脐血间充质干细胞的分离扩增及向成骨及脂肪细胞的分化

目的探讨新生儿脐血间充质干细胞（mesenchymal stem cells，MSCs）体外分离、纯化、扩增，以及向成骨及脂肪细胞定向诱导分化的方法与条件。方法无菌条件下收集新生儿脐血60～120ml，枸橼酸钠抗凝，以Ficoll—Hypaque淋巴细胞分离液密度梯度法、沉降红细胞后密度梯度法及CD34^＋免疫磁珠负选法分离单个核细胞（mononuclear cells，MNCs）。分离获得的MNCs采用L—DMEM培养基或Mesencult^TM培养基／10％胎牛血清进行MSCs培养传代，获得第3代集落生长细胞作流式细胞仪表面抗原测定，并向成骨及脂肪细胞定向诱导分化，成骨细胞钙沉积经茜素红染色鉴定，脂肪细胞胞浆油滴经油红染色鉴定。结果经沉降红细胞后分离的MNCs，使用Mesencult^TM培养基／10％胎牛血清培养成功率高，第3代可出现明显的集落生长，而另两种方法分离培养的细胞则难以形成集落；集落细胞表面抗原测定表达CD29、CD59、CD71而不表达CD34、CD45及HLA—DR等分子。成骨定向诱导分化的集落细胞经茜素红染色胞浆中出现有大量的钙沉积；成脂肪定向诱导分化的集落细胞油红染色示胞浆充满油滴空泡。结论新生儿脐血中可分离出MSCs，并可在体外进行培养扩增。以甲基纤维素沉降红细胞后密度梯度离心分离的MNCs培养较为有效，集落细胞表达基质细胞表面抗原，能够向成骨细胞及成脂肪细胞定向诱导分化。     

5'-氮杂胞苷对激素性股骨头坏死骨髓间充质干细胞分化作用的实验研究

[目的]探讨5'-氮杂胞苷(5'-Azacytidine,5'-Aza-dc)对激素性股骨头坏死患者骨髓间充质干细胞(human mesenchymal stem cells,h MSCs)增殖能力的影响及其对成骨、成脂分化能力的作用。[方法]2012年8月~2013年4月,经患者知情同意和武汉协和医院伦理委员会审批后,选取22例激素性股骨头坏死患者和20例的股骨颈骨折患者,在严格无菌条件下抽取患者股骨骨髓,经密度梯度离心法体外分离、培养h MSCs,贴壁细胞传代,取第3代细胞实验,使用流式细胞仪进行细胞表面抗原标记物检测,确定最适5'-氮杂胞苷干预浓度后干预培养72 h,随后对三组h MSCs进行成骨、成脂分化诱导培养2周,接着对细胞进行碱性磷酸酶含量测定(alkaline phosphatase,ALP)、茜素红和油红O染色,分析实验结果。[结果]激素性股骨头坏死组h MSCs集落形成能力比正常对照组有显著下降(P<0.001)。使用15μmol/L 5'-Aza-dc干预培养后能在一定程度上促进激素性股骨头坏死组h MSCs增殖能力,且干预培养72 h的h MSCs在成脂分化诱导培养2周后,通过油红O染色发现其成脂分化能力接近正常对照组,而5'-Aza-dc干预培养72 h的h MSCs在成骨分化诱导培养2周后,根据碱性磷酸酶检测、茜素红染色发现其成骨分化潜能较干预前明显提高,并接近正常对照组水平。[结论]15μmol/L 5'-氮杂胞苷干预培养激素性骨坏死患者h MSCs,通过改变细胞成骨和成脂分化的平衡,能促进细胞的增殖能力,使成骨能力加强、成脂能力减弱。     

人脐血间充质干细胞的分离培养及其多向分化潜能的实验研究

目的 研究人脐血间充质干细胞(MSCs)分离培养的生物学特性及其向成骨、成脂诱导分化的能力.方法 从人脐血中分离扩增MSCs,显微镜下观察其形态及生长情况,绘制生长曲线,电镜下观察超微结构,流式细胞仪检测细胞表面标志物;成骨、成脂诱导后以碱性磷酸酶(ALP)染色、茜素红染色鉴定MSCs成骨分化潜能,油红O染色鉴定成脂分化潜能.结果 人脐血MSCs为成纤维细胞样,漩涡状贴壁生长排列,传至第110代细胞形态无明显变化;电镜下显示为低分化细胞;细胞表面不表达CD34和CD45,强表达CD29、CD44和CD90;成骨诱导后可检测到ALP表达及钙化结节形成;成脂诱导后可检测到脂滴形成.结论 人脐血中可分离出MSCs,与其他来源的MSCs具有类似的生物学特性及多向分化潜能,脐血有可能成为骨组织工程种子细胞的来源.     

