冻存前后人脐带间充质干细胞对T和B淋巴细胞免疫抑制能力的差异比较

doi:10.3877/cma.j.issn.2095-1221.2021.04.002

中华细胞与干细胞杂志(电子版) ›› 2021, Vol. 11 ›› Issue (04) : 200 -206. doi: 10.3877/cma.j.issn.2095-1221.2021.04.002

论著

冻存前后人脐带间充质干细胞对T和B淋巴细胞免疫抑制能力的差异比较

曾伟杰¹, 廖延², 胡樾², 胡隽源², 曾桂芳², 傅泽钦², 伍世铎², 梁晓³, 谢长峰¹, 刘沐芸^,³

^1. 518000　深圳，个体化细胞治疗技术国家地方联合工程实验室　（深圳）
^2. 518000　深圳，深圳市北科生物科技有限公司
^3. 518000　深圳，个体化细胞治疗技术国家地方联合工程实验室　（深圳）；518000　深圳，深圳市北科生物科技有限公司

收稿日期:2020-07-14 出版日期:2021-08-01
通信作者: 刘沐芸

Comparison of immunosuppressive ability of human umbilical cord mesenchymal stem cells against T and B lymphocytes before and after cryopreservation

Weijie Zeng¹, Yan Liao², Yue Hu², Juanyuan Hu², Guifang Zeng², Zeqin Fu², Shiduo Wu², Xiao Liang³, Changfeng Xie¹, Muyun Liu^,³

^1. Department of National-local Associated Engineering Laboratory for Personalized Cellular Therapy, Shenzhen 518000, China
^2. Shenzhen Beike Biotechnology Company Limited, Shenzhen 518000, China
^3. Department of National-local Associated Engineering Laboratory for Personalized Cellular Therapy, Shenzhen 518000, China; Shenzhen Beike Biotechnology Company Limited, Shenzhen 518000, China

Received:2020-07-14 Published:2021-08-01
Corresponding author: Muyun Liu

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引用本文：

曾伟杰, 廖延, 胡樾, 胡隽源, 曾桂芳, 傅泽钦, 伍世铎, 梁晓, 谢长峰, 刘沐芸. 冻存前后人脐带间充质干细胞对T和B淋巴细胞免疫抑制能力的差异比较[J/OL]. 中华细胞与干细胞杂志(电子版), 2021, 11(04): 200-206.

Weijie Zeng, Yan Liao, Yue Hu, Juanyuan Hu, Guifang Zeng, Zeqin Fu, Shiduo Wu, Xiao Liang, Changfeng Xie, Muyun Liu. Comparison of immunosuppressive ability of human umbilical cord mesenchymal stem cells against T and B lymphocytes before and after cryopreservation[J/OL]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2021, 11(04): 200-206.

目的

探讨冻存前和复苏后人脐带间充质干细胞（hUCMSCs）对T和B淋巴细胞免疫抑制能力的差异。

方法

分离健康人外周血单个核细胞，使用Anti-CD3和Anti-CD28单克隆抗体体外激活T淋巴细胞，使用ODN2395，hCD40L，羊抗人IgM抗体和白介素2体外刺激经过分选的B淋巴细胞，通过冻存前后hUCMSCs进行干预处理，与活化后的T和B淋巴细胞进行共培养，分为单独刺激组（激活后的T或B淋巴细胞单独培养）与hUCMSCs共培养组（激活后的T或B淋巴细胞分别与hUCMSCs共培养），分析hUCMSCs对激活的T和B淋巴细胞的免疫抑制作用，探究冻存前后hUCMSCs免疫调节作用的差异。两组间比较采用独立样本t检验，多组间比较采用重复测量设计方差分析。

结果

与冻存前hUCMSCs对比，冻存后hUCMSCs在抑制CD3⁺T细胞（37.60﹪±0.54﹪比39.40﹪±1.57﹪）、CD4⁺T细胞（36.87﹪±0.54﹪比38.63﹪±1.39﹪）和CD8⁺T细胞增殖能力（40.37﹪±1.14﹪比42.47﹪±1.90﹪）比较，差异无统计学意义（P均> 0.05）；在促进CD3⁺T （18.07﹪±0.66﹪比16.77﹪±1.15﹪）、CD4⁺T（26.47﹪±1.13﹪比24.60﹪±1.47﹪）和CD8⁺T细胞凋亡能力（3.52﹪±0.22﹪比2.72﹪ ± 0.06﹪），差异无统计学意义（P均> 0.05）；增加Treg细胞群比例能力（5.51﹪±0.71﹪比6.87﹪±0.27﹪），差异无统计学意义（P > 0.05）；降低Th1亚群（0.47﹪±0.09﹪比0.33﹪± 0.04﹪）和Th17亚群比例能力（0.21﹪±0.04﹪比0.22﹪±0.03﹪），差异无统计学意义（P均> 0.05）；在对B淋巴细胞增殖能力（38.83﹪±0.25﹪比34.23﹪±4.12﹪），促进B淋巴细胞的凋亡能力（54.95﹪±1.45﹪比55.10﹪±3.80﹪），减少IgA [（1045.09± 99.24）ng/mL比（1014.31±102.55）ng/mL]和IgG抗体的分泌能力[（120.51±4.94） ng/mL比（127.77±7.94） ng/mL]，差异无统计学意义（P均> 0.05）。而在降低Th2细胞亚群比例（0.73﹪ ± 0.07﹪比0.49﹪±0.06﹪）和降低IgM抗体分泌[（739.70±28.39）ng/mL比（560.21±3.81）ng/mL]的能力上，冻存前hUCMSCs的作用能力弱于复苏后hUCMSCs，差异具有统计学意义（P均< 0.05）。

