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

doi:10.3877/cma.j.issn.2095-1221.2020.04.003

Abstract

Abstract:

Objective

To explore the effects of human umbilical cord tissue-derived mesenchymal stem cells (hUCMSCs) on the phenotype shift of microglia induced by lipopolyccharide (LPS) .

Method

Lipopolysaccharide (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.

Result

The 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) .

Conclusions

hUCMSCs could inhibition the inflammatory response of BV-2 induced by LPS and the transformation of microglia from proinflammatory M1 to anti-inflammatory M2.

Key words: Lipopolysaccharides, Mesenchymal stem cells, BV-2 cells

Yabo Mei, Wanqiao Zhang, Chong Chen, Tao Han, Zhichun Feng. Effects of human umbilical cord tissue-derived mesenchymal stem cell on the phenotype of BV-2 cells activated by lipopolysaccharide in vitro[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2020, 10(04): 213-218.

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Figures/Tables 7

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基因	引物序列（5'-3'）		退火温度（℃）
Arginase-1	上游	TTGCGAGACGTAGACCCTGG	60
?	下游	CAAAGCTCAGGTGAATCGGC	?
GAPDH	上游	TTCACCACCATGGAGAAGGC	61
?	下游	GGCATGGACTGTGGTCATGA	?

基因	引物序列（5'-3'）		退火温度（℃）
Arginase-1	上游	TTGCGAGACGTAGACCCTGG	60
?	下游	CAAAGCTCAGGTGAATCGGC	?
GAPDH	上游	TTCACCACCATGGAGAAGGC	61
?	下游	GGCATGGACTGTGGTCATGA	?

分组	NO含量（μg/mL）
分组	24 h	48 h	72 h
未诱导对照组	0.191±0.002	0.507±0.012^e	0.934±0.024^e
LPS诱导组	0.294±0.007	5.183±0.171^ae	12.498±0.168^ae
LPS+低浓度hUCMSCs干预组	0.396±0.009	4.932±0.053^ae	11.852±0.149^abe
LPS+中浓度hUCMSCs干预组	0.190±0.004	3.921±0.066^abce	9.796±0.048^abce
LPS+高浓度hUCMSCs干预组	0.046±0.053^bc	1.202±0.012^abcde	1.876±0.063^abcde

分组	NO含量（μg/mL）
分组	24 h	48 h	72 h
未诱导对照组	0.191±0.002	0.507±0.012^e	0.934±0.024^e
LPS诱导组	0.294±0.007	5.183±0.171^ae	12.498±0.168^ae
LPS+低浓度hUCMSCs干预组	0.396±0.009	4.932±0.053^ae	11.852±0.149^abe
LPS+中浓度hUCMSCs干预组	0.190±0.004	3.921±0.066^abce	9.796±0.048^abce
LPS+高浓度hUCMSCs干预组	0.046±0.053^bc	1.202±0.012^abcde	1.876±0.063^abcde

分组	精氨酸酶1相对表达量
分组	24 h	48 h	72 h
未诱导对照组	1.046±0.057	1.114±0.093	1.139±0.118
低浓度hUCMSCs干预组	0.859±0.081	1.034±0.071	1.105±0.058
中浓度hUCMSCs干预组	1.287±0.084	1.471±0.120	1.382±0.069
高浓度hUCMSCs干预组	19.266±0.641^abc	16.977±0.749^abc	16.959±0.625^abc

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