脐血贴壁层细胞和骨髓基质细胞对脐血单个核细胞增殖能力的影响

Effects of umbilical cord blood adherent cells and bone marrow stromal cells on the proliferation of umbilical cord blood mononuclear cells

目的:比较脐血贴壁层细胞和骨髓的基质细胞对脐血单个核细胞增殖能力的影响。方法:实验于2005-02/07在吉林省肿瘤防治研究所完成。①对象:正常足月顺产儿脐带由长春市妇产医院提供;肋骨取自吉林省肿瘤医院外科手术患者,排除血液疾患。产妇及外科手术患者对治疗及实验均签署知情同意书,实验经医院医学伦理委员会批准。②实验方法:采用红细胞裂解法制备有核细胞,以Ficoll-paque淋巴细胞分层液按密度梯度离心法分离出脐血单个核细胞,体外培养24h后去除悬浮细胞,收集脐血贴壁层细胞,加入到24孔板中,2×106L-1/孔,再加入含12.5%马血清、12.5%胎牛血清、1×10-7mol/L氢化考地松的IMDM培养体系1mL。将肋骨剪成3cm小块,冲洗髓腔收集细胞悬液,用淋巴细胞分层液分离出骨髓单个核细胞,同法进行骨髓基质细胞的培养。分别将两种细胞于37℃、体积分数为0.05的CO2条件下培养3周后,各自加入到24孔板,2×106L-1/孔,同时每孔再加入1×106L-1脐血单个核细胞,培养7d。以未添加脐血贴壁层细胞及骨髓基质细胞的脐血单个核细胞作为空白对照。③实验评估:倒置显微镜下观察脐血贴壁层细胞与骨髓基质细胞的生长状态。采用流式细胞仪测定脐血单个核细胞周期分布。甲基纤维素半固体培养法测定脐血单个核细胞混合集落形成单位情况。结果:①脐血贴壁层细胞与骨髓基质细胞的形态:培养2周时,多数脐血贴壁层细胞呈大小不等的圆形、椭圆形,少部分呈不规则形;骨髓基质细胞主要为梭形,细胞排列成漩涡状、辐射状或相互缠绕成束状。②细胞周期:与空白对照相比,经脐血贴壁层细胞、骨髓基质细胞干预的脐血单个核细胞S+G2+M期所占比例均显著升高(P<0.05)。③混合集落形成单位:经脐血贴壁层细胞、骨髓基质细胞干预后的脐血单个核细胞,其混合集落形成单位的数量显著高于空白对照(P<0.05)。结论:①脐血贴壁层细胞与骨髓基质细胞在形态上具有一定差异,但均能提高脐血单个核细胞体外增殖能力。②脐血贴壁层细胞可替代骨髓基质细胞作为脐血造血细胞体外扩增的辅助条件。

AIM： To compare the effects of umbilical cord blood adherent cells and bone marrow stromal cells on the expansion of umbilical cord blood mononuclear cells. METHODS： Experiments were carded out in Jilin Tumor Prevention Research Institute from February to July 2005. （1）The normal labor baby who was mature was provided by Changchun Hospital for Gynaecology and Obstetrics. The rib was collected from the surgical patient in Jilin Tumor Hospital, whose blood disease was excluded. Both the lying-in woman and the surgical patient signed the informed consent on the treatment and experiment. The experiment was approved by medical ethic committee of the hospital. （2）Red cells produced karyotes by fission. Umbilical cord blood mononuclear cells were separated from Ficoll-paque lymphocyte stratified liquid by density gradient centrifugation, which were treated for 24 hours, and then the suspending cells were got rid of, and the adherent cells were collected. Putting the separated umbilical cord blood adherent cells onto the 24-well plates, 2× 10^6 L^-1/well. l mL IMDM culture system containing 12.5% horse blood serum, 12.5% fetal calf blood serum and 1 × 10^7 mol/L cortisol was used. The rib was cut into 3 cm blocks. After washing the pulp cavity, cells suspension was collected. Marrow mononuclear cells were separated with lymphocyte-stratified liquid, and then the same treatment was performed on the bone marrow stromal as above. All of them were cultured at 37 ℃ and CO2 with the volume fraction of 0.05 for 3 weeks, and then put into 24-well plates, 2 × 10^6 L^-1/well. Umbilical cord blood adherent cells with the amount of l× 10^6 L^-1 was added into each hole for seven days. The control group was the umbilical cord blood mononuclear cells without umbilical cord blood adherent cells and bone marrow stromal cells. （3）The growth morphology of umbilical cord adherent cells and bone marrow stromal cells was observed under an inverted microscope. The distribution of mononuclear cell cycle was determined by a flow cytometry. Half-solid methylcellulose was used to determine the colony forming unit-mixture of umbilical cord blood mononuclear cells. RESULTS： （1）At week 2, most umbilical cord adherent cells were round and ellipse in different sizes, and a few were irregular; Bone marrow stromal cells were fusiform, whirlpool-shape, radiat or bundle-shape. （2）Compared with the blank control group, there were more umbilical cord blood mononuclear cells in the S＋G2＋M phase after intervening with umbilical cord adherent cells and bone marrow stromal cells （P 〈 0.05）. （3）Colony forming unit-mixture of umbilical cord blood mononuclear cells was more after treating with umbilical cord adherent cells and bone marrow stromal cells as compared with the blank control group （P 〈 0.05）. CONCLUSION： （1）Umbilical cord adherent cells and bone marrow stromal cells are different in appearance. Both of them can elevate the proliferation of umbilical cord blood mononuclear cells. （2）Umbilical cord adherent cells, instead of bone marrow stromal cells, can be considered as a subsidiary condition to amplify umbilical cord blood hematopoietic cells in vitro.