结论

冻存后hUCMSCs在降低Th2细胞亚群比例和降低IgM抗体分泌的能力上强于冻存前hUCMSCs，但整体的免疫调节能力相当，因此冻存前后的hUCMSCs均具有很高的临床应用价值。

关键词: 人脐带间充质干细胞, T淋巴细胞, B淋巴细胞, 免疫调节

Objective

To explore the immunosuppressive ability of human umbilical cord mesenchymal stem cells (hUCMSCs) on T and B lymphocytes before and after cryopreservation and provide future for using the clinical application of hUCMSCs.

Methods

Human peripheral blood mononuclear cells were isolated from healthy human peripheral blood. Anti-CD3 and anti-CD8 monoclonal antibodies were used to activate T lymphocytes in vitro. Selected B lymphocytes were stimulated by ODN2395, human CD40 ligand, goat anti-human IgM antibodies and interleukin 2 in vitro. The cells were co-cultured with activated T and B lymphocytes, divided into a single stimulation group and hUCMSCs co-culture group (before and after cryopreservation) . The immunosuppressive ability of hUCMSCs on activated T and B lymphocytes were analyzed. In addition, the differences immunosuppressive ability of hUCMSCs before and after cryopreservation were also studied. The differences between groups were compared by t-test, and the differences among groups were compared by repeated measures design analysis of variance.

Results

Compared with hUCMSCs before cypropreservation, the proliferation of CD3⁺T cells (37.60﹪±0.54﹪vs 39.40﹪±1.57﹪) , CD4⁺T cells (36.87﹪±0.54﹪vs 38.63﹪±1.39﹪) and CD8⁺T cells (40.37﹪±1.14﹪vs 42.47﹪±1.90﹪) (P > 0.05) were not significantly inhibited after cryopreservation. The apoptosis of CD3⁺T cells (18.07﹪±0.66﹪vs 16.77﹪±1.15﹪) , CD4⁺T cells (26.47﹪±1.13﹪vs 24.60﹪±1.47﹪) and CD8⁺T cells (3.52﹪±0.22﹪vs 2.72﹪±0.06﹪) had no significant difference (P > 0.05) . The proportion of regulatory cells (Treg) subsets (5.51﹪±0.71﹪vs 6.87﹪± 0.27﹪) had also no significant difference (P > 0.05) . hUCMSCs did not reduce the proportion of helper T cell (Th) subsets, especially the proportion of Th1 subsets (0.47﹪± 0.09﹪vs 0.33﹪± 0.04﹪) and Th17 subsets (0.21﹪± 0.04﹪vs 0.22﹪± 0.03﹪) (P > 0.05) . For B lymphocytes, hUCMSCs reduced the proportion of Th2 cell subsets (0.73﹪±0.07﹪vs 0.49﹪±0.06﹪) and IgM antibody secretion [ (739.70±28.39) ng/mL vs (560.21±3.81) ng/mL] after cryopreservation (P < 0.05) . There was no significant difference in proliferation and apoptosis of B lymphocytes, and reduced secretion of IgA and IgG antibodies before and after hUCMSCs cytopreservation (P > 0.05) .

Conclusion

After cryopreservation, hUCMSCs were more powerful than those before cryopreservation in reducing the proportion of Th2 cell subsets and the secretion of IgM antibodies. However, the immunosuppressive abilities was same. Both hUCMSCs before and after cryopreservation have high clinical application value.