大鼠骨髓间充质干细胞分离培养及多向分化研究的体外实验

目的 分离培养较高纯度大鼠骨髓间充质干细胞（bone marrow mesenchymal stem cells,BMSCs）,检测其多向分化潜能.方法 采用密度梯度离心结合差速贴壁法分离培养大鼠BMSCs,观察细胞形态,检测表面标志物CD34、CD44、CD45、CD90表达情况,及其成骨、成脂、成神经诱导后茜素红染色、油红O染色和NSE、GFAP免疫荧光染色情况.结果 细胞呈典型的成纤维细胞样形态,CD44和CD90呈阳性表达,CD34和CD45呈阴性表达,细胞纯度＞98%.成骨、成脂、成神经诱导后,茜素红染色、油红O染色、NSE和GFAP免疫荧光均为阳性.结论 密度梯度离心结合差速贴壁法可分离、培养出高纯度的大鼠BMSCs,该细胞具有成脂、成骨、成神经多向分化潜能,是脊髓损伤修复的一种较理想的种子细胞.     

脐血间充质干细胞的分离培养鉴定及诱导分化研究

目的：分离培养人脐血间充质干细胞（human umbilical cord blood-derived mesenchymal stem cells,hUCB-MSC）,体外观察其生长特性,并在特定条件下诱导分化,探讨其成脂成骨分化能力.方法：采用沉降法和密度梯度离心结合贴壁培养法自脐血中分离间充质干细胞,倒置显微镜下观察其形态及生长情况;流式细胞仪分析细胞周期并检测细胞表面标志物;用茜素红染色和油红0染色分别鉴定其成骨成脂分化能力.结果：纯化的hUCB-MSC贴壁生长,呈均一梭形,具有较强的增值能力,流式细胞仪分析P3代hUCB-MSC稳定表达间充质干细胞表面抗原标志CD73,CD105和CD90等,不表达造血标志CD34和CD45;成骨诱导后3周后细胞茜素红染色阳性;成脂诱导3周后细胞油红0染色阳性.结论：本实验分离的hUCB-MSC具有较强的增殖能力,表达间充质干细胞的表面标记,具有成骨成脂分化潜能.     

人骨髓间充质干细胞分离培养及体外诱导分化为成骨细胞

[目的]建立人骨髓来源的间充质干细胞( hBMSCs)分离、培养及传代的方法,观察成hBMSCs体外成骨潜能.[方法]采用全骨髓贴壁筛选法分离培养hBMSCs,流式细胞仪检测细胞表型;所得细胞第3代用含100 nmol/L地塞米松、5mmol/Lβ -甘油磷酸钠,50 μg/ml抗坏血酸的条件培养基进行骨诱导,茜素红染色鉴定.[结果]分离培养的细胞流式细胞术检测显示CD44、CD105阳性,而CD34、CD45阴性,符合MSCs特征;hBMSCs经骨诱导后可形成钙结节,茜素红染色显示阳性.[结论]全骨髓贴壁筛选法可分离获得高纯度的hBMSCs,其在体外具有成骨潜能.     

大鼠SERTOLI细胞对骨髓间充质干细胞体外诱导分化的影响

目的探索Sertoli细胞(SCs)对骨髓间充质干细胞(BMSCs)成脂、成骨和成神经诱导分化的影响。方法密度梯度离心法分离BMSCs,二步酶消化法分离SCs;对第3、4代BMSCs进行成脂、成骨及成神经诱导,并与SCs共培养,观察其形态学变化。结果油红O染色显示BMSCs成脂诱导后,胞浆出现折光性强的脂滴,加入SCs后,脂滴明显减少,染色不明显;茜素红S染色显示BMSCs成骨诱导后可显示钙结节,加入SCs后,钙结节更多,结节内红染更明显;成神经诱导在加入SCs前后没有明显差异。结论 SCs可促进BMSCs成骨分化,而抑制其成脂分化,对成神经分化则没有明显影响。     

