不同来源微血管内皮细胞培养方法的建立及生物学特性比较

发布时间：2018-06-19 01:51

  本文选题：肺 + 脂肪　； 参考：《西南医科大学》2017年硕士论文

【摘要】：目的:分离小鼠脂肪微血管内皮细胞(AMECs)及肺微血管内皮细胞(PMECs),建立微血管内皮细胞(MECs)的培养方法。比较两种MECs生物学特性的差别。为我们下一步研究MECs对造血干细胞(HSCs)体外扩增的影响奠定基础,同时也给MECs的相关实验研究及临床应用提供依据。方法:1、微血管内皮细胞分离培养方法的建立:(1)提取C57小鼠的附睾旁脂肪组织和肺组织,用0.1%I型胶原酶在37℃条件下摇床消化,用70um滤器过滤细胞悬液。(2)寻找分离附睾旁脂肪组织的最佳鼠龄,分组:(1)4w,(2)6w,(3)8w,(4)10w,判断标准:每ml脂肪组织所含种子细胞数量。(3)探索最佳首次换液的时间,分组:(1)24h,(2)48h,(3)72h,判断标准:能否获得原代MECs。(4)寻找原代培养的最佳接种密度,分组:(1)5×105/cm2,(2)1×106/cm2,(3)2×106/cm2,判断标准:培养至第8天的细胞形态、数量。(5)探索明胶包被培养皿的最适宜浓度,分组:(1)0,(2)0.1%,(3)0.5%,(4)1%,判断标准:收获的原代MECs细胞数。2、对AMECs与PMECs的生物学特性进行比较:(1)比较平均每只小鼠获得的脂肪微血管内皮细胞(AMECs)和肺微血管内皮细胞(PMECs)数量。(2)倒置显微镜观察AMECs及PMECs的生长、形态变化。(3)流式细胞分析技术及免疫荧光技术测得AMECs和PMECs的CD31、CD34、CD45、vWF的表达。(4)绘制生长曲线比较AMECs和PMECs的增殖能力。(5)将获得的原代AMECs和PMECs按3:1进行传代,比较两种细胞的传代能力及传代细胞形态。(6)采用10ng/mlVEGF培养两种MECs观察形态特征。结果:1.微血管内皮细胞分离培养的最佳方法:(1)采用酶消法能够成功分离两种组织获得种子细胞。(2)4周龄小鼠单位体积脂肪获得的种子细胞数为4.67±0.58(106),6周组为10.18±0.28(106),8周组为9.94±0.35(106),10周组为6.72±0.39(106);6周组及8周组较4周组、10周组多(p0.05),6周组与8周组比较无差异(p0.05)。(3)24h首次换液可获得原代amecs和pmecs;48h及72h首次换液,杂细胞生长占优势,无法获得原代mecs。(4)1×106/cm2为原代最佳接种密度;5×105/cm2细胞稀疏,mecs增殖不良;2×106/cm2细胞接触抑制,死亡脱壁。(5)培养至第8天,amecs的未包被明胶组的200×镜下细胞数为148.33±13.54,0.1%组为201.83±12.97,0.5%组为191.50±11.52,1%组为193.00±8.67;明胶包被的3组得到的amecs多于未包被组(p0.05),而3种浓度间无差异(p0.05)。培养至第10天,pmecs的未包被明胶组的200×镜下细胞数为182.33±11.03,0.1%组为219.50±11.4,0.5%组为212.00±12.39,1%组为213.00±12.12;明胶包被的3组得到的pmecs多于未包被组(p0.05),而3种浓度间无差异(p0.05)。2.amecs与pmecs的生物学特性比较的结果:(1)原代培养的amecs及pmecs均在第48h-72h形成细胞团,第4天左右细胞呈多角形、卵圆形,amecs在第10天、pmecs在第14天形成“铺路石样”改变,细胞表面可观察到微绒毛。(2)每只小鼠获得的amecs种子细胞为2.08±0.57(×106),pmecs种子细胞为19.07±0.72(×106),二者有差异(p0.05)。每只小鼠获得的amecs原代细胞数为0.26±0.05(×106),pmecs原代细胞数为3.37±0.18(×106),二者有差异(p0.05)。(3)amecs及pmecs均不表达cd45。amecs的cd31、cd34、vwf表达率分别为44.28%、30.15%、76.1%,pmecs的cd31、cd34、vwf表达率分别为45.8%、57.48%、81.39%。(4)传代的两种细胞生长均快于原代细胞,与原代细胞形态相比无明显差异。(5)生长曲线显示PMECs缓慢增殖期较AMECs长;AMECs生长高峰为接种后3-6天,PMECs生长高峰为接种后4-7天。(6)10ng/mlVEGF培养两种细胞,第6天可见细胞伸展、迁移融合形成细胞索,细胞索相互之间连接沟通。结论:1.本研究建立了经济有效、简便、可重复的分离培养AMECs及PMECs的最佳方案:24h首次换液、1×106/cm2接种密度、0.1%明胶、2ng/mlVEGF、6-8周龄小鼠。2.AMECs的细胞形态、特异性标志、细胞传代、生长曲线与PMECs类似;每只小鼠收获的AMECs和PMECs的数量有差异。
[Abstract]:Objective: to isolate the mouse fat microvascular endothelial cells (AMECs) and pulmonary microvascular endothelial cells (PMECs), to establish the culture method of microvascular endothelial cells (MECs), to compare the differences between the two kinds of MECs biological characteristics, and to lay the foundation for the next step to study the effect of MECs on the amplification of hematopoietic stem cells (HSCs) and to study the related experimental research of MECs. Methods: 1, 1, the isolation and culture of microvascular endothelial cells were established: (1) the epididymal adipose tissue and lung tissue of C57 mice were extracted and digested with 0.1%I collagenase at 37 degrees centigrade, and cell suspension was filtered by 70UM filter. (2) the optimal age of para epididymal adipose tissue was found. (1) 4W, (2) 6W, (3) ) 8W, (4) 10W, determine the standard: the number of seed cells in each ml fat tissue. (3) explore the best time for the first change of liquid, grouping: (1) 24h, (2) 48h, (3) 72h, judging whether the original MECs. (4) can find the best inoculation density for primary culture, grouping: (1) 5 x 105/cm2, (2) 1 * 106/cm2, (3) 2 * 106/cm2, criteria: cells cultivated to eighth days Form, quantity. (5) explore the optimum concentration of gelatine coated Petri dish, grouping: (1) 0, (2) 0.1%, (3) 0.5%, (4) 1%, judging standard: the number of primary MECs cells harvested.2, compared with the biological characteristics of AMECs and PMECs: (1) compared the average number of fat microvascular endothelial