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目的:采用细胞共培养模型研究缺氧诱导视网膜新生血管生成过程中周细胞对微血管内皮细胞增生的作用及其相关血管内皮细胞生长因子受体2(KDR/Flk-1)调节机制。方法:免疫磁珠法原代分离培养大鼠视网膜微血管内皮细胞和周细胞,分别采用抗VIII因子、CD31抗体以及抗血小板源性生长因子受体β、结蛋白抗体免疫细胞化学法鉴定内皮细胞和周细胞。通过Millicell小室建立周细胞和内皮细胞共培养模型,使用MTT法和流式细胞仪检测内皮细胞增殖数量及周期,观察缺氧和常氧下周细胞对血管内皮细胞增生的影响,并采用RT-PCR法检测内皮细胞KDR/Flk-1的mRNA水平变化,探讨相关调节机制。结果:经免疫细胞化学法鉴定,免疫磁珠法可获得高纯度的视网膜微血管内皮细胞和周细胞。MTT法显示,缺氧3～9d内皮细胞增生明显,6d增生达高峰(24.9%,P<0.01);共培养时内皮细胞的增生可被周细胞抑制。流式细胞仪检测发现,缺氧6dS期内皮细胞数量明显增加(43.9%,P<0.01);常氧(3.6%,P<0.05)或缺氧(15.1%,P<0.01)共培养时,周细胞均可抑制内皮细胞增生。缺氧下内皮细胞KDR/Flk-1的mRNA水平是常氧下的1.3倍;常氧(45.1%,P<0.05)和缺氧(27.7%,P<0.05)共培养下,周细胞均可下调内皮细胞KDR/Flk-1的mRNA表达。结论:缺氧和常氧共培养时周细胞均可抑制微血管内皮细胞增生,此抑制作用部分是通过下调内皮细胞KDR/Flk-1mRNA表达实现。
OBJECTIVE: To study the effect of pericytes on the proliferation of microvascular endothelial cells and the regulation of KDR / Flk-1 in hypoxia-induced retinal neovascularization using cell co-culture model. Methods: Primary cultured rat retinal microvascular endothelial cells and pericytes were isolated and cultured by immunomagnetic beads method. Endothelial cells were identified by anti-factor VIII, anti-CD31 antibody and anti-platelet-derived growth factor receptor beta, desmin antibody immunocytochemistry Pericytes. The model of pericytes and endothelial cells co-culture was established by Millicell chamber. The number and the period of endothelial cell proliferation were detected by MTT assay and flow cytometry. The effects of hypoxia and normoxic week on the proliferation of vascular endothelial cells were observed. RT- PCR assay of endothelial cell KDR / Flk-1 mRNA levels, to explore the relevant regulatory mechanism. Results: Immunocytochemistry identification, immunomagnetic beads method can obtain high purity of retinal microvascular endothelial cells and pericytes. MTT assay showed that endothelial cells proliferated significantly at 3-9 days of hypoxia and peaked at 6 days (24.9%, P <0.01). The proliferation of endothelial cells was inhibited by pericytes when co-cultured. Flow cytometry showed that the number of endothelial cells was significantly increased in hypoxia 6dS (43.9%, P <0.01), when cultured in normoxia (3.6%, P <0.05) or hypoxia (15.1%, P <0.01) Pericytes can inhibit endothelial cell proliferation. Under hypoxia, the level of KDR / Flk-1 mRNA in endothelial cells was 1.3 times that of normoxia. Under normoxia (45.1%, P <0.05) and hypoxia (27.7%, P <0.05) Downregulates the mRNA expression of KDR / Flk-1 in endothelial cells. CONCLUSION: Pericytes can inhibit the proliferation of microvascular endothelial cells during both hypoxia and normoxia co-culture. The inhibition is partially mediated by the down-regulation of KDR / Flk-1 mRNA in endothelial cells.