CN111662864A - Method for differentiating umbilical cord mesenchymal stem cells into dermal stem cells through in-vitro induction - Google Patents
- ️Tue Sep 15 2020
技术领域technical field
本发明属于干细胞、细胞培养领域，具体涉及体外诱导脐带间充质干细胞分化为真皮干细胞的方法。The invention belongs to the field of stem cells and cell culture, and in particular relates to a method for inducing umbilical cord mesenchymal stem cells to differentiate into dermal stem cells in vitro.
背景技术Background technique
皮肤是人体最大的器官，是人体与外界接触的第一道屏障。烧烫伤、创伤、及慢性疾病如糖尿病并发症造成的大面缺损和深度损伤无法自发修复，需皮肤修补和植皮等人工干预治疗。由于移植皮肤来源和应用受到多重限制，研究实用的皮肤替代物一直方兴未艾。The skin is the largest organ of the human body and the first barrier between the human body and the outside world. Large-scale defects and deep injuries caused by burns, trauma, and chronic diseases such as diabetes complications cannot be repaired spontaneously, and require manual intervention such as skin repair and skin grafting. Due to multiple limitations on the source and application of grafted skin, research into practical skin substitutes has been in the ascendant.
另一方面，作为皮肤病中最常见的脱发，在日常生活中给众多的患者带来很大的烦恼。病理性脱发是指头发异常或过度的脱落。实质上是毛囊细胞长期处于休止期或退行期的表现。随着生活工作压力的增加，脱发年龄越来越年轻。On the other hand, hair loss, which is the most common type of skin disease, causes great trouble for many patients in daily life. Pathological alopecia refers to abnormal or excessive hair loss. In essence, the hair follicle cells are in the telogen phase or degenerative phase for a long time. With the increase of life and work pressure, the age of hair loss is getting younger and younger.
当然治疗脱发要首先从病因上着手，但是许多脱发原因是不能完全去除的。局部对症用药和全身用药，包括促进血液循环和中医中药扶正固本疗法疗效都有限。毛发移植价格昂贵，而且因为毛囊的生存条件没有得到足够改善，移植的毛发难以长期生存。皮肤中存在着多种不同的干细胞。毛囊干细胞(hair follicle stem cells,HFSCs)，存在于毛囊上段的突起区域，能够分化成毛囊结构的所有不同类型细胞，进而形成新的毛发。毛囊干细胞主要来源于分布在表皮基底层的表皮干细胞(Epidermal stem cell)，其数量随着年龄增大而减少；而诱导人表皮干细胞分化成毛囊干细胞的最重要环节是真皮干细胞，又称皮肤前体细胞(skin-derived precursors，SKPs)。SKPs广泛的存在于真皮的乳头层和真皮血管周围区域，这些细胞能够在无血清培养基呈悬浮细胞球生长，研究发现SKPs具有诱导毛囊等皮肤附属器官形成能力。Of course, the treatment of hair loss must first start from the cause, but many causes of hair loss cannot be completely removed. Local symptomatic medication and systemic medication, including promoting blood circulation and traditional Chinese medicine, have limited efficacy. Hair transplantation is expensive, and because the living conditions of the hair follicles are not sufficiently improved, the transplanted hair is difficult to survive for a long time. There are many different types of stem cells in the skin. Hair follicle stem cells (HFSCs), present in the protruding area of the upper part of the hair follicle, can differentiate into all the different types of cells of the hair follicle structure, and then form new hair. Hair follicle stem cells are mainly derived from epidermal stem cells distributed in the basal layer of the epidermis, and their number decreases with age. Somatic cells (skin-derived precursors, SKPs). SKPs are widely present in the papillary layer of the dermis and the perivascular area of the dermis. These cells can grow as suspension cell spheres in serum-free medium. Studies have found that SKPs have the ability to induce the formation of skin appendages such as hair follicles.
美国宾夕法尼亚大学的研究者分析了40岁至65岁男子的头发和头皮组织。结果发现，无论是脱发还是没有脱发的头皮组织中，毛囊干细胞(HFSCs)的数量都是相同的。所不同的是，脱发头皮组织中的毛囊干细胞没有转化成生长头发的毛囊前体细胞，缺乏SKPs细胞诱导活性可能是其中的原因。Researchers at the University of Pennsylvania analyzed the hair and scalp tissue of men between the ages of 40 and 65. It was found that the number of hair follicle stem cells (HFSCs) was the same in both hair loss and non-hair loss scalp tissue. The difference is that the hair follicle stem cells in the alopecia scalp tissue are not transformed into hair follicle precursor cells for growing hair, and the lack of SKPs cell-inducing activity may be the reason.
所以SKPs细胞移植有望成为治疗脱发的一条有效途径。同时，联合移植不同功能的皮肤干细胞，结合3-D组织工程技术制备的人造皮肤，可以替代植皮治疗严重皮肤损伤。Therefore, SKPs cell transplantation is expected to become an effective way to treat hair loss. At the same time, combined transplantation of skin stem cells with different functions, combined with artificial skin prepared by 3-D tissue engineering technology, can replace skin grafting in the treatment of severe skin damage.