不同组织来源间充质干细胞体外成骨分化能力的比较研究

目的比较成人脂肪间充质干细胞(ASCs)、脐带间充质干细胞(UC-MSCs)和成人骨髓间充质干细胞(BMSCs)的成骨能力,选择优势干细胞种类作为应用于骨组织工程治疗骨缺损的种子细胞。方法采用含10%胎牛血清的DMEM/Ham’s F-12培养液培养3种MSCs。取3种MSCs的P3代,通过CCK8方法检测其增殖能力;通过流式细胞仪进行鉴定;通过碱性磷酸酶(ALP)和茜素红染色检测骨分化蛋白ALP的分泌和矿化钙结节的沉积,并对钙结节进行定量分析;通过实时荧光定量PCR(RT-q PCR)方法检测骨再生相关基因的表达。结果 3种P3代MSCs在3~5d之间增殖均处于对数生长期;流式鉴定3种细胞的表面标志物阳性率:CD44、CD90和CD105均高于97%,阴性率:CD14、CD34和CD45均低于1%;ALP染色结果显示3种MSCs成骨诱导9d时,细胞内均表达ALP,茜素红染色结果显示成骨诱导18d时,均呈现较好的矿化能力,BMSCs和UC-MSCs成骨诱导后形成的钙结节无显著性差异;RT-q PCR结果显示3种MSCs成骨诱导组相比较于对照组,成骨再生相关基因Osterix、ALP、I型胶原(COL1)和骨钙素(OCN)均显著性高表达;3种MSCs成骨诱导9d时,UC-MSCs实验组的COLI基因表达显著性高于BMSCs,成骨诱导18d时,ASCs实验组的Osterix基因表达显著性高于BMSCs。结论 ASCs和UC-MSCs具有一定的成骨矿化能力,有望成为骨组织工程治疗骨缺损的种子细胞。     

1例基因芯片筛选青少年特发性脊柱侧凸骨髓间充质干细胞成脂分化相关差异表达基因

[目的]应用基因芯片技术筛选青少年特发性脊柱侧凸(AIS)骨髓间充质干细胞(hBMSCs)成脂分化相关的差异表达基因.[方法]收集AIS及同性别年龄正常对照骨髓标本,密度梯度离心法分离MSCs并培养传代,取第3代MSCs进行成脂诱导2周后,提取各标本的总RNA,反转录成cDNA,并以两种荧光Cy5和Cy3标记后作为探针与人类全基因组表达谱基因芯片杂交,扫描芯片荧光信号图像后用基因图像分析软件进行数字化处理和分析.[结果]筛选出显著表达差异基因1 380条,其中表达上调基因874条,表达下调基因506条.[结论]AIS来源MSCs成脂分化相关差异表达的1 380条基因可能参与了AIS的发生和发展.     

恒河猴骨髓间充质干细胞体外培养及诱导分化成骨细胞的实验研究

[目的]体外分离培养非人类灵长类动物恒河猴骨髓MSCs,研究其生长、扩增及诱导分化为成骨细胞的特性.[方法]梯度离心法分离、纯化恒河猴骨髓源MSCs,观察不同接种密度和换液时间对细胞生长的影响;在体外应用成骨添加剂(含地塞米松10-7mol/L、β-甘油磷酸钠10 mmol/L、维生素C 50 mg/L)或rhBMP-2(100 ng/L)定向诱导分化,对分化后的细胞进行免疫组化染色和ALP、BGP定量测定以鉴定成骨细胞,并比较两种定向诱导分化方法的效果.[结果]梯度离心法分离、纯化得到的恒河猴骨髓源MSCs具有分裂和克隆增殖的能力,种植细胞密度为10×104～30×104/cm2,48 h后半量换液,以后每3 d全量换液的方法较合理,MSCs能少量表达ALP活性,分泌的钙量较少;诱导分化后的细胞具有和体内成骨细胞相同的形态学特征,ALP染色阳性,能表达Ⅰ型胶原,不表达Ⅱ型胶原,细胞融合后3 d,ALP和BGP分泌明显增加,融合后14 d细胞ALP、BGP的分泌水平略有升高,但无明显增加;采用成骨添加剂和rhBMP-2诱导分化MSCs后相同时期成骨细胞ALP活性、BGP含量无明显差异.[结论]密度梯度离心法分离得到的非人类灵长类动物骨髓源MSCs,体外培养具有分裂、克隆增殖的能力,在加有成骨细胞诱导剂或rhBMP-2的培养基里能诱导分化为成骨细胞,化学药物在定向诱导分化过程中具有和生长因子相一致的作用.     

