMEM medium aqueous solution dissolves respectively 100 units of gentamicin, 10 μg of citric acid, 80 μg of pyruvate, 10 μg of fumaric acid and 80 μg of acetyl-CoA, the concentration of the DMEM medium aqueous solution is 16.9 g/L.
实施例18Example 18
一种皮肤精原干细胞保护液，该保护液主要是将庆大霉素、氧合血红蛋白、维生素E、谷胱甘肽、柠檬酸、丙酮酸、延胡索酸和乙酰辅酶A用DMEM培养基水溶液溶解而成，每毫升所述DMEM培养基水溶液分别溶解庆大霉素100单位、氧合血红蛋白600μg、维生素E0.25μmol、谷胱甘肽0.25μmol、柠檬酸1μg、丙酮酸50μg、延胡索酸20μg和乙酰辅酶A 150μg，所述DMEM培养基水溶液的浓度为16.9g/L。A protective solution for skin spermatogonial stem cells, the protective solution is mainly prepared by dissolving gentamicin, oxyhemoglobin, vitamin E, glutathione, citric acid, pyruvic acid, fumaric acid and acetyl-CoA in aqueous solution of DMEM medium 100 units of gentamicin, 600 μg of oxyhemoglobin, 0.25 μmol of vitamin E, 0.25 μmol of glutathione, 1 μg of citric acid, 50 μg of pyruvate, 20 μg of fumaric acid and acetyl-CoA were dissolved in each milliliter of the DMEM medium aqueous solution. 150 μg, the concentration of the DMEM medium aqueous solution is 16.9g/L.
实施例19Example 19
一种皮肤精原干细胞保护液，该保护液主要是将庆大霉素、氧合血红蛋白、维生素E、谷胱甘肽、吐温80、棕榈酰胺、海藻酸钾、月桂酰肌氨酸、柠檬酸、丙酮酸、延胡索酸和乙酰辅酶A用DMEM培养基水溶液溶解而成，每毫升所述DMEM培养基水溶液分别溶解庆大霉素100单位、氧合血红蛋白600μg、维生素E0.25μmol、谷胱甘肽0.25μmol、吐温80 0.5μg、棕榈酰胺60μg、海藻酸钾30μg、月桂酰肌氨酸150μg、柠檬酸4μg、丙酮酸60μg、延胡索酸15μg和乙酰辅酶A 120μg，所述DMEM培养基水溶液的浓度为16.9g/L。A skin essence stem cell protective solution, the protective solution is mainly composed of gentamicin, oxyhemoglobin, vitamin E, glutathione, Tween 80, palmitamide, potassium alginate, lauroyl sarcosine, lemon Acid, pyruvate, fumaric acid and acetyl-CoA are dissolved in DMEM medium aqueous solution, and each milliliter of the DMEM medium aqueous solution dissolves 100 units of gentamicin, 600 μg of oxyhemoglobin, 0.25 μmol of vitamin E, and glutathione 0.25 μmol, Tween 80 0.5 μg, palmitoamide 60 μg, potassium alginate 30 μg, lauroyl sarcosine 150 μg, citric acid 4 μg, pyruvate 60 μg, fumaric acid 15 μg and acetyl-CoA 120 μg, the concentration of the DMEM medium aqueous solution is 16.9g/L.
对照例1Comparative example 1
一种皮肤精原干细胞保护液，该保护液主要是将青霉素用DMEM培养基水溶液溶解而成，每毫升所述DMEM培养基水溶液溶解青霉素100单位，所述DMEM培养基水溶液的浓度为16.9g/L。A skin spermatogonial stem cell protection solution, the protection solution is mainly formed by dissolving penicillin with DMEM medium aqueous solution, 100 units of penicillin are dissolved in each milliliter of the DMEM medium aqueous solution, and the concentration of the DMEM medium aqueous solution is 16.9g/ L.
