3D支架搭载间充质干细胞促进造血干细胞体外生产血小板的研究
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摘要
目的研究3D支架搭载间充质干细胞(MSC)对促进造血干细胞(HSC)体外生产血小板的影响。方法从本院本部干细胞临床研究中心购买种子细胞HSC及MSC。取适量生物材料,环氧乙烷消毒后使用伊思柯夫改良培养液(IMDM)浸泡过夜。将一定数量间MSC贴壁培养至生物材料中,待其长满。设置分组:对照组(24孔板内单独培养HSC)、MSC组(24孔板内单独接种MSC)、实验组(即HSC+3D-MSC+细胞因子),体外共培养7 d,分别于1～7 d时观察HSC的形态变化;比较三组不同时间点HSC活性细胞数量;采用细胞计数试剂盒-8(CCK-8)法测定HSC扩增情况。结果荧光显微镜观察显示:对照组可见HSC细胞呈均一的圆形;MSC组可见MSC呈长梭形;实验组可见圆形细胞和长梭形细胞,在同一视野进行白光和荧光拍照,并经PS软件合成处理后,可见清晰细胞排列。实验组第1、3、5、7天时HSC活性细胞数量分别为(3.00±0.15)、(15.45±2.78)、(133.33±24.68)、(218.88±16.04)×10~4,明显高于对照组的(1.30±0.23)、(3.75±0.66)、(5.60±0.68)、(8.58±0.29)×10~4和MSC组的(1.35±0.60)、(10.18±0.99)、(36.51±2.77)、(56.67±1.88)×10~4,差异均有统计学意义(P<0.05)。对各组培养第1、3、5、7天HSC样本进行CCK-8法检测,从生长曲线可以看出各组HSC数量均随培养时间的延长而增加,第3天开始进入对数生长期,与骨髓MSC共培养对HSC增殖有促进作用, 3D支架搭载较对照组和MSC组对HSC增殖的促进作用更明显。结论 3D支架搭载MSC在体外能够有效促进HSC增殖,显著提高血小板产量。
Objective To study the effect of in vitro platelet production by hematopoietic stem cells(HSC) promoted by mesenchymal stem cells(MSC) on 3 D scaffolds. Methods Seed cells HSC and MSC were purchased from our clinical stem cell research center. After disinfection with ethylene oxide, appropriate biomaterials were soaked overnight in Iscove's modified Dubecco's medium(IMDM). A certain number of MSCs are adherently cultured into biological materials until they are overgrown. The grouping was set up: control group(HSC cultured in 24-well plate alone), MSC group(MSC inoculated separately in 24-well plate), experimental group(HSC+3 DMSC+cytokine), co-cultured for 7 d in vitro. The morphological changes of HSC were observed at 1-7 d, and the number of HSC active cells at different time points was compared among the three groups. HSC amplification was detected by cell counting kit-8(CCK-8). Results Fluorescence microscopy showed that HSC cells were uniform round in the control group, long spindle in the MSC group, and round and long spindle cells in the experimental group. White light and fluorescence photographs were taken in the same field of vision, and clear cell arrangement was observed after PS software synthesis. At 1 st, 3 rd, 5 th and 7 th day, the experimental group had obviously higher number of HSC active cells respectively as(1.30±0.23),(3.75±0.66),(5.60±0.68),(8.58±0.29)×10~4 in the controll group, and(1.35±0.60),(10.18±0.99),(36.51±2.77) and(56.67±1.88)×10~4 in MSC group. Their difference was statistically significant(P<0.05). CCK-8 method was used to detect HSC samples from the 1 st, 3 rd, 5 th and 7 th day of culture in each group. The growth curve showed that the number of HSC in each group increased with the prolongation of culture time. On the 3 rd day, HSC began to enter the logarithmic growth phase. Co-culture with bone marrow MSC promoted the proliferation of HSC. Compared with control group and MSC group, the effect of 3 D scaffolds loading on HSC proliferation was more obvious. Conclusion MSC loaded on 3 D scaffolds can effectively promote HSC proliferation and significantly increase platelet production in vitro.
