人脐带间充质干细胞对1型糖尿病鼠肝脏损伤的保护作用

doi:10.3877/cma.j.issn.2095-1221.2019.01.004

中华细胞与干细胞杂志(电子版) ›› 2019, Vol. 09 ›› Issue (01) : 18 -22. doi: 10.3877/cma.j.issn.2095-1221.2019.01.004

所属专题：文献；

论著

人脐带间充质干细胞对1型糖尿病鼠肝脏损伤的保护作用

赵琳¹, 张涛², 迟静薇³, 李勇⁴, 王韵阳¹, 吕文山¹, 王伟⁵, 王颜刚¹^,^†(

)

^1. 266003　青岛大学附属医院内分泌与代谢性疾病科
^2. 276800　日照市人民医院内分泌科
^3. 266003　青岛大学附属医院甲状腺疾病重点实验室
^4. 266003　青岛大学附属医院心脏超声科
^5. 266003　青岛大学附属医院血液病科

收稿日期:2018-06-23 出版日期:2019-02-01
通信作者: 王颜刚

Protective effect of human umbilical cord mesenchymal stem cells on liver injury in type 1 diabetic mice

Lin Zhao¹, Tao Zang², Jingwei Chi³, Yong Li⁴, Yangang Wang¹, Wenshan Lv¹, Wei Wang⁵, Yangang Wang¹^,^†()

^1. Department of Endocrinology and Metabolism, Affiliated Hospital of Qingdao University, Qingdao 266003, China
^2. Department of Endocrinology, People's Hospital of Rizhao, Rizhao 276800, China
^3. Laboratory of Thyroid Disease , Affiliated Hospital of Qingdao University, Qingdao 266003, China
^4. Department of Cardiac Ultrasound, Affiliated Hospital of Qingdao University, Qingdao 266003, China
^5. Department of Hematology, Affiliated Hospital of Qingdao University, Qingdao 266003, China

Received:2018-06-23 Published:2019-02-01
Corresponding author: Yangang Wang
About author:
Corresponding author: Wang Yangang, Email: wangyg1966@126.com

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引用本文：

赵琳, 张涛, 迟静薇, 李勇, 王韵阳, 吕文山, 王伟, 王颜刚. 人脐带间充质干细胞对1型糖尿病鼠肝脏损伤的保护作用[J]. 中华细胞与干细胞杂志(电子版), 2019, 09(01): 18-22.

Lin Zhao, Tao Zang, Jingwei Chi, Yong Li, Yangang Wang, Wenshan Lv, Wei Wang, Yangang Wang. Protective effect of human umbilical cord mesenchymal stem cells on liver injury in type 1 diabetic mice[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2019, 09(01): 18-22.

目的

探讨人脐带间充质干细胞（hUCMSCs）对初发1型非肥胖型糖尿病（NOD）小鼠肝脏损伤的保护作用。

方法

雌性NOD小鼠共33只，饲养9周后，将成模的21只小鼠随机分为糖尿病组和干细胞组，每组10只，其中干细胞（MSCs）组发病后第3天尾静脉注射hUCMSCs 1?次；另取10只未发病小鼠为正常对照组。各组小鼠每周检测随机血糖（GLU）水平，8周后处死小鼠，取肝脏，HE染色后观察肝脏结构改变，ELISA法检测糖基化终末产物（AGEs）水平，Real-time PCR法检测糖基化终末产物受体（RAGE）、NF-κB P65、白细胞介素6（IL-6）、肿瘤坏死因子α（TNF-α） mRNA的表达水平。采用单因素方差分析和SNK-q检验进行统计学分析。

结果

MSCs治疗8周后，MSCs组小鼠随机血糖（8.46±1.37）mmol/L比T1DM组（32.82±0.59）?mmol/L降低，差异具有统计学意义（P < 0.05）。同时T1DM组肝脏细胞形态异常，炎症细胞浸润，而MSCs组的较T1DM组明显改善。MSCs组小鼠肝脏组织的AGEs浓度（0.72±0.10）μg/ml低于T1DM组（1.35±0.22）μg/ml；同时MSCs组的NF-κB P65、IL-6、TNF-α、RAGE mRNA水平（分别为10.08±1.94、9.31±1.67、11.92±1.82、3.87±0.27），均低于T1DM组（分别为15.46±3.09、18.04±1.69、22.12±3.23、5.12±0.26），差异具有统计学意义（P < 0.05）。

结论

hUCMSCs可以降低糖尿病小鼠血糖水平，改善肝脏微观病理状态，降低AGEs浓度及某些炎性因子的水平以减轻肝脏损伤。

关键词: 人脐带间充质干细胞, 1型糖尿病, 糖基化终末产物, 肝损伤

Objective

To investigate the protective effects of human umbilical cord mesenchymal stem cells (hUCMSCs) on liver injury in type 1 non-obese diabetic (NOD) mice.

