人脐带血单个核细胞联合人脐带间充质干细胞疗法对乙型肝炎肝硬化患者肝功能、炎症程度及免疫功能的影响

韩霞; 云升

doi:10.3969/j.issn.1001-5256.2021.08.016

人脐带血单个核细胞联合人脐带间充质干细胞疗法对乙型肝炎肝硬化患者肝功能、炎症程度及免疫功能的影响

DOI: 10.3969/j.issn.1001-5256.2021.08.016

韩霞,
云升^,

内蒙古医科大学附属医院干细胞实验室，内蒙古医科大学干细胞研究中心，呼和浩特 010050

基金项目:

内蒙古医科大学科技百万工程联合资助项目 (YKD2017KJBW〔LH〕05);

内蒙古自治区科技厅资助项目 (201802150)

利益冲突声明：本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突。

作者贡献声明：韩霞负责申报课题，资料分析，撰写论文；云升负责课题设计，拟定写作思路，修改文章并最后定稿。

详细信息

通信作者:
云升，yunsheng_@163.com

中图分类号: R512.62;R575.2
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出版历程
- 收稿日期: 2020-12-01
- 录用日期: 2021-02-23
- 出版日期: 2021-08-20

Effect of combined treatment with human umbilical cord blood mononuclear cells and human umbilical cord mesenchymal stem cells on liver function, inflammation grade, and immune function in patients with hepatitis B cirrhosis

Xia HAN,
Sheng YUN^,

Stem Cell Laboratory, Affiliated Hospital of Inner Mongolia Medical University, Stem Cell Center of Inner Mongolia Medical University, Hohhot 010050, China

Research funding:

Inner Mongolia Medical University Science and Technology One Million Engineering Joint Joint Venture Project (YKD2017KJBW〔LH〕05);

Inner Mongolia Autonomous Region Science and Technology Department Funded Project (201802150)