但是取材于人体的SKPs细胞很有限，尤其是上年纪的人。而且在体外培养中，SKPs细胞相关标记物的表达和促进毛囊再生的能力随着传代的次数而迅速下降。所以找到一种活力强大的SKPs丰富来源，具有极其重大的临床应用价值。However, the number of SKPs cells derived from the human body is very limited, especially for the elderly. Moreover, in vitro culture, the expression of SKPs cell-related markers and the ability to promote hair follicle regeneration decreased rapidly with the number of passages. Therefore, finding a rich source of SKPs with strong vitality has extremely important clinical application value.
发明内容SUMMARY OF THE INVENTION
本发明目的是提供一种体外诱导脐带间充质干细胞分化为真皮干细胞的方法，用于促进毛囊再生和改善脱发，并进一步和其他干细胞及3-D组织工程材料结合，替代皮肤移植修复损伤。The purpose of the present invention is to provide a method for inducing differentiation of umbilical cord mesenchymal stem cells into dermal stem cells in vitro, which can be used to promote hair follicle regeneration and improve hair loss, and further combine with other stem cells and 3-D tissue engineering materials to replace skin transplantation to repair damage.
一种体外诱导脐带间充质干细胞分化为真皮干细胞的方法，它按以下步骤实现：A method for inducing umbilical cord mesenchymal stem cells to differentiate into dermal stem cells in vitro, which is achieved according to the following steps:
一、人脐带间充质干细胞取材，进行原代培养、消化传代、扩增及冻存；1. The human umbilical cord mesenchymal stem cells are drawn, primary cultured, digested, passaged, expanded and cryopreserved;
二、诱导人脐带间充质干细胞分化为真皮干细胞悬球：人脐带间充质干细胞经过上述消化传代后悬浮于真皮干细胞培养基中，计数，稀释到密度为1×105/ml，单细胞悬液按1.5ml/孔接种在低吸附6孔培养板中，真皮干细胞培养基中加入体积百分含量为1‰的10mM Y-27632，每2-3天换液一次，培养6天，收集真皮干细胞细胞悬球，300rpm离心2min，然后悬浮于真皮干细胞培养基，即完成体外诱导脐带间充质干细胞分化为真皮干细胞。2. Induce human umbilical cord mesenchymal stem cells to differentiate into dermal stem cell suspension spheres: After the above digestion and passage, human umbilical cord mesenchymal stem cells were suspended in dermal stem cell medium, counted, and diluted to a density of 1×10 5 /ml, single cell The suspension was inoculated in a low-adsorption 6-well culture plate at a rate of 1.5ml/well, and 10mM Y-27632 with a volume percentage of 1‰ was added to the dermal stem cell medium, and the medium was changed every 2-3 days for 6 days and collected. Dermal stem cells were suspended in spheres, centrifuged at 300 rpm for 2 min, and then suspended in a dermal stem cell medium to complete in vitro induction of umbilical cord mesenchymal stem cells to differentiate into dermal stem cells.
本发明的优点：Advantages of the present invention:
1、本发明首次采用细胞悬球培养方式，高效诱导MSCs分化诱导成为SKPs细胞，为临床解决真皮干细胞采集困难的问题提供了新出路。本发明诱导SKPs细胞的方法，同时有望解决老年、脱发性患者自体SKPs细胞扩增能力有限，将来无法达到治疗效果的问题。1. The present invention adopts the cell suspension culture method for the first time to efficiently induce MSCs to differentiate into SKPs cells, which provides a new way for clinically solving the problem of difficult collection of dermal stem cells. The method for inducing SKPs cells of the present invention is expected to solve the problem that the autologous SKPs cells of elderly and alopecia patients have limited expansion capacity and cannot achieve therapeutic effects in the future.
2、本发明获得SKPs的细胞表达SKPs特征性标记基因Oct4、Nestin、SOX9、和Vimentin在诱导分化的SKPs细胞悬球中表达量分别是人脐带间充质干细胞(MSCs)的51、21、47和3倍。2. The cells obtained by the present invention express SKPs characteristic marker genes Oct4, Nestin, SOX9, and Vimentin, and the expression levels in the induced differentiated SKPs cell suspensions are 51, 21, and 47 of those of human umbilical cord mesenchymal stem cells (MSCs), respectively. and 3 times.
