大鼠骨髓基质细胞体外定向诱导成骨

将大鼠骨髓单细胞悬液静置培养36h，利用骨髓基质细胞贴壁能力强的特点对其进行纯化和扩增培养。采用爬片培养、HE染色、组化染色以及碱性磷酸酶活性和钙含量测定等手段研究培养骨髓基质细胞的形态、分化和分泌基质情况。结果表明，非诱导培养条件下骨髓基质细胞呈梭形，部分传代细胞中可观察到脂肪细胞和肌细胞。经成骨性诱导培养后，骨髓基质细胞发生明显的形态学变化，碱性磷酸酶活性上升，钙含量增加，最终形成典型的矿化结节。提示培养大鼠骨髓基质细胞具有分化成脂肪细胞和肌细胞的能力，但其分化成骨的潜能最为强大。本实验诱导骨髓基质细胞分化为成骨细胞的模式有可能适用于骨组织工程研究。     

Ⅰ型骨质疏松山羊骨髓基质细胞体外成脂、成骨分化的研究

观察体外定向诱导Ⅰ型骨质疏松山羊骨髓基质细胞（BMSCs）分化为脂肪细胞和成骨细胞.建立去卵巢骨质疏松山羊动物模型,全骨髓法分离培养其骨髓基质细胞.第二代细胞分别成脂、成骨诱导培养,油红O染色、碱性磷酸酶染色、茜素红染色判定其分化结果.成脂诱导21 d,油红O染色有脂滴形成;成骨诱导14 d,碱性磷酸酶染色阳性;连续诱导35 d,茜素红染色证实有钙化结节形成.Ⅰ型骨质疏松山羊骨髓基质细胞在体外可以定向诱导分化为脂肪细胞和成骨细胞.     

Ι型骨质疏松山羊骨髓基质细胞体外成脂、成骨分化的研究

观察体外定向诱导Ι型骨质疏松山羊骨髓基质细胞(BMSCs)分化为脂肪细胞和成骨细胞。建立去卵巢骨质疏松山羊动物模型,全骨髓法分离培养其骨髓基质细胞。第二代细胞分别成脂、成骨诱导培养,油红O染色、碱性磷酸酶染色、茜素红染色判定其分化结果。成脂诱导21d,油红O染色有脂滴形成;成骨诱导14d,碱性磷酸酶染色阳性;连续诱导35d,茜素红染色证实有钙化结节形成。Ι型骨质疏松山羊骨髓基质细胞在体外可以定向诱导分化为脂肪细胞和成骨细胞。     

大鼠骨髓基质干细胞分离培养及向成骨、成脂诱导分化的研究

目的利用细胞原代及传代培养技术将大鼠骨髓基质干细胞诱导分化为成骨细胞和脂肪细胞,为其进一步应用奠定基础.方法全骨髓法分离大鼠骨髓基质干细胞,传代后分别在成骨、成脂诱导条件下继续培养,采用碱性磷酸酶染色、茜素红染色及油红"O"染色观察其成骨及成脂分化结果.结果第2代大鼠骨髓基质干细胞成骨诱导9 d后碱性磷酸酶染色呈阳性细胞,连续诱导14 d后可见矿化结节形成,成脂诱导14 d后可见脂肪细胞形成.结论随着诱导条件的不同,大鼠骨髓基质干细胞在体外可定向分化为成骨细胞和脂肪细胞.     

人胚胎骨髓基质干细胞的长期培养及体外成骨特性

研究了人胚胎骨髓基质干细胞(hBMSCs)在体外长期培养时的基本特性和向成骨细胞的分化能力。结果表明：前三代hBMSCs多数呈长梭形,生长快速,增殖能力强;而后几代细胞变得比较扁平,生长缓慢,增殖能力下降;至第六代,细胞已失去增殖能力。每一代细胞生长均分为延滞期、对数生长期和稳定期,延滞期一般为6～7d,对数生长期为4～5d,最后是稳定期。前三代细胞对数生长期的群体倍增时间(PD71)基本相同,而第四代细胞的PDT略有上升,第五代细胞的PDT最大。在体外扩增能力方面,前三代细胞均可以扩增18倍左右,而第四、第五代细胞则下降至11倍、5倍。实验结果表明扩增后的细胞经过诱导可以形成钙化小结,与未诱导细胞相比碱性磷酸脂酶(ALP)活性显著提高。     

