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

全文 ( ) 下载PDF ( ) 导出引用

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/OL]. 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/OL]. 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

1	Long H,Xu B,Luo Y, et al. Artemisinin protects mice against burn sepsis through inhibiting NLRP3 inflammasome activation[J]. Am J Emerg Med, 2016, 34(5): 772-777.
2	Matsuda N. Alert cell strategy in SIRS-induced vasculitis: sepsis and endothelial cells[J]. J Intensive Care, 2016, 4: 21.
3	张艺森,徐晓涵,孙炳伟. 脓毒症时血管内皮细胞与血管平滑肌细胞相互作用的研究进展[J]. 中华危重病急救医学, 2016, 28(2): 180-183.
4	Luo K,Long H,Xu B, et al. Metallothionein ameliorates burn sepsis partly via activation of Akt signaling pathway in mice: a randomized animal study[J]. World J Emerg Surg, 2015, 10: 53.
5	Fu J,Wang Y,Zhang J, et al. Anti-inflammatory and anti-apoptotic effects of oxysophoridine on lipopolysaccharide-induced acute lung injury in mice[J]. Am J Transl Res, 2015, 7(12): 2672-2682.
6	Yang Y,Chen QH,Liu AR, et al. Synergism of MSC-secreted HGF and VEGF in stabilising endothelial barrier function upon lipopolysaccharide stimulation via the Rac1 pathway[J]. Stem Cell Res Ther, 2015, 6: 250.
7	Jiang CY,Gui C,He AN, et al. Optimal time for mesenchymal stem cell transplantation in rats with myocardial infarction[J]. J Zhejiang Univ Sci B, 2008, 9(8): 630-637.
8	Tan L,Huang Y,Pan X, et al. Administration of bone marrow stromal cells in sepsis attenuates sepsis-related coagulopathy[J]. Ann Med, 2016, 48(4): 235-245.
9	Ma J,Bai J. Protective effects of heparin on endothelial cells in sepsis[J]. Int J Clin Exp Med, 2015, 8(4): 5547-5552.
10	Kang Q,Chen Y,Zhang X, et al. Heat shock protein A12B protects against sepsis-induced impairment in vascular endothelial permeability[J]. J Surg Res, 2016, 202(1): 87-94.
11	Liu L,Yu Y,Hou Y, et al. Human umbilical cord mesenchymal stem cells transplantation promotes cutaneous wound healing of severe burned rats[J]. PLoS One, 2014, 9(2): e88348.
12	Liu L,Li X,Yang J, et al. Comparison of systemic inflammation response and vital organ damage induced by severe burns in different area[J]. Int J Clin Exp Pathol, 2015, 8(6): 6367-6376.
13	Tanaka Y. Human mesenchymal stem cells as a tool for joint repair in rheumatoid arthritis[J]. Clin Exp Rheumatol, 2015, 33(4 Suppl 92): S58-S62.
14	Robinson AM,Sakkal S,Park A, et al. Mesenchymal stem cells and conditioned medium avert enteric neuropathy and colon dysfunction in guinea pig TNBS-induced colitis[J]. Am J Physiol Gastrointest Liver Physiol, 2014, 307(11): G1115-G1129.
15	Bakker AC,Langer B. Cell-based therapies - an innovative therapeutic option in ophthalmology: Treating corneal diseases with stem cells[J]. Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz, 2015, 58(11/12): 1259-1264.
16	Hatakeyama N,Matsuda N. Alert cell strategy: mechanisms of inflammatory response and organ protection[J]. Curr Pharm Des, 2014, 20(36): 5766-5778.
17	Bader AM,Klose K,Bieback K, et al. Hypoxic Preconditioning Increases Survival and Pro-Angiogenic Capacity of Human Cord Blood Mesenchymal Stromal Cells In Vitro[J]. PLoS One, 2015, 10(9): e0138477.
18	Zhang Z,Yang J,Yan W, et al. Pretreatment of Cardiac Stem Cells With Exosomes Derived From Mesenchymal Stem Cells Enhances Myocardial Repair[J]. J Am Heart Assoc, 2016, 5(1).
19	Shen Q,Chen B,Xiao Z, et al. Paracrine factors from mesenchymal stem cells attenuate epithelial injury and lung fibrosis[J]. Mol Med Rep, 2015, 11(4): 2831-2837.
20	Ogata K,Katagiri W,Osugi M, et al. Evaluation of the therapeutic effects of conditioned media from mesenchymal stem cells in a rat bisphosphonate-related osteonecrosis of the jaw-like model[J]. Bone, 2015, 74: 95-105.
21	Ju GQ,Cheng J,Zhong L, et al. Microvesicles derived from human umbilical cord mesenchymal stem cells facilitate tubular epithelial cell dedifferentiation and growth via hepatocyte growth factor induction[J]. PLoS One, 2015, 10(3): e0121534.
22	Zhu J,Liu Q,Jiang Y, et al. Enhanced angiogenesis promoted by human umbilical mesenchymal stem cell transplantation in stroked mouse is Notch1 signaling associated[J]. Neuroscience, 2015, 290: 288-299.
23	聂顺义,周彪,巴特, 等. TNF-α预处理人脐带MSCs的条培对人脐静脉内皮细胞增殖、凋亡的影响[J/CD]. 中华损伤与修复杂志(电子版), 2016, 11(6): 433-437.
24	Yang Jing ,Nie Shunyi ,Liu Lingying , et al. Human umbilical cord mesenchymal stem cells pretreated with Angiotensin-II facilitates angiogenesis by improving the function of endothelial cells[J]. Int J Clin Exp Pathol, 2016, 9(5): 5056-5066.
25	Hou YS,Liu LY,Chai JK, et al. Lipopolysaccharide pretreatment inhibits LPS-induced human umbilical cord mesenchymal stem cell apoptosis via upregulating the expression of cellular FLICE-inhibitory protein[J]. Mol Med Rep, 2015, 12(2): 2521-2528.