Methods

A total of 33 female NOD mice were feeded for 9 weeks. 21 mice with diabetes were randomly divided into diabetic control group and MSC group (10 in each group) . The MSC group was infused with hUCMSCs on the 3rd day after onset. Another 10 mice without diabetes were used as blank control group. The random blood glucose (GLU) level of mice were detected for every week. After 8 weeks, the mice were sacrificed and the liver tissue was harvested for HE staining. AGE was measured by ELISA, and the expression levels of receptor (RAGE) , NF-κB P65, interleukin-6 (IL-6) , and tumor necrosis factor (TNF-α) mRNA were measured by real-time PCR. Statistical analysis was performed by one-way analysis of variance and SNK-q test. P < 0.05 was considered statistically significant.

Results

After 8 weeks of MSCs treatment, the random blood glucose of mice (11.78±4.10) mmol/L was significantly lower in the MSC group than that in the T1DM group (32.30±1.46) mmol/L, (P < 0.05) . And the liver cells were abnormal and inflammatory cells were seen in the T1DM group, which was significantly improved in the MSC group. The AGEs level (0.72±0.10) μg/ml of the liver tissue of the MSC group was significantly lower than that in the T1DM group (1.35±0.22) μg/ml, (P < 0.05) . At the same time, the mRNA levels of NF-κB P65, IL-?6, TNF-α and RAGE of the MSC group (10.08±1.94, 9.31±1.67, 11.92±1.82, and 3.87±0.27 respectively) were significantly lower than those in the T1DM group (15.46±3.09, 18.04±1.69, 22.12±3.23, and 5.12±0.26 respectively) respectively. The difference was statistically significant (P?< 0.05) .

Conclusion

Human umbilical cord mesenchymal stem cells could reduce blood glucose level, improve pathological injury of liver cells, decrease production of AGEs and some inflammatory factors of diabetic mice.

Key words: Human umbilical cord mesenchymal stem cells, Type 1 diabetes, Advanced glycationend products, Liver injury

表1 Real-time qPCR引物序列

基因名称	引物序列
RAGE	上游引物F：5'-ACT ACC GAG TCC GAGTCT ACC--3'下游引物R：5'-GTA GCT TCC CTC AGA CAC ACA-3'扩增片段116bp
NF-κB p65	上游引物F：5'-TTCCCTCAGAGCCAGCCCAGG-3'下游引物R：5'-TAGCGGAATCGCATGCCCCG-3'扩增片段82bp
IL-6	上游引物F：5'-ACAACCACGGCCTTCCCTACTT-3'下游引物R：5'-CACGATTTCCCAGAGAACATGTG-3'扩增片段129bp
TNF-α	上游引物F：5'-GCCAACGGCATGGATCTC-3'下游引物R：5'-GCAGCCTTGTCCCTTGAAGAG-3'扩增片段96bp
β-actin	上游引物F：5'-AGGGTGTGATGGTGGGAAT-3’下游引物R：5'-CTCGGTGAGCAGCACAGG-3'扩增片段200bp

表1 Real-time qPCR引物序列

基因名称	引物序列
RAGE	上游引物F：5'-ACT ACC GAG TCC GAGTCT ACC--3'下游引物R：5'-GTA GCT TCC CTC AGA CAC ACA-3'扩增片段116bp
NF-κB p65	上游引物F：5'-TTCCCTCAGAGCCAGCCCAGG-3'下游引物R：5'-TAGCGGAATCGCATGCCCCG-3'扩增片段82bp
IL-6	上游引物F：5'-ACAACCACGGCCTTCCCTACTT-3'下游引物R：5'-CACGATTTCCCAGAGAACATGTG-3'扩增片段129bp
TNF-α	上游引物F：5'-GCCAACGGCATGGATCTC-3'下游引物R：5'-GCAGCCTTGTCCCTTGAAGAG-3'扩增片段96bp
β-actin	上游引物F：5'-AGGGTGTGATGGTGGGAAT-3’下游引物R：5'-CTCGGTGAGCAGCACAGG-3'扩增片段200bp

