反复冻融与超声波处理制备的干细胞裂解液修复皮肤损伤的效果比较OA
Comparison of the effects of stem cell lysate prepared by repeated freeze-thaw and ultrasonic treatment on skin injury repair
目的:比较反复冻融法与超声波破碎法制备的干细胞裂解液修复皮肤损伤的效果,筛选干细胞裂解液的最优制备方法.方法:将人皮肤成纤维细胞以1×105/mL、1×106/mL、5×106/mL、1×107/mL4种密度重悬于生理盐水,分别采用反复冻融和超声波破碎法处理,通过BCA法检测细胞破碎率和蛋白质含量,筛选两种方法最佳制备条件;采用细胞划痕愈合实验、CCK-8法、Transwell实验分别检测人皮肤成纤维细胞划痕愈合能力、细胞活力、细胞迁移数,ELISA检测裂解液中表皮生长因子(EGF)、血管内皮生长因子(VEGF)、干细胞因子(SCF)蛋白浓度.结果:超声波破碎法最佳条件为细胞浓度5×106/mL、超声处理5 min;反复冻融法最佳条件为细胞浓度5×106/mL、冻融3次(45 min).两种方法细胞破碎率无显著差异(P＞0.05),超声波破碎法制备的裂解液总蛋白含量更高;两种方法制备的裂解液对人皮肤成纤维细胞活力的影响无显著差异(P＞0.05),反复冻融法制备的裂解液能显著促进人皮肤成纤维细胞迁移、提升细胞划痕修复效率(P＜0.05).ELISA结果显示,反复冻融法制备的裂解液中VEGF、SCF蛋白浓度显著高于超声波破碎法(P＜0.05或P＜0.01),EGF浓度无显著差异(P＞0.05).结论:反复冻融法制备的干细胞裂解液修复皮肤损伤的效果优于超声波破碎法.
Objective:To compare the effects of stem cell lysate prepared by repeated freeze-thaw method and ultrasonic disruption method on skin injury repair,and screen the optimal preparation method of stem cell lysate.Methods:Human skin fibroblasts were resuspended in normal saline at four densities(1×105/mL,1×106/mL,5×106/mL,1×107/mL),and treated by repeated freeze-thaw and ultrasonic disruption,respectively.The cell disruption rate and protein content were detected by BCA assay to screen the optimal preparation conditions of the two methods.The scratch healing ability,viability and migration number of human skin fibroblasts were detected by cell scratch healing assay,CCK-8 method and Transwell assay,respectively.The protein concentration of epidermal growth factor(EGF),vascular endothelial growth factor(VEGF)and stem cell factor(SCF)in the lysate were detected by ELISA.Results:The optimal condition of ultrasonic disruption method was cell concentration of 5×106/mL with ultrasonic treatment for 5 min;the optimal condition of repeated freeze-thaw method was cell concentration of 5×106/mL with three freeze-thaw cycles(45 min).There was no significant difference in cell disruption rate between the two methods(P＞0.05),and the total protein content of lysate prepared by ultrasonic disruption method was higher.There was no significant difference in the effect of lysates prepared by the two methods on the viability of human skin fibroblasts(P＞0.05).Compared with ultrasonic disruption method,the lysate prepared by repeated freeze-thaw method could significantly promote the migration of human skin fibroblasts and improve the efficiency of cell scratch repair(P＜0.05).ELISA results showed that the concentrations of VEGF and SCF proteins in the lysate prepared by repeated freeze-thaw method were significantly higher than those by ultrasonic disruption method(P＜0.05 or P＜0.01),and there was no significant difference in EGF content(P＞0.05).Conclusion:The stem cell lysate prepared by repeated freeze-thaw method has a better effect on skin injury repair than that by ultrasonic disruption method.
李凯;徐聪;秦金伟
浙江大学杭州国际科创中心,浙江 杭州 311200||安徽普利药业有限公司杭州分公司,浙江 杭州 310000安徽普利药业有限公司杭州分公司,浙江 杭州 310000浙江大学杭州国际科创中心,浙江 杭州 311200||安徽普利药业有限公司杭州分公司,浙江 杭州 310000
医药卫生
皮肤损伤修复干细胞裂解液反复冻融法超声波破碎法成纤维细胞
skin injury repairstem cell lysaterepeated freeze-thaw methodultrasonic disruption methodfibroblast
《江苏大学学报（医学版）》 2026 (2)
129-135,7
中国博士后科学基金资助项目(2023MZ32157)
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