Circulating plastic adherent mesenchymal stem cells in aged hip fracture patients

We examined the presence of circulating plastic adherent multipotent mesenchymal stem cells (MSCs) in fracture patients. Three patient groups (n = 10–18) were evaluated, including elderly females with a femoral neck fracture treated with cemented hemiarthroplasty, an age‐ and sex‐matched group with hip osteoarthritis (OA) treated with cemented total hip arthroplasty (THA), and younger adults with surgically treated lower extremity fractures. The presence of circulating MSCs pre‐ and postoperatively was compared to bone marrow (BM) MSCs from the same subjects. Criteria for identifying MSCs included cell surface markers (CD105+, CD73+, CD90+, CD45?, CD14?), proliferation through several passages as well as osteogenic, chondrogenic, and adipogenic differentiation. Plastic adherent MSCs were found in peripheral blood (PB) from 22% of hip fracture patients, 46% of younger fracture patients, and in none of 63 pre‐ and postmenopausal women with hip OA. When detectable, circulating MSCs appeared between 39 and 101 h after fracture. PB derived MSCs did not differ from BM derived MSCs, except for a small population (<15%) of CD34+ cells among PB derived MSCs. This initial study indicates mobilization of MSCs into the circulation in response to fracture, even in very old patients, while circulating MSCs were not detectable before or after elective THA. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1634–1642, 2010     

Number and proliferative capacity of osteogenic stem cells are maintained during aging and in patients with osteoporosis.

Decreased bone formation is an important pathophysiological mechanism responsible for bone loss associated with aging and osteoporosis. Osteoblasts (OBs), originate from mesenchymal stem cells (MSCs) that are present in the bone marrow and form colonies (termed colony-forming units-fibroblastic [CFU-Fs]) when cultured in vitro. To examine the effect of aging and osteoporosis on the MSC population, we quantified the number of MSCs and their proliferative capacity in vitro. Fifty-one individuals were studied: 38 normal volunteers (23 young individuals [age, 22-44 years] and 15 old individuals [age, 66-74 years]) and 13 patients with osteoporosis (age, 58-83 years). Bone marrow was aspirated from iliac crest; mononuclear cells were enriched in MSCs by magnetic activated cell sorting (MACS) using STRO-1 antibody. Total CFU-F number, size distribution, cell density per CFU-F, number of alkaline phosphatase positive (ALP+) CFU-Fs, and the total ALP+ cells were determined. In addition, matrix mineralization as estimated by alizarin red S (AR-S) staining was quantified. No significant difference in colony-forming efficiency between young individuals (mean +/- SEM; 87 +/- 12 CFU-Fs/culture), old individuals (99 +/- 19 CFU-Fs/culture), and patients with osteoporosis (129 +/- 13 CFU-Fs/culture; p = 0.20) was found. Average CFU-F size and cell density per colony were similar in the three groups. Neither the percentage of ALP+ CFU-Fs (66 +/- 6%, 65 +/- 7%, and 72 +/- 4% for young individuals, old individuals, and patients with osteoporosis, respectively) nor the percentage of ALP+ cells per culture (34 +/- 5%, 40 +/- 6%, and 41 +/- 4%) differed between groups. Finally, mineralized matrix formation was similar in young individuals, old individuals, and patients with osteoporosis. Our study shows that the number and proliferative capacity of osteoprogenitor cells are maintained during aging and in patients with osteoporosis and that other mechanisms must be responsible for the defective osteoblast (OB) functions observed in these conditions.     

