Isolation and identification brain microvessel pericytes in rats
10.3760/cma.j.issn.1673-4165.2011.07.008
- VernacularTitle:大鼠脑微血管周细胞的分离和鉴定
- Author:
Weiwei QIN
;
Wenbao LU
;
Shuying LIU
;
Hongwei LI
;
Ruijuan XIU
- Publication Type:Journal Article
- Keywords:
Pericytes;
Blood-brain barrier;
Cell separation;
Cell culture techniques;
Rats
- From:
International Journal of Cerebrovascular Diseases
2011;19(7):531-534
- CountryChina
- Language:Chinese
-
Abstract:
Objective To explore the method of primary isolation, cultivation and identification of rat brain microvessel pericytes. Methods The brain tissue of 10 3 week-old Wistar rats was separated sterilely. The brain microvessel fragments were separated using two-step enzyme digestion and one-step gradient centrifugation and were seeded in 35-mm dishes for primary culture. The cell morphology was observed by phase contrast microscopy; the immunofluorescence assay was used to identify the associated antigns, such as the α-smooth muscle actin (α-SMA), neuron-glial antigen 2 (NG2), von Willebrand factor (vWF), and glial fibrillary acidic protein (GFAP). Methyl thiazolyl tetrazolium was used to determine the cell growth curve. Results Pericytes climbed out from the adherent brain microvascular fragments around,showing polygonal, and the cell fusion was 95％ after 12-14 days. Immunofluorescence staining revealed that the molecular markers of the pericytes α-SMA and NG2 related antigens showed double positive, while the vWF and GFAP related antigens showed double negative and the cultured cells were confirmed as brain microvascular pericytes. The growth rate of primary cells was slower. The passage cells entered into logarithmic growth phase after 36 to 60 hours and entered into plateau phase after 72 to 108 hours. Conclusions This method may successfully isolate rat brain microvascular pericytes with higher purity.
