摘要
背景:从以往的文献来看,阴茎海绵体平滑肌细胞的原代培养存在着制作时间长,纯度不高以及易被成纤维细胞污染等诸多缺点。目的:探索兔阴茎海绵体平滑肌细胞的纯化培养技术及纯度鉴定方法。设计、时间及地点:对照实验,于2007-10/2008-03在上海交通大学附属第六人民医院泌尿外科,上海市组织工程研究与开发中心完成。材料:成熟的雄性新西兰大白兔5只,体质量2.5～2.8kg。方法:取雄性新西兰兔新鲜的阴茎组织,利用高浓度的胶原酶消化法结合差速贴壁技术对海绵体平滑肌细胞进行纯化培养。以同期兔成纤维细胞作为对照参考。主要观察指标:以四甲基偶氮唑盐分析细胞生长特性;以平滑肌肌动蛋白、肌球蛋白、结蛋白免疫荧光鉴定海绵体平滑肌细胞;以流式细胞仪检测P2及P5代细胞α-肌动蛋白、肌球蛋白、结蛋白阳性率变化情况。结果:培养细胞四甲基偶氮唑盐显示细胞指数增长期为6d左右,随后进入平台期。α-肌动蛋白、肌球蛋白、结蛋白均呈阳性反应,同期成纤维细胞仅α-肌动蛋白显示阳性结果。第2代细胞表达α-肌动蛋白、肌球蛋白、结蛋白阳性率分别65.3%,42.2%,62.3%。经过反复多次的纯化技术至第5代时,上述指标阳性表达率分别上升为92.8%,72.7%,78.1%。结论:通过高浓度的酶消化法及差速贴壁技术可获得高纯度的阴茎海绵体平滑肌细胞,肌球蛋白、结蛋白相对于α-肌动蛋白可以成为鉴定海绵体平滑肌细胞的特异性指标。
BACKGROUND： Research on corpus cavernosum smooth muscle cells （CSM） culture has the disadvantage of long-time making, low purity and easy contaminated by fibroblasts.
OBJECTIVE： To investigate an effective method on culturing and identification corpus cavernosum smooth muscle cells in rabbits. DESIGN, TIME AND SETTING： The control experiment was performed at the Department of Urinary Surgery, Sixth Hospital Affiliated to Shanghai Jiao Tong University and Shanghai Center for Research and Development of Tissue Engineering between October 2007 and March 2008.
MATERIALS： Five adult, New Zealand white rabbits, with weight of 2.5 2.8 kg. METHODS： The CSM from fresh rabbit penile tissue, were performed culture and purification with collagenase digestion method plus differential velocity adherent technique. The culture of rabbit fibroblast were served as the control group.
MAIN OUTCOME MEASURES： The MTT curve was used t9 analyze the cell growth characteristics, in,addition, the cells were identified by immunoflurescence, which including a -actin, myosin and desmin. Flow cytometry was used to compare the purity of CSM cells between passage 2 and passage 5.
RESULTS： MTT showed the exponential phase of CSM cells was about 6 days, sequentially reached a plat level. The positive expression of α-actin, myosin, desmin could been seen in the CSM cells, however, fibroblast cells only showed positive reactivity in α-actin. The positive expression of α-actin, myosin, and desmin was 65.3%, 42.2%, 62.3%, at the second passage, while, the purification mentioned above increased to 92.8%, 72.7%, 78.1% at fifth passage respectively.
CONCLUSION： High pure CSM could be obtained by high concentrative enzyme digestion method plus differential velocity adherent technique, additionally, myosin and desmin can be served as specific index for identifying the CSM cells.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第50期9848-9852,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