Objective To study the effect of in vitro platelet production by hematopoietic stem cells(HSC) promoted by mesenchymal stem cells(MSC) on 3 D scaffolds. Methods Seed cells HSC and MSC were purchased from our clinical stem cell research center. After disinfection with ethylene oxide, appropriate biomaterials were soaked overnight in Iscove's modified Dubecco's medium(IMDM). A certain number of MSCs are adherently cultured into biological materials until they are overgrown. The grouping was set up: control group(HSC cultured in 24-well plate alone), MSC group(MSC inoculated separately in 24-well plate), experimental group(HSC+3 DMSC+cytokine), co-cultured for 7 d in vitro. The morphological changes of HSC were observed at 1-7 d, and the number of HSC active cells at different time points was compared among the three groups. HSC amplification was detected by cell counting kit-8(CCK-8). Results Fluorescence microscopy showed that HSC cells were uniform round in the control group, long spindle in the MSC group, and round and long spindle cells in the experimental group. White light and fluorescence photographs were taken in the same field of vision, and clear cell arrangement was observed after PS software synthesis. At 1 st, 3 rd, 5 th and 7 th day, the experimental group had obviously higher number of HSC active cells respectively as(1.30±0.23),(3.75±0.66),(5.60±0.68),(8.58±0.29)×10~4 in the controll group, and(1.35±0.60),(10.18±0.99),(36.51±2.77) and(56.67±1.88)×10~4 in MSC group. Their difference was statistically significant(P<0.05). CCK-8 method was used to detect HSC samples from the 1 st, 3 rd, 5 th and 7 th day of culture in each group. The growth curve showed that the number of HSC in each group increased with the prolongation of culture time. On the 3 rd day, HSC began to enter the logarithmic growth phase. Co-culture with bone marrow MSC promoted the proliferation of HSC. Compared with control group and MSC group, the effect of 3 D scaffolds loading on HSC proliferation was more obvious. Conclusion MSC loaded on 3 D scaffolds can effectively promote HSC proliferation and significantly increase platelet production in vitro.
引文
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[4]周燕,李丽兰,钟周琳,等.造血干细胞移植术后抗-CD36抗体介导的血小板输注无效症和相关病例的实验研究.中国实验血液学杂志, 2018, 26(2):541-546.
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[6]孙春红,姚建娜,刘芳,等.不同血细胞分离机采集程序对健康供者造血干细胞采集物细胞成分的影响.中国实验血液学杂志, 2016, 24(6):1869-1872.
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[8]梁雨蒙,王晓娜,邓磊,等.骨髓间充质干细胞微泡生物学特性及其促进造血干细胞体外扩增作用的研究.中国实验血液学杂志, 2017, 25(4):1187-1193.
[2]陆华.体外生产血小板的研究进展.重庆医学, 2011, 40(36):3727-3730.
[3]杨帆,孙雪冬,袁磊,等.聚乙二醇化G-CSF与重组人G-CSF促进恶性血液病异基因造血干细胞移植后造血恢复的对比研究.中华血液学杂志, 2017, 38(10):831-836.
[4]周燕,李丽兰,钟周琳,等.造血干细胞移植术后抗-CD36抗体介导的血小板输注无效症和相关病例的实验研究.中国实验血液学杂志, 2018, 26(2):541-546.
[5]赵昕亚,董林.造血干细胞体外诱导生成血小板研究进展.医学综述, 2016, 22(20):3977-3980.
[6]孙春红,姚建娜,刘芳,等.不同血细胞分离机采集程序对健康供者造血干细胞采集物细胞成分的影响.中国实验血液学杂志, 2016, 24(6):1869-1872.
[7]方洪松,周建林,彭昊,等.不同来源间充质干细胞生物学特性差异.中国组织工程研究, 2015, 19(32):5243-5248.
[8]梁雨蒙,王晓娜,邓磊,等.骨髓间充质干细胞微泡生物学特性及其促进造血干细胞体外扩增作用的研究.中国实验血液学杂志, 2017, 25(4):1187-1193.
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