中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (6): 1031-1036.doi: 10.3969/j.issn.1673-8225.2010.06.017

• 干细胞培养与分化 • 上一篇    下一篇

甲泼尼龙琥珀酸钠对许旺细胞分泌功能的影响

曲  巍，费良健，蒋华军，傅重洋，张卫国，吕德成   

  1. 大连医科大学第一附属医院骨科，辽宁省大连市  116011
  • 出版日期:2010-02-05 发布日期:2010-02-05
  • 作者简介:曲 巍，男，1967年生，辽宁省大连市人，汉族，1999年日本广岛大学毕业，博士，教授，主要从事创伤骨科，显微外科的研究。 quwei@dmu-1.com
  • 基金资助:

    国家自然基金资助项目(30973060)，课题名称“hTERT转染雪旺细胞复合FK506壳聚糖鞘管构建人工神经的研究”。

Effects of methylprednisolone on the secreted function of Schwann cells

Qu Wei, Fei Liang-jian, Jiang Hua-jun, Fu Chong-yang, Zhang Wei-guo, Lü De-cheng   

  1. Department of Orthopedics, First Affiliated Hospital, Dalian Medical University, Dalian   116011, Liaoning Province, China
  • Online:2010-02-05 Published:2010-02-05
  • About author:Qu Wei, Doctor, Professor, Department of Orthopedics, First Affiliated Hospital, Dalian Medical University, Dalian 116011, Liaoning Province, China quwei@dmu-1.com
  • Supported by:

    the National Natural Science Foundation of China, No.30973060*

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摘要：

背景：许旺细胞分泌多种神经营养因子在神经再生中发挥关键作用，但其分泌能力受诸多因素影响，寻找可行方法促进许旺细胞分泌神经生长因子是神经缺损后再生的重要环节。

目的：观察不同浓度甲泼尼龙琥珀酸钠对许旺细胞分泌功能的影响。

方法：酶消化法分离培养SD乳鼠许旺细胞，倒置相差显微镜下观察细胞生长情况；传代后，一部分进行S-100蛋白免疫鉴定许旺细胞的纯度；另一部分用细胞计数板调整细胞浓度为1×109 L-1，移入到6孔平底培养板(接种15个孔)继续培养。培养板中培养4 d后4个孔分别加入不同浓度的甲泼尼龙琥珀酸钠(10-3，10-4，10-6，10-8 mol/L)，并设立1个孔为不加药物的空白对照组，分别作用于细胞24，48，72 h后行RT-PCR检测，观察许旺细胞24，48，72 h神经生长因子mRNA水平的变化。

结果与结论：原代细胞培养至第7天数量明显增加，细胞铺满培养瓶底部80%以上；传代细胞形态为梭形，有2个细长的突起，荧光染色阳性，成纤维细胞为圆形或扁圆形，荧光染色阴性。RT-PCR检测结果显示，10-8 mol/L的甲基强地松龙作用72 h分泌的神经生长因子与空白对照组及其他浓度、时间组相比均表达增加(P < 0.05)；10-3 mol/L甲基强地松龙在各时间段分泌的神经生长因子与空白对照组及其他浓度、时间组相比表达均减少(P < 0.05)。提示高浓度的甲泼尼龙琥珀酸钠抑制许旺细胞分泌神经生长因子，长时间、低浓度的甲泼尼龙琥珀酸钠则促进许旺细胞分泌神经生长因子。

关键词: 许旺细胞, 神经生长因子, 细胞培养, 神经缺损, 甲泼尼龙琥珀酸钠

Abstract:

BACKGROUND: Secretion of various neurotrophic factors by Schwann cells plays important roles in neural regeneration. However, the secretion capability is affected by many factors. To seek a feasible method for promoting nerve growth factor secretion by Schwann cells is a key of regeneraion following neurologic defect.

OBJECTIVE: To explore the effects of methylprednisolone(solu-medrol) on the secreted function of Schwann cells of cultured rats.

 

METHODS: Schwann cells were isolated and cultured by enzyme digestion method. Cell growth was observed under an inverted phase contrast microscope. Following passage, purity of some Schwann cells was identified using S-100 protein immunity. Other Schwann cells were regulated using cell counting plate into 1×109/L, and incubated in a 6-well culture plate (15 wells) for further incubation. Following 4 days of culture, different concentrations of solu-medrol (10-3, 10-4, 10-6, 10-8 mol/L) were administrated to the cell, while blank control group (1 well) was given no drug. 24, 48 and 72 hours after administration, reverse trancription-polymerase chain reaction (RT-PCR) was used in the detection of the levels of nerve growth factor mRNA.

RESULTS AND CONCLUSION: Number of primarily cultured cells was significantly increased at day 7, and 80% cells were confluent. Subcultured cells were spindle-shaped, with 2 thin long processes, showing positive fluorescence staining. Fibroblasts were round or flat, showing negative reaction of fluorescence staining. Reserve transcription-polymerase chain reaction demonstrated that nerve growth factor number at 72 hours affected by 10-8 mol/L radiosone was increased compared with the blank control group and other concentrations and other time points (P < 0.05). Number of nerve growth factor was reduced following treatment of 10-3 mol/L radiosone compared with the blank control group and other concentrations (P < 0.05). These results suggested that high concentration of solu-medrol prohibits secreted function of Schwann’s cells, but long time and low dosage solu-medrol promotes secreted function of Schwann’s cells.

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