间充质干细胞外泌体对海马星形胶质细胞活化的抑制作用研究

doi:10.3877/cma.j.issn.2095-1221.2021.02.005

目的

探讨间充质干细胞外泌体（MSC-Exo）对海马星形胶质细胞活化的抑制作用。

方法

实验通过超速离心法提取脐带MSC-Exo，并使用PKH-26染料标记；MSC-Exo预处理原代海马星形胶质细胞后使用脂多糖（LPS）诱导细胞活化，并分为对照组、LPS组和LPS+MSC-Exo组，进而行免疫细胞化学检测胶质纤维酸性蛋白（GFAP）、C3的表达、免疫印迹实验检测C3、GFAP的蛋白表达，以及酶联免疫吸附测定炎症因子白介素（IL）-1β、IL-6和肿瘤坏死因子（TNF）-α的水平，分别检测各组细胞的抗原表达、表型变化和炎症反应。统计采用单因素方差分析（ANOVA）及最小显著性差异法（LSD）检验。

结果

实验提取的MSC-Exo符合外泌体的一般生物学特性，且能迁移到星形胶质细胞胞体和突起。海马星形胶质细胞经LPS诱导后，其标记性抗原GFAP （1.47±0.15比0.95±0.17，P < 0.01）、A1型标记物C3 （1.32±0.23比0.87±0.16，P < 0.05）、IL-1β [（282.90±5.13）pg/ mL比（252.53±5.51）pg/mL，P < 0.05]、TNF-α [（128.26±1.89） pg/mL比（111.86±2.84）pg/mL，P < 0.01]和IL-6 [（180.95± 3.16） pg/mL比（163.95±3.71）pg/mL，P < 0.05]的表达较正常组明显升高；MSC-Exo预处理后，GFAP（0.97±0.16比1.47±0.15，P < 0.01）、C3 （0.83±0.12比1.32±0.23，P < 0.01）、IL-1β [（262.20±2.64）pg/mL比（282.90±5.13） pg/mL，P < 0.05]和TNF-α [（120.79±0.42）pg/mL比（128.26±1.89）pg/mL，P < 0.05]的表达较LPS组明显降低，IL-6 [（187.25±0.64）pg/mL比（180.95±3.16）pg/mL，P > 0.05]的表达未见明显变化。

结论

MSC-Exo对海马星形胶质细胞活化具有明显的抑制作用。

关键词: 间充质干细胞, 外泌体, 星形胶质细胞, 炎症反应, 细胞活化

Objective

To investigate the inhibitory role of mesenchymal stem cells-derived exosomes (MSC-Exo) in the activation of hippocampal astrocytes.

Methods

Exosomes were isolated from umbilical cord MSCs by ultra-speed centrifugation, and labeled by PKH-26 dye. The primary hippocampal astrocytes were pretreated with MSC-Exo and then stimulated by lipopolysaccharide (LPS) to induce cell activation, as well as divided into control group, LPS group and LPS+MSC- Exo group. Immunocytochemistry and immunoblotting were used to detect the expression of C3 and GFAP, and ELISA was used to test the expression of interleukin (IL) -1β, tumor necrosis factor (TNF) -α and IL- 6. One way ANOVA and least significant difference (LSD) test were used for statistical analysis.

Results

The results showed that the MSC-Exo presented normal biological characteristics of exosomes, as well as exhibited the capacity to integrate into the soma and processes of astrocytes. Compared with the control group, the expression of glial fibrillary acidic protein (GFAP) (1.47±0.15 vs 0.95±0.17, P < 0.01) , A1 phenotype of astrocyte marker C3 (1.32±0.23 vs 0.87±0.16, P < 0.05) , IL-1β [ (282.90±5.13) pg/ mL vs (252.53±5.51) pg/mL, P < 0.05], TNF-α [ (128.26±1.89) pg/mL vs (111.86±2.84) pg/mL, P < 0.01] and IL-6 [ (180.95±3.16) pg/mL vs (163.95±3.71) pg/mL, P < 0.05] were significantly increased after stimulation with LPS. The pre-treatment with MSC-Exo could reduce the expression of GFAP (0.97±0.16 vs 1.47±0.15, P < 0.01) , C3 (0.83±0.12 vs 1.32±0.23, P < 0.01) , IL-1β [ (262.20±2.64) pg/mL vs (282.90±5.13) pg/mL, P < 0.05], and TNF-α [ (120.79±0.42) pg/mL vs (128.26±1.89) pg/mL, P < 0.05], but there was no significant change of IL-6 [ (187.2±0.64) pg/mL vs (180.9±3.16) pg/mL, P > 0.05].

