高表达microRNA-155的骨髓间充质干细胞对免疫调节的影响

doi:10.3877/cma.j.issn.2095-1221.2018.02.004

目的

探讨骨髓间充质干细胞（BMSCs）中miR-155表达水平改变后，通过诱导树突状细胞（DC）实现对免疫调节能力的影响。

方法

实验分为control组、miR-155 agomir NC组、miR-155 agomir组、miR-155 antagomir NC组和miR-155 antagomir组，通过脂质体转染特异性调控BMSC中miR-155表达量后诱导DC 48 h，检测该诱导过程对DC的成熟度和迁移能力的影响；经诱导的DC与T细胞共培养72 h后检测T细胞增殖能力。多组间分析采用One-?way ANOVA进行统计学分析，两组间采用t检验进行统计学分析。

结果

流式柱形直观图可见miR-155 angomir组T细胞增殖能力低于其他组。提高miR-155表达水平后，MSCs诱导的DC细胞成熟的表面标志CD40表达量由100%下降至85%（t = 33.71，P < 0.05）；CD86表达水平由100%下降至75%（t = 57.00，P < 0.05）。miR-155 agomir组的BMSCs诱导的DC的迁移能力较其对照组减弱（t = 7.35，P < 0.05）。提高BMSCs中miR-155表达水平后，其诱导的DC的NF-κβ信号通路蛋白表达下降（t = 23.32，P < 0.05）；AKT信号通路蛋白表达量下降（t?= 22.21，P < 0.05）。

结论

BMSCs高表达miR-155后，可以通过抑制NF-κβ和AKT途径诱导耐受性DC的产生，通过诱导DC减少T细胞的增殖从而对免疫调节进行影响。

关键词: MicroRNA-155, 骨髓间充质干细胞, 树突状细胞, 免疫调节

Objective

To specifically alter the expression level of microRNA-155 (miR-?155) in bone marrow-derived mesenchymal stem cells (BMSC), we investigate the effect of BMSCs on immunoregulation by inducing dendritic cells (DC) after the expression of miR-155 was changed in MSCs.

Methods

The experiment was divided into CON, miR-155 agomir NC, miR-?155 agomir, miR-155 antagmir NC and miR-155 antagomir groups. DCs were induced for 48 hours by coculture with BMSCs transfect with difference miR-155 and then the maturation and migration of DC was detected. Further, the induced DCs were co-cultured with T cells for 72 hours to detect T cell proliferation. One-way ANOVA (and non-parametric) was used for statistical analysis among groups and t tests were used for statistical analysis between two groups.

Results

Flow cytometric visualization showed that the proliferation of T cells was decreased in the miR-155 agomir group. The expression level of CD40, the surface markers of DC maturation decreased from 100% to 85%(t = 33.71, P?< 0.05) after the expression of miR-155 was increased, and the expression of CD86 decreased from 100% to 75% (t = 57.00, P < 0.05). Meanwhile BMSCs overexpressed with miR-?155 decrease the migratory ability of DCs (t = 7.35, P < 0.01). The expression of NF-κβ signaling pathway proteins in DCs induced by BMSCs was significantly decreased (t = 23.32, P < 0.05) after the expression of miR-155 was increased in BMSCs; and the expression of AKT signaling pathway proteins was significantly decreased (t = 22.21, P < 0.05).

Conclusion

By inducing DC, BMSCs highly expressed with miR-155 decreased the proliferation of T cells. The high expression level of miR-155 enhances the immunosuppressive capacity of BMSCs.

Key words: MicroRNA155, Bone marrow derived mesenchymal stem cells, Dendritic cells, Immunoregulation

表1 人工合成microRNA序列

分组	序列（5'-3'）
miR-155-5P agomir组	UUAAUGCUAAUUGUGAUAGGGGU
?	CCCUAUCACAAUUAGCAUUAAUU
miR-155-5P antagomir组	ACCCCUAUCACAAUUAGCAUUAA
agomir NC组	UUC UCC GAA CGU GUC ACG UTT
?	ACG UGA CAC GUU CGG AGA ATT
antagomir NC组	CAGUACUUUUGUGUAGUACAA
agomir NC-Fam组	UUC UCC GAA CGU GUC ACG UTT
?	ACG UGA CAC GUU CGG AGA ATT

表1 人工合成microRNA序列

分组	序列（5'-3'）
miR-155-5P agomir组	UUAAUGCUAAUUGUGAUAGGGGU
?	CCCUAUCACAAUUAGCAUUAAUU
miR-155-5P antagomir组	ACCCCUAUCACAAUUAGCAUUAA
agomir NC组	UUC UCC GAA CGU GUC ACG UTT
?	ACG UGA CAC GUU CGG AGA ATT
antagomir NC组	CAGUACUUUUGUGUAGUACAA
agomir NC-Fam组	UUC UCC GAA CGU GUC ACG UTT
?	ACG UGA CAC GUU CGG AGA ATT

