Integrin-targeted peptide promotes proliferation of bone marrow mesenchymal stem cells in SD rats

doi:10.12307/2022.728

Abstract

Abstract: BACKGROUND: Studies have reported that integrin-targeted peptide dopamine-arginine-glycine-aspatic acid (DOPA-RGD) could inhibit osteoclast activation and promote cell adhesion, while the optimal concentration and optimal time to apply DOPA-RGD peptide to intervene with bone marrow mesenchymal stem cells or modified titanium-based materials are unclear.
OBJECTIVE: To investigate the optimal mass concentration and the optimal intervention time of DOPA-RGD peptide to produce pro-proliferative effect when acting on bone marrow mesenchymal stem cells in rats.
METHODS: SD rat bone marrow mesenchymal stem cells were isolated and cultured in vitro and the cells with the required purity were selected for the next step of intervention. The third-generation bone marrow mesenchymal stem cells were inoculated in 96-well culture plates. Cells in the control group were cultured with simple α-MEM complete medium, while the remaining of the experimental groups were cultured with conditioned medium containing 2.5, 5, 10, 20, 40, and 80 mg/L DOPA-RGD peptide. Cell proliferation was measured by CCK-8 method after 1, 3, 5, and 7 days of DOPA-RGD peptide intervention. The optimal mass concentration and intervention time of DOPA-RGD peptide in bone marrow mesenchymal stem cells were selected, and the optimal mass concentration of DOPA-RGD peptide in bone marrow mesenchymal stem cells was further verified by apoptosis rate using flow cytometry. The effect of optimal mass concentration of DOPA-RGD peptide on the migration ability of bone marrow mesenchymal stem cells was verified by scratch assay.
RESULTS AND CONCLUSION: (1) Using repeated measures analysis of variance, the absorbance values of the cells intervened by DOPA-RGD peptide changed differently between different time length groups, and the best proliferation effect was achieved by the 5th day of intervention, and the difference was significant (F=3 012.618, P < 0.001). Paired comparison of grouping factors by the LSD method showed that there was a significant difference between the 10 mg/L DOPA-RGD peptide group and the three groups with concentrations greater than or equal to 20 mg/L and the control group (P < 0.01). (2) Apoptosis rate assay showed that DOPA-RGD peptide at a mass concentration of 10 mg/L on the fifth day of intervention significantly reduced the apoptosis rate of bone marrow stem cells compared with the rest of the groups (P < 0.001). (3) Compared with the control group, 10 mg/L DOPA-RGD peptide intervention for 24 hours showed some migration ability of bone marrow mesenchymal stem cells and the migration speed was significantly accelerated. (4) The results showed that 10 mg/L DOPA-RGD peptide intervention on day 5 had a significant pro-proliferative effect on bone marrow mesenchymal stem cells.

Key words: integrin, DOPA-RGD peptide, integrin-targeted peptide, bone marrow mesenchymal stem cells, cell proliferation, cell migration, distraction osteogenesis, rats

CLC Number:

Jia Qiyu, Huang Xiaoxia, Guo Jian, Huang Jinyong, Guo Xiaobin, Abdussalam·Alimujiang, Wu Tong, Ma Chuang. Integrin-targeted peptide promotes proliferation of bone marrow mesenchymal stem cells in SD rats[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(30): 4780-4786.

Figures/Tables 7

2.4 不同质量浓度DOPA-RGD肽对骨髓间充质干细胞增殖的影响对所得数据行重复测量方差分析，结果表明经DOPA-RGD肽作用不同时长的骨髓间充质干细胞吸光度值改变不同，存在显著性差异(F=3 012.618，P < 0.001)，说明随着DOPA-RGD肽作用时长增加，细胞增殖情况也随之发生明显改变。不同质量浓度DOPA-RGD肽干预骨髓间充质干细胞的吸光度值改变不同，存在显著性差异 (F=7 421.033，P < 0.001)，说明经不同质量浓度DOPA-RGD肽干预后，各组细胞增殖情况发生明显变化。通过 LSD 法进一步对各组进行两两比较，结果显示： 2.5 mg/L DOPA-RGD肽组与质量浓度≥20 mg/L的3个DOPA-RGD肽组及对照组比较均存在显著性差异(P < 0.05)；5 mg/L DOPA-RGD肽组与质量浓度≥20 mg/L的3个DOPA-RGD肽组均存在显著性差异(P < 0.05)；10 mg/L DOPA-RGD肽组与质量浓度≥20 mg/L的3个DOPA-RGD肽组及对照组比较差异均存在非常显著性差异(P < 0.01)；20 mg/L DOPA-RGD肽组与除对照组外的其他各组比较均存在显著性差异(P < 0.01)；40 mg/L DOPA-RGD肽组与80 mg/L DOPA-RGD肽组之间无显著差异(P > 0.05)，40 mg/L DOPA-RGD肽组与除80 mg/L DOPA-RGD肽组外的其他各组比较均存在显著性差异(P < 0.01)；80 mg/L组与质量浓度≤20 mg/L组的4个DOPA-RGD肽组及对照组比较差异均存在显著性差异(P < 0.001)；对照组与除5，20 mg/L DOPA-RGD肽组外的其他组均存在显著性差异(P < 0.05)。从各组吸光度值汇总数据中可以看出，2.5-10 mg/L DOPA-RGD肽组于第 5 天的吸光度值明显高于其余组，随后整体呈下降趋势，其中10 mg/L DOPA-RGD肽组在第5天和第7天吸光度值均最高。由此可见，当质量浓度≤10 mg/L 的DOPA-RGD肽在干预 5 天时可促进细胞的增殖。DOPA-RGD肽质量浓度≥20 mg/L会促进细胞凋亡，见表1，图4。"

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