豚鼠脑基底动脉平滑肌细胞的原代培养及鉴定
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摘要
目的本研究联合使用直接贴壁法和酶消化法培养细胞,从豚鼠脑基底动脉中提取平滑肌细胞,为相关实验研究提供材料。方法从豚鼠颅内分离出脑基底动脉,用0.125%胰蛋白酶进行消化并将组织块贴壁,细胞用含20%胎牛血清的DMEM/F-12培养基进行培养。采用自然纯化法,并根据不同细胞贴壁能力的差异性对细胞进行纯化。通过形态学观察、免疫荧光、免疫组织化学法及Western blot检测对细胞进行鉴定。结果 85%组织块存活,第4代平滑肌细胞纯度达95%。镜下可见细胞呈典型的"峰-谷"样生长,免疫荧光、免疫组织化学法及Western blot检测显示,平滑肌细胞特异性的肌动蛋白呈阳性表达。结论通过本实验可以得到大量高纯度的豚鼠脑基底动脉平滑肌细胞,有望为颅内动脉粥样硬化等病理生理变化的体外研究及脑血管平滑肌药物评价提供一个理想的实验材料。
Objective To obtain cerebral basilar artery smooth muscle cells from guinea pigs in combination with two common cultural methods, and provide materials for related experimental studies. Methods Cerebral basilar arteries were separated from posterior cranial fossa of guinea pigs first and enzymatically digested with 0.125% trypsin solution, allowed to adhere subsequently, the cells were cultured in DMEM/F-12 with 20% fetal calf serum, then purified based on the comprehensive application of natural purification and differential adherence ability. Finally, they were identified by morphological observation, immunofluorescence and immunocytochemistry analyses, as well as by Western blot. Results 85% of the explants survived. The smooth muscle cells at passage 4 had a purity of over 95%. The cells were observed as a typical "hill-and-valley" growth pattern under microscope. Moreover, immunofluorescent and immunohistochemical staining, and Western blot showed positive expression of α-SM-actin, a smooth muscle cell-specific marker. Conclusions By this protocol, a large number of highly purified cerebral basilar artery smooth muscle cells can be obtained from guinea pigs, which can provide an ideal experimental material for study of pathogenesis of the diseases related to intracranial atherosclerosis in vitro, as well as evaluation of drug effect on cerebral vascular smooth muscle.
Objective To obtain cerebral basilar artery smooth muscle cells from guinea pigs in combination with two common cultural methods, and provide materials for related experimental studies. Methods Cerebral basilar arteries were separated from posterior cranial fossa of guinea pigs first and enzymatically digested with 0.125% trypsin solution, allowed to adhere subsequently, the cells were cultured in DMEM/F-12 with 20% fetal calf serum, then purified based on the comprehensive application of natural purification and differential adherence ability. Finally, they were identified by morphological observation, immunofluorescence and immunocytochemistry analyses, as well as by Western blot. Results 85% of the explants survived. The smooth muscle cells at passage 4 had a purity of over 95%. The cells were observed as a typical "hill-and-valley" growth pattern under microscope. Moreover, immunofluorescent and immunohistochemical staining, and Western blot showed positive expression of α-SM-actin, a smooth muscle cell-specific marker. Conclusions By this protocol, a large number of highly purified cerebral basilar artery smooth muscle cells can be obtained from guinea pigs, which can provide an ideal experimental material for study of pathogenesis of the diseases related to intracranial atherosclerosis in vitro, as well as evaluation of drug effect on cerebral vascular smooth muscle.
引文
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[12]KUCHER A N,NAZARENKO M S,MARKOV A V,et al.Variability of methylation profiles of Cp G Sites in micror NAgenes in leukocytes and vascular tissues of patients with atherosclerosis[J].Biochemistry,2017,82(6):698-706.
[2]MALCOLM K,CHEN J C,JUEN-HAUR H.Risk of peripheral artery occlusive disease in patients with vertigo,tinnitus,or sudden deafness:a secondary case-control analysis of a nationwide,population-based health claims database[J].PLo S One,2016,11(9):DOI:org/10.1371/journal.pone.e0162629.
[3]KUMAR A,KAUR H,DEVI P,et al.Role of coenzyme Q10(Co Q10)in cardiac disease,hypertension and Meniere-like syndrome[J].Pharmacology Therapeutics,2009,124(3):259-268.
[4]王竟悟,于杨,王大新,等.血管平滑肌细胞与动脉粥样硬化关系的研究新进展[J].实用医学杂志,2017,33(21):3661-3663.
[5]杨佳音,简蓉蓉,许雅丽,等.CD73对动脉粥样硬化中血管平滑肌细胞的作用[J].复旦学报(医学版),2015,42(3):300-306.
[6]LAO K H,ZENG L,XU Q.Endothelial and smooth muscle cell transformation in atherosclerosis[J].Current Opinion in Lipidology,2015,26(5):449-456.
[7]朱宁,赵旭勇,向贻佳,等.大鼠肺细小动脉平滑肌细胞培养新方法的建立及血小板衍生因子对其增殖迁移的影响[J].中国应用生理学杂志,2016,32(4):343-346.
[8]梁志浩,吴康,金颖康,等.一种大鼠肺静脉平滑肌细胞快速培养方法的建立及评价[J].国际呼吸杂志,2017,37(1):45-50.
[9]LIU J,ZHANG Y,LI Q,et al.An improved method for guinea pig airway smooth muscle cell culture and the effect of SPFF on intracellular calcium[J].Molecular Medicine Reports,2014,10(3):1309-1314.
[10]SA B,Y S,JP S,et al.Different sensitivity of human endothelial cells,smooth muscle cells and fibroblasts to topography in the nano-micro range[J].Acta Biomaterialia,2009,5(7):2460-2466.
[11]ZHANG J,CHU L F,HOU Z,et al.Functional characterization of human pluripotent stem cell-derived arterial endothelial cells[J].Proc Natl Acad Sci USA,2017,114(30):6072-6078.
[12]KUCHER A N,NAZARENKO M S,MARKOV A V,et al.Variability of methylation profiles of Cp G Sites in micror NAgenes in leukocytes and vascular tissues of patients with atherosclerosis[J].Biochemistry,2017,82(6):698-706.