WO2022052605A1 - 一种增强间充质干细胞分泌功能的方法及应用 - Google Patents

一种增强间充质干细胞分泌功能的方法及应用 Download PDF

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WO2022052605A1
WO2022052605A1 PCT/CN2021/104328 CN2021104328W WO2022052605A1 WO 2022052605 A1 WO2022052605 A1 WO 2022052605A1 CN 2021104328 W CN2021104328 W CN 2021104328W WO 2022052605 A1 WO2022052605 A1 WO 2022052605A1
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stem cells
mesenchymal stem
umbilical cord
sodium hyaluronate
enhancing
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French (fr)
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赵世斗
陈子江
秦莹莹
焦文林
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山东大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/90—Polysaccharides
    • C12N2501/905—Hyaluronic acid

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  • the invention belongs to the field of biotechnology, and in particular relates to a method and application for enhancing the secretion function of mesenchymal stem cells.
  • MSCs Mesenchymal stem cells
  • mesenchymal stem cells can function in various ways such as cell differentiation, paracrine and cell contact regulation.
  • paracrine effect means that mesenchymal stem cells act on target cells and exert regulatory functions by secreting cytokines, extracellular vesicles, microRNAs and other substances, and it is an important way for stem cells to function.
  • Stem cells can promote cell proliferation, inhibit cell apoptosis, promote angiogenesis and resist fibrosis through paracrine, thereby promoting the repair and regeneration of damaged tissue.
  • MSCs can promote the repair of gastric mucosa, skin, ovary and other tissue damage through paracrine activation of PI3K-AKT and other signaling pathways.
  • the present invention provides a method and application for enhancing the secretory function of mesenchymal stem cells.
  • the present invention finds through research that by using sodium hyaluronate (Hyaluronic acid sodium, HA) to pretreat umbilical cord mesenchymal stem cells (Umbilical cord mesenchymal stem cells, UCMSCs) in vitro, the secretion function of umbilical cord mesenchymal stem cells can be significantly promoted.
  • the method is simple, convenient, low in cost and high in safety, so it has good practical application value.
  • the first aspect of the present invention provides the application of sodium hyaluronate in enhancing the secretion function of mesenchymal stem cells.
  • the research of the present invention finds that the in vitro pretreatment of sodium hyaluronate has a good effect on promoting the secretion of mesenchymal stem cells, and hyaluronic acid is an important component of the extracellular matrix and has good biological safety and histocompatibility.
  • the application includes at least any one or more of the following:
  • the second aspect of the present invention provides a method for enhancing the secretory function of mesenchymal stem cells, the method comprising: pre-culturing the mesenchymal stem cells in a medium containing sodium hyaluronate.
  • the in vitro pretreatment of stem cells with a suitable concentration of sodium hyaluronate can significantly enhance the secretory function of stem cells.
  • Enhanced paracrine components can further activate the PI3K-AKT signaling pathway and promote germ cell survival after ovarian injury in mice.
  • the method is simple, safe, feasible and has great application potential.
  • the above technical solution provides a simple, safe and effective method for promoting the secretory function of stem cells in vitro. It has been found through research that adding an appropriate concentration of sodium hyaluronate during in vitro culture and treating it for a certain period of time can enhance the secretory function of stem cells and promote HGF. , SCF, EGF, VEGF and other factors secretion, further promote the activation of PI3K-AKT signaling pathway, and enhance the repair of damage, so it has good practical application value.
  • FIG. 1 is the scatter plot result of detecting 440 cytokines in the supernatant of stem cells by sandwich antibody array method in Example 1 of the present invention.
  • FIG. 2 is the analysis result of KEGG pathway of cytokine chip differential protein in Example 1 of the present invention.
  • FIG. 3 is the result of ELISA method for verifying the differential cytokines of the chip in Example 2 of the present invention.
  • Fig. 4 shows that the supernatant of UCMSCs was pretreated with HA in Example 3 of the present invention to promote the activation of PI3K-AKT pathway in mouse ovary after 4 days of in vitro culture.