对照例2Comparative example 2
一种皮肤精原干细胞保护液，该保护液主要是将庆大霉素和转铁蛋白用DMEM培养基水溶液溶解而成，每毫升所述DMEM培养基水溶液分别溶解庆大霉素100单位、转铁蛋白400μg，所述DMEM培养基水溶液的浓度为16.9g/L。A skin spermatogonial stem cell protection solution, the protection solution is mainly formed by dissolving gentamicin and transferrin with DMEM medium aqueous solution, and each milliliter of the DMEM medium aqueous solution dissolves 100 units of gentamicin and transferrin respectively. 400 μg of ferritin, the concentration of the DMEM medium aqueous solution is 16.9 g/L.
对照例3Comparative example 3
一种皮肤精原干细胞保护液，该保护液主要是将庆大霉素、维生素E和谷胱甘肽用DMEM培养基水溶液溶解而成，每毫升所述DMEM培养基水溶液分别溶解庆大霉素100单位、维生素E0.1μmol、谷胱甘肽0.9μmol，所述DMEM培养基水溶液的浓度为16.9g/L。A skin spermatogonial stem cell protection solution, the protection solution is mainly formed by dissolving gentamicin, vitamin E and glutathione with DMEM medium aqueous solution, and each milliliter of the DMEM medium aqueous solution dissolves gentamicin respectively 100 units, vitamin E 0.1 μmol, glutathione 0.9 μmol, the concentration of the DMEM medium aqueous solution is 16.9 g/L.
对照例4Comparative example 4
一种皮肤精原干细胞保护液，该保护液主要是将庆大霉素、司盘80、棕榈酰胺、海藻酸钾和月桂酰肌氨酸用DMEM培养基水溶液溶解而成，每毫升所述DMEM培养基水溶液分别溶解庆大霉素100单位、司盘80 0.5μg、棕榈酰胺80μg、枸橼酸钾50μg、月桂酰肌氨酸100μg，所述DMEM培养基水溶液的浓度为16.9g/L。A skin spermatogonial stem cell protective solution, the protective solution is mainly formed by dissolving gentamicin, Span 80, palmitamide, potassium alginate and lauroyl sarcosine with DMEM medium aqueous solution, and each milliliter of the DMEM 100 units of gentamicin, 0.5 μg of Span 80, 80 μg of palmitamide, 50 μg of potassium citrate, and 100 μg of lauroyl sarcosine were respectively dissolved in the aqueous medium solution, and the concentration of the aqueous DMEM medium solution was 16.9 g/L.
对照例5Comparative example 5
一种皮肤精原干细胞保护液，该保护液主要是将庆大霉素、吐温80、棕榈酰胺和海藻酸钾用DMEM培养基水溶液溶解而成，每毫升所述DMEM培养基水溶液分别溶解庆大霉素100单位、吐温80 0.5μg、棕榈酰胺80μg、海藻酸钾50μg，所述DMEM培养基水溶液的浓度为16.9g/L。A skin spermatogonial stem cell protection solution, the protection solution is mainly formed by dissolving gentamicin, Tween 80, palmitamide and potassium alginate with DMEM medium aqueous solution, and each milliliter of the DMEM medium aqueous solution dissolves gentamicin respectively. Amamicin 100 units, Tween 80 0.5 μg, palmitamide 80 μg, potassium alginate 50 μg, the concentration of the DMEM medium aqueous solution is 16.9 g/L.
对照例6Comparative example 6
一种皮肤精原干细胞保护液，该保护液主要是将庆大霉素、柠檬酸钾、丙酮酸钠、延胡索酸和乙酰辅酶A用DMEM培养基水溶液溶解而成，每毫升所述DMEM培养基水溶液分别溶解庆大霉素100单位、柠檬酸钾10μg、丙酮酸钠80μg、延胡索酸10μg和乙酰辅酶A 80μg，所述DMEM培养基水溶液的浓度为16.9g/L。A skin spermatogonial stem cell protection solution, the protection solution is mainly formed by dissolving gentamicin, potassium citrate, sodium pyruvate, fumaric acid and acetyl-CoA with DMEM medium aqueous solution, and each milliliter of the DMEM medium aqueous solution 100 units of gentamicin, 10 μg of potassium citrate, 80 μg of sodium pyruvate, 10 μg of fumaric acid and 80 μg of acetyl-CoA were dissolved respectively, and the concentration of the DMEM medium aqueous solution was 16.9 g/L.
