免疫磁珠法分离纯化乳鼠肾血管周细胞

Isolation and purification of sucking mice pericytes by immunomagnetic bead techniques

目的通过建立肾原代细胞培养,探讨免疫磁珠分离纯化乳鼠肾血管周细胞的方法。方法选取健康BALB/c乳鼠肾脏,剪碎、消化和过滤制成细胞悬液,原代培养并传代(传3代)。免疫磁珠法分离出神经胶质细胞2型硫酸软骨素糖蛋白(NG-2);倒置相差显微镜观察细胞形态特点,免疫细胞化学检测NG-2、平滑肌肌动蛋白(SMA)和CD31表达,Brd U法检测细胞增殖活性,鉴定是否符合周细胞特征及纯化后的细胞是否具有增殖活性。结果纯化后获取的细胞宽大扁平,形态呈梭形、三角形或多边形并有突起。细胞核椭圆居中,多为单核,偶为双核,细胞质丰富,无接触性抑制生长,符合周细胞形态及生长特征。NG-2和SMA免疫细胞化学染色阳性表达,CD31免疫细胞化学染色阴性表达,符合周细胞特点。分离纯化后的细胞48、72 h细胞增殖率分别为(0.37±0.11)%、(0.42±0.10)%。结论免疫磁珠法可使周细胞从细胞成分复杂的肾组织中分离出来,并具有生物活性,可继续体外培养,为后续研究提供参考依据。

Objective To establish the primary culture by renal cells and to investigate the isolation and purification method of sucking mice renal vessel pericytes by the imunomagnetic bead technique. Methods The kidney of healthy BALB/c sucking mice was selected,cut into pieces,digested and filtered for preparing the cellular suspension. The primary culture and passage（3 generations）were performed;the mimunomagnetic bead technique isolated neurogliocytes（NG-2）;the inverted phase contrast microscope was adopted to observe the cellular morphological characteristics,the immunohistochemistry was used to detect NG-2,SMA and CD31 expression by joint judgement,the cellular proliferation activity was detected by using the Brd U method.Whether meeting pericytes characteristics and whether purified cells having the proliferation activity were identified. Results The cells obtained by purification were wide and flat,the morphologies were fusiform, triangle or polygon with prominence. The cellular nucleus was ellipse and in the middle,the majority were mononucleus,occasionally bi-neucleus,with plentiful cytoplasma,without contacting inhibition growth,which conformed to the pericytes morphology and growth characteristics. NG-2 and SMA were positively expressed by immunocytochemical staining,CD31 was negatively expressed,which conformed to the pericytes characteristics. The 48,72 h proliferation activities of isolated and purified cells were（0.37±0.11）% and（0.42±0.10）% respectively. Conclusion The imunomagnetic bead method can make the pericytes to be isolated from the kidney with complex cellular compositions,which have biological activity,can continuously culture in vitro and provide the reference basis for subsequent study.