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目的:探讨单细胞海藻(MPPT)提取物对人脐带间充质干细胞(hUC-MSCs)的抗衰老作用。方法:取剖宫产新生儿脐带,分离、传代培养hUC-MSCs,显微镜下观察细胞形态,收集第3、10、15代细胞,qRT-PCR分析不同代细胞中p16、p21、p53、PCNA、Sirt2 mRNA的表达情况。CCK-8法检测不同质量浓度MPPT提取物对第15代hUCMSCs增殖的影响;流式细胞术检测MPPT提取物对hUC-MSCs细胞周期和凋亡的影响;β-半乳糖苷酶染色法检测MPPT提取物对hUC-MSCs衰老的影响;qRT-PCR检测MPPT提取物对hUC-MSCs中p16、p21、p53、PCNA、Sirt2 mRNA表达的影响。结果:第3代细胞呈梭形贴壁细胞,第15代细胞呈不规则状态,易脱壁。随着传代次数的增加,hUC-MSCs中PCNA、Sirt2 mRNA表达减少(F=76.944、25.649,P<0.001),而p16、p21和p53 mRNA表达增加(F=46.714、77.645、845.676,P<0.001);与第15代细胞相比,1 g/L的MPPT提取物可促进第15代hUC-MSCs增殖,促进细胞周期进入S期,抑制细胞凋亡(P均<0.05);MPPT提取物处理组衰老细胞率降低(F=773.557,P<0.001),细胞中PCNA、Sirt2 mRNA表达增加,p16、p21和p53 mRNA表达降低(P均<0.05)。结论:MPPT提取物可能通过调节hUC-MSCs中p16、p21、p53、PCNA和Sirt2等衰老相关基因的表达发挥抗衰老作用。
Objective: To investigate the anti-aging effect of MPPT on human umbilical cord mesenchymal stem cells (hUC-MSCs). Methods: Umbilical cord cesarean section neonatal umbilical cord, hUC-MSCs were isolated and subcultured. Cell morphology was observed under the microscope. The 3rd, 10th and 15th generation cells were harvested. The expression of p16, p21, p53 and PCNA were detected by qRT- Sirt2 mRNA expression. The effects of different MPPT extracts on the proliferation of hUCMSCs on passage 15 were examined by CCK-8 assay. The effects of MPPT extracts on the cell cycle and apoptosis of hUC-MSCs were detected by flow cytometry. The effects of MPPT on β-galactosidase The effect of extract on the senescence of hUC-MSCs was detected by qRT-PCR. The effect of MPPT extract on the expression of p16, p21, p53, PCNA and Sirt2 mRNA in hUC-MSCs was detected by qRT-PCR. Results: The third generation cells were spindle-shaped adherent cells, 15th generation cells were irregular, easy to detached. With the increase of passage times, the expression of PCNA and Sirt2 mRNA in hUC-MSCs was decreased (F = 76.944, 25.649, P <0.001), while the expression of p16, p21 and p53 mRNA increased (F = 46.714,77.645,845.676, P <0.001 ); Compared with the 15th generation cells, 1 g / L MPPT extract can promote the proliferation of the 15th generation hUC-MSCs, promote the cell cycle to enter the S phase and inhibit the apoptosis (all P <0.05); MPPT extract treatment The number of senescent cells was decreased (F = 773.557, P <0.001). The expression of PCNA and Sirt2 mRNA was increased and the expressions of p16, p21 and p53 mRNA were decreased (all P <0.05). Conclusion: MPPT extract may play an anti-aging role by regulating the expression of aging-related genes p16, p21, p53, PCNA and Sirt2 in hUC-MSCs.