对照例7Comparative example 7
一种皮肤精原干细胞保护液，该保护液主要是将庆大霉素、柠檬酸、丙酮酸和延胡索酸用DMEM培养基水溶液溶解而成，每毫升所述DMEM培养基水溶液分别溶解庆大霉素100单位、柠檬酸10μg、丙酮酸80μg和延胡索酸10μg，所述DMEM培养基水溶液的浓度为16.9g/L。A skin spermatogonial stem cell protection solution, the protection solution is mainly formed by dissolving gentamicin, citric acid, pyruvic acid and fumaric acid with DMEM medium aqueous solution, and each milliliter of the DMEM medium aqueous solution dissolves gentamicin respectively 100 units, 10 μg of citric acid, 80 μg of pyruvic acid and 10 μg of fumaric acid, the concentration of the DMEM medium aqueous solution is 16.9 g/L.
试验例1Test example 1
取存储于4℃冰箱的灭菌保护液，与刚采集的皮肤细胞或精原细胞按1:1体积比例混合均匀，置于4℃环境，不同的时间点各采样一次，以MTT法测定细胞活力，以第一次取样的活力为100％。Take the sterilized protective solution stored in the refrigerator at 4°C, mix it with the skin cells or spermatogonia just collected at a volume ratio of 1:1, place it in an environment at 4°C, take samples at different time points, and measure the cells by MTT method For vitality, take the vitality of the first sampling as 100%.
本组试验主要考察氨基糖苷类抗生素和DMEM浓度的影响，以对照例1保护液分别保存的皮肤细胞和精原细胞为对照1组，以实施例2-6保护液分别保存的皮肤细胞和精原细胞为实验1-5组，各实验组和对照组的皮肤细胞和精原细胞分别在24h后进入试验程序，调整皮肤细胞和精原细胞的密度均为1×106cells/mL。按细胞悬液：0.4％台盼蓝＝3:1(v:v)充分混匀，取20μL细胞悬液加入细胞计数板中，用Countstar细胞计数器检测各实验组和对照组保护液内表皮干细胞和精原干细胞的活率，结果如表1所示。This group of experiments mainly investigates the influence of the concentration of aminoglycoside antibiotics and DMEM. The skin cells and spermatogonia preserved respectively in the protective solution of Control Example 1 are used as the control group 1, and the skin cells and spermatogonia preserved respectively in the protective solutions of Examples 2-6 The original cells were in the experimental groups 1-5. The skin cells and spermatogonia of each experimental group and control group entered the test procedure after 24 hours, and the density of the skin cells and spermatogonia was adjusted to be 1×106 cells/mL. According to the cell suspension: 0.4% trypan blue = 3:1 (v:v), mix thoroughly, take 20 μL of the cell suspension and add it to the cell counting plate, and use the Countstar cell counter to detect the epidermal stem cells in the protection solution of each experimental group and control group and the activity rate of spermatogonial stem cells, the results are shown in Table 1.
表1试验例1各实施例与对照例的细胞活率结果The cell viability result of each embodiment and control example of table 1 test example 1
由表1可知，实施例1-6的保护液分别保存的皮肤细胞和精原细胞的细胞活率要比使用对照例1组使用的保护液分别保存的皮肤细胞和精原细胞的细胞活率高；实施例4使用的保护液分别保存的皮肤细胞和精原细胞的细胞活率要比使用实施例1-3、5-6使用的保护液分别保存的皮肤细胞和精原细胞的细胞活率高。As can be seen from Table 1, the cell viability of the skin cells and spermatogonia preserved respectively by the protective solutions of Examples 1-6 is higher than the cell viability of the skin cells and spermatogonial cells preserved respectively by the protective solution used in the control group 1 High; the cell viability of the skin cells and spermatogonia that the protection solution that embodiment 4 uses preserves respectively than uses the skin cells that the protection solution that embodiment 1-3,5-6 uses preserves respectively and the cell viability of spermatogonia High rate.
