不同传代倍数胎儿间充质干细胞的成骨分化能力观察  

Observation of the capability of osteogenic differentiation from fetus mesenchyme stem cells in distinct transfer of culture power

作　　者：李敏霞[1] 孟保福 龚红梅[1] 王利霞[1] 

机构地区：[1]安阳市人民医院检验科,河南省安阳市455000

出　　处：《中国临床康复》2005年第30期48-50,i0001,共4页Chinese Journal of Clinical Rehabilitation

摘　　要：目的:观察不同传代倍数胎儿骨髓间充质干细胞定向诱导分化为成骨细胞的能力,为骨髓间充质干细胞在骨组织工程中的应用提供部分实验参数。方法:实验于2004-03/10在安阳市人民医院检验科和安阳肿瘤医院临床实验室完成。采用全髓直接接种法分离培养13～18周胎龄水囊引产新鲜胎儿股骨骨髓(获提供者知情同意标本用于此实验)。贴壁细胞达90%以上融合时消化传代。传代细胞部分以1×109L-1的细胞密度接种于含体积分数为0.1的胎牛血清的L-DMEM的培养基中继续培养,传代;部分在培养基中添加成骨诱导剂地塞米松,β-甘油磷酸钠及抗坏血酸向成骨细胞诱导分化。如此连续传10代。倒置显微镜下观察细胞形态、细胞化学染色检测成熟成骨细胞的标志酶碱性磷酸酶的表达、全自动生化分析仪测定碱性磷酸酶活性,鉴定不同代次培养细胞成骨分化能力。结果:①碱性磷酸酶染色阳性百分率:传代7代以内胎儿骨髓间充质干细胞成骨分化能力无显著差异,均在87.5%以上(P>0.05);以第3代最强达93.4%。第8代后逐渐减弱,为72.7%～51.3%。②碱性磷酸酶活性:传代7代以内无差异(P>0.05),以第3代最高,为(2307.1±15.0)nkat/L,第8代后逐渐降低,至第10代仅为(905.2±10.0)nkat/L。结论:不同代次的骨髓间充质干细胞诱导分化为成骨细胞的能力不同,8代以后明显减弱。提示脱离了体内环境后,骨髓间充质干细胞逐渐老化。做为组织工程的种子细胞,骨髓间充质干细胞体外培养不宜超过7代。AIM： To observe the capability of fetus bone marrow meseochyme stem cells in distinct transfer of culture power orientation inductive differentiation into osteoblast, which provides part of test parameter for the application of bone marrow mesenchyme stem cells in the bone tissue engineering. METHODS： The experiment was conducted in Department of Clinical Laboratory, Anyang People＇s Hospital and Clinical Laboratory, Anyang Tumor Hospital from March to October 2004. The fresh femoral bone marrow of 13-18 weeks conceptus age embryo by induction of labor with water bag were fractional cultivation by holo-marrow direct inoculation （the sample used in this experiment with the knowledge and agreement of provider）.When the adherence cells were confluence above 90%,then those were digested and went down to posterity.All passage cells were separated into 1×10^9L^-1 cell density, and then were inoculated in L-DMEM culture contained 0. I-volume fraction of fetus ox blood serum （FBS） to go on cultivation and passage. The osteogenic inducer desamethasone,β-sodium glycerophosphate and ascorbic acid were added in part of the culture medium, which were used to induce differentiation into osteoblast.Ten generations were passed on in this way. The formation of cells was observed under the invert microscope; The expression of alkaline phosphatase （ALP）, the marker enzyme of mature osteoblast, was detected by the cytochemistry staining＇, The activity of ALP was ascertained by automatic biochemistry analyzer, by means of evaluating the capability of osteogenic differentiation from aniso-generation times cultivation cells. RESULTS： ① The positive percentage of ALP staining： In 7 generation times the capability of osteogenic differentiation from fetal bone marrow mesenchyme stem cells had insignificant difference, all over 87.5% （P 〉 0.05）; It was 93.4% of the 3^rd generation that was the most powerful. It was 72.7-51.3% from the 8^th generation which reduced gradually.② The activity of ALP： There

关 键 词：骨髓细胞 干细胞 成骨细胞 细胞分化 

分 类 号：R714[医药卫生—妇产科学]