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

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

中华细胞与干细胞杂志(电子版) ›› 2020, Vol. 10 ›› Issue (04) : 213 -218. doi: 10.3877/cma.j.issn.2095-1221.2020.04.003

所属专题：文献；

论著

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

梅亚波¹, 张万巧¹, 陈冲¹, 韩涛¹, 封志纯¹^,^†(

)

^1. 100700　北京，中国人民解放军总医院儿科医学部　中国人民解放军总医院第七医学中心八一儿童医院　出生缺陷防控关键技术国家工程实验室　儿童器官功能衰竭北京市重点实验室

收稿日期:2020-03-23 出版日期:2020-08-01
通信作者: 封志纯

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

Yabo Mei¹, Wanqiao Zhang¹, Chong Chen¹, Tao Han¹, Zhichun Feng¹^,^†()

^1. Faulty of Pediatrics, Chinese PLA General Hospital, BaYi Children's Hospital, the Seventh Medical Center of Chinese PLA General Hospital, National Engineering Laboratory for Birth Defects Prevention and Control of Key Technology, Beijing Key Laboratory of Pediatric Organ Failure, Beijing 100700, China

Received:2020-03-23 Published:2020-08-01
Corresponding author: Zhichun Feng
About author:
Corresponding author:Feng Zhichun, Email:fengzc81@sina.com

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引用本文：

梅亚波, 张万巧, 陈冲, 韩涛, 封志纯. 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[J]. 中华细胞与干细胞杂志(电子版), 2020, 10(04): 213-218.

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.

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1.0 μg/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。

关键词: 脂多糖, 间充质干细胞, BV-2细胞

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

表1 小鼠小胶质细胞精氨酸酶1和GAPDH的引物序列

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	?

表1 小鼠小胶质细胞精氨酸酶1和GAPDH的引物序列

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	?

图1 光镜下观察静息状态下BV-2细胞形态（×100）

图2 CD11b流式细胞学鉴定

图3 倒置显微镜下观察不同培养条件下的BV-2细胞形态

表2 hUCMSCs与BV-2直接共培养上清液NO含量（±s，n = 3）

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

表2 hUCMSCs与BV-2直接共培养上清液NO含量（±s，n = 3）

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

表3 hUCMSCs与BV-2直接共培养细胞的精氨酸酶1相对表达量（±s，n = 3）

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

表3 hUCMSCs与BV-2直接共培养细胞的精氨酸酶1相对表达量（±s，n = 3）

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

表4 LPS刺激条件下hUCMSCs与BV-2直接共培养细胞的精氨酸酶1相对表达量（±s，n = 3）

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
LPS诱导组	0.000^a	0.000^a	0.000^a
LPS+低浓度hUCMSCs干预组	0.039±0.016	0.189±0.170^b	0.000±0.000
LPS+中浓度hUCMSCs干预组	0.095±0.045	0.017±0.020	0.100±0.030
LPS+高浓度hUCMSCs干预组	0.879±0.077^bcd	1.023±0.081^bcd	1.121±0.078^bcd

表4 LPS刺激条件下hUCMSCs与BV-2直接共培养细胞的精氨酸酶1相对表达量（±s，n = 3）

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
LPS诱导组	0.000^a	0.000^a	0.000^a
LPS+低浓度hUCMSCs干预组	0.039±0.016	0.189±0.170^b	0.000±0.000
LPS+中浓度hUCMSCs干预组	0.095±0.045	0.017±0.020	0.100±0.030
LPS+高浓度hUCMSCs干预组	0.879±0.077^bcd	1.023±0.081^bcd	1.121±0.078^bcd

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