由此得出，本发明提供的保护液中的氨基糖苷类抗生素换成了青霉素等抗生素后，皮肤细胞或精原细胞的活率显著降低，并且氨基糖苷类抗生素采用庆大霉素、卡那霉素和妥布霉素的混合物后，保存的皮肤细胞和精原细胞的细胞活率更高。It can thus be concluded that after the aminoglycoside antibiotics in the protective solution provided by the present invention are replaced by antibiotics such as penicillin, the viability of skin cells or spermatogonia is significantly reduced, and the aminoglycoside antibiotics are gentamicin, kanamycin, etc. The cell viability of preserved skin cells and spermatogonia was higher after treatment with a mixture of tobramycin and tobramycin.
试验例2Test example 2
本组试验主要考察氧合血红蛋白的影响，以对照例2保护液分别保存的皮肤细胞和精原细胞为对照2组，以实施例1、7和8保护液分别保存的皮肤细胞和精原细胞为实验7-9组，检测过程如试验例1，保存24h后的检测结果如表2。This group of experiments mainly investigated the influence of oxyhemoglobin. The skin cells and spermatogonia preserved in the protective solution of Comparative Example 2 were used as the control group 2, and the skin cells and spermatogonia preserved in the protective solutions of Examples 1, 7 and 8 were used respectively. It is the experimental group 7-9, the detection process is as in the test example 1, and the detection results after storage for 24 hours are shown in Table 2.
表2试验例2各实施例与对照例的细胞活率结果The cell viability result of table 2 test example 2 each embodiment and control example
由表2可知，实施例7、8的保护液分别保存的皮肤细胞和精原细胞的细胞活率要比使用对照例2组使用的保护液分别保存的皮肤细胞和精原细胞的细胞活率高；实施例8使用的保护液分别保存的皮肤细胞和精原细胞的细胞活率要比使用实施例7使用的保护液分别保存的皮肤细胞和精原细胞的细胞活率高。As can be seen from Table 2, the cell viability of the skin cells and spermatogonia preserved respectively by the protective solution of Examples 7 and 8 is higher than the cell viability of the skin cells and spermatogonia preserved respectively by the protective solution used in the control group 2 High; the cell viability of the skin cells and spermatogonia preserved by the protective solution used in Example 8 is higher than the cell viability of the skin cells and spermatogonia preserved by the protective solution used in Example 7.
由此得出，本发明提供的保护液中的氧合血红蛋白换成了转铁蛋白后，皮肤细胞或精原细胞的活率显著降低，而氧合血红蛋白的浓度为600μg/mL时，保护液分别保存的皮肤细胞和精原细胞的细胞活率高更高。It can thus be concluded that after the oxyhemoglobin in the protective solution provided by the present invention is replaced by transferrin, the viability of skin cells or spermatogonia is significantly reduced, and when the concentration of oxyhemoglobin is 600 μg/mL, the protective solution The cell viability of skin cells and spermatogonia preserved separately is higher.
试验例3Test example 3
本组试验主要考察抗氧化剂的影响，以对照例3分别保存的皮肤细胞和精原细胞为对照组，实施例7、10-12为实验组，检测过程如试验例1，保存48h后的检测结果如表3。This group of experiments mainly investigates the influence of antioxidants. The skin cells and spermatogonia preserved in Control Example 3 are used as the control group, and Examples 7 and 10-12 are used as the experimental group. The results are shown in Table 3.
表3试验例3各实施例与对照例的细胞活率结果Table 3 test example 3 each embodiment and the cell viability result of control example
由表3可知，实施例10-12的保护液分别保存的皮肤细胞和精原细胞的细胞活率要比使用实施例7、对照例3组使用的保护液分别保存的皮肤细胞和精原细胞的细胞活率高；实施例12使用的保护液分别保存的皮肤细胞和精原细胞的细胞活率要比使用实施例10、11使用的保护液分别保存的皮肤细胞和精原细胞的细胞活率高。As can be seen from Table 3, the cell viability of the skin cells and spermatogonia preserved in the protective solution of Examples 10-12 is higher than that of the skin cells and spermatogonia preserved in the protective solution used in Example 7 and Comparative Example 3. The cell viability rate is high; the cell viability rate of the skin cells and spermatogonia preserved respectively by the protective solution used in Example 12 is higher than the cell viability of the skin cells and spermatogonia preserved respectively by the protective solution used in Example 10 and 11. High rate.