  • Fig. 5 shows that the supernatant of UCMSCs was pretreated with HA in Example 4 of the present invention to promote the survival of germ cells in mouse ovary after 8 days of in vitro culture.
  • FIG. 6 is a volcano plot result of detecting the changes in mRNA level and expression of UCMSCs gene after HA pretreatment by RNA sequencing method (RNA-seq) in Example 5 of the present invention.
  • the research of the present invention finds that the secretory function of stem cells can be significantly enhanced by using appropriate concentration of sodium hyaluronate to pretreat stem cells in vitro.
  • Enhanced paracrine components can further activate the PI3K-AKT signaling pathway and promote germ cell survival after ovarian injury in mice.
  • the method is simple, safe, feasible and has great application potential.
  • the application of sodium hyaluronate in enhancing the secretion function of mesenchymal stem cells is provided.
  • the research of the present invention finds that the in vitro pretreatment of sodium hyaluronate has a good effect on promoting the secretion of mesenchymal stem cells, and hyaluronic acid is an important component of the extracellular matrix and has good biological safety and histocompatibility.
  • the sodium hyaluronate is a commercial cross-linked sodium hyaluronate gel.
  • the application includes at least any one or more of the following:
  • the cytokines include at least HGF, SCF, EGF and VEGF.
  • enhancing the repairing effect on damage includes enhancing the repairing effect on ovarian tissue; more specifically, promoting the survival of germ cells after ovarian injury in mice.
  • the concentration of the sodium hyaluronate is 0.1-0.3 mg/ml (preferably 0.3 mg/ml).
  • the sodium hyaluronate at this concentration can not only play the function of promoting the secretion of stem cells, but also has no obvious negative effect on the cultured stem cells in vitro.
  • the mesenchymal stem cells can be derived from umbilical cord, bone marrow, fat, umbilical cord blood, endometrium, amniotic membrane, placenta or dental pulp; preferably, the mesenchymal stem cells are human umbilical cord source of mesenchymal stem cells.
  • a method for promoting the secretory function of stem cells comprising pre-conditioning and culturing mesenchymal stem cells in a medium containing sodium hyaluronate.
  • the in vitro pretreatment of stem cells with a suitable concentration of sodium hyaluronate can significantly enhance the secretory function of stem cells.
  • Enhanced paracrine components can further activate the PI3K-AKT signaling pathway and promote germ cell survival after ovarian injury in mice.
  • the method is simple, safe, feasible and has great application potential.
  • the mesenchymal stem cells can be derived from umbilical cord, bone marrow, fat, umbilical cord blood, endometrium, amniotic membrane, placenta or dental pulp; preferably, the mesenchymal stem cells are mesenchymal stem cells derived from human umbilical cord.
  • the concentration of sodium hyaluronate is 0.1 to 0.3 mg/ml, preferably 0.3 mg/ml.
  • the pretreatment culture time is 3 to 5 days, preferably 5 days.
  • Gong Ankang cross-linked sodium hyaluronate gel for uterine cavity, specification: 5mg/ml, manufacturer: Changzhou Bai Ruiji Bio-Pharmaceutical Co., Ltd., medical device registration certificate number: National Machinery Note 20153641542); 2.MEM -alpha medium (GIBCO, 12571-063); 3. Serum substitute (Helios, HPCFDCRL05); 4. ELISA kit (Thermo, ebioscience); 5. Ultrafiltration centrifuge tube (3KDa, Millipore, UFC900308); 6. p-AKT, AKT antibody (Cell Signaling Technology, #9271, #9272); DDX4 antibody (Abcam, ab27591); 7.
  • Embedded cell culture chamber (Sigma, PICMORG50); 8. VCD (4-vinylcyclonhexene diepoxide, Sigma, 94956); 9. DMEM/F12 (1:1) medium (GIBCO, 11320-033); 10. Leibovitz's L-15 medium (GIBCO, 11415064); 11. ITS liquid medium supplement (Sigma, I3146) ; 12. Protein extraction kit (Invent, SD-001/SN-002); 13. T75 culture flask (Corning, 430641); 14. Trizol (Ambion, 15596018).
