中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (10): 1755-1759.doi: 10.3969/j.issn.1673-8225.2010.10.009

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

骨髓间充质干细胞旁分泌途径与阿霉素损伤心肌细胞凋亡：怎样证明其抑制效应？

郭俊芳，张赢予，陈  蓉，周艳芳，王  好，张国辉   

  1. 镇江市第一人民医院，江苏大学附属人民医院心内科，江苏省镇江市 212002
  • 出版日期:2010-03-05 发布日期:2010-03-05
  • 通讯作者: 张国辉，博士，主任医师，硕士生导师，镇江市第一人民医院，江苏大学附属人民医院心内科，江苏省镇江市 212002 13338812776@ e165.com
  • 作者简介:郭俊芳，女，1973年生，江苏省丹阳市人，汉族，2009年江苏大学毕业，硕士，主治医师，主要从事心血管介入方面的研究。 guojunfang@ medmail.com.cn

Paracrine effects of bone marrow mesenchymal stem cells on the apoptosis of adriamycin-injured cardiomyocytes: How to verify the inhibitory effects?

Guo Jun-fang, Zhang Ying-yu, Chen Rong, Zhou Yan-fang, Wang Hao, Zhang Guo-hui   

  1. Department of Cardiology, First People’s Hospital of Zhenjiang City, Affiliated People’s Hospital, Jiangsu University, Zhenjiang   212002, Jiangsu Province, China
  • Online:2010-03-05 Published:2010-03-05
  • Contact: Zhang Guo-hui, Doctor, Chief physician, Master’s supervisor, Department of Cardiology, First People’s Hospital of Zhenjiang City, Affiliated People’s Hospital, Jiangsu University, Zhenjiang 212002, Jiangsu Province, China 13338812776@ e165.com
  • About author:Guo Jun-fang, Master, Attending physician, Department of Cardiology, First People’s Hospital of Zhenjiang City, Affiliated People’s Hospital, Jiangsu University, Zhenjiang 212002, Jiangsu Province, China guojunfang@ medmail.com.cn

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摘要：

背景：近来研究发现骨髓间充质干细胞移植后短期心功能受益主要是因为其可分泌多种细胞因子，促进了内生性修复过程，而不是心肌细胞的再生。

目的：观察骨髓间充质干细胞旁分泌途径对阿霉素损伤心肌细胞凋亡的影响。

方法：体外培养的乳鼠心肌细胞按3×108 L-1浓度加入6孔培养板，3 mL/孔，培养72 h后分为3组：阿霉素损伤组、共培养组加入1 mg/L阿霉素作用4 h建立心肌细胞损伤模型，正常对照组不进行任何干预。共培养组取培养至第3代大鼠骨髓间充质干细胞，调整细胞浓度为3×108 L-1，以3 mL/孔加入共培养插件Millicell装置中，预培养24 h，在造模后将Millicell装置插入到预先培养心肌细胞的6孔培养板中，建立共培养体系。检测条件培养基内细胞因子的质量浓度变化，骨髓间充质干细胞对阿霉素损伤心肌细胞Caspase-9和Caspase-3活性、细胞凋亡、Bcl-2和Bax蛋白表达的影响。

结果与结论：与正常对照组比较，阿霉素损伤组胰岛素样生长因子1及肝细胞生长因子的质量浓度、心肌细胞Caspase-3及Caspase-9活性、心肌细胞凋亡率、心肌细胞Bax蛋白表达均明显升高(P < 0.05)，Bcl-2蛋白表达明显降低(P < 0.05)。与阿霉素损伤组比较，共培养组胰岛素样生长因子1及肝细胞生长因子的质量浓度、心肌细胞Bcl-2蛋白表达均明显升高   (P < 0.05)，心肌细胞Caspase-3及Caspase-9活性、心肌细胞凋亡率、心肌细胞Bax蛋白表达均明显降低(P < 0.05)。结果证实骨髓间充质干细胞在损伤环境下细胞因子分泌增加，并可通过旁分泌途径抑制阿霉素诱导的心肌细胞凋亡。

关键词: 旁分泌, 阿霉素, 凋亡, 胰岛素样生长因子1, 肝细胞生长因子, Bcl-2蛋白, Bax蛋白, 骨髓间充质干细胞, 损伤, 心肌细胞

Abstract:

BACKGROUND: Several studies have shown the improvement of heart function through the introduction of mesenchymal stem cells(MSCs) from bone marrow, which may be attributed to secretion of various cytokines that accelerate endogenous reparative process, but not regeneration of cardiomyocytes.

OBJECTIVE: To observe the anti-apoptotic effects of MSCs on adriamycin(ADR)- injured cardiomyocytes apoptosis in vitro through paracrine pathway.

METHODS: In vitro cultured neonatal rat cardiomyocytes (3×108/L) were incubated into a 6-well plate, 3 mL/well. 72 hours later, these cells were assigned into 3 groups. The primary cultured neonatal rat cardiomyocytes in the ADR-injured and coculture groups were exposed to 1 mg/L ADR for 4 hours to establish experimental models of toxic cardiomyocytes. The normal control group was left intact. In the coculture group, rat bone marrow MSCs (BMSCs) at passage 3 were regulated to 3×108/L, 3 mL/well was added into Millicell device for 24 hours. Following model induction, the Millicell device was inseted into above-mentioned 6-well plate to establish coculture system. The levels of cytokines were measured in the conditioned medium from three cardiomyocytes groups. Effects of BMSCs on Caspase-9 and Caspase-3 activities, apoptosis and Bcl-2 and Bax protein expression in ADR-injured cardiomyocytes were measured.

RESULTS AND CONCLUSION: Compared with the normal control group,the level of cytokine including insulin-like growth factor (IGF-1) and hepatocyte growth factor (HGF) was significantly higher in the medium from ADR-injured group,the activity of caspase-9, caspase-3 and the apoptosis rate increased significantly, the expression of Bax protein was higher and Bcl-2 Protein was lower in ADR-injured group(P < 0.05). Compared with the ADR-injured group, the level of IGF-1 and HGF in co-cultured group increased significantly, the apoptosis rate, Caspase-3 and Caspase-9 activities decreased significantly,the expression of Bax protein was lower while Bcl-2 Protein was higher than ADR-injured group (P < 0.05). Results indicated that BMSCs show increased cytokine secretion and significant anti-apoptotic effects on ADR-injured cardiomyocytes through paracrine pathway.

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