Key words: Human Umbilical cord mesenchymal stem cells, T lymphocytes, B lymphocytes, Immunoregulation

表1 冻存前后P3代hUCMSCs与PBMC共培养对T细胞亚群增殖的影响（ ± s，﹪）

分组	实验次数	CD3⁺	CD4⁺	CD8⁺
单独刺激	3	80.00±1.58	79.63±1.51	81.55±1.30
hUCMSCs共培养（P3）	3	37.60±0.54^a	36.87±0.54^a	40.37±1.14^a
hUCMSCs共培养（冻存后P3）	3	39.40±1.57^a	38.63±1.39^a	42.47±1.90^a
F值		1002.000	1168.000	877.700
P值		< 0.001	< 0.001	< 0.001

表1 冻存前后P3代hUCMSCs与PBMC共培养对T细胞亚群增殖的影响（ ± s，﹪）

分组	实验次数	CD3⁺	CD4⁺	CD8⁺
单独刺激	3	80.00±1.58	79.63±1.51	81.55±1.30
hUCMSCs共培养（P3）	3	37.60±0.54^a	36.87±0.54^a	40.37±1.14^a
hUCMSCs共培养（冻存后P3）	3	39.40±1.57^a	38.63±1.39^a	42.47±1.90^a
F值		1002.000	1168.000	877.700
P值		< 0.001	< 0.001	< 0.001

表2 冻存前后P3代hUCMSCs与PBMC共培养对T细胞亚群凋亡的影响（ ± s，﹪）

分组	实验次数	CD3⁺	CD4⁺	CD8⁺
单独刺激	3	8.25±0.96	14.05±1.31	4.03±0.72
hUCMSCs共培养（P3）	3	18.07±0.66^a	26.47±1.13^a	3.52±0.22
hUCMSCs共培养（冻存后P3）	3	16.77±1.15^a	24.60±1.47^a	2.72±0.06^a
F值		106.700	87.550	4.735
P值		< 0.001	< 0.001	0.039

表2 冻存前后P3代hUCMSCs与PBMC共培养对T细胞亚群凋亡的影响（ ± s，﹪）

分组	实验次数	CD3⁺	CD4⁺	CD8⁺
单独刺激	3	8.25±0.96	14.05±1.31	4.03±0.72
hUCMSCs共培养（P3）	3	18.07±0.66^a	26.47±1.13^a	3.52±0.22
hUCMSCs共培养（冻存后P3）	3	16.77±1.15^a	24.60±1.47^a	2.72±0.06^a
F值		106.700	87.550	4.735
P值		< 0.001	< 0.001	0.039

表3 冻存前后P3代hUCMSCs与PBMC共培养后对Th1、Th2和Th17亚群的影响（ ± s，﹪）

分组	实验次数	Th1	Th2	Th17
单独刺激	3	4.64±0.27	0.91±0.09	0.55±0.03
hUCMSCs共培养（P3）	3	0.47±0.09^a	0.73±0.07	0.21±0.04^a
hUCMSCs共培养（冻存后P3）	3	0.33±0.04^a	0.49±0.06^ab	0.22±0.03^a
F值		399.500	18.540	68.980
P值		< 0.001	0.003	< 0.001

表3 冻存前后P3代hUCMSCs与PBMC共培养后对Th1、Th2和Th17亚群的影响（ ± s，﹪）

分组	实验次数	Th1	Th2	Th17
单独刺激	3	4.64±0.27	0.91±0.09	0.55±0.03
hUCMSCs共培养（P3）	3	0.47±0.09^a	0.73±0.07	0.21±0.04^a
hUCMSCs共培养（冻存后P3）	3	0.33±0.04^a	0.49±0.06^ab	0.22±0.03^a
F值		399.500	18.540	68.980
P值		< 0.001	0.003	< 0.001

表4 冻存前后P3代hUCMSCs与B淋巴细胞共培养后对B细胞分泌Ig抗体的影响（ ± s，ng/mL）

分组	实验次数	IgA	IgM	IgG
单独刺激	3	2183.35±273.70	859.16±45.56	161.64±5.87
hUCMSCs共培养（P3）	3	1045.09± 99.24^a	739.70±28.39	120.51±4.94^a
hUCMSCs共培养（冻存后P3）	3	1014.31±102.55^a	560.21± 3.81^ab	127.77±7.94^a
F值		27.950	27.260	23.710
P值		< 0.001	< 0.001	< 0.001

表4 冻存前后P3代hUCMSCs与B淋巴细胞共培养后对B细胞分泌Ig抗体的影响（ ± s，ng/mL）

分组	实验次数	IgA	IgM	IgG
单独刺激	3	2183.35±273.70	859.16±45.56	161.64±5.87
hUCMSCs共培养（P3）	3	1045.09± 99.24^a	739.70±28.39	120.51±4.94^a
hUCMSCs共培养（冻存后P3）	3	1014.31±102.55^a	560.21± 3.81^ab	127.77±7.94^a
F值		27.950	27.260	23.710
P值		< 0.001	< 0.001	< 0.001

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Comparison of immunosuppressive ability of human umbilical cord mesenchymal stem cells against T and B lymphocytes before and after cryopreservation

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Comparison of immunosuppressive ability of human umbilical cord mesenchymal stem cells against T and B lymphocytes before and after cryopreservation