  • Wild-type C57BL/6 suckling mice were purchased from the Experimental Animal Center of Shandong University. Human umbilical cord mesenchymal stem cells were prepared in our laboratory.
  • Example 1 Cytokine chip detects the effect of HA pretreatment on the secretory function of stem cells
  • the two groups of cells were replaced with 13ml of serum-free basal medium, and the two groups of supernatants were collected after culturing in an incubator for 48 hours.
  • the concentrated cell supernatants of the two groups were sent to Raybiotech Co., Ltd. for cytokine chip (Raybiotech, GSH-CAA-440) detection, and scatter plot analysis and differential protein KEGG pathway enrichment analysis were performed.
  • cytokine chip Raybiotech, GSH-CAA-440
  • Example 2 ELISA method to verify the results of cytokine chip
  • the process of collecting the concentrated supernatant was as described in Example 1.
  • the mesenchymal stem cell supernatant (Mesenchymal stem cells-conditioned medium, MSC-CM) of the control group and the mesenchymal stem cell supernatant (HA/MSC-CM) of the HA treatment group were collected.
  • MSC-CM mesenchymal stem cells-conditioned medium
  • Example 3 In vitro ovarian culture to detect the effect of stem cell supernatant after HA pretreatment on the activation of PI3K-AKT pathway
  • Ovarian dissection solution containing 10% FBS and 1% penicillin-streptomycin was prepared in L15 medium.
  • Postnatal day 4 (PD4) suckling mouse ovaries were isolated in freshly prepared dissection solution under a stereo microscope. Gently peel off the surrounding tissue and capsule of the ovary, taking care to move quickly and not to damage the ovarian tissue.
  • Example 4 In vitro ovarian culture to detect the effect of stem cell supernatant on germ cell survival after HA pretreatment
  • the step of collecting the supernatant is as in Example 1, and the step of culturing the ovary in vitro is as in Example 3.
