乳酸诱导巨噬细胞M2型极化对黑色素瘤细胞增殖、凋亡、迁移和侵袭的影响

Effects of lactic acid-induced M2-type polarization of macrophages on proliferation,apoptosis,migration and invasion of melanoma cells

目的探究乳酸(LA)是否可通过调控巨噬细胞M2型极化影响黑色素瘤细胞增殖、凋亡、迁移和侵袭。方法2021年1月至2022年2月采用细胞计数试剂盒(CCK-8)法检测乳酸对细胞的毒性;使用乳酸干预巨噬细胞后的细胞上清液培养黑色素瘤细胞,经CCK-8法和流式细胞术检测细胞增殖和凋亡;经划痕愈合实验和Transwell法检测细胞迁移和侵袭。通过光学显微镜对乳酸干预后的巨噬细胞的形态进行观察,并通过ELISA检测巨噬细胞极化相关因子的表达情况。实时荧光定量逆转录聚合酶链式反应(qRT-PCR)检测乳酸对巨噬细胞中肿瘤蛋白翻译调节因子1(Tpt1)、肺腺癌转移相关转录本1(Malat1)表达的影响。结果乳酸对巨噬细胞Raw264.7和黑色素瘤细胞B16F10均无明显毒性(P>0.05)。5、10、20 mmol/L乳酸干预Raw264.7细胞后的上清液后,B16F10细胞活力[(125.36±7.88)%、(144.36±8.65)%、(167.28±10.12)%比(100.00±0.00)%]、划痕愈合率[(39.21±3.15)%、(52.65±3.87)%、(64.24±6.12)%比(25.26±2.23)%]、侵袭细胞数[(89.36±8.12)个、(113.05±10.03)个、(131.25±11.14)个比(45.36±4.21)个]增高,凋亡率[(41.23±4.02)%、(29.26±3.14)%、(18.64±2.09)%比(57.26±4.25)%]下降(P<0.05)。5、10、20 mmol/L乳酸作用Raw264.7细胞后,细胞形态出现M2型形态变化,M1型相关分子表达差异无统计学意义(P>0.05),M2型相关因子血管内皮生长因子(VEGF)、白细胞介素-10(IL-10)[(47.26±4.02)ng/L、(65.32±5.48)ng/L、(79.36±7.12)ng/L比(27.25±2.21)ng/L]、CD163表达均显著增多(P<0.05)。乳酸对Raw264.7细胞中Tpt1、Malat1表达具有明显的促进作用(P<0.05)。结论乳酸诱导巨噬细胞M2型极化促进黑色素瘤恶性生物学发展,且Tpt1、Malat1表达可能参与此过程。

Objective To investigate whether lactic acid(LA)can affect melanoma cell proliferation,apoptosis,migration and invasion by regulating macrophage M2-type polarization.Methods The toxicity of LA to cells was detected by CCK-8 method from January 2021 to February 2022;the melanoma cells were cultured with the cell supernatant after LA intervening macrophages,and cell proliferation and apoptosis were detected by CCK-8 method and flow cytometry;the cell migration and invasion were detected by scratch healing assay and Transwell assay.The morphology of macrophages after LA intervention was observed by light microscope,and the expression of macrophage polarization-related factors was detected by ELISA.The effects of LA on the expressions of tumor protein translation regulator 1(Tpt1)and lung adenocarcinoma metastasis-associated transcript 1(Malat1)in macrophages were measured by qRTPCR.Results LA had no obvious toxicity to macrophage Raw264.7 and melanoma cell B16F10(P>0.05).After the supernatant of Raw264.7 cells was treated with 5,10 and 20 mmol/L LA,B16F10 cell activity[(125.36±7.88)%,(144.36±8.65)%,(167.28±10.12)%vs.(100.00±0.00)%],scratch healing rate[(39.21±3.15)%,(52.65±3.87)%,(64.24±6.12)%vs.(25.26±2.23)%]and invasion cell number[(89.36±8.12)individual,(113.05±10.03)individual,(131.25±11.14)individual vs.(45.36±4.21)individual]increased,the apoptosis rate[(41.23±4.02)%,(29.26±3.14)%,(18.64±2.09)%vs.(57.26±4.25)%]decreased(P<0.05).After treating Raw264.7 cells with 5,10 and 20 mmol/L LA,the cell morphology showed M2-type morphological changes,and there was no significant difference in the expression of M1-type-related molecules(P>0.05),but the expressions of M2-type-related factors vascular endothelial growth factor(VEGF),interleukin-10(IL-10)[(47.26±4.02)ng/L,(65.32±5.48)ng/L,(79.36±7.12)ng/L vs.(27.25±2.21)ng/L]and CD163 were greatly increased(P<0.05).LA greatly promoted the expressions of Tpt1 and Malat1 in Raw264.7 cells(P<0.05).Conclusion LA induces M2-type polar ization of macrophages to promote the malignant biological development of melanoma,and the expressions of Tpt1 and Malat1 may be involved in this process.