冲击波对糖尿病慢性创面微环境下HaCat细胞增殖及迁移的影响

康靖汶; 盛炜; 瓮长水; 李美蓉; 罗芸; 张丽

doi:10.3969/j.issn.2095-5227.2022.12.010

冲击波对糖尿病慢性创面微环境下HaCat细胞增殖及迁移的影响

Effects of shock wave on proliferation and migration of HaCat cells in chronic diabetic wound cell model

摘要

摘要:
背景体外冲击波疗法可促进糖尿病慢性创面愈合，其机制有待深入研究。
目的观察不同剂量冲击波干预对糖尿病慢性创面微环境中HaCat细胞增殖和迁移能力的影响，尝试从细胞水平揭示体外冲击波疗法促进糖尿病慢性创面愈合的作用机制。
方法以人表皮角质形成细胞(HaCat细胞)为研究对象，分为对照组、糖尿病慢性创面模型(chronic diabetic wound，CDW)组以及不同剂量冲击波(shock wave， SW)干预组(SW1组：0.05 mJ/mm²，500脉冲；SW2组：0.10 mJ/mm²，500脉冲；SW3组：0.10 mJ/mm²，1 000脉冲)，通过高糖低氧孵育条件建立糖尿病慢性创面细胞模型，采用倒置显微镜观察细胞形态学改变，CCK-8法检测细胞增殖能力，细胞划痕试验检测细胞迁移能力，观察不同能流密度和脉冲输出的冲击波对糖尿病慢性创面微环境中HaCat细胞增殖和迁移能力的影响。
结果与对照组比较，冲击波干预后HaCat细胞形态未见明显改变。CCK-8实验显示，HaCat细胞增殖活性呈现“先增加、后下降”的趋势，其OD₄₅₀值：Day6＞Day4＞Day8＞Day2＞Day1，Day2及其后各时点与Day1比较差异有统计学意义(P均＜0.01)。各组HaCat细胞增殖活性：SW2组＞SW1组＞SW3组＞对照组＞CDW组(以Day6为例)(P均＜0.05)。倒置相差显微镜观察显示，各组HaCat细胞划痕愈合率均随时间延长逐渐增加，划痕愈合率：SW2组＞SW1组＞SW3组＞对照组＞CDW组(P均＜0.05)。
结论离体条件下，冲击波可促进糖尿病慢性创面微环境中功能受损的HaCat细胞的增殖和迁移能力；0.10 mJ/mm²、500脉冲的冲击波干预剂量对HaCat细胞的促增殖效应更为明显。

Abstract:
Background Extracorporeal shock wave therapy (ESWT) is an effective and noninvasive method for improving the healing of chronic wounds. More researches on mechanisms of ESWT are needed.
Objective To investigate the effect of shock wave (SW) with different doses on the proliferation and migration of HaCat cells in chronic diabetic wound (CDW) cell model, and reveal the mechanism of extracorporeal shock wave therapy in promoting the healing of chronic diabetic wounds at the cellular level.
Methods HaCat cells were divided into control group, CDW group, SW1 group (0.05 mJ/mm², 500 pulses), SW2 group (0.10 mJ/mm², 500 pulses), and SW3 group (0.10 mJ/mm², 1000 pulses). The CDW cell model was established by high-glucose and hypoxia incubation conditions. The morphological changes of HaCat cells were observed using an inverted microscope, the proliferation ability of HaCat cells was quantitatively detected by CCK-8, and the migration ability of HaCat cell was evaluated by scratch assay. The effects of shock waves with different energy flux and pulse output on the proliferation and migration of HaCat in the microenvironment of chronic diabetic wounds were observed.
Results There were no obvious morphological changes of HaCat cells in the SW groups when compared to the control group. CCK-8 test showed that the proliferation activity of HaCat cells had a trend of "first increasing, then decreasing". The OD₄₅₀ increased over time (day6 > day4 > day8 > day2 > day1), with significant difference between different timepoints (all P＜0.01). The effects of different interventions on the proliferation activity of HaCat cells in day6 were as follows: SW2 group＞SW1 group＞SW3 group＞control group > CDW group, with statistically significant differences between each group (all P＜0.05). Scratch assay results showed that the scratch healing rates of HaCat cells in different groups increased by time. The healing rates in different groups were as follows: SW2 group＞SW1 group＞SW3 group＞control group > CDW group, and the differences between groups were statistically significant (all P＜0.05).
Conclusion In vitro, shock wave can promote the proliferation and migration abilities of HaCat cells in chronic diabetic wound cell model. The effect of shock wave (0.10 mJ/mm², 500 pules) is more obvious.

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