小鼠骨髓高增殖潜能内皮祖细胞的培养与促增殖因子研究

发布时间：2018-05-19 14:16

本文选题：内皮祖细胞 + 体外扩增　；参考：《湖南师范大学》2009年硕士论文

【摘要】： 目的:用含小鼠骨髓内皮细胞系条件培养液(BMEC-CM)的培养体系,进行骨髓高增殖潜能内皮祖细胞(HPP-EPC)及其后代细胞的培养和纯化,建立这些细胞的一种扩增培养方法。检测小鼠骨髓内皮细胞系细胞的血管内皮细胞生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)、胰岛素样生长因子-1(IGF-1)mRNA表达水平,分析BMEC-CM促进EPC增殖的分子机理。方法和结果:本实验分为以下三个部分: 1.总EPC集落形成实验制备小鼠骨髓单细胞悬液,骨髓细胞预贴壁培养6h后,吸出含未贴壁细胞的上悬液,移入新的培养瓶,并添加BMEC-CM,进行二次贴壁培养。培养5d后用胰酶法将贴壁细胞消化下来,洗涤后计数。以每mm~2孔底面积1个细胞(1/mm~2)的密度,将这些细胞种入培养板,培养体系中仍添加BMEC-CM。观察贴壁细胞集落的形成,计数集落数和计算集落形成率。数码显微拍摄集落,计数集落细胞数,用集落平均细胞数绘制细胞生长曲线,并计算细胞群体倍增时间(DT)。按原代骨髓细胞种植后的天数计,9～12d可见EPC细胞簇,2～3d内形成集落(细胞数≥50),集落形成率为每10~6骨髓有核细胞生成600.0±52.4个集落(个/10~6NBMCs)。集落细胞大多呈椭圆形、圆形和纺锤形。大部分集落约在1周内生长较快,以后生长减慢,至30d左右,集落平均细胞数为504.6±126.0。在原代骨髓细胞种植后10～13d、14～17d、18～21d、22～25d四个时间段的细胞群体倍增时间依次是58.0h、74.4h、131.2h、358.2h。 2.HPP-EPC集落形成实验及单集落源细胞的扩增培养和鉴定用上述消化和洗涤的贴壁细胞,以每mm~2孔底面积50个细胞(50/mm~2)的密度种入培养板,培养体系同前,每周换液。观察贴壁细胞集落的形成,计数细胞数达5000左右的集落,计算集落形成率。进行单集落细胞扩增培养,每4d做细胞计数,按单个集落绘制细胞生长曲线和计算DT。用扩增细胞做CD31和vWF免疫荧光染色、FITC-UEA-1结合实验和Dil-Ac-LDL摄取实验。按原代骨髓细胞种植后的天数计,28～35d时细胞数5000左右的HPP-EPC集落形成,其细胞紧密聚集,细胞形态大多为椭圆形和圆形,集落形成率为3.0±0.6个/10~6NBMCs。单集落源细胞传代扩增培养显示,细胞在低密度时可形成索状和网状排列,高密度时细胞呈铺路石状排列。在原代骨髓细胞种植后50～53d,DT最短,平均为51.2h;在74～77d时间段,DT延长至324.2h。至原代骨髓细胞种植后80d左右,集落的平均细胞数达8.0×10~6±1.2×10~5个,这些细胞为CD31、vWF免疫荧光染色阳性,能结合FITC-UEA-1和吞噬Dil-Ac-LDL。 3.mBMEC细胞中三种细胞因子mRNA水平的检测Trizol法提取小鼠骨髓内皮细胞系细胞RNA,进行荧光实时定量逆转录PCR,检测VEGF、bFGF、IGF-1 mRNA的表达水平。PCR结果显示mBMEC细胞高水平表达VEGF mRNA和bFGF mRNA,而IGF-1 mRNA的表达水平较低。结论:添加BMEC-CM的培养体系十分适合小鼠骨髓EPC的生长,用这种体系进行HPP-EPC单集落源细胞培养能大量获得其后代细胞。骨髓内皮细胞系细胞高水平表达VEGF和bFGF,这可能是BMEC-CM促进EPC增殖的重要分子基础。这些结果还为内皮细胞与EPC之间的反馈调控机制提供了新的实验证据。
[Abstract]:Objective : To investigate the expression of vascular endothelial growth factor ( VEGF ) , basic fibroblast growth factor ( bFGF ) and insulin - like growth factor - 1 ( IGF - 1 ) mRNA in bone marrow endothelial cells of mice and to analyze the molecular mechanism of BMEC - CM .

Methods and Results : This experiment is divided into the following three parts :

1 . The mouse bone marrow mononuclear cell suspension was prepared by EPC colony formation experiment . After 6 hours of pre - adherent culture of bone marrow cells , the supernatant containing unadherent cells was aspirated into the new culture flask , and BMEC - CM was added to culture the cells . After 5 days of culture , BMEC - CM was added . The colony formation , colony count and colony formation rate were counted .

The cell population doubling time was 54.6 卤 126.0 . The doubling time of colony was 54.6 卤 126.0 . The doubling time of colony was 58.0h , 74.4 h , 131.2 h , 358.2 h after the first generation of bone marrow cells .

2 . The colony forming experiment and the amplification culture of single colony source cells were carried out and the adherent cells were cultured and identified with the above digested and washed adherent cells . The colony formation rate was calculated by using the density of 50 cells ( 50 / mm ~ 2 ) per mm ~ 2 hole bottom area . The colony formation rate was calculated . The colony formation rate was calculated . The cell growth curve and the calculation DT were drawn on the basis of single colony . The cells were used as CD31 and von Wilms immunofluorescence staining , FITC - UEA - 1 binding experiment and Dil - Ac - LDL uptake experiment .

The colony formation rate was 3.0 卤 0.6 / 10 ~ 6NB12.The average number of cells was 8.0 脳 10 ~ 6 卤 1.2 脳 10 ~ 5 after the growth of primary bone marrow cells . The cells were CD31 and VWF immunofluoresence staining , which could bind to FITC - UEA - 1 and phagocytose Dil - Ac - LDL .

3 . The mRNA levels of three cytokines mRNA in mBMEC cells were detected by Trizol method . The expression of VEGF , bFGF and IGF - 1 mRNA was detected by real - time quantitative reverse transcription polymerase chain reaction ( RT - PCR ) , and the expression level of VEGF , bFGF and IGF - 1 mRNA was detected in mBMEC cells .

Conclusion : The culture system of BMEC - CM is very suitable for the growth of mouse bone marrow EPC , and its progeny cells can be obtained by using this system . The high level expression of VEGF and bFGF in bone marrow endothelial cell line cells may be an important molecular basis for BMEC - CM to promote EPC proliferation . These results also provide new experimental evidence for the feedback regulation mechanism between endothelial cells and EPC .
【学位授予单位】：湖南师范大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

【参考文献】