人脐带间充质干细胞对脂多糖活化的小胶质细胞BV-2表型的影响

目的探讨人脐带间充质干细胞(hUCMSCs)对脂多糖(LPS)活化的小胶质细胞功能表型的影响。方法实验设未诱导对照组(加入PBS无LPS诱导的BV-2细胞),LPS诱导组(加入1.0μg/mL的LPS诱导BV-2细胞向M1型分化),按比例加入不同浓度hUCMSCs进行干预(LPS+低、中、高浓度hUCMSCs干预组hUCMSCs与BV-2细胞比例分别为:1:100、1:10、1:1),分别于24、48、72 h观察BV-2形态变化,Griess法检测细胞培养上清中M1表型产物一氧化氮(NO)的浓度;将hUCMSCs与BV-2细胞在不同条件下(LPS+/LPS-)共培养,qRT-PCR检测BV-2细胞M2表型标记物精氨酸酶1表达变化。数据分析采用重复测量资料的方差分析,组间比较采用Tukey分析。结果BV-2细胞经LPS诱导后活化,细胞变大,呈"煎饼状"、"阿米巴状"变化,呈经典的M1表型分化;与未诱导对照组相比,LPS诱导组48、72 h BV-2细胞NO含量升高48 h:(0.507±0.012)μg/mL比(5.183±0.171)μg/mL;72 h:(0.934±0.024)μg/mL比(12.498±0.168)μg/mL,P均<0.01],与LPS诱导组比较,LPS+低、中、高浓度hUCMSCs干预组72 h(12.498±0.168)μg/mL比(11.852±0.149)μg/mL、(9.796±0.048)μg/mL、(1.876±0.063)μg/mL]及LPS+中、高浓度hUCMSCs干预组48 h NO含量(5.183±0.171)μg/mL比(3.921±0.066)μg/mL、(1.202±0.012)μg/mL]降低,且呈干预浓度依赖性NO含量下降,差异均有统计学意义(P均<0.01)。精氨酸酶1 qRT-PCR结果显示:与未诱导组比较,单纯高浓度hUCMSCs干预组3个时间点精氨酸酶1的相对表达量均升高(1.046±0.057比19.266±0.641,1.114±0.093比16.977±0.749,1.139±0.118比16.959±0.625),与LPS诱导对照组(0.000)比较,未诱导对照组(1.046±0.057,1.114±0.093,1.139±0.118)及LPS+高浓度hUCMSCs干预组精氨酸酶1表达(0.879±0.077,1.023±0.081,1.121±0.078)升高,差异具有统计学意义(P均<0.01)。结论LPS可诱导小胶质细胞BV-2炎症反应,而hUCMSCs可抑制活化小胶质细胞的炎症反应,抵消LPS对BV-2的诱导效应,促进小胶质细胞由促炎的M1型向抗炎的M2型转变。

Effects of human umbilical cord tissue-derived mesenchymal stem cell on the phenotype of BV-2 cells activated by lipopolysaccharide in vitro

ObjectiveTo explore the effects of human umbilical cord tissue-derived mesenchymal stem cells (hUCMSCs) on the phenotype shift of microglia induced by lipopolyccharide (LPS) . MethodLipopolysaccharide (LPS) was used to stimulate BV-2 microglial cell line to establish microglial M1 phenotypic differentiation model in vitro, while the BV-2 microglial cell was treated by PBS without LPS as control group. Then hUCMSCs were co-cultured with BV-2 at different ratios (hUCMSCs to BV-2 cell ratio at 1:100, 1:10, 1:1). After 24, 48 and 72 h, the morphology of BV-2 were observed, and the M1 phenotype product NO in the cell culture supernatants was detected by Griess reagent. The expression of arginase1, a M2 phenotype marker in BV-2 cells under different conditions (LPS+/LPS-) was determined by qRT-PCR analysis. The data was analyzed by variance of repeated measurement data, and Tukey analysis was used for comparison. ResultThe cells became larger and "pancake-like" and "amemeba-like" cells were observed after treated by LPS, showing classical M1 phenotype differentiation. Compared with control group, the NO concentration of cell culture supernatants was increased significantly after LPS stimulation for more than 48 hours 48 h: (0.507±0.012) μg/mL vs (5.183±0.171) μg/mL, 72 h: (0.934±0.024) μg/mL vs (12.498±0.168) μg/mL] (P < 0.01). In addition, the NO content in cells treated by LPS+hUCMSCs was significantly lower than that in cells treated by LPS alone 48 h: (5.183±0.171) μg/ mL vs (3.921±0.066) μg/mL and (1.202±0.012) μg/mL, 72h: (12.498 ± 0.168) μg/mL vs (11.852±0.149) μg/mL, (9.796±0.048) μg/mL and (1.876±0.063) μg/mL], which presented in concentration-dependent manner (P < 0.01). The expression of arginase1 was increased significantly in the cells treated by hUCMSCs in a concentration-dependent manner under no LPS induction. When the ratio of hUCMSCs to BV-2 was 1:1, a statistical difference in the expression of arginase 1 was found between cells treated by LPS+hUCMSCs and untreated cells (24 h: 1.046±0.057 vs 19.266±0.641, 48 h: 1.114±0.093 vs 16.77±0.749 and 72 h: 1.139±0.118 vs 16.959±0.625, P < 0.01). The expression of arginase1 was not detected in the BV-2 cells under LPS induction (0.000), while the expression of arginase 1 could be detected at three time points both in BV-2 treated with LPS alone or LPS+hUCMSCs (LPS: 1.046±0.057, 1.114±0.093, 1.139±0.118, LPS+hUCMSCs: 0.879±0.077, 1.023±0.081, 1.121±0.078) (P < 0.01) . ConclusionshUCMSCs could inhibition the inflammatory response of BV-2 induced by LPS and the transformation of microglia from proinflammatory M1 to anti-inflammatory M2.