  • the ovaries were collected, fixed in Bouin's solution, dehydrated by gradient, embedded and sliced.
  • the sections of each group were stained with immunohistochemical staining, labeled with germ cell marker DDX4, and the sections of each group were observed and photographed under a microscope.
  • Example 5 RNA-seq to detect the effect of HA pretreatment on stem cell gene mRNA expression
  • UCMSCs The same amount of UCMSCs was cultured in normal medium or medium supplemented with 0.3mg/ml HA for 5 days, and the medium was changed on the 3rd day, and the culture was continued after the medium change.

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Abstract

提供了一种增强间充质干细胞分泌功能的方法及应用。通过使用透明质酸钠(Hyaluronic acid sodium，HA)在体外预处理脐带间充质干细胞，能够促进脐带间充质干细胞分泌功能，促进HGF、SCF、EGF、VEGF等因子的分泌，进一步促进PI3K-AKT信号通路的激活，增强对损伤的修复作用，该方法简单、方便、成本低廉，安全性高，具有良好的实际应用价值。

Description

一种增强间充质干细胞分泌功能的方法及应用 技术领域
本发明属于生物技术领域，具体涉及一种增强间充质干细胞分泌功能的方法及应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
干细胞在组织工程、疾病治疗等领域具有巨大的发展潜力和临床应用价值，其中间充质干细胞(Mesenchymal stem cells，MSCs)广泛存在于全身结缔组织和器官间质中，是目前研究和应用较多的一种成体干细胞。近年来已成为生物医学领域重要的研究方向之一。
现有研究表明，间充质干细胞能够通过细胞分化、旁分泌及细胞接触调控等多种方式发挥作用。其中，旁分泌作用是指间充质干细胞通过分泌细胞因子、胞外囊泡、microRNAs等多种物质作用于靶细胞并发挥调控功能，是干细胞发挥作用的重要途径。干细胞通过旁分泌可以发挥促进细胞增殖、抑制细胞凋亡、促进血管再生及抗纤维化等作用，进而促进损伤组织修复再生。有研究报道，MSCs可以通过旁分泌激活PI3K-AKT等信号通路促进胃粘膜、皮肤、卵巢等多种组织损伤修复。
目前，高质量MSCs的标准之一是细胞具有较强的旁分泌能力。研究人员已尝试通过细胞因子、生长因子、低氧、基因修饰等途径预处理干细胞，以增强其旁分泌功能，从而在一定程度上提高了干细胞的治疗效果。但是发明人发现，这些方式仍处于研究阶段，有些因子价格比较昂贵，合适的浓度和处理时间还 需要进一步探索；低氧预处理需要特殊的培养设备，条件要求比较高；而基因修饰会改变细胞的基因组，其安全性需进一步明确。因此，目前仍需要寻找简便、安全、有效的能够增强间充质干细胞分泌功能的方法。
发明内容
针对上述现有技术的不足，本发明提供一种增强间充质干细胞分泌功能的方法及应用。本发明通过研究发现，通过使用透明质酸钠(Hyaluronic acid sodium，HA)在体外预处理脐带间充质干细胞(Umbilical cord mesenchymal stem cells，UCMSCs)，能够显著促进脐带间充质干细胞分泌功能，该方法简单、方便、成本低廉，安全性高，因此具有良好的实际应用之价值。
本发明是通过如下技术方案实现的：