3、本发明中SKPs与人表皮细胞共培养结果显示，分化诱导的SKPs细胞能促进皮服细胞向表皮干细胞/毛囊干细胞方向转化，培养后的细胞表达表皮干细胞/毛囊干细胞特征性标记基因CD200、Sox9和角质蛋白19分别是单独培养的人皮肤细胞的3.8、1.5和4.5倍。因此成功验证了其促进毛囊干细胞分化的作用。3. The results of co-culture of SKPs and human epidermal cells in the present invention show that differentiation-induced SKPs cells can promote the transformation of skin cells into epidermal stem cells/hair follicle stem cells, and the cultured cells express epidermal stem cells/hair follicle stem cells characteristic marker genes CD200, Sox9 and keratin 19 were 3.8, 1.5 and 4.5 times higher than those of human skin cells cultured alone. Therefore, its effect on promoting the differentiation of hair follicle stem cells was successfully verified.
4、本发明充分利用人脐带间充质干细胞丰富来源，和多种组织细胞分化，尤其是真皮结缔组织中各种细胞的潜能，诱导其分化为SKPs细胞，用以替代来源极为受限的皮肤中的真皮干细胞；在临床用于促进毛囊再生和改善脱发。并进一步和其他干细胞及3-D组织工程材料结合，替代皮肤移植修复损伤。4. The present invention makes full use of the rich sources of human umbilical cord mesenchymal stem cells and the differentiation of various tissue cells, especially the potential of various cells in the dermal connective tissue, and induces them to differentiate into SKPs cells to replace skin with extremely limited sources. Dermal stem cells in the dermis; used clinically to promote hair follicle regeneration and improve hair loss. It is further combined with other stem cells and 3-D tissue engineering materials to replace skin grafts to repair damage.
本发明应用于体外诱导脐带间充质干细胞分化为真皮干细胞。The present invention is applied to induce differentiation of umbilical cord mesenchymal stem cells into dermal stem cells in vitro.
附图说明Description of drawings
图1为实施例中诱导分化前人脐带间充质干细胞(MSCs)贴附培养的显微镜图；Fig. 1 is a microscope image of the adherent culture of human umbilical cord mesenchymal stem cells (MSCs) before induction of differentiation in the Examples;
图2为实施例中接种时的MSCs单细胞的显微镜图；Fig. 2 is the microscope picture of the MSCs single cell when seeding in the embodiment;
图3为实施例中接种MSCs单细胞第3天形成SKPs细胞悬球的显微镜图；Fig. 3 is the microscope picture of the SKPs cell suspension formed on the third day after seeding MSCs single cells in the embodiment;
图4为实施例中接种MSCs单细胞第6天形成SKPs细胞悬球的显微镜图；Fig. 4 is the microscope picture of the SKPs cell suspension formed on the 6th day after seeding MSCs single cells in the embodiment;
图5为实施例中诱导分化6天的SKPs细胞悬球表现很强的SKPs特征性标记物Nestin的免疫荧光染色图；Fig. 5 is the immunofluorescence staining diagram of Nestin, a characteristic marker of SKPs, which was induced to differentiate for 6 days in the embodiment of SKPs cell suspension;
图6为实施例中诱导分化6天的SKPs细胞悬球表现很强的SKPs特征性标记物Vimentin的免疫荧光染色图；Fig. 6 is the immunofluorescence staining diagram of Vimentin, a characteristic marker of SKPs, which was induced to differentiate for 6 days in SKPs cell suspension for 6 days;
图7为实施例中诱导分化前人脐带间充质干细胞(MSCs)的Nestin的免疫荧光染色图；Figure 7 is an immunofluorescence staining diagram of Nestin of human umbilical cord mesenchymal stem cells (MSCs) before induction of differentiation in the Example;
图8为实施例中诱导分化前人脐带间充质干细胞(MSCs)的Vimentin的免疫荧光染色图；Fig. 8 is the immunofluorescence staining diagram of Vimentin of human umbilical cord mesenchymal stem cells (MSCs) before induction of differentiation in the Example;
图9为实施例中实时定量PCR检查相关基因表达水平的柱状图；Fig. 9 is the bar graph of real-time quantitative PCR checking the expression level of related genes in the embodiment;
图10为实施例中SKPs细胞悬球共同培养的人皮肤细胞生成表皮干细胞/毛囊干细胞特征性标记物CD200的显微镜图；Figure 10 is a microscope image of CD200, a characteristic marker of epidermal stem cells/hair follicle stem cells generated by human skin cells co-cultured with SKPs cell suspensions in the Example;
图11为实施例中SKPs细胞悬球共同培养的人皮肤细胞生成表皮干细胞/毛囊干细胞特征性标记物Sox9阳性的细胞显微镜图；Figure 11 is a microscope image of cells that are positive for Sox9, a characteristic marker of epidermal stem cells/hair follicle stem cells from human skin cells co-cultured with SKPs cell suspensions in the Example;
图12实施例中单独培养的人皮肤细胞生成表皮干细胞/毛囊干细胞特征性标记物CD200的显微镜图；Figure 12 is a microscope image of the characteristic marker CD200 of epidermal stem cells/hair follicle stem cells generated by human skin cells cultured alone in the embodiment;
图13为实施例中单独培养的人皮肤细胞生成表皮干细胞/毛囊干细胞特征性标记物Sox9阳性的细胞显微镜图；Figure 13 is a microscopic picture of cells that are positive for Sox9, a characteristic marker of epidermal stem cells/hair follicle stem cells from human skin cells cultured alone in the Example;
图14为实施例中实时定量PCR检查表皮干细胞/毛囊干细胞特征性标记物CD200、Sox9和角质蛋白19表达水平的柱状图。14 is a bar graph of the expression levels of epidermal stem cells/hair follicle stem cells characteristic markers CD200, Sox9 and keratin 19 examined by real-time quantitative PCR in the Example.