大鼠骨髓基质细胞培养的实验研究

目的探讨体外培养大鼠骨髓基质细胞的可行性,为细胞移植治疗疾病奠定基础.方法采用淋巴细胞分离液(1.077 g/L)离心分离MSCs.加入碱性成纤维生长因子(bFGF)进行培养,并用流式细胞仪进行荧光三标检测鉴定.结果分离后的MSCs在生长因子的作用下出现增殖性生长,经流式细胞仪检测显示CD90,CD106阳性,CD45阴性.结论可在体外培养出较大密度大鼠骨髓基质细胞.     

骨髓间充质细胞联合细胞外基质成血管能力的研究

选用8周龄的雄性SD大鼠,处死后分离骨髓单个核细胞,用DMEM培养基和细胞外基质进行培养,观察细胞集落.选用8周龄雌性SD大鼠,结扎一侧股动脉引起下肢缺血,随机分为4组,A组进行细胞外基质联合细胞移植;B组进行单纯的细胞移植;C组进行细胞外基质移植;D组进行生理盐水注射;2周后测定大鼠下肢活动能力、小血管数目、Y染色体SRY基因量评估细胞外基质和细胞联合移植的效果.在细胞外基质中培养得到的细胞集落形态学上呈血岛样外观,而在普通DMEM培养基中得到的细胞集落呈铺路石样外观.进行细胞移植的动物中,A组大鼠跛行距离最长(P<0.05);小血管密度最高(P<0.05);Y染色体SRY的DNA量最高(P<0.05).     

纳米氧化铈颗粒对骨髓基质细胞成骨分化和成脂分化的影响

以原代骨髓基质细胞(BMSCs)为模型,在细胞水平上,研究50nm氧化铈颗粒对BMSCs的活性、成骨分化和成脂分化的影响.结果表明,氧化铈颗粒促进BMSCs的活性,并呈现很好的剂量及时间依赖性,但其对BMSCs的碱性磷酸酶(ALP)活性、胶原的合成、矿化结节的形成以及成脂分化的影响是复杂的,作用浓度和时间是影响其分化的关键因素.这些实验结果为阐明纳米氧化铈的生物学效应机理及其在生物医学领域的研究应用提供科学依据.     

成人骨髓基质细胞的体外培养

本研究从成人的髋骨抽取骨髓液，离心后将骨髓基质细胞悬液接种培养，待细胞贴壁融合后，进行传代扩增培养．并采用流式细胞术进行细胞周期分析，结果表明原代培养和传代培养的细胞均为贴壁、形态不一的骨髓基质细胞，且此体外培养的骨髓基质细胞具有很强的增殖能力．本研究成功地建立了一套完整、简单、可行的体外分离、长期培养扩增人骨髓基质细胞(Human Bone Marrow Stromal Cells hBMSCs)的系统，证明成人骨髓基质细胞能够在体外长期增殖，为人的骨髓基盾细胞的理论研究和临床应用奠定了基础.     

骨髓基质细胞研究进展

骨髓基质细胞在多种疾病的治疗中起着重要的作用,对于其培养、分化、鉴定等问题目前众说纷纭,特别是它能否转化成神经细胞更是神经科学探讨的焦点所在,文章就以上问题进行综述.     