本发明的第一个方面，提供透明质酸钠在增强间充质干细胞分泌功能中的应用。本发明研究发现，透明质酸钠体外预处理对于促进间充质干细胞分泌有良好效果，而且透明质酸是细胞外基质的重要成分，具有良好的生物安全性和组织相容性。
所述应用至少包括如下任意一种或多种：
(1)促进细胞因子分泌；
(2)促进PI3K-AKT信号通路的激活；
(3)增强对损伤的修复作用。
本发明的第二个方面，提供一种增强间充质干细胞分泌功能的方法，所述方法包括：将间充质干细胞在含有透明质酸钠的培养基中进行预处理培养。本发明研究发现，通过运用适宜浓度的透明质酸钠对干细胞进行体外预处理能够显著增强干细胞的分泌功能。增强的旁分泌成分可以进一步激活PI3K-AKT信号通路，促进小鼠卵巢损伤后的生殖细胞存活。该方法简便、安全、可行，应用潜力大。
上述一个或多个技术方案的有益技术效果：
上述技术方案提供一种简便、安全、有效的体外促进干细胞分泌功能的方法，经过研究发现，通过体外培养时添加适宜浓度的透明质酸钠并处理一定时间，能够增强干细胞的分泌功能，促进HGF、SCF、EGF、VEGF等因子的分泌，进一步促进PI3K-AKT信号通路的激活，增强对损伤的修复作用，因此具有良好的实际应用之价值。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。
图1为本发明实施例一中夹心抗体阵列法检测干细胞上清中440个细胞因子芯片散点图结果。
图2为本发明实施例一中细胞因子芯片差异蛋白KEGG通路分析结果。
图3为本发明实施例二中ELISA法验证芯片差异细胞因子结果。
图4为本发明实施例三中利用HA预处理UCMSCs上清促进体外培养4天后小鼠卵巢中PI3K-AKT通路激活。
图5为本发明实施例四中利用HA预处理UCMSCs上清促进体外培养8天后小鼠卵巢中生殖细胞存活。
图6为本发明实施例五中RNA测序法(RNA-seq)检测经HA预处理后UCMSCs基因mRNA水平表达变化火山图结果。
具体实施方式
应该指出，以下详细说明都是例示性的，旨在对本发明提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本发明的示例性实施方式。如在这里所使用的，除非上下文另外明确 指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。下列具体实施方式中如果未注明具体条件的实验方法，通常按照本领域技术内的分子生物学的常规方法和条件，这种技术和条件在文献中有完整解释。参见例如Sambrook等人，《分子克隆：实验手册》中所述的技术和条件，或按照制造厂商所建议的条件。
如前所述，促进间充质干细胞分泌功能的方法有限且具有一定的局限性和安全隐患。
有鉴于此，本发明研究发现，通过运用适宜浓度的透明质酸钠对干细胞进行体外预处理能够显著增强干细胞的分泌功能。增强的旁分泌成分可以进一步激活PI3K-AKT信号通路，促进小鼠卵巢损伤后的生殖细胞存活。该方法简便、安全、可行，应用潜力大。
本发明的一个典型实施方式中，提供透明质酸钠在增强间充质干细胞分泌功能中的应用。本发明研究发现，透明质酸钠体外预处理对于促进间充质干细胞分泌有良好效果，而且透明质酸是细胞外基质的重要成分，具有良好的生物安全性和组织相容性。在本发明的一个具体实施方式中，透明质酸钠选用的是商品化的交联透明质酸钠凝胶。
所述应用至少包括如下任意一种或多种：
(1)促进细胞因子分泌；
(2)促进PI3K-AKT信号通路的激活；
(3)增强对损伤的修复作用。
本发明的又一具体实施方式中，所述(1)中，细胞因子至少包括HGF、SCF、EGF和VEGF。
本发明的又一具体实施方式中，所述(3)中，增强对损伤的修复作用包括增强对卵巢组织的损伤修复作用；更具体表现为促进小鼠卵巢损伤后的生殖细胞存活。
本发明的又一具体实施方式中，所述透明质酸钠浓度为0.1～0.3mg/ml(优选为0.3mg/ml)。该浓度下的透明质酸钠既能发挥促进干细胞分泌的功能，同时对体外培养干细胞无明显负面影响。
本发明的又一具体实施方式中，所述间充质干细胞可以来源于脐带、骨髓、脂肪、脐带血、子宫内膜、羊膜、胎盘或牙髓；优选的，所述间充质干细胞为人脐带来源的间充质干细胞。
本发明的又一具体实施方式中，提供一种促进干细胞分泌功能的方法，所述方法包括将间充质干细胞在含有透明质酸钠的培养基中进行预处理培养。本发明研究发现，通过运用适宜浓度的透明质酸钠对干细胞进行体外预处理能够显著增强干细胞的分泌功能。增强的旁分泌成分可以进一步激活PI3K-AKT信号通路，促进小鼠卵巢损伤后的生殖细胞存活。该方法简便、安全、可行，应用潜力大。