表2 实时荧光定量PCR体系

定量PCR 10 μl体系	加样量
2×Real-time PCR Master Mix（SYBR）	5.0 μl
miRNA/U6 snRNA specific Primer set	0.2 μl
ROX reference dye	1.0 μl
Taq DNA polymerase	0.1 μl
miRNA RT product	1.0 μl
Sterilized H₂O	2.7 μl

表2 实时荧光定量PCR体系

定量PCR 10 μl体系	加样量
2×Real-time PCR Master Mix（SYBR）	5.0 μl
miRNA/U6 snRNA specific Primer set	0.2 μl
ROX reference dye	1.0 μl
Taq DNA polymerase	0.1 μl
miRNA RT product	1.0 μl
Sterilized H₂O	2.7 μl

表3 转染后miR-155表达结果（±s）

分组	样品数	miR-155表达水平
control组	3	1.19± 0.19
miR-155 agomir NC组	3	2.95± 0.30
miR-155 agomir组	3	12219.51±1001.83^ab
miR-155 antagomir NC组	3	6.01± 0.45
miR-155 antagomir组	3	2.54± 0.80^c

表3 转染后miR-155表达结果（±s）

分组	样品数	miR-155表达水平
control组	3	1.19± 0.19
miR-155 agomir NC组	3	2.95± 0.30
miR-155 agomir组	3	12219.51±1001.83^ab
miR-155 antagomir NC组	3	6.01± 0.45
miR-155 antagomir组	3	2.54± 0.80^c

图1 BMSC表型的鉴定

图2 倒置相差显微镜下观察转染后的间充质干细胞（×100）

图3 流式细胞检测Fam组荧光提示转染效率为100﹪

表4 DC表面标志表达结果（﹪，±s）

表面标志	转染前	转染后	t值	P值
MHC II	99.57±0.39	97.96±0.57	2.224	0.09
CD40	99.69±034	85.18±0.66	33.710	<0.001
CD86	99.58±0.45	75.26±0.64	57.000	<0.001

表4 DC表面标志表达结果（﹪，±s）

表面标志	转染前	转染后	t值	P值
MHC II	99.57±0.39	97.96±0.57	2.224	0.09
CD40	99.69±034	85.18±0.66	33.710	<0.001
CD86	99.58±0.45	75.26±0.64	57.000	<0.001

表5 Transwell小室中迁移DC数目（个，±s）

分组	样本数	迁移DC的数目
control组	5	40.00±3.54
MSCs组	5	27.00±1.58^a
miR-155 agomir NC组	5	27.00±1.58
miR-155 agomir组	5	18.00±2.24^b
miR-155 antagomir NC组	5	30.00±0.71
miR-155 antagomir组	5	35.00±2.24^c

表5 Transwell小室中迁移DC数目（个，±s）

分组	样本数	迁移DC的数目
control组	5	40.00±3.54
MSCs组	5	27.00±1.58^a
miR-155 agomir NC组	5	27.00±1.58
miR-155 agomir组	5	18.00±2.24^b
miR-155 antagomir NC组	5	30.00±0.71
miR-155 antagomir组	5	35.00±2.24^c

表6 AKT和NF-κβ信号通路蛋白相对表达情况结果（±s）

分组	样本数	AKT	NF-κβ
DC only	3	0.64±0.00	0.60±0.01
miR-155 agomir NC	3	0.65±0.02	0.48±0.01
miR-155 agomir	3	0.33±0.02^ab	0.31±0.01^ab
miR-155 antagomir NC	3	0.55±0.04	0.49±0.02
miR-155 antagomir	3	0.62±0.01^c	0.51±0.00^c
F值	?	134.047	230.5
P值	?	< 0.0001	< 0.0001

表6 AKT和NF-κβ信号通路蛋白相对表达情况结果（±s）

分组	样本数	AKT	NF-κβ
DC only	3	0.64±0.00	0.60±0.01
miR-155 agomir NC	3	0.65±0.02	0.48±0.01
miR-155 agomir	3	0.33±0.02^ab	0.31±0.01^ab
miR-155 antagomir NC	3	0.55±0.04	0.49±0.02
miR-155 antagomir	3	0.62±0.01^c	0.51±0.00^c
F值	?	134.047	230.5
P值	?	< 0.0001	< 0.0001

图4 转染前后DC表面标志表达情况

图5 倒置相差显微镜下观察DC迁移能力（×100）

图6 Western Blot检测信号通路蛋白表达情况

图7 流式细胞检测T细胞增殖能力

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Effect of bone marrow mesenchymal stem cells modified with miR-155 on immunoregulation

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Effect of bone marrow mesenchymal stem cells modified with miR-155 on immunoregulation