具体实施方式Detailed ways
本发明技术方案不局限于以下所列举具体实施方式，还包括各具体实施方式间的任意组合。The technical solutions of the present invention are not limited to the specific embodiments listed below, but also include any combination of specific embodiments.
具体实施方式一：本实施方式一种体外诱导脐带间充质干细胞分化为真皮干细胞的方法，它按以下步骤实现：Specific embodiment 1: This embodiment is a method for inducing umbilical cord mesenchymal stem cells to differentiate into dermal stem cells in vitro, which is achieved according to the following steps:
一、人脐带间充质干细胞取材，进行原代培养、消化传代、扩增及冻存；1. The human umbilical cord mesenchymal stem cells are drawn, primary cultured, digested, passaged, expanded and cryopreserved;
二、诱导人脐带间充质干细胞分化为真皮干细胞悬球：人脐带间充质干细胞经过上述消化传代后悬浮于真皮干细胞培养基中，计数，稀释到密度为1×105/ml，单细胞悬液按1.5ml/孔接种在低吸附6孔培养板中，真皮干细胞培养基中加入体积百分含量为1‰的10mM Y-27632，每2-3天换液一次，培养6天，收集真皮干细胞细胞悬球，300rpm离心2min，然后悬浮于真皮干细胞培养基，即完成体外诱导脐带间充质干细胞分化为真皮干细胞。2. Induce human umbilical cord mesenchymal stem cells to differentiate into dermal stem cell suspension spheres: After the above digestion and passage, human umbilical cord mesenchymal stem cells were suspended in dermal stem cell medium, counted, and diluted to a density of 1×10 5 /ml, single cell The suspension was inoculated in a low-adsorption 6-well culture plate at a rate of 1.5ml/well, and 10mM Y-27632 with a volume percentage of 1‰ was added to the dermal stem cell medium, and the medium was changed every 2-3 days for 6 days and collected. Dermal stem cells were suspended in spheres, centrifuged at 300 rpm for 2 min, and then suspended in a dermal stem cell medium to complete in vitro induction of umbilical cord mesenchymal stem cells to differentiate into dermal stem cells.
本实施方式步骤一中人脐带间充质干细胞原代培养后可进行冻存，需要时再复苏培养，过程如下：将从液氮中取出人脐带间充质干细胞，在37℃水浴中解冻，悬浮在MSCs完全培养基，1000rpm离心5min，然后悬浮于MSCs完全培养基，以1×104/cm2密度接种，再置于37℃、5％CO2培养箱中培养。In step 1 of this embodiment, human umbilical cord mesenchymal stem cells can be frozen and stored after primary culture, and then re-cultivated when necessary. The process is as follows: human umbilical cord mesenchymal stem cells will be taken out from liquid nitrogen, thawed in a 37°C water bath, Suspended in MSCs complete medium, centrifuged at 1000 rpm for 5 min, then suspended in MSCs complete medium, inoculated at a density of 1×10 4 /cm 2 , and cultured in a 37°C, 5% CO 2 incubator.
