【摘要】：目的： 通過(guò)構(gòu)建殼聚糖-明膠三維支架，并用三維支架與旋轉(zhuǎn)培養(yǎng)系統(tǒng)(RCCS)形成的三維培養(yǎng)體系(3D)來(lái)培養(yǎng)臍血分離的CD34+細(xì)胞，觀察臍血干細(xì)胞向血小板分化的情況。 方法： 1、冷凍干燥法制備不同濃度配比的殼聚糖-明膠支架，掃描電鏡觀察支架表面特征，測(cè)量支架的孔徑大小、并計(jì)算孔隙率、吸水率以及相容性測(cè)定，選取適合臍血干細(xì)胞生長(zhǎng)的支架。 2、用磁珠分選的方法對(duì)人臍血CD34+細(xì)胞進(jìn)行分離鑒定，將臍血CD34+細(xì)胞附著在殼聚糖-明膠支架上，放入RCCS系統(tǒng)進(jìn)行3D培養(yǎng)，倒置顯微鏡觀察細(xì)胞形態(tài)，掃描電鏡觀察細(xì)胞在支架上的粘附生長(zhǎng)狀況，MTT法檢測(cè)細(xì)胞活性，流式細(xì)胞儀檢測(cè)細(xì)胞標(biāo)志物，以及對(duì)分化所得的血小板進(jìn)行形態(tài)及功能鑒定，并與二維培養(yǎng)體系(2D)進(jìn)行比較。 結(jié)果： 1、成功構(gòu)建殼聚糖-明膠支架，選用殼聚糖、明膠各0.5g，預(yù)凍溫度為-30℃條件下制備支架，孔徑為(86±21)μm、吸水率為(87.62±10.19)%、孔隙率為(92.56±1.12)%，細(xì)胞相容性好，無(wú)毒副作用，，適宜臍血干細(xì)胞培養(yǎng)用三維支架。 2、磁珠分選出臍血CD34+細(xì)胞，將細(xì)胞以5×104/ml的種植率種植在2D和3D體系中。掃描電鏡觀察細(xì)胞粘附于支架上，生長(zhǎng)狀況良好；3D培養(yǎng)第8d CD34+細(xì)胞可增殖(87.02±4.35)倍，2D培養(yǎng)到第5d達(dá)到峰值，細(xì)胞可增殖(20.78±5.03)倍，兩者間具有統(tǒng)計(jì)學(xué)意義(P 0.05)；流式檢測(cè)細(xì)胞膜表面標(biāo)志物CD41+CD61+，培養(yǎng)到第7d、14d，3D體系測(cè)得的的結(jié)果為((28.70±1.75)、(82.40±2.91))%，2D體系中測(cè)得的結(jié)果為((27.18±2.63)、(80.33±3.56))%；3D培養(yǎng)系統(tǒng)所得類(lèi)血小板顆粒較2D多，3D培養(yǎng)系統(tǒng)所得類(lèi)血小板顆粒細(xì)胞數(shù)為(31.60±0.74)×106/ml，2D培養(yǎng)條件所得類(lèi)血小板顆粒細(xì)胞數(shù)為(7.91±0.93)×106/ml，其黏附率為(43.05±1.93)%，2D為(41.37±1.06)%，兩者無(wú)統(tǒng)計(jì)學(xué)意義；兩種培養(yǎng)系統(tǒng)所收集的類(lèi)血小板顆粒在鏡下觀察都具有聚集能力，流式細(xì)胞儀檢測(cè)類(lèi)血小板細(xì)胞膜有活化后標(biāo)志物CD62p的表達(dá)，陽(yáng)性率分別為((73.82±1.01)、(75.26±2.57))%，差異不顯著，無(wú)統(tǒng)計(jì)學(xué)差異。 結(jié)論： 1、通過(guò)冷凍干燥法成功構(gòu)建了適合臍血干細(xì)胞生長(zhǎng)的殼聚糖-明膠三維支架。 2、殼聚糖-明膠支架和RCCS系統(tǒng)構(gòu)建的三維培養(yǎng)體系利于臍血干細(xì)胞的增殖生長(zhǎng)。
[Abstract]:Objective: to construct a three-dimensional scaffold of chitosan gelatin and culture CD34 cells isolated from umbilical cord blood by using three-dimensional scaffold and three-dimensional culture system (3D) formed by rotating culture system (RCCS). The differentiation of umbilical cord blood stem cells into platelets was observed. Methods: 1. Chitosan gelatin scaffolds with different concentrations were prepared by freeze-drying method. The surface characteristics of the scaffolds were observed by scanning electron microscope (SEM), the pore size of the scaffolds was measured, and the porosity, water absorption and compatibility were calculated. The scaffold suitable for cord blood stem cell growth was selected. 2. Human umbilical cord blood CD34 cells were isolated and identified by magnetic bead sorting. The CD34 cells were attached to chitosan gelatin scaffold and placed in RCCS system for 3D culture. The morphology of the cells was observed by inverted microscope. The adhesion and growth of cells on scaffolds were observed by scanning electron microscope (SEM), cell activity was detected by MTT assay, cell markers were detected by flow cytometry, and morphology and function of platelets were identified. And compared with 2 D culture system (2 D). Results: 1. Chitosan and gelatin scaffolds were successfully constructed. The scaffolds were prepared by using chitosan, gelatin 0.5 g and pre-freezing temperature -30 鈩

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