【作者】 马兰兰；

【导师】 庞希宁；

【作者基本信息】 中国医科大学 ， 发育生物学， 2010， 硕士

【摘要】 目的间充质干细胞(mesenchymal stem cells, MSCs)是来源于发育早期的中胚层和外胚层,具有多向分化潜能、造血支持和促进干细胞植入和自我复制等特点的一类细胞,在体内或体外特定的诱导条件下,可分化为骨、软骨、肌肉、脂肪、内皮等多种表型组织细胞,传代培养和冷冻保存后仍具有多向分化潜能,可作为理想的种子细胞用于衰老和病变引起的组织器官损伤修复。MSCs多取材于骨髓,属侵袭性取材,制约其应用于临床。脐带血(umbilical cord blood, UCB)是胎儿娩出、脐带结扎并离断后残留在胎盘和脐带中的血液,含有丰富的干细胞,临床常采集脐带血中造血干细胞用于治疗血液类疾病。最近几年来有研究表明,脐带血中也存在MSCs[2]。由于脐带血来源丰富,采集方便,排斥小,无伦理争议等优点,脐血来源MSCs作为移植用细胞越来越受到重视。脐带血MSCs缺少特异性表面标志物,此类细胞表达CD29、CD44、CD73、CD90和HLA-ABC, CD 105和CD106,不表达CD14、CD34、CD45、CD133和HLA-DR,并可分化为成骨、软骨、成脂和成肌样细胞。有学者应用流式细胞学单管多色染色技术分析脐带血内MSCs的数量,每百万个有核细胞当中含有8.8±4.3个MSCs细胞,而骨髓样本当中每百万个有核细胞当中含有13.2±5.8个MSC细胞。分析了脐血MSCs表面抗原表达情况,建立了应用流式细胞学单管多色染色分析脐带血内MSCs数量的方法。有报道说脐带血中MSCs数量变化较大.脐带血中MSCs的数量能有多少,随着胎儿成熟,其数量是否发生变化,]MSCs体外培养其生长速度如何,培养的成功率如何,脐带血中MSCs在胎儿发育中是扮演了什么样的角色,发挥了怎样的作用都是值得探讨的问题。本实验应用单管多色荧光染色技术分析了不同胎龄脐带血中CD44、CD105阳性而CD34、CD45阴性细胞的数量,以比较不同胎龄脐带血中MSCs数量的差别,并分析脐带血成功培养出MSCs的影响因素,比较不同胎龄脐带血获得的MSCs的生长特性,为进一步探讨脐带血MSCs生物学特性打下基础。方法1、单管多色染色行流式细胞学分析脐带血中MSCs的数量(1)无菌条件下取正常脐带血52份,胎龄从28周～42周,产妇及胎儿无血液系统疾病及其他影响造血功能的病变,(2)每份各取适量血进行单管多色染色行流式细胞学分析,(3)比较不同胎龄脐带血中MSCs的数量。2、培养(1)无菌条件下取37周～40周脐带血12份,28周～36周脐带血12例,用含抗凝保护液的采血袋抽取,分别作为足月组和非足月组。(2)每组各例分别取适量血进行单管多色染色行流式细胞学分析(3)将脐血稀释后,直接用Ficoll分离单个核细胞,采用含10%血清浓度的L-DMEM常规间充质干细胞贴壁培养；(4)对贴壁细胞类型、生长速度进行观察记录；(5)对各组成功培养出间充质干细胞的例数进行统计分析；(6)对分离出的脐血间充质干细胞进行鉴定。3、统计学分析。结果1、流式细胞学结果：52例脐带血中CD44(+)、CD105(+)/CD34(-)、CD45(-)细胞在10例28周～30周脐带血中的数量为15±0.2个／百万有核细胞；在12例31周～33周脐带血中为11±0.3个／百万有核细胞；在10例34周～36周脐带血中为10±0.1个／百万有核细胞；在10例37周～39周脐带血中为6±0.5个／百万有核细胞；在10例40周～42周脐带血中,为2±0.3个／百万有核细胞。可见随胎龄增加,检测到的MSC数量减少。在用来培养的脐带血足月组中CD44(+)、CD105(+),而CD34(-)、CD45(-)的细胞数量为6.2±0.2个／百万有核细胞,非足月组为10.3±0.2个／百万有核细胞,两组间存在显著性差异(P<0.01)。2、足月组与非足月组间充质干细胞生长特性比较足月组单个核细胞培养96小时后贴壁细胞均出现梭形纤维状的细胞和圆形细胞两种形态细胞,传代后有1／12份脐血出现旋涡状排列的间充质样的细胞,其余11／12份传代后只有少量梭形细胞混杂,胰酶难以消化下来,最终老化死亡。非足月组单个核细胞培养72小时后,贴壁细胞多为纤维状的间充质样细胞,传代后3／12份脐带血分离出了间充质样梭形细胞,且生长速度明显快于足月组。3、脐血分离培养间充质干细胞成功率足月组分离培养间充质千细胞总的成功率为8.3%,非足月组为25.0%。4、对分离出的脐血MSCs进行鉴定两组培养所获得梭形细胞免疫细胞化学染色检测MSCs标志物表达：CD45(-),CD90(+)。结论1、脐带血中含有MSCs,随胎龄增加数量减少；2、非足月产脐带血间充质干细胞接种成功率高于足月产脐带血的接种成功率；3、非足月产脐带血间充质干细胞生长速度快于足月产脐带血间充质干细胞生长速度。更多还原