具体实施方式二：本实施方式与具体实施方式一不同的是，步骤一中所述人脐带间充质干细胞取材，进行原代培养、消化传代、扩增及冻存的具体操作如下：Embodiment 2: The difference between this embodiment and Embodiment 1 is that the human umbilical cord mesenchymal stem cells described in step 1 are obtained, and the specific operations of primary culture, digestion and passage, expansion and cryopreservation are as follows:
a、新生儿脐带经体积百分含量为75％的酒精消毒处理，然后用生理盐水清洗2次；a. The neonatal umbilical cord is disinfected with 75% alcohol by volume, and then washed twice with normal saline;
b、将清洗后的脐带剪成长度为2-3cm的段，在平皿内剥离华通式胶；b. Cut the cleaned umbilical cord into segments with a length of 2-3cm, and peel off the Huatong glue in the plate;
c、将华通式胶置于离心管内，用手术剪剪碎至1-2mm3的块状；c. Put the Huatong glue in a centrifuge tube, and cut it into pieces of 1-2mm 3 with surgical scissors;
d、按照2g/T75培养瓶接种于脐带间充质干细胞完全培养基，在37℃、5％CO2培养箱中培养，4-5天后换液，以后每隔3天换液一次，培养至细胞融合度达到70％以上完成原代培养；d. Inoculate the complete medium of umbilical cord mesenchymal stem cells according to the 2g/T75 culture flask, cultivate in a 37°C, 5% CO 2 incubator, change the medium after 4-5 days, and then change the medium every 3 days, and culture until The primary culture is completed when the cell confluence reaches more than 70%;
e、人脐带间充质干细胞消化传代和扩增：吸除培养基，PBS洗一次，然后用体积百分浓度为0.25％的胰酶消化4-6min，吹打悬浮，DMEM/F12洗，1000rpm离心3min，再以1×104/cm2密度悬浮于脐带间充质干细胞完全培养基中进行传代和扩增培养；e. Digestion, passage and expansion of human umbilical cord mesenchymal stem cells: remove the medium by suction, wash once with PBS, then digest with 0.25% trypsin by volume for 4-6 min, suspend by pipetting, wash with DMEM/F12, and centrifuge at 1000 rpm 3min, and then suspended in the complete medium of umbilical cord mesenchymal stem cells at a density of 1×10 4 /cm 2 for passage and expansion culture;
f、冻存：细胞悬液加DMSO到10％，混匀，分装在冻存管中，使用程序降温盒在-80℃冷冻过夜后转移到液氮罐。其它步骤及参数与具体实施方式一相同。f. Cryopreservation: add DMSO to the cell suspension to 10%, mix well, distribute it in cryopreservation tubes, use a programmed cooling box to freeze at -80°C overnight, and then transfer it to a liquid nitrogen tank. Other steps and parameters are the same as in the first embodiment.
具体实施方式三：本实施方式与具体实施方式二不同的是，所述脐带间充质干细胞完全培养基(即MSCs完全培养基)的成分：95％alphaMEM培养基、添加5％血小板裂解液和5IU/ml肝素钠。其它步骤及参数与具体实施方式二相同。Embodiment 3: The difference between this embodiment and Embodiment 2 is that the components of the umbilical cord mesenchymal stem cell complete medium (ie, the MSCs complete medium) are: 95% alphaMEM medium, 5% platelet lysate and 5IU/ml heparin sodium. Other steps and parameters are the same as in the second embodiment.
具体实施方式四：本实施方式与具体实施方式一至三之一不同的是，步骤二中所述真皮干细胞培养基(即SKPs培养基)的成分：DMEM:F12＝1:1基础培养基加1/50B27、20ng/ml EGF、20ng/ml bFGF、0.2mM抗坏血酸和2μg/ml庆大霉素。其它步骤及参数与具体实施方式一至三之一相同。具体实施方式五：本实施方式与具体实施方式一至四之一不同的是，步骤二中培养的条件:37℃，5％CO2。其它步骤及参数与具体实施方式一至四之一相同。Embodiment 4: The difference between this embodiment and one of Embodiments 1 to 3 is that the composition of the dermal stem cell medium (ie SKPs medium) described in step 2: DMEM:F12=1:1 basal medium plus 1 /50B27, 20ng/ml EGF, 20ng/ml bFGF, 0.2mM ascorbic acid and 2μg/ml gentamicin. Other steps and parameters are the same as one of the first to third embodiments. Embodiment 5: The difference between this embodiment and one of Embodiments 1 to 4 is that the culture conditions in step 2 are: 37° C., 5% CO 2 . Other steps and parameters are the same as one of the first to fourth embodiments.
通过以下实施例验证本发明的有益效果：The beneficial effects of the present invention are verified by the following examples:
实施例：Example:
一种体外诱导脐带间充质干细胞分化为真皮干细胞的方法，它按以下步骤实现：A method for inducing umbilical cord mesenchymal stem cells to differentiate into dermal stem cells in vitro, which is achieved according to the following steps:
一、人脐带间充质干细胞取材，进行原代培养、消化传代、扩增及冻存；1. The human umbilical cord mesenchymal stem cells are drawn, primary cultured, digested, passaged, expanded and cryopreserved;
二、诱导人脐带间充质干细胞分化为真皮干细胞悬球：人脐带间充质干细胞经过上述消化传代后悬浮于真皮干细胞培养基中，计数，稀释到密度为1×105/ml，单细胞悬液按1.5ml/孔接种在低吸附6孔培养板中，真皮干细胞培养基中加入体积百分含量为1‰的10mM Y-27632，每2-3天换液一次，培养6天，收集真皮干细胞细胞悬球，300rpm离心2min，然后悬浮于真皮干细胞培养基，即完成体外诱导脐带间充质干细胞分化为真皮干细胞。2. Induce human umbilical cord mesenchymal stem cells to differentiate into dermal stem cell suspension spheres: After the above digestion and passage, human umbilical cord mesenchymal stem cells were suspended in dermal stem cell medium, counted, and diluted to a density of 1×10 5 /ml, single cell The suspension was inoculated in a low-adsorption 6-well culture plate at a rate of 1.5ml/well, and 10mM Y-27632 with a volume percentage of 1‰ was added to the dermal stem cell medium, and the medium was changed every 2-3 days for 6 days and collected. Dermal stem cells were suspended in spheres, centrifuged at 300 rpm for 2 min, and then suspended in a dermal stem cell medium to complete in vitro induction of umbilical cord mesenchymal stem cells to differentiate into dermal stem cells.