其中，所述间充质干细胞可以来源于脐带、骨髓、脂肪、脐带血、子宫内膜、羊膜、胎盘或牙髓；优选的，所述间充质干细胞为人脐带来源的间充质干细胞。
透明质酸钠浓度为0.1～0.3mg/ml，优选为0.3mg/ml。
预处理培养时间为3～5天，优选为5天。
以下通过实施例对本发明做进一步解释说明，但不构成对本发明的限制。应理解这些实施例仅用于说明本发明而不用于限制本发明的范围。另外，实施例中未详细说明的分子生物学方法均为本领域常规的方法，具体操作可参看分子生物学指南或产品说明书。下述实施例中的细胞培养条件为37℃、5％CO 2、 饱和湿度。
各实施例所用主要材料如下：
1.宫安康(宫腔用交联透明质酸钠凝胶，规格：5mg/ml，厂家：常州百瑞吉生物医药有限公司，医疗器械注册证号：国械注准20153641542)；2.MEM-alpha培养基(GIBCO，12571-063)；3.血清替代物(Helios，HPCFDCRL05)；4.ELISA试剂盒(Thermo，ebioscience)；5.超滤离心管(3KDa，Millipore，UFC900308)；6.p-AKT、AKT抗体(Cell Signaling Technology，#9271、#9272)；DDX4抗体(Abcam，ab27591)；7.嵌入型细胞培养小室(Sigma，PICM0RG50)；8.VCD(4-vinylcyclonhexene diepoxide，Sigma，94956)；9.DMEM/F12(1:1)培养基(GIBCO，11320-033)；10.Leibovitz's L-15培养基(GIBCO，11415064)；11.ITS液体培养基添加物(Sigma，I3146)；12.蛋白提取试剂盒(Invent，SD-001/SN-002)；13.T75培养瓶(Corning，430641)；14.Trizol(Ambion，15596018)。
野生型C57BL/6乳鼠购买于山东大学实验动物中心。人脐带间充质干细胞为本实验室制备。
实施例一：细胞因子芯片检测HA预处理对干细胞分泌功能的影响
S1.将同等数量的UCMSCs培养于正常培养基或添加0.3mg/ml HA的培养基中5天，其中第3天时换液，换液后继续培养。
S2.5天后将两组细胞更换无血清替代物的基础培养基13ml，于培养箱中培养48h后收集两组上清。
S3.将细胞上清1500×g离心后，经0.22μM滤器过滤，经3KDa超滤离心管在常温下5000×g离心50min，收集浓缩后的上清进行后续实验。
S4.将两组浓缩后的细胞上清送瑞博奥生物科技有限公司进行细胞因子芯片(Raybiotech，GSH-CAA-440)检测，并进行散点图分析及差异蛋白KEGG通路富集分析。
结果表明：两组细胞因子变化结果如图1所示，间充质干细胞经HA处理后，分泌功能显著增强。KEGG通路富集分析结果如图2所示，HA处理后，UCMSCs分泌的差异细胞因子富集于PI3K-AKT、JAK-STAT、MAPK等信号通路。
实施例二：ELISA法验证细胞因子芯片结果
收集浓缩上清过程如实施例一中所述，将对照组间充质干细胞上清(Mesenchymal stem cells-conditioned medium，MSC-CM)及HA处理组间充质干细胞上清(HA/MSC-CM)送至北京北方生物技术研究所有限公司进行ELISA检测，验证因子包括HGF、VEGF、EGF、SCF。
结果表明：如图3所示，经过HA处理后，细胞因子HGF、VEGF、EGF、SCF分泌增加，与芯片结果一致。
实施例三：体外卵巢培养检测HA预处理后干细胞上清对PI3K-AKT通路激活的影响
S1.收集间充质干细胞上清过程如实施例一所述。
S2.配制卵巢解剖液。用L15培养基配制含有10％FBS以及1％青霉素-链霉素的卵巢解剖液。
S3.进行卵巢培养准备。配制对照组、VCD(30μM)组、VCD+MSC-CM组以及VCD+HA/MSC-CM组培养基，将嵌入型培养小室放入6孔板培养基(每孔1.5ml)中平衡2h。
S4.体视镜下在新鲜配置的解剖液中分离出生后4天(PD4)的乳鼠卵巢。轻轻剥离卵巢周围组织及包膜，注意动作迅速并且不要损伤卵巢组织。
S5.将取得的卵巢用解剖液清洗三次，尽快转移至无菌操作台。在置于各组小室之前，用各组相应培养基再次清洗三次，清洗结束后，放置于各组培养小室中间位置，间隔放置，隔天换液，体外培养4天后收集卵巢组织提蛋白。