Conclusion

MSC-Exo has inhibitory effect on the activation of hippocampal astrocytes.

Key words: Mesenchymal stem cells, Exosomes, Astrocytes, Inflammatory response, Cell activation

图1 激光共聚焦显微镜下观察间充质干细胞外泌体标记性蛋白CD63和CD81 （免疫荧光染色，×1 000倍）

图2 Western blot检测间充质干细胞和间充质干细胞外泌体中CD63和CD81蛋白表达

图3 透射电子显微镜观察间充质干细胞外泌体的形态及大小（×50 000倍）

图4 激光共聚焦显微镜下显示间充质干细胞外泌体在LPS处理的原代海马星形胶质细胞中的分布情况（免疫荧光染色）

表1 星形胶质细胞的GFAP和C3的免疫印迹定量结果（±s）

分组	实验次数	GFAP	C3
对照	3	0.95±0.17	0.87±0.16
LPS	3	1.47±0.15 ^a	1.32±0.23 ^a
LPS+MSC-Exo	3	0.97±0.16 ^b	0.83±0.12 ^b
F值		10.150	7.172
P值		0.012	0.026

表1 星形胶质细胞的GFAP和C3的免疫印迹定量结果（±s）

分组	实验次数	GFAP	C3
对照	3	0.95±0.17	0.87±0.16
LPS	3	1.47±0.15 ^a	1.32±0.23 ^a
LPS+MSC-Exo	3	0.97±0.16 ^b	0.83±0.12 ^b
F值		10.150	7.172
P值		0.012	0.026

表2 星形胶质细胞炎症因子IL-1β、TNF-α和IL-6的ELISA检测结果（pg/mL，±s）

分组	实验次数	IL-1β	TNF-α	IL-6
对照	3	252.53 ± 5.51	111.86 ± 2.84	163.95 ± 3.71
LPS	3	282.90 ± 5.13^a	128.26 ± 1.89^a	180.95 ± 3.16^a
LPS+MSC-Exo	3	262.20 ± 2.64^b	120.79 ± 0.42^b	187.25 ± 0.64
F值		11.342	17.215	18.057
P值		0.009	0.003	0.003

表2 星形胶质细胞炎症因子IL-1β、TNF-α和IL-6的ELISA检测结果（pg/mL，±s）

分组	实验次数	IL-1β	TNF-α	IL-6
对照	3	252.53 ± 5.51	111.86 ± 2.84	163.95 ± 3.71
LPS	3	282.90 ± 5.13^a	128.26 ± 1.89^a	180.95 ± 3.16^a
LPS+MSC-Exo	3	262.20 ± 2.64^b	120.79 ± 0.42^b	187.25 ± 0.64
F值		11.342	17.215	18.057
P值		0.009	0.003	0.003

图5 共聚焦显微镜下观察对照组，LPS组和LPS+MSC-Exo组星形胶质细胞（免疫荧光染色）

图6 Western blot检测A1型星形胶质细胞标记物C3、GFAP的蛋白表达水平

图7 Western blot后灰度值分析星形胶质细胞对照组、LPS组和LPS+MSC-Exo组的GFAP和C3表达水平

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Inhibitory effect of mesenchymal stem cell-derived exosomes on the activation of hippocampal astrocytes

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Inhibitory effect of mesenchymal stem cell-derived exosomes on the activation of hippocampal astrocytes