本实施例步骤一中人脐带间充质干细胞原代培养后可进行冻存，需要时再复苏培养，过程如下：将从液氮中取出人脐带间充质干细胞，在37℃水浴中解冻，悬浮在MSCs完全培养基，1000rpm离心5min，然后悬浮于MSCs完全培养基，以1×104/cm2密度接种，再置于37℃、5％CO2培养箱中培养。In step 1 of this example, the human umbilical cord mesenchymal stem cells can be frozen and stored after primary culture, and then re-cultivated when necessary. Suspended in MSCs complete medium, centrifuged at 1000 rpm for 5 min, then suspended in MSCs complete medium, inoculated at a density of 1×10 4 /cm 2 , and cultured in a 37°C, 5% CO 2 incubator.
本实施例步骤一中所述人脐带间充质干细胞取材，进行原代培养、消化传代、扩增及冻存的具体操作如下：The specific operations of the human umbilical cord mesenchymal stem cells described in step 1 of this example, primary culture, digestion and passage, expansion and cryopreservation are as follows:
a、新生儿脐带经体积百分含量为75％的酒精消毒处理，然后用生理盐水清洗2次；a. The neonatal umbilical cord is disinfected with 75% alcohol by volume, and then washed twice with normal saline;
b、将清洗后的脐带剪成长度为2-3cm的段，在平皿内剥离华通式胶；b. Cut the cleaned umbilical cord into segments with a length of 2-3cm, and peel off the Huatong glue in the plate;
c、将华通式胶置于离心管内，用手术剪剪碎至1-2mm3的块状；c. Put the Huatong glue in a centrifuge tube, and cut it into pieces of 1-2mm 3 with surgical scissors;
d、按照2g/T75培养瓶接种于脐带间充质干细胞完全培养基，在37℃、5％CO2培养箱中培养，4-5天后换液，以后每隔3天换液一次，培养至细胞融合度达到70％以上完成原代培养；d. Inoculate the complete medium of umbilical cord mesenchymal stem cells according to the 2g/T75 culture flask, cultivate in a 37°C, 5% CO 2 incubator, change the medium after 4-5 days, and then change the medium every 3 days, and culture until The primary culture is completed when the cell confluence reaches more than 70%;
e、人脐带间充质干细胞消化传代和扩增：吸除培养基，PBS洗一次，然后用体积百分浓度为0.25％的胰酶消化4-6min，吹打悬浮，DMEM/F12洗，1000rpm离心3min，再以1×104/cm2密度悬浮于脐带间充质干细胞完全培养基中进行传代和扩增培养；e. Digestion, passage and expansion of human umbilical cord mesenchymal stem cells: remove the medium by suction, wash once with PBS, then digest with 0.25% trypsin by volume for 4-6 min, suspend by pipetting, wash with DMEM/F12, and centrifuge at 1000 rpm 3min, and then suspended in the complete medium of umbilical cord mesenchymal stem cells at a density of 1×10 4 /cm 2 for passage and expansion culture;
f、冻存：细胞悬液加DMSO到10％，混匀，分装在冻存管中，使用程序降温盒在-80℃冷冻过夜后转移到液氮罐；f. Cryopreservation: add DMSO to the cell suspension to 10%, mix well, distribute it in cryopreservation tubes, use a programmed cooling box to freeze at -80 °C overnight, and then transfer it to a liquid nitrogen tank;
其中所述脐带间充质干细胞完全培养基(即MSCs完全培养基)的成分：95％alphaMEM培养基、添加5％血小板裂解液和5IU/ml肝素钠。The components of the umbilical cord mesenchymal stem cell complete medium (ie the MSCs complete medium) are: 95% alphaMEM medium, 5% platelet lysate and 5IU/ml heparin sodium.
本实施例步骤二中所述真皮干细胞培养基(即SKPs培养基)的成分：DMEM:F12＝1:1基础培养基加1/50B27、20ng/ml EGF、20ng/ml bFGF、0.2mM抗坏血酸和2μg/ml庆大霉素。The composition of the dermal stem cell medium (ie SKPs medium) described in step 2 of this example: DMEM:F12=1:1 basal medium plus 1/50B27, 20ng/ml EGF, 20ng/ml bFGF, 0.2mM ascorbic acid and 2 μg/ml gentamicin.
本实施例步骤二中培养的条件:37℃，5％CO2。The culture conditions in step 2 of this example: 37°C, 5% CO 2 .
本实施例步骤二中完成体外诱导脐带间充质干细胞分化为真皮干细胞，可作为诱导分化的第一代真皮干细胞，还可以进一步传代。In step 2 of this example, the umbilical cord mesenchymal stem cells are induced to differentiate into dermal stem cells in vitro, which can be used as the first-generation dermal stem cells induced to differentiate, and can be further passaged.
