血管紧张素Ⅱ对人脐带间充质干细胞生物学特性的影响

doi:10.3877/cma.j.issn.1673-9450.2015.05.005

中华损伤与修复杂志(电子版) ›› 2015, Vol. 10 ›› Issue (05) : 395 -401. doi: 10.3877/cma.j.issn.1673-9450.2015.05.005

所属专题：文献；

论著

血管紧张素Ⅱ对人脐带间充质干细胞生物学特性的影响

杨靖¹, 刘玲英², 尹会男², 柴家科³^,^†(

)

^1. 300270　天津医科大学
^2. 100048　北京，解放军总医院第一附属医院烧伤整形科　全军烧伤研究所
^3. 300270　天津医科大学；100048　北京，解放军总医院第一附属医院烧伤整形科　全军烧伤研究所

收稿日期:2015-07-16 出版日期:2015-10-01
通信作者: 柴家科
基金资助:
国家自然科学基金面上项目(81372052)

Effect of angiotensin Ⅱ on biological characteristics of human umbilical cord mesenchymal stem cells

Jing Yang¹, Lingying Liu², Huinan Yin², Jiake Chai³^,^†()

^1. Tianjin Medical University, Tianjin 300270, China
^2. Department of Burn and Plastic Surgery, the First Affiliated to General Hospital of People′s Liberation Army, Beijing 100048, China
^3. Tianjin Medical University, Tianjin 300270, China; Department of Burn and Plastic Surgery, the First Affiliated to General Hospital of People′s Liberation Army, Beijing 100048, China

Received:2015-07-16 Published:2015-10-01
Corresponding author: Jiake Chai
About author:
Corresponding author: Chai Jiake, Email: cjk304@126.com

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引用本文：

杨靖, 刘玲英, 尹会男, 柴家科. 血管紧张素Ⅱ对人脐带间充质干细胞生物学特性的影响[J]. 中华损伤与修复杂志(电子版), 2015, 10(05): 395-401.

Jing Yang, Lingying Liu, Huinan Yin, Jiake Chai. Effect of angiotensin Ⅱ on biological characteristics of human umbilical cord mesenchymal stem cells[J]. Chinese Journal of Injury Repair and Wound Healing(Electronic Edition), 2015, 10(05): 395-401.

目的

体外观察不同浓度血管紧张素Ⅱ(Ang-Ⅱ)对人脐带间充质干细胞(hUCMSC)凋亡、增殖和旁分泌的影响，筛选适宜浓度Ang-Ⅱ预处理hUCMSC。

方法

照笔者单位实验室建立的方法，体外分离、培养并鉴定hUCMSC，取3～8代hUCMSC接种于培养板，将细胞按随机数字表法分为4组(每组3个复孔)：空白对照组、100 ng/mL Ang-Ⅱ组、500 ng/mL Ang-Ⅱ组及1000 ng/mL Ang-Ⅱ组，后3组分别于含有100ng/mL、500 ng/mL及1000 ng/mL Ang-Ⅱ的培养基、空白对照组使用不含有Ang-Ⅱ的培养基培养，各组于培养24、48、72 h后分别于倒置显微镜镜下观察细胞形态和细胞融合的情况，吖啶橙/溴化乙锭(AO/EB)染色法检测细胞凋亡情况，CCK-8法检测细胞增殖活性，流式细胞仪检测细胞周期(S期)，酶联免疫吸附试验(ELISA)检测各组细胞上清液中血管内皮生长因子(VEGF)、成纤维细胞生长因子(bFGF)、肝细胞生长因子(HGF)的含量。

结果

培养24、48、72 h后，倒置显微镜下观察各组细胞形态类似，均呈长梭形，少数细胞呈多角形，100 ng/mL Ang-Ⅱ组细胞的融合率分别为40%～45%、70%～80%、90%～95%，融合速度显著增加。AO/EB结果显示：仅100 ng/mL Ang-Ⅱ组与空白对照组未出现凋亡细胞或死亡细胞。CCK-8的结果同样显示100 ng/mL Ang-Ⅱ组细胞增殖速度显著快于空白对照组，差异有统计学意义(P<0.05)。流式细胞仪检测结果显示为100 ng/mL Ang-Ⅱ组所处的增殖周期(S)期细胞数量的比例显著增加。ELISA检测结果是100 ng/mL Ang-Ⅱ组细胞的VEGF、bFGF、HGF含量最高，与空白对照组比较，差异均有统计学意义(P均小于0.05)。

