Liposome prepared from human umbilical cord mesenchymal stem cell conditioned medium for repair of skin wound in rats

doi:10.3969/j.issn.2095-4344.1587

Abstract

Abstract:

BACKGROUND: The stem cell conditioned medium contains a large number of cell secretory factors contributing to wound repair. Liposome encapsulation facilitates the sustained release of cytokines at the wound site.
OBJECTIVE: To study the effect of liposomes prepared from the conditioned medium of human umbilical cord mesenchymal stem cells on the healing of skin wounds in rats.
METHODS: Human umbilical cord mesenchymal stem cells were isolated and cultured by tissue explant method, and cytokine detection was then performed. Twenty Sprague-Dawley rats were provided by the Experimental Animal Center of Shanxi Medical University in China to make skin wound models of 1 cm in diameter. Membrane dispersion technique was used to prepare the conditioned medium liposomal vesicles used for skin wounds in the rats. The skin and surrounding tissues of the repair site were taken for hematoxylin-eosin staining. The histological scores were used to judge the repair of wounds.
RESULTS AND CONCLUSION: (1) The human umbilical cord mesenchymal stem cells were positive for CD73 (99.7%), CD90 (95.7%), CD105 (99.4%), CD45 (1.1%) and CD34 (0.1%). The cells could be induced to differentiate into osteocytes and adipocytes. (2) The concentrations of vascular endothelial growth factor, interleukin-8, and monocyte chemotactic protein 1 in the conditioned medium were 558.60 ng/L, 5 236.48 ng/L, and 2 972.55 ng/L, respectively. (3) The average particle size of liposome in the conditioned medium prepared by membrane dispersion method was 262.3 nm. Conditional culture of human umbilical cord mesenchymal stem cells and its liposome could increase the hair follicles and glands at the site of rat skin defects, the epidermis was thickened, and the histological score was markedly increased. There was no difference between the conditioned medium and conditioned medium liposome groups at 2 days of culture (P=0.730), but significant differences existed among the other groups (P < 0.05). To conclude, the human umbilical cord mesenchymal stem cell conditioned medium and its liposomes can promote the repair of skin defects in rats.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Mesenchymal Stem Cell Transplantation, Cell Culture Techniques, Liposomes, Tissue Engineering

CLC Number:

R459.9，R392.4

Liu Meilin, Fu Songtao, Wang Peisen, Hao Ran, Chang Bingmei, Hou Xiuying. Liposome prepared from human umbilical cord mesenchymal stem cell conditioned medium for repair of skin wound in rats[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(5): 734-740.

Figures/Tables 2

2.1 人脐带间充质干细胞的原代培养原代培养第10天可见脐带组织块周围有大量贴壁细胞爬出，细胞大小不一，呈短棒状或多角形。待生长达80%融合时去除组织块，进行消化传代。传至第3代时，细胞呈形态大小均一的长梭形漩涡状。见图2。 2.2 人脐带间充质干细胞表面标志物检测第3代人脐带间充质干细胞经流式细胞仪检测细胞表面抗原，结果显示CD105、CD44、CD29的阳性率分别为99.7%、95.7%、99.4%，CD45、CD34的阳性率分别为1.1%、0.1%，见图3。 2.3 人脐带间充质干细胞成骨细胞、成脂肪细胞诱导如图4，5所示：人脐带间充质干细胞成骨细胞诱导28 d后，经茜素红染色可见红色颗粒物；成脂细胞诱导21 d后，油红O染色呈可见大量油样的红色空泡。表明人脐带间充质干细胞可向成骨细胞、成脂细胞方向分化。 2.4 人脐带间充质干细胞条件培养基SDS-PAGE考马斯亮蓝染色第5代人脐带间充质干细胞的条件培养液蛋白质量浓度为0.145 g/L，经考马斯亮蓝染色SDS-PAGE，70 kD有蛋白条带出现，而空白培养基没有蛋白条带出现，见图6。人脐带间充质干细胞条件培养基中含有细胞分泌因子，用无血清的基础培养基培养人脐带间充质干细胞可获得无动物源性的条件培养基(干细胞分泌因子)。 2.5 人脐带间充质干细胞条件培养基中相关因子蛋白浓度经ELISA测定，条件培养液中血管内皮生长因子、白细胞介素8、单核细胞趋化蛋白1的质量浓度分别为558.60 ng/L、5 236.48 ng/L、2 972.55 ng/L。 2.6 人脐带间充质干细胞条件培养基脂质体电镜观察电镜下，用薄膜分散法制备得到人脐带间充质干细胞分泌因子脂质体呈不规则的圆球状，大小不均一，但分散比较均匀，见图7。 2.7 脐带间充质干细胞分泌因子脂质体囊泡粒径用薄膜分散法制备得到人脐带间充质干细胞条件培养基脂质体，其粒径呈拟正态分布，粒径范围在37.84-3 580.00 nm，平均粒径为262.3 nm，见图8。 2.8 观察大鼠伤口愈合情况及病理组织切片判断其修复效果大鼠皮肤创口修复实验表明，人脐带间充质干细胞条件培养基和其脂质体制剂均可以促进创伤的修复，创口及周边组织石蜡切片苏木精-伊红染色后，光镜下观察可见：空白对照组与空白脂质体对照组，创面修复缓慢，真皮层及创面深层未见腺体和毛囊生成，有组织水肿；人脐带间充质干细胞条件培养基组，早期大鼠皮肤表皮层加厚，创面下腺体与毛囊增多；人脐带间充质干细胞条件培养基脂质体组，早期大鼠皮肤表皮层加厚，创面下及皮肤深层的腺体与毛囊增多，后期毛囊和皮脂腺细胞发生迁移和上皮分化。干细胞分泌因子及其脂质体处理伤口，可促进了创口的愈合和修复，表现为促进伤口处毛囊和皮脂腺生成、迁移和上皮分化，从而促进创面缩小，加快损伤组织的修复。与单纯的人脐带间充质干细胞条件培养基组相比，人脐带间充质干细胞条件培养基脂质体组在皮肤深层也发现大量腺体和毛囊生成的变化，说明人脐带间充质干细胞条件培养基脂质体制剂具有渗透到皮肤深层来促进损伤修复的作用，见图9。对伤口组织病理切片进行组织学评分，见图10。第2天的条件培养基与条件培养基脂质体差异无显著性意义(P=0.730)，其他各组之间有显著差异(P < 0.05)。"

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