本实施例步骤二完成后，进行接种人皮肤细胞：After the second step of this example is completed, inoculate human skin cells:
取复苏或消化传代后的人皮肤细胞，按1.5×105/ml接种在Insert嵌套式12孔培 养板中，皮肤细胞培养基中加入体积百分含量为1‰的10mM Y-27632，培养4h，然后接种入300μl的步骤二中所得悬浮于真皮干细胞培养基的真皮干细胞细胞悬球，进行共培养，每2天换液一次，培养6天后，分别收集共培养物中的真皮干细胞和人皮肤细胞，提取RNA，做实时定量PCR分析，或4％多聚甲醛固定后做免疫荧光染色。用以鉴定真皮干细胞对于人皮肤细胞的作用。The human skin cells recovered or digested and passaged were taken and seeded in the Insert nested 12-well culture plate at 1.5×10 5 /ml, and 10mM Y-27632 with a volume percentage of 1‰ was added to the skin cell culture medium. After culturing for 4 hours, inoculate 300 μl of the dermal stem cell suspension pellets suspended in the dermal stem cell medium obtained in step 2 for co-cultivation. The medium was changed every 2 days. After 6 days of culture, the dermal stem cells and Human skin cells, extract RNA, do real-time quantitative PCR analysis, or fix with 4% paraformaldehyde and do immunofluorescence staining. To identify the effect of dermal stem cells on human skin cells.
上述皮肤细胞培养基的成分：DMEM:F12＝1:1的基础培养基加5％胎牛血清、1/100B27、1/200ITS、2mM谷氨酰氨、10ng/ml EGF和10ng/ml bFGF。Composition of the above skin cell culture medium: DMEM:F12=1:1 basal medium plus 5% fetal bovine serum, 1/100B27, 1/200ITS, 2mM glutamine, 10ng/ml EGF and 10ng/ml bFGF.
上述共培养的条件:37℃，5％CO2。Conditions for the above co-cultivation: 37°C, 5% CO 2 .
RNA、cDNA制备和实时定量PCR分析：RNA, cDNA preparation and real-time quantitative PCR analysis:
各步实验按照各个试剂盒的说明操作。Each step of the experiment was performed according to the instructions of each kit.
1)Trizel方法提纯RNA。测定RNA含量；1) The Trizel method is used to purify RNA. Determination of RNA content;
2)东洋纺反转录试剂盒制备cDNA。测定cDNA含量；2) cDNA was prepared by Toyobo reverse transcription kit. Determination of cDNA content;
3)用TaKaRa TB Green Fast qPCR Mix做实时定量PCR分析。3) Real-time quantitative PCR analysis was performed with TaKaRa TB Green Fast qPCR Mix.
SKPs悬浮培养标本作免疫荧光染色：SKPs suspension culture specimens for immunofluorescence staining:
1)多聚鸟氨酸(PLO)包被24孔(有爬片)>2hr，PBS洗；1) Polyornithine (PLO) coated 24 wells (with slides) > 2hr, washed with PBS;
2)接种SKPs细胞悬球在PLO孔中；2) Seed the SKPs cell suspension in the PLO well;
3)一天后细胞团贴附好。PBS洗一次，4％多聚甲醛(PFA)固定。部分SKPs细胞悬球包埋之后冰冻切片；3) After one day, the cell pellets were well attached. Washed once with PBS and fixed with 4% paraformaldehyde (PFA). Some SKPs cells were frozen and sectioned after being embedded in suspension beads;
4)PBS洗X1。0.1％Triton X-100破膜30min；4) Wash X1 with PBS. 0.1% Triton X-100 breaks the membrane for 30min;
5)5％正常羊血清(NGS)–PBS封闭30min；5) Block with 5% normal goat serum (NGS)-PBS for 30min;
6)加各种一抗(溶于0.3％BSA-PBS)。4℃孵育过夜；6) Add various primary antibodies (dissolved in 0.3% BSA-PBS). Incubate overnight at 4°C;
7)PBS洗X1，再PBS洗(摇床)15min X2；7) PBS wash X1, then PBS wash (shaker) 15min X2;
8)加二抗(Alexa Fluor594-山羊抗小鼠，Alexa Fluor 488山羊抗兔，1:500)。室温孵育2h；8) Add secondary antibody (Alexa Fluor594-goat anti-mouse, Alexa Fluor 488 goat anti-rabbit, 1:500). Incubate at room temperature for 2h;
9)PBS洗X1，DAPI核染色10min。PBS洗(摇床)15min；9) Wash X1 with PBS, and stain the nucleus with DAPI for 10 min. PBS wash (shaker) 15min;
10)ProlongGold封片；10) ProlongGold coverslip;
11)显微镜下观察、照相；11) Observation and photography under the microscope;
12)照片图像分析，细胞计数，统计学分析。12) Photo image analysis, cell count, statistical analysis.
