脂多糖预处理人脐带间充质干细胞的条件培养基对人脐静脉内皮细胞增殖、凋亡的影响

doi:10.3877/cma.j.issn.1673-9450.2017.03.003

中华损伤与修复杂志(电子版) ›› 2017, Vol. 12 ›› Issue (03) : 169 -175. doi: 10.3877/cma.j.issn.1673-9450.2017.03.003

所属专题：文献；

论著

脂多糖预处理人脐带间充质干细胞的条件培养基对人脐静脉内皮细胞增殖、凋亡的影响

聂顺义¹, 巴特²^,^†(

), 夏成德³, 周彪², 金盼盼³, 兰斌斌², 王琼², 李武²

^1. 014010　包头，内蒙古医科大学第三临床医学院　内蒙古烧伤研究所；450003　郑州市第一人民医院烧伤中心
^2. 014010　包头，内蒙古医科大学第三临床医学院　内蒙古烧伤研究所
^3. 450003　郑州市第一人民医院烧伤中心

收稿日期:2017-03-06 出版日期:2017-06-01
通信作者: 巴特
基金资助:
内蒙古自治区自然科学基金(2015MS0815); 内蒙古医科大学科技百万工程联合项目(YKD2016KJBW（LH）041)

Effect of conditioned medium from lipopolysaccharide pretreatment human umbilical cord mesenchymal stem cells on proliferation and apoptosis of human umbilical vein endothelial cells

Shunyi Nie¹, Te Ba²^,^†(), Chengde Xia³, Biao Zhou², Panpan Jin³, Binbin Lan², Qiong Wang², Wu Li²

^1. Department of Burns, the Third Clinical Medical College of Inner Mongolia Medical University, Inner Mongolia Burn Medical Research Institute, Baotou 014010, China;Burn Center of the Medical Group of Zhengzhou First People′s Hospital, Zhengzhou 450003, China
^2. Department of Burns, the Third Clinical Medical College of Inner Mongolia Medical University, Inner Mongolia Burn Medical Research Institute, Baotou 014010, China
^3. Burn Center of the Medical Group of Zhengzhou First People′s Hospital, Zhengzhou 450003, China

Received:2017-03-06 Published:2017-06-01
Corresponding author: Te Ba
About author:
Corresponding author: Ba Te, Email: bgyybate@126.com

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引用本文：

聂顺义, 巴特, 夏成德, 周彪, 金盼盼, 兰斌斌, 王琼, 李武. 脂多糖预处理人脐带间充质干细胞的条件培养基对人脐静脉内皮细胞增殖、凋亡的影响[J]. 中华损伤与修复杂志(电子版), 2017, 12(03): 169-175.

Shunyi Nie, Te Ba, Chengde Xia, Biao Zhou, Panpan Jin, Binbin Lan, Qiong Wang, Wu Li. Effect of conditioned medium from lipopolysaccharide pretreatment human umbilical cord mesenchymal stem cells on proliferation and apoptosis of human umbilical vein endothelial cells[J]. Chinese Journal of Injury Repair and Wound Healing(Electronic Edition), 2017, 12(03): 169-175.

目的

探讨脂多糖预处理人脐带间充质干细胞(hUCMSC)的条件培养基对人脐静脉内皮细胞(HUVEC)增殖和凋亡的影响。

方法

本实验按随机数字表法将HUVEC分为对照组、条件培养基组和预处理条件培养基组。首先使用100 ng/mL脂多糖的内皮细胞培养基刺激3组HUVEC 12 h后，对照组更换为原培养基，条件培养基组更换为hUCMSC的条件培养基，预处理条件培养基组更换为脂多糖预处理hUCMSC的条件培养基。分别于培养24、48、72 h时点采用MTT法测定HUVEC的增殖活性；台盼蓝排除染色法检测细胞增殖的数量，流式细胞仪检测细胞的增殖周期，Hoechst染色和流式细胞仪定性定量检测细胞凋亡的情况。方差分析统计分析比较数据。

