刮取法改良人原代大隐静脉内皮细胞的提取方法

doi:10.6040/j.issn.1671-7554.0.2022.0805

摘要/Abstract

摘要： 目的改良人原代大隐静脉内皮细胞的提取方法,为冠状动脉旁路移植术后静脉移植物衰败的研究提供细胞模型。方法传统酶消法(酶消组)和刮取法(刮取组,刮取酶消法后的大隐静脉内皮并进一步酶消)从人大隐静脉中提取内皮细胞。获得的内皮细胞在含10%胎牛血清+1%内皮细胞生长补充剂+100 U/mL青霉素+100 mg/mL链霉素的内皮细胞培养基中传代培养。倒置显微镜观察内皮细胞形态和贴壁情况;血管性血友病因子(vWF)的免疫荧光实验鉴定内皮细胞特异性抗原;CD31流式细胞计数检测内皮细胞纯度;绘制不同观测日的内皮细胞生长曲线测定内皮细胞活性。结果显微镜下观察到两组大隐静脉内皮细胞贴壁汇合并且呈典型的鹅卵石样排列;免疫荧光实验证实培养的细胞vWF阳性表达;流式细胞计数结果显示,酶消组99.9%、刮取组89.2%的细胞阳性表达CD31;不同观测日的生长曲线结果显示,两组内皮细胞活性的主效应不显著(P=0.057)。两组内皮细胞均能够传至第7~8代。结论在传统酶消法的基础上增加刮取再消化的步骤,能够提取出数量更多且满足实验需要的原代人大隐静脉内皮细胞。

关键词: 人大隐静脉, 内皮细胞, 提取方法, 血管性血友病因子, 静脉移植物衰败

Abstract: Objective To improve the extraction method of primary endothelial cells from human saphenous vein and to provide a cell model for the study of vein graft failure after coronary artery bypass grafting. Methods Endothelial cells were extracted from abandoned human saphenous vein by enzymatic digestion and scraping(scraping the vascular intima after enzymatic digestion for further enzymatic digestion). The extracted endothelial cells were cultured in the endothelial cell culture medium containing 10% fetal bovine serum +1% endothelial cell growth supplement +100 U/mL penicillin +100 mg/mL streptomycin. The morphology and adhesion of endothelial cells were observed under an inverted microscope. The specific antigen was identified with immune fluorescence assay of von Will brand Factor(vWF). The purity was detected with flow cytometry of CD31. The activity of endothelial cells was determined with the cell growth curve plotted on different observation days. Results Under the inverted microscope, endothelial cells converged on the wall and arrayed in a typical cobblestone shape. Immune fluorescence assay confirmed the positive expression of vWF. The results of flow cytometry showed that 99.9% and 89.2% of cultured cells in the enzymatic group and scraping group were positive for CD31. Data of the cell growth curve demonstrated that there was no significant difference in endothelial cell activity between the two groups(P=0.057). Endothelial cells from both groups were able to transmit to the 7th-8th generation. Conclusion Based on the traditional enzymatic digestion method, adding the scraping and re-digestion step can extract more primary human saphenous vein endothelial cells.

Key words: Human saphenous vein, Endothelial cell, Extraction method, von Willebrand factor, Vein graft failure

中图分类号:

R574

于瑞明,邴卫东,刘春晓,孟祥斌,毕研文. 刮取法改良人原代大隐静脉内皮细胞的提取方法[J]. 山东大学学报 (医学版), 2023, 61(2): 43-48.

YU Ruiming, BING Weidong, LIU Chunxiao, MENG Xiangbin, BI Yanwen. Scraping method improves the extraction of primary endothelial cells from human saphenous vein[J]. Journal of Shandong University (Health Sciences), 2023, 61(2): 43-48.

