脂肪源性干细胞直径、表型及向脂肪细胞分化的研究

发布时间：2018-06-07 01:50

本文选题：脂肪源性干细胞 + 细胞直径　；参考：《辽宁医学院》2012年硕士论文

【摘要】：目的探讨脂肪源性干细胞(Adipose-derived stem/stromal cells, ASCs）的直径、表面表型及向成脂诱导分化的生物学特性。方法 1、细胞取材培养：脂肪组织取材于沈阳军区总医院整形外科吸脂患者，共5例。无菌条件下吸取脂肪组织，0.1%Ⅰ型胶原酶消化45min。密度梯度离心法分离细胞，得到基质血管成分（stromal vascular fraction, SVF），其中含有ASCs。接种至25cm2培养瓶,37℃、5%CO2孵箱中培养。48h后换液除去未贴壁细胞。3天换一次液，细胞达到80%~90%融合时传代。 2、细胞直径检测：采用Cellometer细胞计数仪检测原代至第三代（P0至P3代）细胞直径，每次检测重复5次，实验数据结果采用平均数±标准差表示。统计分析采用t检验。 3、细胞表型检测：第3代细胞胰蛋白酶消化为单个细胞悬液，分别加入CD31、CD34、CD45、CD49d、CD105和CD106抗体各20ul，以不加抗体作为对照，避光孵育15min。200×g离心力下离心15min。PBS洗涤以去除未结合抗体，200×g离心力下离心3min，流式细胞仪检测细胞表型。 4、向脂肪细胞诱导分化：P3代细胞培养48h后换成成脂诱导培养液（DMEM、10%FBS、1umol/L地塞米松、200umol/L吲哚美辛，0.5umol/L3-异丁基-1-甲基黄嘌呤（IBMX），10ug/ml胰岛素）培养。培养7天后，油红O染色液室温染色15min，倒置显微镜下观察随机采集10张图像。分别计算视野中全部细胞与成脂细胞数，计算成脂诱导率。计算方法为：成脂诱导率=全部成脂细胞数/全部细胞数×100%。结果 1、细胞形态：SVF接种于25cm2培养瓶中24~48h后出现贴壁，10天左右可铺满瓶底。传代后细胞生长速度加快，原代培养细胞一般5~7天增殖达80%融合。第一代细胞形态多样化，一部分为三角形样细胞，另一部分为梭形细胞，可见梭形细胞中间类圆形的胞质区。第二代细胞形态部分为多角形，另一部分为长梭形，二者比例相近。第三代细胞形态大部分为长梭形，胞体较宽，胞质折光性好。 2、细胞直径变化：细胞未贴壁时平均细胞直径范围为9.40±1.89μm，最小细胞直径为7.45μm，最大细胞直径为29.18μm，其中76.15%细胞直径为7.45~9μm。第一代平均细胞直径为13.26±1.64μm，最小细胞直径为7.42μm，最大细胞直径为28.66μm,其中60.81%细胞直径为7.42~15μm。第二代平均细胞直径为13.66±0.97μm,最小细胞直径为7.22μm，最大细胞直径为25.65μm，其中91.67%细胞直径范围为7.22~19.07μm。第三代平均细胞直径为15.44±0.97μm,最小细胞直径为7.47μm，最大细胞直径为29.50μm，其中76.92%细胞直径范围为7.47~18.49μm。 3、细胞表型检测：流式细胞仪分析P3代细胞表面表型为CD31-CD34-CD45-CD49d+CD105+CD106-。具体检测结果CD31为0.67±0.56%,CD34为2.45±1.68%，CD45为1.19±0.61%，CD105为32.33±33.79%。CD49d31.94±14.37%，CD106为1.41±0.73%。 4、成脂诱导变化和鉴定：成脂诱导48~72h后，细胞形态逐渐发生变化，由长梭形细胞变为多角形、类圆形细胞，可见胞质内出现透亮、高折光性、“分房”状的小脂滴，最初位于细胞核外周。脂滴的数量和体积随诱导时间的延长而增加变大，相互融合，，最终7天后油红O染色显示有大量红色脂质沉积。同时，细胞形态以圆形为主，长梭形细胞少见。200倍倒置显微镜下随机采集10个视野计数，P3代细胞7天成脂诱导率为51.12±8.01%。结论本研究体外分离人脂肪源性干细胞并培养，细胞于接种后24h开始贴壁，逐渐由圆形透亮细胞转变为长梭形细胞。采用Cellometer细胞计数仪证实，体外分离的细胞大小存在差异，随着培养时间延长而发生变化。流式细胞仪检测证实，第三代细胞表型为CD31-CD34-CD45-CD49d+CD105+CD106-。体外培养细胞在成脂诱导条件下可向脂肪细胞分化。
[Abstract]:objective
Objective to investigate the diameter, surface phenotype and biological characteristics of adipose derived stem cells (Adipose-derived stem/stromal cells) (ASCs).
Method
1, cell culture: adipose tissue was obtained from the liposuction patients in the General Hospital of Shenyang military district general hospital, 5 cases. The adipose tissue was absorbed under aseptic conditions, and the cells were separated by 0.1% type collagenase digestion 45min. density gradient centrifugation, and the components of stromal vascular fraction (SVF) were obtained, including ASCs. inoculation to 25cm2 culture bottle, 37 When incubated in 5%CO2 incubator,.48h was changed to remove the non adherent cells for.3 days and the cells were replaced by 80%~90% fusion.
2, cell diameter detection: the diameter of the primary to third generation (P0 to P3) cell diameter was detected by Cellometer cell count instrument, and 5 repeated tests were repeated each time. The results of the experimental data were expressed with the average number of standard deviation. The statistical analysis was tested by t test.
3, cell phenotype detection: the third generation of cell trypsin was digested as a single cell suspension, adding CD31, CD34, CD45, CD49d, CD105 and CD106 antibodies in each 20ul, with no antibody as the control, the non light incubating 15min.200 * g centrifugal force centrifugation 15min.PBS washing to remove unconjugated antibodies, 200 x g centrifugal force under centrifugal 3min, flow cytometry fine detection Cell phenotype.