结果

MTT测定结果示：培养24 h时，对照组、条件培养基组和预处理条件培养基组细胞吸光度值为0.46、0.52和0.56；培养48 h时，3组的吸光度值为0.35、0.46和0.51；培养72 h时，3组的吸光度值为0.21、0.38和0.47；在3个时间点上，条件培养基组、预处理条件培养基组的吸光度值均高于对照组，且预处理条件培养基组的细胞量高于条件培养基组。台盼蓝染色计数的结果与MTT的结果趋势类似。培养24 h时，对照组、条件培养基组和预处理条件培养基组的细胞数量为(4.554±0.103)×10³、(5.251±0.091)×10³、(5.585±0.038)×10³个；培养48 h时，3组的细胞数量分别为(3.465±0.087)×10³、(4.659±0.116)×10³、(5.347±0.099)×10³个；培养72 h时，3组的细胞数量是(2.079±0.127)×10³、(4.063±0.052)×10³、(4.950±0.104)×10³个；在3个时间点上，条件培养基组、预处理条件培养基组细胞的数量均显著高于对照组，差异有统计学意义(P值均小于0.05)，且预处理条件培养基组的细胞数量显著高于条件培养基组，差异有统计学意义(P值均小于0.05)。流式细胞仪检测细胞增殖周期(G2/M＋S期)的结果示：培养24 h时，3组细胞处于G2/M＋S期的比例是(17.07±1.15)%，(22.52±1.61)%和(26.19±2.25)%；培养48 h时分别是(13.46±1.74)%，(18.67±2.07)%和(24.58±2.54)%；培养72 h时分别是(8.73±1.61)%，(14.31±1.93)%和(18.43±2.02)%；在培养的各个时间点上，条件培养基组、预处理条件培养基组细胞处于增殖周期的比例均显著高于对照组，差异有统计学意义(P值均小于0.05)，预处理条件培养基组细胞处于增殖周期的比例显著高于条件培养基组，差异有统计学意义(P值均小于0.05)。Hoechst染色结果和流式细胞仪检测细胞凋亡结果趋势类似，具体为：培养24 h时，对照组、条件培养基组和预处理条件培养基组细胞的凋亡率分别为(30.78±3.20)%、(23.21±1.72)%和(17.69±1.81)%；培养48 h时分别为(26.02±2.06)%、(20.04±1.83)%和(15.03±1.56)%; 培养72 h时分别为(22.41±1.63)%、(15.27±1.14)%和(11.25±1.07)%；在培养24、48、72 h时间点，3组中对照组细胞的凋亡率最高，而条件培养基组和预处理条件培养基组细胞的凋亡率显著低于对照组，差异均有统计学意义(P值均小于0.05)，预处理条件培养基组细胞的凋亡率显著低于条件培养基组，差异均有统计学意义(P值均小于0.05)。

结论

脂多糖预处理hUCMSC的条件培养基发挥了显著促进HUVEC增殖活性和抑制HUVEC凋亡的作用。

关键词: 脂多糖类, 间质干细胞, 内皮细胞, 预处理, 细胞增殖, 细胞凋亡

Objective

To investigate the effect of conditioned medium of lipopolysaccharide pretreatment human umbilical cord mesenchymal stem cells (hUCMSC) on proliferation and apoptosis of human umbilical vein endothelial cells (HUVEC).

Methods

HUVEC was randomly divided into 3 groups: control group, conditioned medium group (CM group) and pretreatment-conditioned medium group (pretreatment-CM group). After 100 ng/mL lipopolysaccharide stimulating HUVEC 12 h, media in control group was replaced for the original medium, and conditioned medium and preconditioning medium were replaced for the CM and lipopolysaccharide pretreatment-CM, respectively. Cell proliferation was detected by MTT. Cell counting were detected by trypan blue exclusion staining. Cell cycle was detected by flow cytometry. Cell apoptosis was assessed by Hoechst staining and flow cytometry. Statistic was analyzed by variance analysis.