参考文献

[1] Head SJ, Milojevic M, Daemen J, et al. Mortality after coronary artery bypass grafting versus percutaneous coronary intervention with stenting for coronary artery disease: a pooled analysis of individual patient data[J].Lacent, 2018, 391(10124): 939-948.
[2] Caliskan E, Souza DR, Böning A, et al. Saphenous vein grafts in contemporary coronary artery bypass graft surgery[J]. Nat Rev Cardiol, 2020, 17(3): 155-169.
[3] Gaudino M, Benedetto U, Fremes S, et al. Association of radial artery graft vs saphenous vein graft with long-term cardiovascular outcomes among patients undergoing coronary artery bypass grafting: a systematic review and meta-analysis[J]. JAMA, 2020, 324(2): 179-187.
[4] Chiu JJ, Chien S. Effects of disturbedflow on vascular endothelium: pathophysiological basis and clinical perspectives[J]. Physiol Rev, 2011, 91(1): 327-387.
[5] Dobrin PB, Littooy FN, Endean ED. Mechanical factors predisposing to intimal hyperplasia and medial thickening in autogenous vein grafts[J]. Surgery, 1989, 105(3): 393-400.
[6] Khaleel MS, Dorheim TA, Duryee MJ, et al. High-pressure distentionof the saphenous vein during preparation results in increased markersof inflammation: a potential mechanism for graft failure[J]. Ann Thorac Surg, 2012, 93(2): 552-558.
[7] Ward AO, Caputo M, Angelini GD, et al. Activation and inflammation of the venous endothelium in vein graft disease[J]. Atherosclerosis, 2017, 265: 266-274. doi: 10.1016/j.atherosclerosis.2017.08.023.
[8] Guida G, Ward AO, Bruno VD, et al. Saphenous vein graft disease, pathophysiology, prevention, and treatment. A review of the literature[J]. J Card Surg, 2020, 35(6): 1314-1321.
[9] Mosquera DA, Goldman M. Endothelial cell seeding[J]. Br J Surg, 1991, 78: 656-660. doi: 10.1002/bjs.1800780608.
[10] Kent KC, Shindo S, Ikemoto T, et al. Species variation and the success of endothelial cell seeding[J]. J Vasc Surg, 1989, 9: 271-276. doi: 10.1016/0741-5214(89)90046-3.
[11] Belair DG, Khalil AS, Miller MJ, et al. Serum-dependence of affinity-mediated VEGF release from biomimetic microspheres[J]. Biomacromolecules, 2014, 15(6): 2038-2048.
[12] James SL, Abate D, Abate KH, et al. Global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990—2017: a systematic analysis for the Global Burden of Disease Study 2017[J]. Lancet, 2018, 392(10159): 1789-1858.
[13] Mohr FW, Morice MC, Kappetein AP, et al. Coronary artery bypass graft surgery versus percutaneous coronary intervention in patients with three-vessel disease and left main coronary disease: 5-year follow-up of the randomised, clinical SYNTAX trial[J]. Lancet, 2013, 381(9867): 629-638.
[14] Jaffe EA, Nachman RL, Becker CG, et al. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria[J]. J Clin Invest, 1973, 52(11): 2745-2756.
[15] 白雪晶, 冯磊, 徐文波, 等. 人脐静脉内皮细胞培养技术的研究进展[J].吉林医学, 2018, 39(5): 960-962.
[16] 刘洋洋, 冉林武, 范学文, 等. 人原代脐静脉内皮细胞体外模型的建立及鉴定[J].宁夏医科大学学报, 2016, 38(2): 223-225.
[17] 肖紫春, 谭金海, 周益. 兔主动脉血管内皮细胞原代改良提取法[J].中国组织工程研究, 2016, 20(7):1019-1024. XIAO Zichun, TAN Jinhai, ZHOU Yi. Improved extraction of primary vascular endothelial cells from the rabbit aorta[J].Chinese Journal of Tissue Engineering Research, 2016, 20(7): 1019-1024.
[18] 陈小翠, 陈邦党, 杨毅宁, 等. 人脐静脉内皮细胞分离培养和鉴定[J].细胞与分子免疫学杂志, 2016, 32(3): 328-331. CHEN Xiaocui, CHEN Bangdang, YANG Yining, et al. Isolation, culture and identification of human umbilical vein endothelial cells[J]. Chinese Journal of Cellular and Molecular Immunology, 2016, 32(3): 328-331.
[19] Siow RC. Culture of human endothelial cells from umbilical veins[J]. Methods Mol Biol, 2012, 806:265-274. doi: 10.1007/978-1-61779-367-7_18.
[20] Jiménez N, Krouwer VJ, Post JA. A new, rapid and reproducible method to obtain high quality endothelium in vitro[J].Cytotechnology, 2013, 65(1): 1-14.
[21] 李刚,萧晗,唐军建,等. 胶原酶消化刮取再消化法分离培养犬静脉内皮细胞的实验研究[J].河北医科大学学报, 2021, 42(1): 7-11. LI Gang, XIAO Han, TANG Junjian, et al. Experimental study on isolation and culture of canine vascular endothelial cells by collagenase digestion scrape and repeat digestion techniques[J]. Journal of Hebei Medical University, 2021, 42(1): 7-11.
[22] Baudin B, Bruneel A, Bosselut N, et al. A protocol for isolation and culture of human umbilical vein endothelial cells[J]. Nat Protoc, 2007, 2(3): 481-485. doi: 10.1038/nprot.2007.54.
[23] Kadam SS, Tiwari S, Bhonde RR. Simultaneous isolation of vascular endothelial cells and mesenchymal stem cells from the human umbilical cord[J]. In Vitro Cell Dev Biol Anim, 2009, 45(1-2): 23-27.
[24] Hofbauer P, Riedl S, Witzeneder K, et al. Human platelet lysate is a feasible candidate to replace fetal calf serum as medium supplement for blood vascular and lymphatic endothelial cells[J]. Cytotherapy, 2014, 16(9): 1238-1244.
[25] 于世勇,黄岚,李丽,等. 人脐静脉内皮细胞分离培养及不同消化酶效力比较[J].华南国防医学杂志, 2007, 21(6): 1-3. YU Shiyong, HUANG Lan, LI Li, et al. Different enzyme Digestion in isolation and culture of Human umbilical vein endothelial cells[J]. Military Medical Journal of South China, 2007, 21(6): 1-3.
[26] 卞杰勇,周岱. 人脐静脉内皮细胞体外培养的影响因素[J].中国细胞生物学学报, 1997, 19(2): 66-69.

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