中国药物警戒 ›› 2015, Vol. 12 ›› Issue (8): 454-457.

• 基础研究 • 上一篇    下一篇

中药血清对体外缺氧条件下纯化培养的RGCs的保护作用研究

李颖, 邸红莲   

  1. 保定市第一中心医院眼一科,河北 保定 071000
  • 收稿日期:2015-09-02 修回日期:2015-09-02 出版日期:2015-08-08 发布日期:2015-09-02
  • 作者简介:李颖,女,主治医师,眼科临床。

Study on Protective Effect of Chinese Herb Serum on RGCs Purified Cultured under the Condition of Hypoxia in Vitro

LI Ying, DI Hong-lian   

  1. No.1 Department of Ophthalmology, First Central Hospital of Baoding, Hebei Baoding 071000, China
  • Received:2015-09-02 Revised:2015-09-02 Online:2015-08-08 Published:2015-09-02

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摘要： 目的 探讨补肾活血中药血清对体外缺氧条件下纯化培养的视网膜神经节细胞（RGCs）的保护作用及其机制。方法 选取出生1 d的新生乳鼠30只,采用“两步筛选法”纯化培养SD新生乳鼠视网膜神经节细胞（RGCs）,选取出生7 d的SD大鼠6只,采用“改进的酶消化法”纯化培养新生SD大鼠视网膜Muller细胞,然后按照1:25的比例将RGCs接种于被视网膜Muller细胞包被的培养基中建立共同培养模型,在体外培养基中分别加入葡萄糖（50 mmol·L-1）和连二亚硫酸钠（1.0 mmol·L-1）（高糖缺氧组）;仅加入葡萄糖（50 mmol·L-1）（高糖组）;加入连二亚硫酸钠（1.0 mmol·L-1）（缺氧组）;在上述基础分别加入制定好的20%体积分数的补肾活血中药血清进行干预,分别测定24、48、72 h培养模型外液中的乳酸脱氢酶（LDH）漏出量和谷氨酰胺合成酶（GS）活性。结果 在第48 h、72 h正常对照组、高糖组、缺氧组3组的LDH漏出量较第24 h均有显著的增高趋势（P <0.05）,正常对照组、高糖组、缺氧组、高糖缺氧组在同一时刻与各自对应的中药干预组比较,可以发现LDH漏出量均显著的高于各中药干预组（P <0.05）。在第48 h、72 h正常对照组、高糖组、缺氧组、高糖缺氧组及其中药干预组的GS活性较第24 h均有显著的增高趋势（P <0.05）,正常对照组、高糖组、缺氧组、高糖缺氧组随着时间的延长其GS活性均显著的低于中药干预组。结论 高糖或缺氧条件下可以造成RGCs、视网膜Muller细胞的细胞膜稳定性变差,通透性增加,补肾活血中药可以改善细胞膜的稳定性,提高GS的活性,从而达到保护RGCs细胞的目的。

关键词: 补肾活血中药, 缺氧, 视网膜神经节细胞, 视网膜Muller细胞

Abstract: Objective To explore the protective effect and mechanism of Bushenhuoxue(invigorating the kidney and promoting blood circulation) TCM serum on retinal ganglion cells (RGCs) purified cultured under the condition of hypoxia in vitro. Methods 30 neonatal rats 1d after birth were selected. Through the two step screening system, the RGCs of SD neonatal rats were purified cultured. 6 SD rats 7d after birth were selected and through improved enzymatic digestion technique, retinal Muller cells of neonatal SD rats were purified cultured. Then, according to the ratio of 1:25, RGCs were inoculated in the medium coated by retinal Muller cells to establish a common culture model. Glucose (50 mmol·L-1) and sodium dithionite (1.0 mmol·L-1) (high glucose hypoxia group), only glucose (50 mmol·L-1) (high glucose group) and sodium dithionite (1.0 mmol·L-1) (hypoxia group) were respectively added into the medium in vitro. Based on this, formulated Bushenhuoxue TCM serum of 20% volume fraction was respectively added for intervention. And the leakage of lactate dehydrogenase (LDH) and activity of glutamine synthetase (GS) in extracellular fluid of the model were respectively determined in 24 h, 48 h and 72 h. Results In 48 h, 72 h, the leakages of LDH in the normal control group, high glucose group and hypoxia group significantly increased, compared with that in 24 h (P <0.05). Comparing normal control group, high glucose group, hypoxia group and high glucose hypoxia group with their respectively corresponding TCM intervention in the same time, it could be found that the leakages of LDH were significantly higher than those TCM intervention groups (P <0.05). When it was 48 h and 72 h, the activity of GS in normal control group, high glucose group, hypoxia group, high glucose hypoxia group and their corresponding TCM intervention group increased significantly, compared with that in 24 h (P <0.05). With extension of time, the activity of GS in normal control group, high glucose group, hypoxia group, high glucose hypoxia group was significantly lower than that of the TCM intervention group. Conclusion High glucose or anoxic conditions can cause deviation of cell membrane stability of RGCs and retinal Muller cells and increase of permeability. Bushenhuoxue TCM can improve the stability of the cell membrane and increase GS activity, so as to protect the RGCs cells.

Key words: Bushenhuoxue TCM, hypoxia, retinal ganglion cells, retinal Muller cells

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