本文選題：脂肪源性干細胞 + 細胞直徑��； 參考：《遼寧醫(yī)學院》2012年碩士論文

【摘要】：目的 探討脂肪源性干細胞(Adipose-derived stem/stromal cells, ASCs）的直徑、表面表型及向成脂誘導分化的生物學特性。 方法 1、細胞取材培養(yǎng)：脂肪組織取材于沈陽軍區(qū)總醫(yī)院整形外科吸脂患者，共5例。無菌條件下吸取脂肪組織，0.1%Ⅰ型膠原酶消化45min。密度梯度離心法分離細胞，得到基質(zhì)血管成分（stromal vascular fraction, SVF），其中含有ASCs。接種至25cm2培養(yǎng)瓶,37℃、5%CO2孵箱中培養(yǎng)。48h后換液除去未貼壁細胞。3天換一次液，細胞達到80%~90%融合時傳代。 2、細胞直徑檢測：采用Cellometer細胞計數(shù)儀檢測原代至第三代（P0至P3代）細胞直徑，每次檢測重復5次，實驗數(shù)據(jù)結(jié)果采用平均數(shù)±標準差表示。統(tǒng)計分析采用t檢驗。 3、細胞表型檢測：第3代細胞胰蛋白酶消化為單個細胞懸液，分別加入CD31、CD34、CD45、CD49d、CD105和CD106抗體各20ul，以不加抗體作為對照，避光孵育15min。200×g離心力下離心15min。PBS洗滌以去除未結(jié)合抗體，200×g離心力下離心3min，流式細胞儀檢測細胞表型。 4、向脂肪細胞誘導分化：P3代細胞培養(yǎng)48h后換成成脂誘導培養(yǎng)液（DMEM、10%FBS、1umol/L地塞米松、200umol/L吲哚美辛，0.5umol/L3-異丁基-1-甲基黃嘌呤（IBMX），10ug/ml胰島素）培養(yǎng)。培養(yǎng)7天后，油紅O染色液室溫染色15min，倒置顯微鏡下觀察隨機采集10張圖像。分別計算視野中全部細胞與成脂細胞數(shù)，計算成脂誘導率。計算方法為：成脂誘導率=全部成脂細胞數(shù)/全部細胞數(shù)×100%。 結(jié)果 1、細胞形態(tài)：SVF接種于25cm2培養(yǎng)瓶中24~48h后出現(xiàn)貼壁，10天左右可鋪滿瓶底。傳代后細胞生長速度加快，原代培養(yǎng)細胞一般5~7天增殖達80%融合。第一代細胞形態(tài)多樣化，一部分為三角形樣細胞，另一部分為梭形細胞，可見梭形細胞中間類圓形的胞質(zhì)區(qū)。第二代細胞形態(tài)部分為多角形，另一部分為長梭形，二者比例相近。第三代細胞形態(tài)大部分為長梭形，胞體較寬，胞質(zhì)折光性好。 2、細胞直徑變化：細胞未貼壁時平均細胞直徑范圍為9.40±1.89μm，最小細胞直徑為7.45μm，最大細胞直徑為29.18μm，其中76.15%細胞直徑為7.45~9μm。第一代平均細胞直徑為13.26±1.64μm，最小細胞直徑為7.42μm，最大細胞直徑為28.66μm,其中60.81%細胞直徑為7.42~15μm。第二代平均細胞直徑為13.66±0.97μm,最小細胞直徑為7.22μm，最大細胞直徑為25.65μm，其中91.67%細胞直徑范圍為7.22~19.07μm。第三代平均細胞直徑為15.44±0.97μm,最小細胞直徑為7.47μm，最大細胞直徑為29.50μm，其中76.92%細胞直徑范圍為7.47~18.49μm。 3、細胞表型檢測：流式細胞儀分析P3代細胞表面表型為CD31-CD34-CD45-CD49d+CD105+CD106-。具體檢測結(jié)果CD31為0.67±0.56%,CD34為2.45±1.68%，CD45為1.19±0.61%，CD105為32.33±33.79%。CD49d31.94±14.37%，CD106為1.41±0.73%。 4、成脂誘導變化和鑒定：成脂誘導48~72h后，細胞形態(tài)逐漸發(fā)生變化，由長梭形細胞變?yōu)槎嘟切�、類圓形細胞，可見胞質(zhì)內(nèi)出現(xiàn)透亮、高折光性、“分房”狀的小脂滴，最初位于細胞核外周。脂滴的數(shù)量和體積隨誘導時間的延長而增加變大，相互融合，，最終7天后油紅O染色顯示有大量紅色脂質(zhì)沉積。同時，細胞形態(tài)以圓形為主，長梭形細胞少見。200倍倒置顯微鏡下隨機采集10個視野計數(shù)，P3代細胞7天成脂誘導率為51.12±8.01%。 結(jié)論 本研究體外分離人脂肪源性干細胞并培養(yǎng)，細胞于接種后24h開始貼壁，逐漸由圓形透亮細胞轉(zhuǎn)變?yōu)殚L梭形細胞。采用Cellometer細胞計數(shù)儀證實，體外分離的細胞大小存在差異，隨著培養(yǎng)時間延長而發(fā)生變化。流式細胞儀檢測證實，第三代細胞表型為CD31-CD34-CD45-CD49d+CD105+CD106-。體外培養(yǎng)細胞在成脂誘導條件下可向脂肪細胞分化。
[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.
【學位授予單位】：遼寧醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R329

【參考文獻】

相關(guān)期刊論文 前2條

1 陳希哲,林云鋒,喬鞠,田衛(wèi)東,閆征斌,李聲偉;人體脂肪基質(zhì)細胞分離培養(yǎng)及其成骨潛能[J];實用口腔醫(yī)學雜志;2004年01期

2 王棟;鹿均先;熊傳芝;;人脂肪干細胞分離培養(yǎng)鑒定及向成骨細胞誘導分化的實驗研究[J];現(xiàn)代醫(yī)學;2008年03期

本文編號：1989155