结论

100 ng/mL Ang-Ⅱ具有抑制细胞凋亡，促进细胞增殖的功能，这可能与其使hUCMSC大量分泌VEGF、bFGF、HGF有关。

关键词: 血管紧张素Ⅱ, 脐带, 间质干细胞, 细胞增殖, 细胞凋亡, 旁分泌

Objective

To investigate the effects of different concentrations of angiotensin Ⅱ on the apoptosis, proliferation and paracrine of human umbilical cord mesenchymal stem cells in vitro and select the appropriate concentration of angiotensin Ⅱ to pretreat mesenchymal stem cells.

Methods

According to the method of the author′s laboratory established for isolate, culture and identification of the human umbilical cord mesenchymal stem cells, the 3-8th human umbilical cord mesenchymal stem cells were cultured in plates. The human umbilical cord mesenchymal stem cells were randomly divided into 4 groups (3 in each group): control group, 100 ng/mL angiotensin Ⅱ group, 500 ng/mL angiotensin Ⅱ group and 1000 ng/mL angiotensin Ⅱ group. The culture media containing 100 ng/mL, 500 ng/mL and 1000 ng/mL angiotensin Ⅱ were used to treat experimental groups, respectively, the control group was cultured with normal medium without Ang-Ⅱ. When the human umbilical cord mesenchymal stem cells were cultured for 24, 48 and 72 h, the cell morphology and density were observed by inverted microscope. The apoptosis was assessed by acridineorange/ethidium bromide staining. The cell proliferation activity was measured by Cell Counting Kit-8, and the cell cycle (S period) was tested by flow cytometry after propidium iodide staining. The levels of vascular endothelial growth factor, basic fibroblast growth factor, and hepatocyte growth factor in each group were detected by enzyme linked immunosorbent assay.

Results

When the human umbilical cord mesenchymal stem cells was cultured for 24 h, 48 h, 72 h, cell morphology under inverted microscope in each group was long-shuttle shape. A few cells were polygonal. The fusion rate of 100 ng/mL angiotensin Ⅱ group were 40%-45%, 70%-80%, 90%-95%, the fusion rate was significantly increased. Acridineorange/ethidium bromide staining showed that no apoptosis cells were observed in group of 100 ng/mL angiotensin Ⅱ and the group of control. The results of Cell Counting Kit-8 also showed that the cells value-added of 100 ng/mL angiotensin Ⅱ group was significantly faster than that in control group; The results of flow cytometry showed that the ratio of the number of cells in 100 ng/mL angiotensin Ⅱ group was significantly increased. Enzyme linked immunosorbent assay analysis showed that the contents of the vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor of 100 ng/mL angiotensin Ⅱ group were highest, and compared with the control group, the difference was statistically significant (P<0.05).

Conclusion

100 ng/mL angiotensin Ⅱ can inhibit apoptosis and promote cell proliferation, which may be related to a large number of secretion of vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor.

Key words: Angiotensin Ⅱ, Umbilical cord, Mesenchymal stem cells, Cell proliferation, Apoptosis, Paracrine

图1 各组hUCMSC培养24、48、72 h后形态学观察，均呈长梭形，少数细胞呈多角形(倒置相差显微镜×100)。1A－C、1D－F、1G－I、1J－L分别为空白对照组、100 ng/mL Ang－Ⅱ组、500 ng/mL Ang－Ⅱ组、1000 ng/mL Ang－Ⅱ组培养24、48、72 h；hUCMSC：人脐带间充质干细胞，Ang－Ⅱ：血管紧张素Ⅱ,

图2 各组hUCMSC培养24、48、72 h后染色结果观察(AO/EB染色×100)。2A－C、2D－F、2G－I、2J－L分别为空白对照组、100 ng/mL Ang－Ⅱ组、500 ng/mL Ang－Ⅱ组、1000 ng/mL Ang－Ⅱ组培养24、48、72 h；hUCMSC：人脐带间充质干细胞，AO/EB：吖啶橙/溴化乙锭，Ang－Ⅱ：血管紧张素Ⅱ