4, induced differentiation to adipocytes: P3 cells were cultured for 48h and changed into lipid induced culture fluid (DMEM, 10%FBS, 1umol/L dexamethasone, 200umol/L indomethacin, 0.5umol/L3- isobutyl -1- methylxanthine (IBMX), 10ug/ml insulin) culture. 7 days after culture, oil red O dyeing liquid was stained 15min at room temperature, and 10 maps were collected under inverted microscope. Figure. Calculate the number of all cells and adipocytes in the field of vision and calculate the lipid induction rate. The calculation method is: the percentage of lipid induction = all the number of adipocyte / all cell number x 100%.
Result
1, cell morphology: SVF was inoculated with 24~48h in 25cm2 culture bottle. The cell growth rate was accelerated after 10 days. The growth rate of cells in the primary culture cells was 80% fusion. The first generation cells were diversified, some were triangular like cells, the other part was spindle cells, and the middle round circle of spindle cells could be seen. The cell morphology of the second generation is polygonal, the other part is long spindle shape, and the proportion of the two is similar. The third generation of cell morphology is mostly long spindle shape, the cell body is wider, and the cytoplasm is well refracted.
2, cell diameter change: the average cell diameter of cell was 9.40 + 1.89 mu m, the smallest cell diameter was 7.45 mu m and the maximum cell diameter was 29.18 micron m, and the diameter of 76.15% cells was 13.26 + 1.64 u m, the smallest cell diameter was 7.42 mu m, and the maximum cell diameter was 28.66 mu m, of which 60.81% fine. The diameter of the cell diameter was 7.42~15 mu m. second generation average diameter of 13.66 + 0.97 mu m, the minimum cell diameter was 7.22 m and the maximum cell diameter was 25.65 mu m. The diameter of 91.67% cells was 7.22~19.07 mu m. third generation average diameter of 15.44 + 0.97 mu m, the smallest cell diameter was 7.47 u m and the maximum cell diameter was 29.50 micron m, among which 76.92% cells were straight. The diameter range is 7.47~18.49 mu m.
3, cell phenotype detection: flow cytometry analysis of P3 cell surface phenotype is CD31-CD34-CD45-CD49d+CD105+CD106-. specific detection results, CD31 is 0.67 + 0.56%, CD34 is 2.45 + 1.68%, CD45 is 1.19 + 0.61%, CD105 is 32.33 + 33.79%.CD49d31.94 + 14.37%, CD106 is 1.41 + 0.73%.
4, lipid induced changes and identification: after the lipid induction of 48~72h, the cell morphology changes gradually, from long spindle cells into polygons, round cells, visible light, high refraction, "room" like small fat droplets, initially located outside the nucleus. The number and volume of lipid droplets increase with the prolongation of the induction time. In the final 7 days, oil red O staining showed a large number of red lipid deposits. At the same time, the morphology of the cells was round, and the long spindle cells were rare under the.200 double inversion microscope to collect 10 visual fields at random. The 7 days of P3 generation cells were 51.12 + 8.01%..
conclusion
In this study, human adipose derived stem cells were isolated and cultured in vitro. After inoculation, 24h began to stick to the wall and gradually changed from round and bright cells to long spindle cells. Cellometer cell counting apparatus proved that the size of cells separated in vitro was different and changed with the prolongation of culture time. Flow cytometry confirmed that the third generation of cells were fine. The cell phenotype is CD31-CD34-CD45-CD49d+CD105+CD106-.. In vitro culture cells can differentiate into adipocytes under adipogenic induction.
【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

【参考文献】