改良前处理的原代组织块贴壁法培养大鼠肺动脉平滑肌细胞

Modified pretreatment tissue explants adherent method for primary rat pulmonary artery smooth muscle cells culture

目的建立改良前处理的原代组织块贴壁法培养大鼠肺动脉平滑肌细胞(PASMCs)。方法将SD大鼠经颈椎脱臼处死断头后放入盛有75%酒精的烧杯中浸泡消毒。在无菌条件下剖开胸腹腔,取出大鼠的心肺组织,然后用非体式显微镜分离方法分离出肺动脉血管段,去除肺动脉血管外层纤维结缔组织、破坏血管内膜层内皮细胞后,用组织块贴壁法培养PASMCs,并传代、冻存和复苏,倒置相差显微镜观察细胞形态,普通免疫细胞化学法和免疫荧光法进行细胞鉴定。结果大鼠PASMCs呈典型的"峰-谷"样生长状态,细胞形态呈梭形。α-SM actin蛋白在培养的大鼠PASMCs为阳性表达,传至第4代的PASMCs成活率可达98%,生长形态未见异常改变。PASMCs细胞从-80℃冰箱中取出复苏后,生长状态良好,形态、结构稳定。结论用改良前处理的组织块贴壁法能够成功培养PASMCs,较传统组织块贴壁法更简单,培养效率高。

Objective To set up a modified pretreatment tissue explants adherent method for primary rat pulmonary artery smooth muscle cells（ PASMCs） culture. Methods SD rat was sacrificed by cervical dislocation,and then was put into the beaker containing 75% alcohol for immersion disinfection. The chest was opened and the heart and lung were taken out under aseptic condition. The pulmonary artery segments were acquired with a non- stereo microscope separation method. The outer membrane of pulmonary artery trunk was separated and endothelial cells were damaged. PASMCs were cultured by the modified tissue explants adherent method. The PASMCs were then passaged,cryopreserved and resuscitated. The cellular morphology and typical growth conditions were observed with the inverted phase contrast microscope. Cell specificity was identified by methods of immunocytochemistry and immunofluorescence. Results PASMCs showed a typical ＂ peak- valley＂ growth state and presented spindle shape. The protein expression of α- SM actin in the isolated cells was positive by the method of immunocytochemistry and immunofluorescence. The survival rate of the 4th generation of PASMCs was up to 98% by the cell counting detection method. The growth of passaged PASMCs was normal. The good growth state,stable morphology and structure of PASMCs were presented when cells were recovered from- 80 ℃ freezer. Conclusion PASMC could be successfully cultured using the modified pretreatment tissue explants adherent method,and this method is easier and more efficient than the traditional method.