Comparison of the osteogenic capacity of minipig and human bone marrow‐derived mesenchymal stem cells

Minipigs are a recommended large animal model for preclinical testing of human orthopedic implants. Mesenchymal stem cells (MSCs) are the key repair cells in bone healing and implant osseointegration, but the osteogenic capacity of minipig MSCs is incompletely known. The aim of this study was to isolate and characterize minipig bone marrow (BM) and peripheral blood (PB) MSCs in comparison to human BM‐MSCs. BM sample was aspirated from posterior iliac crest of five male Göttingen minipigs (age 15 ± 1 months). PB sample was drawn for isolation of circulating MSCs. MSCs were selected by plastic‐adherence as originally described by Friedenstein. Cell morphology, colony formation, proliferation, surface marker expression, and differentiation were examined. Human BM‐MSCs were isolated and cultured from adult fracture patients (n = 13, age 19–60 years) using identical techniques. MSCs were found in all minipig BM samples, but no circulating MSCs could be detected. Minipig BM‐MSCs had similar morphology, proliferation, and colony formation capacities as human BM‐MSCs. Unexpectedly, minipig BM‐MSCs had a significantly lower ability than human BM‐MSCs to form differentiated and functional osteoblasts. This observation emphasizes the need for species‐specific optimization of MSC culture protocol before direct systematic comparison of MSCs between human and various preclinical large animal models can be made. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1019–1025, 2012     

人羊水来源c-kit~+间充质干细胞的生物学特征和心肌诱导分化

目的探讨人羊水来源c-kit~+间充质干细胞(c-kit~+human amniotic fluid-derived mesenchymal stemcells,c-kit~+HAFMSCs)的生物学特征及心肌诱导分化能力。方法通过产前诊断或自愿引产获取50份孕中期羊水标本,经流式细胞仪分选c-kit~+HAFMSCs,MTT法观察细胞增殖情况,并通过流式细胞仪、细胞免疫化学染色观察行细胞表型鉴定。在体外经成骨及成脂诱导分化,于诱导培养后21 d采用yon Kossa染色观察钙沉积颗粒,油红O染色观察脂滴形成情况。实时荧光定量PCR检测细胞经5氮-2’脱氧胞苷心肌诱导前后,NKx2.5、Tbx5、GATA-4、α-MHC 4种心肌特异性基因表达情况。结果经流式细胞仪分选,人孕中期羊水中c-kit~+HAFMSCs含量约为贴壁细胞的3.07%±1.03%;体外增殖迅速,表达MSCs表面标志CD29、CD44、CD73、CD90、CD105,不表达造血干细胞表面标志CD34、CD45;农达人类白细胞抗原(human leukocyte antigen,HLA)-ABC,不表达HLA-DR。细胞免疫化学染色示CD29、CD44、CD73、CD90、CD105、Oct-4染色呈阳性,CD34、CD45染色呈阴性。MTT检测示第5、10、15代c-kit~+HAFMSCs具有相似的生长曲线。成骨和成脂诱导培养后21 d,细胞内有钙盐沉积和脂滴形成。实时荧光定量PCR检测示,心肌诱导后14 d,NKx2.5、Tbx5、GATA-4、α-MHC 4种心肌特异性基因表达均较诱导前明显增加,差异有统计学意义(P<0.05)。结论通过流式细胞仪分选的c-kit~+HAFMSCs在体外可以诱导分化为心肌样细胞,可能成为心肌再生性治疗的种子细胞。     

转基因骨髓间质干细胞在组织工程承载体中生长及成骨转化的研究

[目的]观察经携带人骨形态发生蛋白-2(BMP-2)基因的复制缺陷重组腺病毒(Ad-BMP-2)转染的兔骨髓间质干细胞(MSCs)在以兔脱细胞脱钙骨基质作为组织工程承载体中的生长情况及转基因前后细胞成骨能力的变化。[方法]用携带有人BMP-2基因片段的复制缺陷重组腺病毒(Ad-BMP-2)转染兔骨髓间质干细胞并将其种植在兔脱细胞脱钙骨基质支架中,扫描电镜观察转基因细胞在支架中的黏附生长情况;并通过检测培养上清中的碱性磷酸酶(ALP)、骨钙素(BGP)含量及Ⅰ型胶原(CollagenⅠ)的分泌量来评估转基因对MSCs成骨能力的影响。[结果]转基因骨髓间质干细胞在组织工程支架中黏附生长良好,转基因组细胞的ALP、BGP含量及Ⅰ型胶原的分泌量与对照组比较差异有显著性意义。[结论]转基因骨髓间质干细胞在兔脱细胞脱钙骨基质支架中能很好的黏附并立体生长,转基因细胞在目的基因所表达的BMP-2蛋白作用下成骨能力明显增强。