表2 小鼠不同时点血糖浓度（mmol/L， ± s）

分组	动物数（只）	0 d	7 d	14 d	21 d	28 d
正常对照组	10	5.93±0.67	6.69±1.09	7.05±1.69	7.08±0.83	6.76±1.72
干细胞组	10	23.02±1.66	14.42±5.03	11.15±5.68	9.08±2.16	8.84±2.51
糖尿病组	10	23.37±1.04^a	31.66±1.68^ab	33.18±0.10^ab	32.78±0.68^ab	32.63±0.67^ab
F值	?	696.34	169.45	168.54	950.19	640.49
P值	?	< 0.05	< 0.05	< 0.05	< 0.05	< 0.05

表2 小鼠不同时点血糖浓度（mmol/L， ± s）

分组	动物数（只）	0 d	7 d	14 d	21 d	28 d
正常对照组	10	5.93±0.67	6.69±1.09	7.05±1.69	7.08±0.83	6.76±1.72
干细胞组	10	23.02±1.66	14.42±5.03	11.15±5.68	9.08±2.16	8.84±2.51
糖尿病组	10	23.37±1.04^a	31.66±1.68^ab	33.18±0.10^ab	32.78±0.68^ab	32.63±0.67^ab
F值	?	696.34	169.45	168.54	950.19	640.49
P值	?	< 0.05	< 0.05	< 0.05	< 0.05	< 0.05

表3 小鼠不同时点血糖浓度（mmol/L， ± s）

分组	动物数（只）	35 d	42 d	49 d	56 d
正常对照组	10	6.96±1.47	5.96±1.13	6.36±1.26	7.18±1.62
干细胞组	10	7.14±1.14	7.20±1.08	7.30±1.37	8.46±1.37
糖尿病组	10	32.65±0.69^ab	32.62±0.72^ab	32.72±0.63^ab	32.82±0.59^ab
F值	?	1671.28	2287.84	1743.24	1233.08
P值	?	< 0.05	< 0.05	< 0.05	< 0.05

表3 小鼠不同时点血糖浓度（mmol/L， ± s）

分组	动物数（只）	35 d	42 d	49 d	56 d
正常对照组	10	6.96±1.47	5.96±1.13	6.36±1.26	7.18±1.62
干细胞组	10	7.14±1.14	7.20±1.08	7.30±1.37	8.46±1.37
糖尿病组	10	32.65±0.69^ab	32.62±0.72^ab	32.72±0.63^ab	32.82±0.59^ab
F值	?	1671.28	2287.84	1743.24	1233.08
P值	?	< 0.05	< 0.05	< 0.05	< 0.05

图1 光学显微镜下观察各组小鼠肝脏形态（HE染色，×200）

表4 小鼠肝脏组织AGEs，IL-6、NF-κB p65、TNF-α及RAGE mRNA表达水平（ ± s）

分组	动物数（只）	AGEs（ug/ml）	IL6（×10^-2）	NF-κB p65（×10^-2）	TNF-α（×10^-2）	RAGE（×10^-2）
正常对照组	10	0.50±0.11	6.19±0.36	7.05±0.20	7.81±0.31	0.95±0.16
干细胞组	10	0.72±0.10	9.31±1.67	10.08±1.94	11.92±1.82	3.87±0.27
糖尿病组	10	1.35±0.22^ab	18.04±1.69^ab	15.46±3.09^ab	22.12±3.23^ab	5.12±0.26^ab
F值	?	85.91	92.82	128.33	141.23	186.82
P值	?	< 0.05	< 0.05	< 0.05	< 0.05	< 0.05

表4 小鼠肝脏组织AGEs，IL-6、NF-κB p65、TNF-α及RAGE mRNA表达水平（ ± s）

分组	动物数（只）	AGEs（ug/ml）	IL6（×10^-2）	NF-κB p65（×10^-2）	TNF-α（×10^-2）	RAGE（×10^-2）
正常对照组	10	0.50±0.11	6.19±0.36	7.05±0.20	7.81±0.31	0.95±0.16
干细胞组	10	0.72±0.10	9.31±1.67	10.08±1.94	11.92±1.82	3.87±0.27
糖尿病组	10	1.35±0.22^ab	18.04±1.69^ab	15.46±3.09^ab	22.12±3.23^ab	5.12±0.26^ab
F值	?	85.91	92.82	128.33	141.23	186.82
P值	?	< 0.05	< 0.05	< 0.05	< 0.05	< 0.05

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