由此得出，本发明提供的保护液中的抗氧化剂可以显著提高皮肤细胞或精原细胞的活率。It can thus be concluded that the antioxidants in the protection solution provided by the present invention can significantly increase the viability of skin cells or spermatogonia.
试验例4Test example 4
以对照例4-7分别保存的皮肤细胞和精原细胞为对照组，实施例4、实施例7、实施例10、实施例14、16、实施例17和实施例19为实验组，各实验组和对照组的皮肤细胞和精原细胞分别在24h、48h、96h、240h、480h后进入试验程序，调整皮肤细胞和精原细胞的密度均为1×106cells/mL。按细胞悬液：0.4％台盼蓝＝3:1(v:v)充分混匀，取20μL细胞悬液加入细胞计数板中，用Countstar细胞计数器检测各实验组和对照组保护液内表皮干细胞和精原干细胞的活率，结果如表4所示。The skin cells and spermatogonia preserved respectively in Control Example 4-7 are as the control group, and Example 4, Example 7, Example 10, Example 14, 16, Example 17 and Example 19 are the experimental group, and each experiment The skin cells and spermatogonia of the group and the control group entered the test program after 24h, 48h, 96h, 240h, and 480h respectively, and the density of skin cells and spermatogonia was adjusted to be 1×106 cells/mL. According to the cell suspension: 0.4% trypan blue = 3:1 (v:v), mix thoroughly, take 20 μL of the cell suspension and add it to the cell counting plate, and use the Countstar cell counter to detect the epidermal stem cells in the protection solution of each experimental group and control group and the viability of spermatogonial stem cells, the results are shown in Table 4.
表4试验例4各实施例与对照例的细胞活率结果The cell viability result of table 4 test example 4 each embodiment and control example
由表4可知，实施例14、16-17和实施例19的保护液分别保存的皮肤细胞和精原细胞的细胞活率要比使用实施例1、实施例7和实施例10对照例4-7、使用的保护液分别保存的皮肤细胞和精原细胞的细胞活率高；实施例17和实施例19使用的保护液分别保存的皮肤细胞和精原细胞的时间要比使用实施例14和16的保存时间长。As can be seen from Table 4, the cell viability of the skin cells and spermatogonia preserved respectively by the protective solution of Examples 14, 16-17 and Example 19 is higher than that using Example 1, Example 7 and Example 10 Comparative Example 4- 7. The cell viability of the skin cells and spermatogonia preserved respectively by the protective solution used is high; the time of the skin cells and spermatogonia preserved by the protective solution used in Example 17 and Example 19 is higher than that of using Example 14 and 16 has a long storage time.
由此得出，本发明提供的保护液中的吐温80、棕榈酰胺、海藻酸钾和月桂酰肌氨酸组合物可以显著提高皮肤细胞或精原细胞的活率，柠檬酸、丙酮酸、延胡索酸和乙酰辅酶A组合物可以显著提高保护液对皮肤细胞或精原细胞的保存时间。Thus, the composition of Tween 80, palmitamide, potassium alginate and lauroyl sarcosine in the protective solution provided by the invention can significantly improve the viability of skin cells or spermatogonia, and citric acid, pyruvic acid, The combination of fumaric acid and acetyl coenzyme A can significantly improve the preservation time of the protective solution on skin cells or spermatogonia.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610622068.2ACN107668023A (en) | 2016-08-01 | 2016-08-01 | A kind of skin spermatogonium protects liquid |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610622068.2ACN107668023A (en) | 2016-08-01 | 2016-08-01 | A kind of skin spermatogonium protects liquid |
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| CN107668023Atrue CN107668023A (en) | 2018-02-09 |
| Application Number | Title | Priority Date | Filing Date |
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| CN201610622068.2APendingCN107668023A (en) | 2016-08-01 | 2016-08-01 | A kind of skin spermatogonium protects liquid |
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| US20130040281A1 (en)* | 2011-08-11 | 2013-02-14 | Robert A. Dracker | Procurement of Placental Stem Cells |
| CN104396940A (en)* | 2014-10-11 | 2015-03-11 | 张炳强 | Tissue sample preservative solution and preparation method thereof |
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