cells (AMECs) and pulmonary microvascular endothelial cells (PMECs) obtained in each mouse. (2) the growth and morphological changes of AMECs and PMECs were observed by inverted microscope. (3) flow cytometry and immunofluorescence techniques were used to detect the expression of CD31, CD34, CD45, vWF of AMECs and PMECs. (4) the growth curve was plotted to compare the proliferation ability of AMECs and PMECs. (5) the primary AMECs and PMECs were subpassable and the passages of the two cells were compared. Force and subcellular morphology. (6) the morphological characteristics of two kinds of MECs were observed by 10ng/mlVEGF. Results: the best method of isolation and culture of 1. microvascular endothelial cells: (1) the seed cells were successfully isolated from two tissues by enzyme digestion. (2) the number of seed cells obtained by unit volume fat of 4 week old mice was 4.67 + 0.58 (106) and 6 weeks was 10.18. 0.28 (106), 8 weeks group was 9.94 + 0.35 (106), 10 week group was 6.72 + 0.39 (106), and 6 week group and 8 week group were more than 4 week group (P0.05), and there was no difference (P0.05) in 8 weeks group. The density of 5 x 105/cm2 cells was sparse and MECs was poorly proliferating; 2 x 106/cm2 cells were exposed to contact inhibition, and death was removed. (5) culture to eighth days. The number of cells in the 200 x group of the unwrapped group of AMECS was 201.83 + 12.97,0.5% and 191.50 + 11.52,1% in group 201.83 was 193 + 8.67, and the AMECS of gelatin bag was more than that of the unwrapped group. P0.05), and there was no difference between the 3 concentrations (P0.05). For tenth days, the number of cells in the 200 x mirror group of the unwrapped gelatin group of PMECs was 182.33 + 11.03,0.1% and 213 + 12.12 in the 219.50 + 11.4,0.5% group, and the 3 groups of the gelatin bag were more than the unwrapped group (P0.05), and 3 concentrations were no difference (P0.05).2.amecs and pmec. The results of biological characteristics of s were compared: (1) the primary culture of AMECS and PMECs formed cell clusters in 48h-72h, fourth days or so, the cells were polygonal, oval, AMECS was tenth days, PMECs formed the "pave stone like" change on the fourteenth day, and the cell surface could be observed microvilli. (2) the AMECS seed cells of each mouse were 2.08 + 0.57 (x 106). The PMECs seed cells were 19.07 + 0.72 (x 106) and two were different (P0.05). The number of primary AMECS cells in each mouse was 0.26 + 0.05 (x 106), the number of primary cells of PMECs was 3.37 + 0.18 (x 106), and there was a difference between them (P0.05). (3) AMECS and PMECs did not express cd45.amecs's CD31, CD34, vWF expression rate, respectively, PMECs The expression rate of vWF was 45.8%, 57.48%, and 81.39%. (4) of the two cells were faster than the original cells. (5) the growth curve showed that the slow proliferation period of PMECs was longer than that of AMECs; the peak of AMECs growth was 3-6 days after inoculation, and the peak of PMECs growth was 4-7 days after inoculation. (6) 10ng/mlVEGF culture two cells sixth. Sixth Days can be seen that cell extension, migration and fusion form cell cables, and cell lines connect and communicate with each other. Conclusion: 1. this study established the best economic, simple, repeatable isolation and culture of AMECs and PMECs: 24h first liquid, 1 x 106/cm2 inoculation density, 0.1% gelatin, 2ng /mlVEGF, 6-8 week old mouse.2.AMECs cell morphology, specific markers The cell growth curve was similar to that of PMECs, and the number of AMECs and PMECs harvested per mouse was different.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R457.7

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