【Abstract】 PurposeMesenchymal stem cells (mesenchymal stem cells, MSC) is derived from the development of the early mesoderm and ectoderm, with multi-differentiation potential of hematopoietic stem cells implanted to support and promote and self-replication features of a class of cells in vivo or in vitro Induction of specific conditions, differentiate into bone, cartilage, muscle, fat, endothelium and other tissue cells phenotype, Subculture and cryopreservation after a multi-directional differentiation potential can be used as an ideal seed cells for the aging and disease caused by injury and repair tissues and organs. Mesenchymal stem cells, many drawn from the bone marrow, an invasive subjects, restricting its clinical application. Umbilical cord blood (umbilical cord blood, UCB) is the baby is delivered, the umbilical cord ligation and away from the fractured left in the placenta and umbilical cord blood is rich in stem cells, umbilical cord blood collected in regular clinical hematopoietic stem cells for the treatment of blood diseases. In recent years studies have shown that cord blood there are also MSC. Because cord blood is rich in easily collected, exclusion of small, non-ethical arguments for the advantages of cord blood derived mesenchymal stem cells as transplant cells to more attention.The lack of specificity of cord blood MSCS surface markers, these cells expressed CD29, CD44, CD73, CD90 and HLA-ABC, CD105, and CD106, did not express CD14, CD34, CD45, CD133, and HLA-DR, and can be divided as bone, cartilage, fat into muscle-like cells and adult. Some scholars have applied single-tube multi-color flow cytometry analysis of the blood staining the number of MSCs,for every one million nucleated cells, which contain 8.8±4.3 MSC cells, bone marrow samples for every one million nucleated cells were containing 13.2±5.8 MSC cells. MSC also reported in the literature the number of cord blood varied affected by many factors. We apply single-tube multi-color fluorescence staining technical analysis of different gestational age cord blood CD44, CD 105 positive and CD34, CD45-negative cells, and colony forming capacity of cultured cells, made after the analysis to compare the number of different gestational age cord blood MSC difference, and analysis of umbilical cord blood successfully cultivated MSC related factors, in order to further explore the biological characteristics of umbilical cord blood MSC basis.Methods1. single-tube multi-color flow cytometry analysis of stained(1)sterile conditions, take a normal (28 weeks to 42 weeks) umbilical cord blood 52 cases of umbilical cord blood.(2)each amount of blood were collected from all cases for single-tube multi-color flow cytometry analysis of stained.(3)Compare the number of MSCs with different terms.2.Cell culture(1)sterile conditions, take a normal full-term (37 weeks to 40 weeks) umbilical cord blood of 12 non-full-term (28 weeks to 36 weeks) 12 cases of umbilical cord blood, CAPD-1 compound anticoagulant anticoagulant, respectively, As a full-term group and non-term group.(2)each amount of blood were collected from all cases for single-tube multi-color flow cytometry analysis of stained(3)the cord blood dilution, the direct use of Ficoll mononuclear cells containing 10%serum with the concentration of L-DMEM conventional mesenchymal stem cells in adherent culture;(4) pairs of two the number of mononuclear cells was compared; wall cell types, the growth rate of observation records; right to successfully cultivate the umbilical cord blood mesenchymal stem cells, the number of cases for statistical analysis;(5)colony-forming ability of analysis and comparison;(6)pairs of isolated umbilical cord blood mesenchymal stem cells were identified.3.statistical analysis.Results1. the results of flow cytometry:CD44 (+), CD105 (+), and CD34 (-), CD45 (-) cell content in 28weeks～30 weeks was 15±0.2/1 million nucleated cells, in 31weeks～33 weeks was 11 i 0.3, in 34 weeks～36 weeks was 10±0.1/1 million nucleated cells, in 37 weeks～39 weeks was 6±0.5/1 million nucleated cells, in 40 weeks～42weeks was 2±0.3/1 million nucleated cells;in full-term group was 6.2±0.2/1 million nucleated cells, the non-full-term group was 10.3±0.2/1 million nucleated cells, there was a significant difference (P<0.01);2. two separate comparison of cord blood mononuclear cellsFull-term group and non-term group received the number of cord blood nucleated cells, respectively ((2.8±0.3) X 106, (3.5±0.5) X 107, there was a significant difference (P<0.05); two methods of separation nucleated cells, no significant difference in mortality (P> 0.05); two groups of cells cultured for 7 days later in 30cm2 culture bottle cloning of the number of adherent cells were 18±0,27±8, there was a significant difference (P<0.05).3. full-term group and non-term group of mesenchymal stem cells ComparisonFull-term group of mononuclear cells cultured adherent cells after 96 hours there were spindle cells and round cells, fibrous two forms of cells after passage 1/12 cord blood units appear ordered spiral-shaped mesenchymal-like cells The remaining 11/12 were passaged after only a few spindle cells intermingled, trypsin digestion down hard to the final aging death. Non-term group of mononuclear cells cultured for 72 hours, adherent cells, mostly fibrous mesenchymal-like cells, after passage 3/12 were isolated from the umbilical cord blood mesenchymal-like spindle cells, and the growth rate markedly higher than in full-term group. 4. isolated and cultured umbilical cord blood mesenchymal stem cells, the success rate of non-full-term group of isolated and cultured mesenchymal stem cells the overall success rate was 8.3%, full-term group was 25.0%.5. pairs of isolated umbilical cord blood mesenchymal stem cells for identification of obtained two sets of spindle cells cultured immune cells staining MSCs marker expression:CD45 (-), CD90 (+).Conclusion1. cord blood contains mesenchymal stem cells, the non-full-term umbilical cord blood mesenchymal stem cells in the middle of content is more than full-term umbilical cord blood mesenchymal stem cells in the middle of content.2. non-full-term umbilical cord blood mesenchymal stem cells combined with the success rate is higher than full-term umbilical cord blood vaccination success rate.3. non-full-term umbilical cord blood mesenchymal stem cells grow faster than full-term umbilical cord blood mesenchymal stem cells in the growth rate.更多还原