Effects of genistein, daidzein and glycitein on the osteogenic and adipogenic differentiation of bone marrow stromal cells and on the adipogenic trans-differentiation of osteoblasts

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), alkaline phosphatase (ALP) activity and oil red O assays were used to examine the effects of genistein, daidzein and glycitein on the osteogenic and adipogenic differentiation of primary mouse bone marrow stromal cells (MSCs) and the adipogenic trans-differentiation of primary mouse osteoblasts. The results indicated that daidzein, genistein and glycitein at concentrations from 1×10^-8 mol/L to 1×10^-5 mol/L promoted the proliferation of MSCs and osteoblasts; genistein, daidzein and glycitein promoted osteogenic differentiation and inhibited adipogenic differentiation of MSCs, and inhibited adipocytic transdifferentiation of osteoblasts at appropriate concentrations as 17β-estradiol. It suggests that genistein, daidzein and glycitein regulate a dual differentiational process of MSCs into the osteogenic and adipogenic lineages, and trans-differentiational process of primary osteoblasts into the adipocyte lineages, causing a lineage shift toward osteoblast. Protective effects of them on bone may be mediated by a reversal of adipogenesis which may promote the proliferation, differentiation and mineralization of osteoblasts, and make adipocytes secrete less cytokines which may promote osteoclast formation and activation. In addition, the results also indicated that genistein, daidzein and glycitein may be helpful in preventing the development of steroid induced osteonecrosis.     

碱性成纤维细胞生长因子对骨髓基质细胞的生物学效应

目的探讨碱性成纤维细胞生长因子(bFGF)对骨髓基质细胞(BMSC)粘附、增殖和分化等生物学效应的影响.方法利用体视学计数、MTT法及ALP试剂盒分别测定不同浓度bFGF诱导一定时间后BMSG的粘附特性、增殖和分化情况的变化.结果10n/mLbFGF明显促进BMSG的粘附,但是200ng/mLbFGF反而不利于细胞粘附;在细胞增殖和分化测定中,100ng/mLbFGF明显促进细胞增殖,但细胞碱性磷酸酶含量也最低.结论碱性成纤维细胞生长因子对骨髓基质细胞的生物效应是复杂和多方面的,可以作用于骨髓基质细胞的粘附、增殖和分化等多个环节,这种影响与碱性成纤维细胞生长因子的剂量有关.     

Effect of Dy3+on osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells and adipocytic trans-differentiation of mouse primary osteoblasts

A series of experimental methods including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test, alkaline phosphatase (ALP) activity measurement, mineralized function, Oil Red O stain and measurement were employed to assess the effect of Dy³⁺ on the osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells (BMSCs) and the adipogenic trans-differentiation of mouse primary osteoblasts (OBs). The results showed that Dy³⁺ had no effect on BMSC proliferation at concentrations of 1×10⁻⁸ and 1×10⁻⁵ mol/L, but inhibited BMSC proliferation at other concentrations. Dy³⁺ had no effect on OB proliferation at concentrations of 1×10⁻¹⁰ and 1×10⁻⁹ mol/L, but inhibited OB proliferation at other concentrations. Dy³⁺ had no effect on the osteogenic differentiation of BMSCs at concentrations of 1×10⁻⁹ and 1×10⁻⁷ mol/L, and promoted osteogenic differentiation of BMSCs at other concentrations at the 7th day. The osteogenic differentiation of BMSCs was inhibited by Dy³⁺ at concentration of 1×10⁻⁵ mol/L at the 14th day, but promoted osteogenic differentiation of BMSCs at concentrations of 1×10⁻⁹, 1×10⁻⁸, 1×10⁻⁷ and 1×10⁻⁶ mol/L with the maximal effect at concentration of 10⁻⁶ mol/L. Dy³⁺ promoted mineralized function of BMSCs at any concentration. Dy³⁺ had no effect on adipogenic differentiation of BMSCs at concentration of 1×10⁻⁷ mol/L, but inhibited adipogenic differentiation of BMSCs at other concentrations. Dy³⁺ inhibited adipocytic trans-differentiation of OBs at any concentration, suggesting that Dy³⁺ had protective effect on bone and the protective effect on bone may be mediated by modulating differentiation of BMSCs away from the adipocyte and inhibiting adipocytic trans-differentiation of OBs which may promote differentiation and mineralization of OBs. These results may be valuable for better understanding the mechanism of the effect of Dy³⁺ on pathogenesis of osteoporosis. Supported by the Foundation for Key Program of Ministry of Education of China (Grant No. 208018)     

