改良Dexter法与IMDM培养基培养骨髓基质细胞的比较

Bone marrow stromal cells cultured by modified Dexter method versus IMDM medium method

背景：体外分离培养骨髓基质细胞,并于骨髓移植后将其注入严重联合免疫缺陷鼠体内,对于促进造血及免疫重建尤为必要。目的：比较骨髓基质细胞在不同培养条件下的生长状况,寻找一种体外分离培养扩增骨髓基质细胞简单而实用的方法。设计、时间及地点：体外对比观察实验,于2006-07/2007-01在太原市中心医院皮肤科实验室完成。材料：人骨髓取自太原市中心医院血液科经筛选的正常骨髓（取材均经患者同意）。方法：分离人骨髓单个核细胞,对照组采用改良Dexter法培养：将骨髓单个核细胞用5×10^-7mol/L氢化可的松的RPMI1640培养基调整细胞浓度为1×10^9L^-1,以每孔1mL接种于24孔培养板,培养2d后全量换液,去除非贴壁细胞,之后每3d半量换液1次。细胞生长至半融合状态时传代,传两三代后用胰酶消化,收集细胞,液氮冷冻保存。实验组用IMDM培养基培养：用不含氢化可的松的IMDM培养基培养,其余成分及培养条件均与对照组相同。2d后全量换液,去除非贴壁细胞,之后每4天半量换液1次。传代及收集方法同对照组。主要观察指标：通过倒置显微镜观察细胞贴壁情况、形态变化及增殖状态,MTT比色法检测细胞增殖活性。结果：倒置显微镜下,两组培养细胞在接种24h后细胞大量贴壁并伸展开,随着培养时间延长,贴壁细胞数量增多,14d左右细胞铺满板底达融合状态。传代细胞经消化后变形,24h后恢复原来形态,并贴壁增殖,形态和原代细胞相似,4.0-5.0d后呈融合状态。两种方法培养的骨髓基质细胞增殖活性差异无显著性意义（P〉0.05）。结论：改良Dexter法及IMDM培养基培养均可使骨髓基质细胞在短期内贴壁及增殖。IMDM培养基由于不需加氢化可的松,故比改良Dexter法更易于操作。

BACKGROUND： Bone marrow stromal cells were isolated and cultured in vitro. After bone marrow transplantation, bone marrow stromal cells were injected into rats with severe combined immuno-deficiency, which is significant for enhancing hematogenesis and immunologic reconstitution. OBJECTIVE： To compare the growth condition of bone marrow stromal cells under different culture conditions, and to search for a simple and practical in vitro culture method of bone marrow stromal cells. DESIGN, TIME AND SETTING： The in vitro control observation experiment was performed at the Laboratory of Department of Dermatology, Taiyuan Central Hospital from July 2006 to January 2007. MATERIALS： Human bone marrow was obtained from Department of Hematology, Taiyuan Central Hospital after the agreement of patients. METHODS： Human bone marrow mononuclear cells were harvested and cultured by the modified Dexter method. Bone marrow mononuclear cells were adjusted into 1 × 10^-9/L in RPMI 1640 medium containing 5×10^-7 mol/L dermacort, and then incubated in 24-well plate, 1 mL in each well. Medium was changed after 2 days. Non-adhered cells were removed. From then on, half of medium was changed once every three days. When half celts were confluent, passage began. At passages 2-3, cells were digested in trypsin, collected and frozen in liquid nitrogen. Cells in the experimental group were cultured in IMDM medium without dermacort. The medium contained the same component as control group under the same condition. Two days later, medium was changed completely, and non-adhered cells were removed. Subsequently, the medium was changed once every 4 days. Passage and collection method were the same as the control group. MAIN OUTCOME MEASURES： Cell adherence, morphology and proliferation were observed under an inverted microscope. Cell proliferation activity was detected by MTT. RESULTS： Under the inverted microscope, cells were adhered and expanded 24 hours after incubation in both groups. Adhered cells became more over time. At 14 days, cells expanded on the bottom of the flask till confluence. After digestion, passaged cells were deformed, and recovered 24 hours later, and then adhered, proliferated in a similar morphology as primary cells. At 4 5 days, cells were confluent. No significant difference was detected in the bone marrow stromal cell proliferation by both methods （P〉 0.05）. CONCLUSION： Both modified Dexter method and IMDM medium method can promote the adherence and proliferation of bone marrow stromal cells in a short time. It is the simple and practical culture method of bone marrow stromal cells by culturing the cells in the medium of IMDM compared to the modified Dexter method.