中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (46): 8648-8652.doi: 10.3969/j.issn.1673-8225.2011.46.023

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

近似体腔温度不宜体外培养大鼠A型精原细胞

邓呈亮1，王达利1，李三华2，朱晶晶1，唐修俊1，龚德彰1   

  1. 1遵义医学院附属医院烧伤整形外科，贵州省遵义市 563003
    2遵义医学院中心实验室  贵州省遵义市  563003
  • 收稿日期:2011-05-16 修回日期:2011-06-15 出版日期:2011-11-12 发布日期:2011-11-12
  • 通讯作者: 王达利，主任医师，教授，硕士生导师，遵义医学院附属医院烧伤整形外科，贵州省遵义市 563003 daliwangzy@sina.com
  • 作者简介:邓呈亮★，男，1984年生，湖南省娄底市人，汉族，遵义医学院在读硕士，主要从事创伤修复方面研究。 dengchengliang00@163.com
  • 基金资助:

    贵州省优秀青年科技人才培养对象基金(黔科合人字(2009)26号)；遵义市重大科技项目培育与科技人才培养计划基金(遵科培字(2009)05号)

Effects of body cavity-like temperature unsuitable for in vitro culture on rat type A spermatogonial cells

Deng Cheng-liang1, Wang Da-li1, Li San-hua2, Zhu Jing-jing1, Tang Xiu-jun1, Gong De-zhang1   

  1. 1Department of Plastic Surgery, Affiliated Hospital of Zunyi Medical College, Zunyi  563003, Guizhou Province, China
    2Central Laboratory of Zunyi Medical College, Zunyi 563003, Guizhou Province, China
  • Received:2011-05-16 Revised:2011-06-15 Online:2011-11-12 Published:2011-11-12
  • Contact: Wang Da-li, Chief physician, Professor, Master’s supervisor, Department of Plastic Surgery, Affiliated Hospital of Zunyi Medical College, Zunyi 563003, Guizhou Province, China daliwangzy@sina.com
  • About author:Deng Cheng-liang★, Studying for master’s degree, Department of Plastic Surgery, Affiliated Hospital of Zunyi Medical College, Zunyi 563003, Guizhou Province, China dengchengliang00@163.com
  • Supported by:

    Guizhou Province Foundation for Fostering Excellent Young Science and Technology Scholars, No.(2009)26*; Chongqing Major Science and Technology Program Construction and Science and Technology Scholars Fostering Plan, No. (2009)05*

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364

被引次数

2

摘要：

背景：温度会影响体外培养精原细胞的增殖与凋亡。
目的：观察不同体外培养温度对A型精原细胞细胞周期及增殖相关基因c-kit、CyclinD3的影响。
方法：通过两步酶消化法分离大鼠生精细胞，分别置于32，37 ℃条件下与支持细胞进行体外共培养，观察其形态及细胞数变化等生长特性；培养第8天采用非连续密度梯度离心、差异性贴壁分离富集A型精原细胞。
结果与结论：体外培养第1天，32，37 ℃组均可见悬浮的呈圆形或卵圆形的生精细胞和贴壁的呈长梭形或不规则形的支持细胞；从第4天开始，37 ℃组生精细胞数、生长状态逐渐降低，第8天时，37 ℃生精细胞数明显低于32 ℃组 (P < 0.05)；体外培养第8天，32 ℃组处于S期A型精原细胞所占比例明显高于37 ℃组(P < 0.05)，32 ℃组A型精原细胞c-kit、CyclinD3 mRNA及蛋白的相对表达量均高于37 ℃组(P < 0.05)。说明在37℃这种近似体腔的温度下体外培养生精细胞会抑制精原细胞的增殖。

关键词: A型精原细胞, 体外培养, 细胞周期, c-kit, 大鼠

Abstract:

BACKGROUND: Temperature would influence the proliferation and apoptosis of spermatogonial cells cultured in vitro.
OBJECTIVE: To investigate the effects of temperature on cell cycle and proliferation-related gene c-kit and cyclinD3 of type A spermatogonial cells cultured in vitro.
METHODS: The isolated spermatogenic cells by two-step enzymatic digestion were co-cultured with sertoli cells in an incubator at 32 ℃ and 37 ℃ to observe the morphological changes and growth characteristics every day. On the 8th day of in vitro culture, type A spermatogenic cells were separated by gradient centrifugation and differential adhesion method.
RESULTS AND CONCLUSION: On the 1st day of in vitro culture, suspending spermatogenic cells which were round or oval and the adherent sertoli cells that were fusiform or irregular shape were observed in 32 ℃ group and 37 ℃ group. Starting from the 4th day, the number and growth state of spermatogenic cells were gradually decreased in the 37 ℃ group, at the 8th day, the number of spermatogenic cells in the 37 ℃ group was significantly lower than that in the 32 ℃ group (P < 0.05). Flow cytometry results showed that at the 8th day of in vitro culture, the proportion of type A spermatogonial cells at S phase in the 32 ℃ group was significantly higher than that in the 37 ℃ group (P < 0.05); the relative mRNA and protein expression levels of c-kit, cyclinD3 in type A spermatogonial cells were significantly higher in the 32 ℃ group than in the 37 ℃ group (P < 0.05). Results indicate that spermatogenic cells cultured in vitro would inhibit the proliferation of type A spermatogonial cells at body cavity-like temperature (37 ℃).

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