Establishment and characterization of murine bone marrow stromal macrophage line

The authors established a murine bone marrow stromal cell line QXMSC1 by discarding the suspensible hematopoietic cells and passaging the adherent stromal cells many times in long-term bone marrow culture. QXMSC1 cells have been passaged 65 times for 15 months and immortalized. The mean number of QXMSC1 chromosomes is 66 ± 4. The appearance of the cells is elliptical. There are many pseudopods and mononuclei under the optical microscope. Many lisosomes and phagosomes in cytoplasm exist under the transmission electron microscope. There are no desmosome junction between the cells, no lipid drops, no intermediate filament. Vimentin is positive and keratin is negative by stain of immunocytochemistry. Nonspecific lipase staining is positive. These results indicate that QXMSC1 cells are murine bone marrow macrophages.     

Role of Notch expression in premature senescence of murine bone marrow stromal cells

The aim of the present study was to investigate the role of the Notch signaling pathway in premature senescence of murine bone marrow stromal cells in vitro. The intracellular domain of Notch 1 (ICN) was transfected into cultured murine bone marrow stromal cells by lipofectamine transfection. After three days, the proliferation of transfected cells was measured by MTT assay. Cell cycle distribution was analyzed by flow cytometry. Senescence-associated beta-galactosidase (SA-beta-gal) was measured, and the percentage of positive cells was evaluated by assessing 1000 cells in random fields of view. The expressions of p53 and p21Cip1/Waf1 were analyzed by both RT-PCR and Western blot analysis. The results showed that activation of Notch signaling inhibited proliferation of murine bone marrow stromal cells with induction of G1 arrest, increased the percentage of SA-beta-gal positive cells, and upregulated p53 and p21Cip1/Waf1 mRNA and protein expression levels. Thus, the activated Notch signaling could induce premature senescence of bone marrow stromal cells through the p53-p21Cip1/Waf1 pathway.     

Effect of La^3＋ on osteoblastic differentiation of rat bone marrow stromal cells

Lanthanides are increasingly used in industry and agriculture in recent years. These applications increase the accumulation of lanthanides in the human body, especially in bone[1]. Thus, people paid extensive atten-tion to the effect of lanthanides on bon…     

KDR基因和Sema3基因在再障和正常人骨髓基质细胞中的表达

目的：了解激酶插入区域受体(Kinase insert domain receplor,KDR)基因和脑信号蛋白Ⅲ(Semaphorin3,Sema3)基因在再生障碍性贫血(AA)和正常人的骨髓基质细胞(BMSC)和骨髓造血细胞中的表达情况。方法：收集9例AA和33例正常骨髓标本,分离单个核细胞(MNC)后体外长期培养扩增BMSC,并收集悬浮细胞(骨髓细胞)。RT-PCR-ELISA检测KDR基因和Sema3基因在BMSC和造血细胞中的表达,分析表达率,并以管家基因β2微球蛋白(β2M)为内参照进行半定量分析。结果：KDR基因在正常对照BMSC中的表达率(97．0％)明显高于对应的骨髓细胞(70．8％,P=0．07)。I①R基因在AA的BMSC和骨髓细胞中的表达率和表达水平与正常对照比较均无显差异。Sema3基因在正常BMSC中的表达水平明显低于其对应的骨髓细胞(P=0．035)。Sema3基因在AA的BMSC和骨髓细胞中的表达率和表达水平与正常对照组比较均无显差异。KDR基因和Sema3基因的表达水平在正常造血细胞中呈显正直线相关(r=0．703,P=0．002)。结论：KDR基因在BMSC中的高表达提示其可能在维持造血微环境中有重要作用。Sema3基因在造血细胞中的高表达状态提示其可能具有维持造血细胞生存的作用。KDR基因和Sema3基因在AA中的表达变化不大。