法医学杂志

法医学杂志 ›› 2006, Vol. 0 ›› Issue (1): 24-27+9.

• 论文 • 上一篇    下一篇

兔阴茎海绵体平滑肌细胞培养及其RyRs的表达

刘洪国;朱广友;赵子琴;刘志萍;   

  1. 复旦大学上海医学院法医学系,淄博市中级人民法院 山东淄博255037,司法部司法鉴定科学技术研究所,复旦大学上海医学院法医学系,复旦大学上海医学院法医学系,,上海200032司法部司法鉴定科学技术研究所,上海200063,上海200063,上海200032,上海200032
  • 发布日期:2006-02-25 出版日期:2006-02-28

Primary Study in Rabbit Penile Corpus Cavernosum Smooth Muscle Cell Culture and RyRs Expression

LIU HONG-GUO1,2,3, ZHU GUANG-YOU2, ZHAO ZI-QIN1, LIU ZHI-PING1 (1.DEPARTMENT OF FORENSIC MEDICINE, SHANGHAI MEDICAL COLLEGE, FUDAN UNIVERSITY, SHANGHAI 200032,CHINA; 2. INSTITUTE OF FORENSIC SCIENCES, MINISTRY OF JUSTICE, P.R.CHINA, SHANGHAI 200063,CHINA;   

  • Online:2006-02-25 Published:2006-02-28

被引次数

31

摘要： 目的探讨兰尼碱受体(RyRs)在兔阴茎海绵体平滑肌细胞(CCSM)的正常表达。方法取雄性性成熟新西兰兔CCSM细胞,用组织块法进行细胞培养,差速贴壁法进行细胞纯化,利用倒置显微镜观察、免疫荧光染色法鉴定细胞纯度;取三代以内培养细胞,通过Genebank查出RyRscDNA序列,针对RyRs3种不同亚型的非同源部分用PrimerPremier5.0软件设计3对引物,以逆转录PCR方法分别检测RyRs3种亚型mRNA的表达,凝胶成像系统分析结果。结果培养7d开始有细胞从组织块长出,15～20d后长满瓶底,呈典型的“峰-谷”现象,经倒置显微镜观察见培养的细胞呈现典型的梭形,免疫荧光染色可见细胞内染为绿色的肌丝,证实培养的细胞为CCSM细胞,经差速贴壁法纯化后,细胞纯度可接近100%。经RT-PCR检测,CCSM只表达RyRs1,未见RyRs其余亚型的表达,提示CCSM表达RyRs1蛋白。结论CCSM表达RyRs中的1亚型,提示其可能参与CCSM细胞的钙调节,进而能调节阴茎勃起功能。

关键词: 兰尼碱受体, 阴茎海绵体平滑肌, 细胞培养, 游离钙

Abstract: Objective To investigate expression of ryanodine receptors (RyRs) in rabbit penile corpus cavernosum smooth muscle(CCSM) cells. Methods New Zealand White Rabbit CCSM cells were cultured by primary tissue culture. Using CCSM cells and fibroblast have different adherence velocity, CCSM cell can be purified. Identification of CCSM cell was by inverted microscope and immunofluorescence technique. The cDNA sequence of RyRs was found out by searching genebank. Three pair of primers were designed by Primer Premier 5.0. The RyRs subunits mRNA was detected by reverse transcription PCR in cultured CCSM cells. Results After 7 d, we found growth of cultured cells. While 15～20 d, cells filled the bottom of culture flask. They were identified by inverted microscope and immunofluorescence technique. After purification, purity of CCSM cells was near to 100%. It suggested only RyRs1 subunit was expressive in CCSM by RT-PCR. Conclusion RyRs1 subunit is expressed in CCSM cells. It suggests that RyRs contribute to the regulation of Ca2+ in CCSM cells.

Key words: ryanodine receptors, penile corpus cavernosum smooth muscle, cell culture, [Ca2+]i