中国组织工程研究

中国组织工程研究 ›› 2018, Vol. 22 ›› Issue (13): 1976-1980.doi: 10.3969/j.issn.2095-4344.0491

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

大鼠骨髓来源内皮祖细胞体外增殖和凋亡及阿托伐他汀的干预

张日霖1，陈淑玲1，李上海2，宁奕明3，李庆军1，叶小敏1，梁伟均2   

  1. 1湛江中心人民医院，广东省湛江市 524000；2广东医科大学附属医院，广东省湛江市 524000；3吴川市妇幼保健院，广东省吴川市 524500
  • 修回日期:2018-02-09 出版日期:2018-05-08 发布日期:2018-05-08
  • 通讯作者: 梁伟均，硕士，主任医师，广东医科大学附属医院，广东省湛江市 524000
  • 作者简介:张日霖，男，1979年生，广东省茂名市人，汉族，2011年广东医科大学毕业，硕士，主治医师，主要从事心血管疾病的诊治。
  • 基金资助:

    广东省医学科学技术研究基金(A2017494)；广东省科技计划项目(2009B030801337)

Atorvastatin effects on proliferation and apoptosis of rat bone marrow-derived endothelial progenitor cells in vitro

Zhang Ri-lin1, Chen Shu-ling1, Li Shang-hai2, Ning Yi-ming3, Li Qing-jun1, Ye Xiao-min1, Liang Wei-jun2   

  1. 1Central People’s Hospital of Zhanjiang, Zhanjiang 524000, Guangdong Province, China; 2Affiliated Hospital of Guangdong Medical University, Zhanjiang 524000, Guangdong Province, China; 3Maternal and Child Health Hospital of Wuchuan, Wuchuan 524500, Guangdong Province, China
  • Revised:2018-02-09 Online:2018-05-08 Published:2018-05-08
  • Contact: Liang Wei-jun, Master, Chief physician, Affiliated Hospital of Guangdong Medical University, Zhanjiang 524000, Guangdong Province, China
  • About author:Zhang Ri-lin, Master, Attending physician, Central People’s Hospital of Zhanjiang, Zhanjiang 524000, Guangdong Province, China
  • Supported by:

    the Medical Science and Technology Research Fund of Guangdong Province, No. A2017494; the Scientific Research Plan Program of Guangdong Province, No. 2009B030801337

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摘要：

文章快速阅读：

文题释义：
内皮祖细胞：是血管内皮细胞的前体，亦称成血管细胞。该细胞最早于1997年被Asahara等证明存在于循环外周血中并能分化为血管内皮细胞，在缺血刺激下可通过动员到外周血，迁移、归巢到血管新生部位，并在迁移部位增殖、分化为内皮细胞，在体内具有明显的内皮修复及促血管新生作用。多数研究认为内皮祖细胞是表达CD34、CD133、VEGFR-2、Flk -1、vWF的细胞，但具体的表达标志尚存在颇多争议，因此，其表达标志并无特异性。
内皮祖细胞移植：①内皮祖细胞的提取、鉴定、扩增上仍需一个具体和规范的方法与标准，未扩增的内皮祖细胞含量极低，而扩增后的细胞归巢能力下降，因此内皮祖细胞向心肌细胞和血管内皮细胞的分化概率也很低；②缺血缺氧状态下内皮祖细胞动员、募集和归巢受众多化学趋化因子和细胞因子的调节，它们之间的相互作用机制目前仍需进一步明确；③细胞移植时机的选择直接影响到细胞的存活。移植时间过早可能由于局部微环境恶劣致使大量移植的细胞死亡；移植时间过晚则可能导致局部损伤已不可逆；④如何保证移植后的定向分化，如分化为心肌细胞或血管内皮细胞等，且不会分化为肿瘤，这些都待进一步研究。

 

