大气压冷等离子体对人脐带间充质干细胞增殖和分化的影响

Effects of Cold Atmospheric Plasma on Proliferation and Differentiation of Human Umbilical Cord Mesenchymal Stem Cells

人脐带间充质干细胞(hUC-MSCs)是一种具有多向分化潜能的干细胞,具有广阔的应用前景,但是由于其体外大量增殖和定向分化等问题尚未解决,制约了其进一步应用。既往研究表明,大气压冷等离子体(CAP)可促进离体培养的脂肪或骨髓来源MSCs的增殖和分化,但对hUC-MSCs的影响国内外尚未见报道。因此,本研究的目标是探讨CAP对hUC-MSCs增殖和成骨分化的影响。在电源频率(17 kHz)和气体流量(4 slpm)保持不变的条件下,原代培养的hUC-MSCs经不同放电电压下产生的氦(He)等离子体处理后,在不同时间点收集细胞,采用CCK-8法检测细胞活力,微板法检测碱性磷酸酶(ALP)的活性,茜红素染色检测钙化结节的形成。结果显示,与对照组相比,纯氦气组上述指标无明显变化,而CAP组的细胞活力显著降低,并且与等离子体放电电压和处理时间呈负相关; CAP处理10 s和30 s后,ALP活性则无明显变化,而处理60 s后ALP活性显著下降,同时CAP处理30 s后钙化结节面积无显著变化。以上结果表明,本试验条件下的CAP处理可抑制hUCMSCs的增殖,但对其体外成骨分化无明显影响。

Human umbilical cord mesenchymal stem cell (hUC-MSC) is a kind of stem cell with multidirectional differentiation potential,which is widely used in tissue engineering and disease treatment.However,the limited proliferation and orientation differentiation of hUC-MSCs have restricted its further application.Previous studies showed that cold atmospheric plasma (CAP) could promote the proliferation and differentiation of MSCs from adipose and bone marrow in vitro,but the effects on hUC-MSCs have not been reported.Therefore,the purpose of this study is to explore the effects of CAP on the proliferation and osteogenic differentiation of hUC-MSCs.The primary cultured hUC-MSCs were treated by the helium CAPs produced under different discharge voltages with the power supply driving frequency of 17 kHz and gas flow rate of 4 slpm.The cell viability was measured by CCK-8 kit for different treatment time.The activity of alkaline phosphatase (ALP) was measured by the microplate method.Alizarin red staining was used to measure the formation of calcified nodules.The results showed that compared with the control group,the above indexes in the He group showed no obvious changes,while the cell viability of hUC-MSCs in the CAP group was markedly reduced,and was negatively correlated with the discharge voltage and the treatment time.After CAP treatment for 10 s and 30 s,ALP activity showed no significant change,while ALP activity decreased significantly after treatment for 60 s.Meanwhile,there was no significant change in the area of calcified nodules after CAP treatment for 30 s.The above results indicated that under this experimental condition,CAP treatment could inhibit the proliferation of hUC-MSCs,but it had no obvious effect on osteogenic differentiation in vitro.