摘要

摘要: 目的探讨人脐带血单个核细胞(hUCB-MNCs)联合人脐带间充质干细胞(hUC-MSCs)对乙型肝炎相关失代偿期肝硬化患者的肝功能、炎症程度及免疫功能的影响。方法选取2016年11月—2019年6月在内蒙古医科大学附属医院诊治的11例肝硬化患者，所纳入患者第1周输注1次hUCB-MNCs(＞18×10⁹/次)，第2、3、4周每周输注1次hUC-MSCs，每次输注1×10⁶/kg，在治疗结束的第4、8、12周进行复查，对比治疗前后肝功能、血氨、凝血因子、血清细胞因子及淋巴细胞亚群的变化，同时观察记录神经、精神症状的改变。计量资料组间比较采用单因素重复测量方差分析。结果 hUCB-MNCs联合hUC-MSCs治疗后的11例患者其精神、神经症状与细胞输注前相比较有所改善；肝功能各项指标及凝血功能基本趋于正常(P值均＜0.05)；细胞联合输注后的第12周，血氨水平显著下降(P＜0.05)；炎性细胞因子IL-6、TNFα水平降低(F值分别为49.497、37.071，P值均＜0.05)；而抗炎细胞因子TGFβ和IL-10水平显著上升(F值分别为35.843、15.918，P值均＜0.05)；同时CD3⁺CD8⁺T淋巴细胞和CD19⁺B淋巴细胞均降低(F值分别为52.242、89.097，P值均＜0.05)；免疫调节性T淋巴细胞CD4⁺CD25⁺Treg明显升高(F=17.337，P＜0.05)。结论 hUCB-MNCs联合hUC-MSCs治疗肝硬化，与细胞输注前相比较，可改善肝功能，减少炎性细胞因子产生，减轻肝脏炎症反应以及肝细胞的破坏，升高免疫调节性T淋巴细胞，从而影响其自身免疫功能。
- 乙型肝炎 /
- 肝硬化 /
- 胎血 /
- 单核细胞 /
- 间充质基质细胞
Abstract: Objective To retrospectively investigate the effect of combined treatment with human umbilical cord blood mononuclear cells (hUCB-MNCs) and human umbilical cord mesenchymal stem cells (hUC-MSCs) on liver function, inflammation grade, and immune function in patients with hepatitis B-related decompensated cirrhosis. Methods 11 patients with liver cirrhosis who were admitted to the Affiliated Hospital of Inner Mongolia Medical University from November 2016 to June 2019 were enrolled in this clinical study and were given infusion of hUCB-MNCs (> 18×10⁹/time) once in the first week and infusion of hUC-MSCs (1×10⁶/kg) once a week in the second, third, and fourth weeks. Reexamination was performed at 4, 8, and 12 weeks after treatment to compare the changes in liver function, blood ammonia, blood coagulation factors, serum levels of cytokines, and lymphocyte subsets after treatment, and the changes in neurological and psychiatric symptoms were observed and recorded. A one-way repeated-measures analysis of variance was used for comparison of continuous data between groups. Results After the combined treatment with hUCB-MNCs and hUC-MSCs, all 11 patients achieved certain improvements in psychiatric and neurological symptoms after infusion, and liver function parameters and blood coagulation function basically returned to normal (all P < 0.05). At 12 weeks after the combined infusion of cells, blood ammonia level returned to the normal level (P < 0.05), and there were significant reductions in the levels of the inflammatory cytokines interleukin-6 and tumor necrosis factor (F=49.497 and 37.071, both P < 0.05) and significant increases in the levels of the anti-inflammatory cytokines transforming growth factor-β and interleukin-10 (F=35.843 and 15.918, both P < 0.05). There were also significant reductions in the percentages of the cytotoxic cells CD3⁺CD8⁺ T and CD19⁺ B cells (F=52.242 and 89.097, both P < 0.05) and a significant increase in the regulatory T cells CD4⁺CD25⁺ (F=17.337, P < 0.05). Conclusion Combined treatment with hUCB-MNCs and hUC-MSCs for liver cirrhosis can optimize liver function, reduce the production of inflammatory cytokines, alleviate liver inflammation and liver cell destruction, and increase regulatory T cells, thereby affecting the body's immune function.
- Hepatitis B /
- Liver Cirrhosis /
- Fetal Blood /
- Monocytes /
- Mesenchymal Stromal Cells

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图 1 hUC-MSCs形态(×100)

注：a，P3代hUC-MSCs培养1 d，细胞为短杆状或梭形；b，培养第3天，细胞呈梭形，巢状或单极辐射生长。

下载: 全尺寸图片幻灯片

图 2 hUC-MSCs表面标记表达

注：a，阴性对照组；b，CD44和CD105表达为98.59%；c，CD44和CD90表达为99.06%；d，CD34和HLA-DR表达为0.08%；e，山东脐血库CD34表达为1.66%。

下载: 全尺寸图片幻灯片

图 3 hUC-MSCs分化诱导的肝细胞及神经细胞形态(×400)

注：a，hUC-MSCs分化诱导的肝细胞；b，hUC-MSCs分化诱导的神经细胞。

下载: 全尺寸图片幻灯片

表 1 入组患者的信息和条件

患者	年龄(岁)	性别	腹水	TBil(μmol/L)	Alb(g/L)	PT(s)	Child-Pugh分级
No.1	50	男	-	48.1	26.5	19.3	B级
No.2	48	男	-	47.9	22.9	19.9	B级
No.3	55	男	Ⅰ	47.4	24.8	22.5	B级
No.4	56	男	-	34.9	26.0	22.9	B级
No.5	60	男	Ⅰ	54.4	22.8	20.5	C级
No.6	52	女	-	48.5	31.9	20.2	B级
No.7	41	男	-	43.8	40.8	19.4	B级
No.8	49	男	-	50.2	23.9	17.2	B级
No.9	51	男	Ⅰ	34.8	27.4	16.2	B级
No.10	53	男	Ⅱ	67.6	22.6	19.4	C级
No.11	51	男	Ⅰ	48.7	21.9	19.4	B级