[1]	张刚, 秦勇, 黄超, 薛震, 吕松岑. 基于骨关节炎软骨细胞表型转化的新兴治疗靶点[J/OL]. 中华关节外科杂志(电子版), 2024, 18(03): 352-362.
[2]	钟雅雯, 王煜, 王海臻, 黄莉萍. 肌苷通过抑制线粒体通透性转换孔开放缓解缺氧/复氧诱导的人绒毛膜滋养层细胞凋亡[J/OL]. 中华妇幼临床医学杂志(电子版), 2024, 20(05): 525-533.
[3]	孙鸿坤, 艾虹, 陈正. 内质网应激介导的牙周炎骨改建失衡的研究进展[J/OL]. 中华口腔医学研究杂志(电子版), 2024, 18(04): 211-218.
[4]	黄福, 王黔, 金相任, 唐云川. VEGFR2、miR-27a-5p在胃癌组织中的表达与临床病理参数及预后的关系研究[J/OL]. 中华普外科手术学杂志(电子版), 2024, 18(05): 558-561.
[5]	祝炜安, 林华慧, 吴建杰, 黄炯煅, 吴婷婷, 赖文杰. RDM1通过CDK4促进前列腺癌细胞进展的研究[J/OL]. 中华腔镜泌尿外科杂志(电子版), 2024, 18(06): 618-625.
[6]	郑俊, 吴杰英, 谭海波, 郑安全, 李腾成. EGFR-MEK-TZ三联合分子的构建及其对去势抵抗性前列腺癌细胞增殖与凋亡的影响[J/OL]. 中华腔镜泌尿外科杂志(电子版), 2024, 18(05): 503-508.
[7]	赵蒙蒙, 黄洁, 余荣环, 王葆青. 过表达小GTP酶Rab32抑制非小细胞肺癌细胞侵袭性生长[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(04): 512-518.
[8]	黄程鑫, 陈莉, 刘伊楚, 王水良, 赖晓凤. OPA1 在乳腺癌组织的表达特征及在ER阳性乳腺癌细胞中的生物学功能研究[J/OL]. 中华细胞与干细胞杂志(电子版), 2024, 14(05): 275-284.
[9]	季加翠, 孙春斌, 罗恩丽. 姜黄素通过调节NF-κB/NLRP3通路减轻LPS诱导小胶质细胞神经炎症损伤[J/OL]. 中华细胞与干细胞杂志(电子版), 2024, 14(04): 193-203.
[10]	曾聿理, 雷发容, 肖慧, 邱德亮, 谢静, 吴寻. 氯普鲁卡因通过调控circRNA-ZKSCAN1表达抑制肝癌Huh-7细胞体外生长和转移的研究[J/OL]. 中华细胞与干细胞杂志(电子版), 2024, 14(04): 220-228.
[11]	李晶, 潘侠, 周芳, 汪晶, 洪佳. 普鲁卡因通过上调lncRNA DGCR5抑制胃癌细胞增殖、迁移和侵袭[J/OL]. 中华细胞与干细胞杂志(电子版), 2024, 14(03): 151-158.
[12]	李博, 马秀岩, 孙杰. lncRNA TINCR对滋养层HTR-8/SVneo细胞生物学行为的影响及其机制[J/OL]. 中华细胞与干细胞杂志(电子版), 2024, 14(03): 167-172.
[13]	杨兴业, 彭旭云, 曾倩, 梁伟铖, 肖翠翠, 郑俊, 姚嘉. LMO7通过靶向铁死亡促进肝细胞癌生长[J/OL]. 中华肝脏外科手术学电子杂志, 2024, 13(03): 370-376.
[14]	杜霞, 马梦青, 曹长春. 造影剂诱导的急性肾损伤的发病机制及干预靶点研究进展[J/OL]. 中华肾病研究电子杂志, 2024, 13(05): 279-282.
[15]	冯盼, 梁秋华. 细胞间相互作用及代谢微环境在动脉钙化中的作用机制研究进展[J/OL]. 中华诊断学电子杂志, 2024, 12(03): 193-198.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

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

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

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