摘要
背景：研究发现，阿托伐他汀具有心血管保护作用，能显著改善内皮功能，促进内皮祖细胞的动员、迁移和分化。现阶段在细胞体外培养水平上进行阿托伐他汀药物浓度筛选所进行的研究较少。
目的：探讨不同浓度阿托伐他汀对大鼠骨髓来源内皮祖细胞体外生长特性的影响。
方法：取SD大鼠长骨骨髓，用密度梯度离心法结合差异贴壁法提取单核细胞，用选择性培养液诱导培养内皮祖细胞，采用免疫荧光染色法鉴定细胞表面标志物。将内皮祖细胞分为对照组和4个不同浓度阿托伐他汀组(0.01，0.1，1，10 μmol/L)进行培养，光镜下观察和MTT法检测细胞生长增殖情况，流式细胞术检测细胞凋亡率，硝酸还原酶法化学比色法检测培养液中一氧化氮和内皮型一氧化氮合酶水平。
结果与结论：①对照组和阿托伐他汀组的细胞数量均表现为增长趋势，1 μmol/L阿托伐他汀组增长最明显，10 μmol/L阿托伐他汀组在7 d后出现下降趋势；②1 μmol/L阿托伐他汀组凋亡率低于其他各组，10 μmol/L阿托伐他汀组凋亡率高于其他各组；③0.01，0.1，1.0 μmol/L阿托伐他汀组一氧化氮和内皮型一氧化氮合酶水平明显高于空白对照组(P < 0.01)，10 μmol/L阿托伐他汀组一氧化氮和内皮型一氧化氮合酶水平明显低于其他各组(P < 0.01)；④结果表明，阿托伐他汀可通过增加内皮型一氧化氮合酶和一氧化氮的生成，促进内皮祖细胞增殖并减少凋亡，其中1 μmol/L浓度的阿托伐他汀最适合内皮祖细胞培养。

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程
ORCID: 0000-0001-5131-2950(张日霖)

关键词: 内皮祖细胞, 阿托伐他汀, 细胞增殖, 细胞凋亡, 一氧化氮合酶, 一氧化氮, 干细胞

Abstract:

BACKGROUND: Atorvastatin has a cardiovascular protective effect that significantly improves endothelial function and promotes the mobilization, migration, and differentiation of endothelial progenitor cells. However, the screening of atorvastatin concentration for in vitro cell culture is not well documented.
OBJECTIVE: To investigate the effects of different concentrations of atorvastatin on rat bone marrow-derived EPCs growth characteristics.
METHODS: Bone marrow mononuclear cells from Sprague-Dawley rats were induced in selective culture fluid to culture EPCs. Immunofluorescence staining was used to identify cell surface markers. Harvested EPCs were divided into control group and atorvastatin groups with four different concentrations (0.01, 0.1, 1, and 10 μmol/L) for culture. The growth and proliferation of EPCs were observed under light microscope and MTT assay. Flow cytometry was used to detect apoptosis in EPCs. Nitric oxide and endothelial nitric oxide synthase levels in the culture fluid were measured by nitrate reductase method.
RESULTS AND CONCLUSION: The number of cells tended to increase in the control and atorvastatin groups, and it was highest in the
1 μmol/L atorvastatin group. The cell number in the 10 μmol/L atorvastatin group began to decrease at 7 days of culture. Among the five groups, the apoptotic rate of cells was lowest in the 1 μmol/L atorvastatin group and highest in the 10 μmol/L atorvastatin group. The levels of nitric oxide and endothelial nitric oxide synthase were significantly higher in the 0.01, 0.1 and 1.0 μmol/L atorvastatin groups compared with the control group (P < 0.01), but lower in the 10 μmol/L atorvastatin group compare with the other groups (P < 0.01). Overall, atorvastatin can promote the proliferation of endothelial progenitor cells and reduce apoptosis by increasing the production of endothelial nitric oxide synthase and nitric oxide, and 1 μmol/L atorvastatin is most suitable for the EPCs culture.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Endothelial Cells, Cell Proliferation, Apoptosis, Nitric Oxide, Nitric Oxide Synthase, Tissue Engineering

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