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表 2 细胞输注前后肝功能变化

输注时间	ALT(U/L)	AST(U/L)	ALP(U/L)	GGT(U/L)	TBil(μmol/L)	Alb(g/L)	PTA(%)	PT(s)
输注前	88.4±15.3¹⁾	99.8±25.0¹⁾	123.8±39.4¹⁾	305.1±83.4¹⁾	47.8±8.1¹⁾	26.5±6.4¹⁾	45.8±7.9¹⁾	16.2±1.3¹⁾
输注(4周)	77.9±22.7	85.5±31.3	97.5±40.1	189.1±66.4	23.2±9.8	30.5±3.6	66.0±6.0	12.6±0.5
输注(8周)	59.1±21.5	26.8±7.9	58.1±17.6	86.6±39.9	9.9±5.3	36.0±4.2	74.9±6.9	12.2±1.8
输注(12周)	42.7±12.3	24.2±8.3	59.5±28.0	43.2±19.8	6.7±1.8	40.8±2.4	87.6±3.0	11.3±0.2
F值	14.496	41.163	10.941	45.740	79.730	20.496	86.289	1 029.600
P值	＜0.05	＜0.05	＜0.05	＜0.05	＜0.05	＜0.05	＜0.05	＜0.05
注：与输注后的第4、8、12周比较, 1)P＜0.05。

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表 3 hUCB-MNCs联合hUC-MSCs输注前后血氨值变化

输注时间	血氨(μmol/L)
输注前	166.6±47.7¹⁾
输注(4周)	115.6±32.8
输注(8周)	87.2±36.3
输注(12周)	66.2±32.5
F值	104.6
P值	P＜0.05
注：与输注后的第4、8、12周比较, 1)P＜0.05。

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表 4 hUCB-MNCs联合hUC-MSCs输注前后Child-Pugh评分变化

输注时间	Child-Pugh评分
输注前	10.10±2.43¹⁾
输注(4周)	8.36±2.94
输注(8周)	6.82±2.27
输注(12周)	6.09±1.58
F值	68.4
P值	P＜0.05
注：与输注后的第4、8、12周比较, 1)P＜0.05。

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表 5 hUCB-MNCs联合HUC-MSCs输注前后细胞因子和淋巴细胞亚群变化

输注时间	IL-6(ng/L)	TNFα(ng/L)	IL-10(pg/L)	TGFβ(pg/L)	CD3⁺	CD3⁺CD4⁺	CD3⁺CD8⁺	CD4⁺CD25⁺	CD19⁺
细胞输注前	198.8±54.0¹⁾	300.6±73.5¹⁾	18.3±7.8¹⁾	25.9±4.3¹⁾	75.8±11.0¹⁾	23.3±4.2¹⁾	48.3±9.3¹⁾	1.3±1.2¹⁾	32.1±4.2¹⁾
输注后(4周)	96.2±19.6	198.9±106.4	48.9±15.7	49.3±7.1	74.8±8.9	53.1±10.9	20.4±6.8	18.3±9.5	12.7±3.6
输注后(8周)	58.9±16.9	92.2±26.1	47.3±8.7	43.9±4.4	76.9±12.4	54.0±7.7	20.4±3.4	18.0±5.0	11.7±5.3
输注后(12周)	59.8±16.4	92.8±13.7	49.6±15.9	35.5±6.4	80.1±10.9	55.9±6.4	16.7±6.0	19.1±8.3	6.1±2.4
F值	49.497	37.071	15.918	35.843	0.493	44.755	52.242	17.337	89.097
P值	＜0.01	＜0.01	＜0.01	＜0.01	＞0.05	＜0.01	＜0.01	＜0.01	＜0.01
注：与输注后的第4、8、12周比较, 1)P＜0.05。

下载: 导出CSV

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