Effects of mesenchymal stem cell conditioned media from different sources on intrinsic aging cells

doi:10.3969/j.issn.2095-4344.1765

Abstract

Abstract:

BACKGROUND: With the economic development, people have an increasing pursuit of beauty, and anti-aging research has become a hot research topic. Stem cells have self-renewal and multi-directional differentiation ability, and mesenchymal stem cells are one of them. Studies have shown that mesenchymal stem cells can repair aging cells through paracrine action, but little has been reported on the anti-aging effects of mesenchymal stem cell conditioned media from different sources.

OBJECTIVE: To compare the anti-aging effects of human adipose-, amniotic fluid-, placenta- and umbilical cord-derived mesenchymal stem cell conditioned media on aging skin fibroblasts.
METHODS: Human adipose, amniotic fluid, placenta and umbilical cord-derived mesenchymal stem cell conditioned media were prepared and used to culture aging skin fibroblasts. Cell viability was detected by cell counting kit-8 and cell senescence rate measured by β-galactosidase staining kit. DCFH-DA probe was used to detect the content of reactive oxygen species. qPCR was used to detect the expression levels of p16, p53, COL1 and KLOTHO genes.
RESULTS AND CONCLUSION: (1) Four sources of mesenchymal stem cell conditioned media could increase the cell viability of skin fibroblasts, among which the adipose-derived mesenchymal stem cell conditioned medium had the best effect and the lowest β-galactosidase staining rate. (2) Four sources of mesenchymal stem cell conditioned media could reduce reactive oxygen species content, and adipose- and amniotic fluid-derived mesenchymal stem cell conditioned media had the lowest content of reactive oxygen species. (3) p16 and p53 levels in the placenta and umbilical cord-derived mesenchymal stem cell conditioned media were higher than those in the control group. The expression of p53 in the amniotic fluid-derived mesenchymal stem cell conditioned medium was lower than that in the control group, but there was no difference in the p16 expression between the two groups. Compared with the control group, the expression of p16 was lower and the expression of p53 was higher in the adipose-derived mesenchymal stem cell conditioned medium. (4) Compared with the control group, the expression of COL1 gene was significantly higher in the different culture media except for the amniotic fluid-derived mesenchymal stem cell conditioned medium. Compared with the control group, the expression of KLOTHO was significantly higher in different culture media except for placenta-derived mesenchymal stem cell conditioned medium, and highest in the adipose-derived mesenchymal stem cell conditioned medium. To conclude, mesenchymal stem cell conditioned media from four different sources can delay cell senescence to a certain extent, though its mechanism remains to be further studied.

Key words: skin fibroblasts, endogenous senescence, adipose-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, umbilical cord mesenchymal stem cells, amniotic fluid mesenchymal stem cells, mesenchymal stem cell conditioned medium, Natural Science Foundation of Guangdong Province

CLC Number:

R459.9

Zheng Guichun, Zhao Shucan, Huang Lizhen, Zhang Xiaofang, Wang Bingyun, Chen Shengfeng, Chen Zhisheng. Effects of mesenchymal stem cell conditioned media from different sources on intrinsic aging cells[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(21): 3357-3363.

Figures/Tables 1

2.1 衰老人皮肤成纤维细胞的形态及β-半乳糖苷酶染色结果当人皮肤成纤维细胞传至45代以上，细胞呈扁平状，逐渐排列不规则，胞核增大，细胞增殖速度明显减缓，见图1A。经β-半乳糖苷酶染色后，人皮肤成纤维细胞被染成蓝绿色，综合人皮肤成纤维细胞形态的改变可证明细胞发生了复制性衰老，见图1B。 2.2 四种间充质干细胞条件培养基对衰老人皮肤成纤维细胞增殖的影响 CCK-8检测结果见图2。在24-72 h，4种间充质干细胞条件培养基组的细胞活性显著高于对照组(P < 0.01)，说明人脂肪、羊水、脐带、胎盘来源间充质干细胞条件培养基能提高衰老人皮肤成纤维细胞的活力，从结果可看出，脂肪间充质干细胞条件培养基的效果最好。 2.3 衰老人皮肤成纤维细胞β-半乳糖苷酶染色结果培养72 h后，SA-β-gal染色结果显示，脂肪间充质干细胞条件培养基组和胎盘间充质干细胞条件培养基组蓝绿色的衰老细胞较少，与对照组比较差异有显著性意义(P < 0.01)，见图3，说明上述2种条件培养基具有显著抗细胞衰老作用。 2.4 衰老人皮肤成纤维细胞活性氧水平 4种间充质干细胞条件培养基组细胞活性氧荧光强度均低于对照组(P < 0.01)，其中胎盘间充质干细胞条件培养基组与脐带间充质干细胞条件培养基组差异无显著性意义(P > 0.05)；脂肪间充质干细胞条件培养基组和羊水间充质干细胞条件培养基组差异也无显著性意义(P > 0.05)，但这2组细胞荧光强度显著低于胎盘间充质干细胞条件培养基组和脐带间充质干细胞条件培养基组(P < 0.05)，说明脂肪、羊水来源间充质干细胞条件培养基降低衰老人皮肤成纤维细胞活性氧荧光强度的效果最好，见图4。 2.5 实时荧光定量PCR检测衰老基因表达衰老基因p16和p53在胎盘间充质干细胞条件培养基组和脐带间充质干细胞条件培养基组表达较高，且高于对照组(P < 0.01)；脂肪间充质干细胞条件培养基组p16基因表达低于对照组，差异有显著性意义(P < 0.05)，但p53基因表达较高；羊水间充质干细胞条件培养基组p16表达量与对照组差异无显著性意义，p53的表达显著低于对照组(P < 0.01)，见图5A。与对照组相比，脂肪间充质干细胞条件培养基组COL1和KLOTHO基因表达最高，见图5B。"

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