图3 各组hUCMSC培养24、48、72 h后细胞增殖活性(以吸光度值表示)测定。hUCMSC：人脐带间充质干细胞，Ang－Ⅱ：血管紧张素Ⅱ

表1 各组hUCMSC培养不同时间后处于增殖周期(S期)的细胞数量比例(%, ±s)

组别	24 h	48 h	72 h
空白对照组	10.32±1.62	14.96±1.02	17.26±2.73
100 ng/mLAng-Ⅱ组	13.84±2.87^a	18.84±2.67^a	22.77 ±1.87^a
500 ng/mLAng-Ⅱ组	8.48±2.30^a	11.05±2.42^a	14.00±2.63^a
1000 ng/mLAng-Ⅱ组	7.74±1.45^a	10.05±1.75^a	12.69±1.92^a

表1 各组hUCMSC培养不同时间后处于增殖周期(S期)的细胞数量比例(%, ±s)

组别	24 h	48 h	72 h
空白对照组	10.32±1.62	14.96±1.02	17.26±2.73
100 ng/mLAng-Ⅱ组	13.84±2.87^a	18.84±2.67^a	22.77 ±1.87^a
500 ng/mLAng-Ⅱ组	8.48±2.30^a	11.05±2.42^a	14.00±2.63^a
1000 ng/mLAng-Ⅱ组	7.74±1.45^a	10.05±1.75^a	12.69±1.92^a

表2 各组hUCMSC培养不同时间后细胞上清液中VEGF、bFGF、HGF的含量(pg/mL, ±s)

组别	VEGF			bFGF			HGF
组别	24 h	48 h	72 h	24 h	48 h	72 h	24 h	48 h	72 h
空白对照组	85.0±1.3	123.0±2.6	150.0±1.4	800.0±2.1	1080.0±2.3	1300.0±2.2	138.0±1.3	194.0±1.4	211.0±1.3
100 ng/mLAng-Ⅱ组	123.0±2.3^a	194.0±2.5^a	228.0±1.1^a	1225.0±1.2^a	1600.0±1.4^a	2061.0±1.1^a	215.0±1.3^a	305.0±1.7^a	320.0±1.3^a
500 ng/mLAng-Ⅱ组	60.0±2.2^a	84.0±2.1^a	110.0±2.1^a	600.0±2.3^a	800.0±2.5^a	1000.0±1.3^a	102.0±1.3^a	151.0±2.2^a	200.0±2.4^a
1000 ng/mLAng-Ⅱ组	50.0±2.3^a	86.0±2.2^a	100.0±1.3^a	500.0±1.3^a	737.0±1.2^a	908.0±1.3^a	87.0±1.2^a	137.0±2.5^a	142.0±2.2^a

表2 各组hUCMSC培养不同时间后细胞上清液中VEGF、bFGF、HGF的含量(pg/mL, ±s)

组别	VEGF			bFGF			HGF
组别	24 h	48 h	72 h	24 h	48 h	72 h	24 h	48 h	72 h
空白对照组	85.0±1.3	123.0±2.6	150.0±1.4	800.0±2.1	1080.0±2.3	1300.0±2.2	138.0±1.3	194.0±1.4	211.0±1.3
100 ng/mLAng-Ⅱ组	123.0±2.3^a	194.0±2.5^a	228.0±1.1^a	1225.0±1.2^a	1600.0±1.4^a	2061.0±1.1^a	215.0±1.3^a	305.0±1.7^a	320.0±1.3^a
500 ng/mLAng-Ⅱ组	60.0±2.2^a	84.0±2.1^a	110.0±2.1^a	600.0±2.3^a	800.0±2.5^a	1000.0±1.3^a	102.0±1.3^a	151.0±2.2^a	200.0±2.4^a
1000 ng/mLAng-Ⅱ组	50.0±2.3^a	86.0±2.2^a	100.0±1.3^a	500.0±1.3^a	737.0±1.2^a	908.0±1.3^a	87.0±1.2^a	137.0±2.5^a	142.0±2.2^a

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Effect of angiotensin Ⅱ on biological characteristics of human umbilical cord mesenchymal stem cells

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Effect of angiotensin Ⅱ on biological characteristics of human umbilical cord mesenchymal stem cells