结果：result:
1成功诱导人脐带间充质干细胞(hUC-MSCs)分化成高度表达SKPs特征性标记物的细胞悬球。图1至图4，显示培养中的脐带间充质干细胞(MSCs)，和从接种时的MSCs单细胞(SKPs 0天)，到第3天和第6天形成的SKPs细胞悬球。第六天的SKPs细胞悬球用来做免疫荧光染色和实时定量PCR分析；同皮肤细胞做共培养；以及动物移植实验。比例尺200μm。1. Human umbilical cord mesenchymal stem cells (hUC-MSCs) were successfully induced to differentiate into cell suspensions highly expressing markers characteristic of SKPs. Figures 1 to 4 show umbilical cord mesenchymal stem cells (MSCs) in culture, and SKPs cell suspensions formed from MSCs single cells (SKPs day 0) at the time of seeding to days 3 and 6. The SKPs cell suspension on the sixth day was used for immunofluorescence staining and real-time quantitative PCR analysis; co-culture with skin cells; and animal transplantation experiments. Scale bar 200 μm.
诱导分化6天的SKPs细胞悬球表现很强的SKPs特征性标记物(Nestin、Vimentin)免疫荧光染色(见图5和图6)。而诱导分化前人脐带间充质干细胞(MSCs)的Nestin和Vimentin的免疫荧光染色为阴性(见图7和图8，相同曝光条件)。比例尺100μm。Vimentin计数结果显示，SKP细胞悬球中大于90％的细胞为阳性。SKPs cell suspensions induced to differentiate for 6 days showed strong immunofluorescence staining of SKPs characteristic markers (Nestin, Vimentin) (see Figures 5 and 6). In contrast, the immunofluorescence staining of Nestin and Vimentin of human umbilical cord mesenchymal stem cells (MSCs) before differentiation was negative (see Figures 7 and 8, the same exposure conditions). Scale bar 100 μm. Vimentin counting results showed that more than 90% of the cells in the SKP cell suspension were positive.
实时定量PCR检测相关基因表达：如图9所示，SKPs特征性标记物Oct4、Nestin、、SOX9、和Vimentin在诱导分化的SKPs细胞悬球中表达量分别是人脐带间充质干细胞(MSCs)的51、21、47和3倍。Real-time quantitative PCR detection of related gene expression: As shown in Figure 9, the expression levels of SKPs characteristic markers Oct4, Nestin, SOX9, and Vimentin in the induced differentiation of SKPs cell suspensions are respectively the same as those of human umbilical cord mesenchymal stem cells (MSCs) 51, 21, 47 and 3 times.
2、SKPs细胞悬球在共同培养中促进人皮肤细胞生成表皮干细胞/毛囊干细胞，与单独培养的人皮肤细胞相比，和SKPs细胞悬球共同培养的人皮肤细胞生成更多的表皮干细胞/毛囊干细胞。2. SKPs cell suspension promotes human skin cells to generate epidermal stem cells/hair follicle stem cells in co-culture. Compared with human skin cells cultured alone, human skin cells co-cultured with SKPs cell suspensions generate more epidermal stem cells/hair follicles stem cell.
显示和SKPs细胞悬球共同培养的人皮肤细胞(见图10和图11)比单独培养的人皮肤细胞(见图12和图13)生成更多表皮干细胞/毛囊干细胞特征性标记物CD200和Sox9阳性的细胞。比例尺200μm。阳性细胞的百分比见表1。showed that human skin cells co-cultured with SKPs cell suspensions (see Figures 10 and 11) produced more epidermal stem cell/hair follicle stem cell markers CD200 and Sox9 than human skin cells cultured alone (see Figures 12 and 13) positive cells. Scale bar 200 μm. The percentage of positive cells is shown in Table 1.
表1免疫组化分析共培养后毛囊干细胞标志蛋白阳性表达率Table 1 Immunohistochemical analysis of the positive expression rate of hair follicle stem cell marker proteins after co-culture
皮肤细胞和SKPs细胞共培养Co-culture of skin cells and SKPs cells 皮肤细胞单独培养skin cells alone CD200CD200 8.7％8.7% 0％0% SOX9SOX9 12.2％12.2% 4.1％4.1%
实时定量PCR结果如图14所示，和SKPs细胞悬球共同培养的人皮肤细胞，比单独培养的人皮肤细胞表达更大量的表皮干细胞/毛囊干细胞特征性标记物CD200、Sox9和角质蛋白19。其比例分别是3.8、1.5和4.5倍。The real-time quantitative PCR results are shown in Figure 14. Human skin cells co-cultured with SKPs cell suspensions expressed a greater amount of epidermal stem cell/hair follicle stem cell characteristic markers CD200, Sox9 and keratin 19 than human skin cells cultured alone. The ratios are 3.8, 1.5 and 4.5 times, respectively.