Results

At 24 h post treatment, the optical density values of control group, CM group and pretreatment-CM group were 0.46, 0.52 and 0.56, respectively. At 48 h post treatment, the optical density values of control group, CM group and pretreatment-CM group were 0.35, 0.46 and 0.51, respectively. At 72 h post treatment, the optical density values of control group, CM group and pretreatment-CM group were 0.21, 0.38 and 0.47, respectively. At the same time point post treatment, the proliferation numbers in CM group and pretreatment-CM group were markedly higher than that in control group, as well as that in pretreatment-CM group was higher than that in CM group. The result of cell counting was similar with MTT result. At 24 h post treatment, the cell counting of control group, CM group and pretreatment-CM group were (4.554±0.103)×10^3, (5.251±0.091)×10³ and (5.585±0.038)×10^3, respectively. At 48 h post treatment, the cell counting of control group, CM group and pretreatment-CM group were (3.465±0.087)×10^3, (4.659±0.116)×10³ and (5.347±0.099)×10^3, respectively. At 72 h post treatment, the cell counting of control group, CM group and pretreatment-CM group were (2.079±0.127)×10^3, (4.063±0.052)×10³ and (4.950±0.104)×10^3, respectively. At the same time point post treatment, the cells numbers in CM group and pretreatment-CM group were markedly higher than that in control group, the differences were statistically significant (P<0.05), as well as that in pretreatment-CM group was higher than that in CM group, the difference was statistically significant (P<0.05). Meanwhile, the results of cell cycle were assayed by flow cytometry. At 24 h post treatment, the G2/M+ S ratio of control group, CM group and pretreatment-CM group were (17.07±1.15)%, (22.52±1.61)% and (26.19±2.25)%, respectively. At 48 h post treatment, the G2/M+ S ratio of control group, CM group and pretreatment-CM group were (13.46±1.74)%, (18.67±2.07)% and (24.58±2.54)%, respectively. At 72 h post treatment, the G2/M+ S ratio of control group, CM group and pretreatment-CM group were (8.73±1.61)%, (14.31±1.93)% and (18.43±2.02)%, respectively. At the same time point post treatment, the G2/M+ S ratio in CM group and pretreatment-CM group were markedly higher than that in control group, the differences were statistically significant (P<0.05), as well as that in pretreatment-CM group was higher than that in CM group, the difference was statistically significant (P<0.05). Furthermore, the results of Hoechst staining and flow cytometry were showed that cell apoptosis rates of control group, CM group and pretreatment-CM group were (30.78±3.20)%, (23.21±1.72)% and (17.69±1.81)% at 24 h post treatment. At 48 h post treatment, the G2/M+ S ratio of control group, CM group and pretreatment-CM group were (26.02±2.06)%, (20.04±1.83)% and (15.03±1.56)%, respectively. At 72 h post treatment, the G2/M+ S ratio of control group, CM group and pretreatment-CM group were (22.41±1.63)%, (15.27±1.14)% and(11.25±1.07)%, respectively. At the same time point post treatment, the apoptosis rates in CM group and pretreatment-CM group were markedly lower than that in control group, the differences were statistically significant (P<0.05), as well as that in pretreatment-CM group was lower than that in CM group, the difference was statistically significant (P<0.05).

Conclusion

The conditioned medium from 100 ng/mL lipopolysaccharide pretreatment hUCMSC significantly increased HUVEC proliferation activities and deceased cell apoptosis numbers and apoptosis rates, which were induced by lipopolysaccharide.

Key words: Lipopolysaccharides, Mesenchymal stem cells, Endothelial cells, Pretreatment, Cell proliferation, Apoptosis

图1 不同培养时间点3组HUVEC的增殖曲线

图2 不同培养时间点3组HUVEC的增殖数量(台盼蓝染色)

表1 不同培养时间点3组细胞HUVEC处于G2/M＋S期的比例(n＝6，%，±s)

组别	培养24 h	培养48 h	培养72 h
对照组	17.07±1.15	13.46±1.74	8.73±1.61
条件培养基组	22.52±1.61^a	18.67±2.07^a	14.31±1.93^a
预处理条件培养基组	26.19±2.25^ab	24.58±2.54^ab	18.43±2.02^ab
F值	31.220	25.816	23.987
P值	<0.05	<0.05	<0.05

表1 不同培养时间点3组细胞HUVEC处于G2/M＋S期的比例(n＝6，%，±s)

组别	培养24 h	培养48 h	培养72 h
对照组	17.07±1.15	13.46±1.74	8.73±1.61
条件培养基组	22.52±1.61^a	18.67±2.07^a	14.31±1.93^a
预处理条件培养基组	26.19±2.25^ab	24.58±2.54^ab	18.43±2.02^ab
F值	31.220	25.816	23.987
P值	<0.05	<0.05	<0.05

图3 不同培养时间点3组HUVEC的凋亡情况(倒置荧光显微镜 ×100)

表2 不同培养时间点3组细胞HUVEC的凋亡率(n＝6，%，±s)

组别	培养24 h	培养48 h	培养72 h
对照组	30.78±3.20	26.02±2.06	22.41±1.63
条件培养基组	23.21±1.72^a	20.04±1.83^a	15.27±1.14^a
预处理条件培养基组	17.69±1.81^ab	15.03±1.56^ab	11.25±1.07^ab
F值	21.107	20.094	44.526
P值	<0.05	<0.05	<0.05

表2 不同培养时间点3组细胞HUVEC的凋亡率(n＝6，%，±s)

组别	培养24 h	培养48 h	培养72 h
对照组	30.78±3.20	26.02±2.06	22.41±1.63
条件培养基组	23.21±1.72^a	20.04±1.83^a	15.27±1.14^a
预处理条件培养基组	17.69±1.81^ab	15.03±1.56^ab	11.25±1.07^ab
F值	21.107	20.094	44.526
P值	<0.05	<0.05	<0.05

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Effect of conditioned medium from lipopolysaccharide pretreatment human umbilical cord mesenchymal stem cells on proliferation and apoptosis of human umbilical vein endothelial cells

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Effect of conditioned medium from lipopolysaccharide pretreatment human umbilical cord mesenchymal stem cells on proliferation and apoptosis of human umbilical vein endothelial cells