S6.利用Western blot方法检测各组p-AKT及AKT蛋白水平变化。
结果表明：如图4所示，对照组卵巢有一定水平p-AKT的表达，经过VCD损伤后，p-AKT表达水平显著降低；干细胞上清能够一定程度上逆转VCD损伤引起的p-AKT降低，而与未经过HA处理的干细胞上清相比，经HA预处理之后的干细胞上清对VCD损伤的逆转作用更强。各组AKT蛋白表达水平基本一致。
实施例四：体外卵巢培养检测HA预处理后干细胞上清对生殖细胞存活的影响
收集上清步骤如实施例一，卵巢体外培养步骤如实施例三。卵巢经体外培养8天后，收集卵巢，Bouin氏液固定，梯度脱水后，包埋，切片。各组切片进行免疫组织化学染色，标记生殖细胞标志物DDX4，各组切片显微镜下观察拍照。
结果表明：如图5所示，正常培养的卵巢中存在数量较多的生殖细胞；经VCD损伤后，生殖细胞数目明显减少且卵巢体积减小；与未经过HA处理的干细胞上清相比，HA预处理进一步增强了干细胞上清对VCD损伤生殖细胞的挽救，生殖细胞存活增加。
实施例五：RNA-seq检测HA预处理对干细胞基因mRNA表达的影响
S1.将同等数量的UCMSCs培养于正常培养基或添加0.3mg/ml HA的培养基 中5天，其中第3天时换液，换液后继续培养。
S2.第5天时，弃培养基，用PBS清洗细胞三遍后加入1ml胰酶消化，30s后加入5ml培养基终止消化。1000rpm离心5min，在细胞沉淀中加入1ml Trizol送山东修越生物技术有限公司利用Illumina novaseq 6000平台进行RNA测序。
结果表明：如图6所示，经RNA测序，在mRNA水平一共检测到29207个表达基因，按照p adj＜0.05，|log 2FC|≥1标准进行分析，其中两组差异表达基因只有2个(FOSB和NR4A1)，均为HA预处理后表达上调基因(FOSB的fold change为2.03；NR4A1的fold change为4.70)，并且这两个基因表达丰度均较低，提示HA预处理对干细胞基因mRNA水平表达没有明显影响。
应注意的是，以上实例仅用于说明本发明的技术方案而非对其进行限制。尽管参照所给出的实例对本发明进行了详细说明，但是本领域的普通技术人员可根据需要对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围。

Claims (15)

  1. 透明质酸钠在增强间充质干细胞分泌功能中的应用。
  2. 如权利要求1所述应用，其特征在于，所述应用至少包括如下任意一种或多种：
    (1)促进细胞因子分泌；
    (2)促进PI3K-AKT信号通路的激活；
    (3)增强对损伤的修复作用。
  3. 如权利要求2所述应用，其特征在于，细胞因子包括HGF、SCF、EGF和VEGF。
  4. 如权利要求2所述应用，其特征在于，增强对损伤的修复作用包括增强对卵巢组织的损伤修复作用。
  5. 如权利要求2所述应用，其特征在于，增强对损伤的修复作用具体为促进小鼠卵巢损伤后的生殖细胞存活。
  6. 如权利要求1所述应用，其特征在于，所述透明质酸钠浓度为0.1～0.3mg/ml。
  7. 如权利要求6所述应用，其特征在于，所述透明质酸钠浓度为0.3mg/ml。
  8. 如权利要求1所述应用，其特征在于，所述间充质干细胞来源于脐带、骨髓、脂肪、脐带血、子宫内膜、羊膜、胎盘或牙髓。
  9. 如权利要求8所述应用，其特征在于，所述间充质干细胞为人脐带来源的间充质干细胞。
  10. 一种促进干细胞分泌功能的方法，其特征在于，所述方法包括将间充质干细胞在含有透明质酸钠的培养基中进行预处理培养。
  11. 如权利要求10所述方法，其特征在于，所述间充质干细胞来源于脐带、骨髓、脂肪、脐带血、子宫内膜、羊膜、胎盘或牙髓。
  12. 如权利要求11所述方法，其特征在于，所述间充质干细胞为人脐带来 源的间充质干细胞。
  13. 如权利要求10所述方法，其特征在于，透明质酸钠浓度为0.1～0.3mg/ml。
  14. 如权利要求13所述方法，其特征在于，透明质酸钠浓度为0.3mg/ml。
  15. 如权利要求10所述方法，其特征在于，预处理培养时间为3～5天，优选为5天。
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