长白猪骨髓和脂肪间充质干细胞复制性衰老过程中生物学特性和抗氧化损伤能力的比较

为研究对比长白猪骨髓间充质干细胞(bone marrow derived mesenchymal stem cells,BMSCs)和脂肪间充质干细胞(adiposederivedmesenchymalstemcells,AMSCs)在体外衰老过程中的生物学特性和抗氧化损伤能力,分别利用贴壁筛选法、Ⅰ型胶原酶消化法分离获得BMSCs和AMSCs,利用流式细胞术鉴定细胞,EdU检测细胞增殖能力,实时荧光定量聚合酶链反应(RT-qPCR)检测基因表达,β-半乳糖苷酶(β-galactosidase,β-gal)染色检测细胞衰老情况,油红O和茜素红染色检测细胞的成脂和成骨分化能力,TUNEL检测细胞的凋亡情况．分离的BMSCs和AMSCs均高表达CD44和CD90,不表达CD45和CD34．与AMSCs相比,P8代BMSCs增殖能力强,OCT4和Ki67表达高,β-gal阳性细胞数少,成骨分化能力高．但在成脂分化能力方面,AMSCs强于BMSCs．进一步发现H2O2处理P8代细胞时,AMSCs比BMSCs对氧化损伤更敏感,细胞凋亡率显著增加,究其原因可能与抗氧化损伤基因表达的显著降低有关．     

人骨髓间充质干细胞的分离培养及生物性状研究

分离培养人骨髓间充质干细胞，研究其在体外生长增殖的生物特性。冲洗手术弃骨骨髓，获取骨髓间充质干细胞进行培养，倒置相差显微镜观察细胞生长情况，绘制生长曲线，免疫细胞化学法鉴定细胞表面抗原，进行染色体核型分析。冷冻保存细胞复苏后的生长状况观察。结果显示，原代和传代培养的细胞呈现梭形外观，具有较强的生长增殖能力；细胞CD44，CD54抗原表达阳性，CD34表达阴性。染色体核型分析表明是正常的人二倍体细胞。复苏细胞生物学特性无明显改变。     

大鼠骨髓间质干细胞的培养和生物学特征

为探索大鼠骨髓间充质干细胞的最佳培养条件及表型,用大鼠股骨抽取骨髓,密度梯度离心分离骨髓间充质干细胞。通过测定生长曲线和克隆形成能力,比较不同培养条件、不同融合度传代及不同生长基质对MsC的影响。免疫组化双标法检测增殖MSC的表型。结果Matrigel包被培养器皿,含10％胎牛血清、10ng／mLEGF的α-MEM培养基,50％融合时消化传代,是MSC保持低分化状态、体外大量扩增的较佳条件。绝大部分MSC表达CD44,部分MSC表达c-kit,MSC几乎不表达CD34。说明以上培养条件是MSC保持低分化状态又大量增殖的较好培养条件,为MSC用于细胞移植和组织工程打下了良好基础。     

液晶仿生材料与骨髓间充质干细胞体外培养的实验

目的:研究骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)与液晶仿生材料复合后体外培养的生长、增殖、形态等变化,探讨液晶仿生材料与BMSCs的生物相容性.方法:从仿生学的角度,合成在生理温度下呈现胆甾醇液晶态的羟丙基纤维素衍生物液晶材料,利用偏光显微镜、扫描电镜和表面静态水接触角等对改变性质后的液晶的物化性质进行表征,与BMSCs复合后在光学显微及电镜下观察培养3 d、6 d的细胞生长、增殖、形态学改变,流式细胞仪检测其表面抗原CD29、CD31、CD44、CD45表达情况.结果:BMSCs在液晶材料中生长、增殖良好,在整个过程都保持存活,表面抗原正常表达.结论:液晶仿生材料与BMSCs生物相容性良好,是可选的干细胞复合载体.     

补肾和解复方对再障患者CD34+细胞造血干/祖细胞调节作用的实验研究

目的:探索补肾和解方对再障患者CD34 造血干/祖细胞的刺激增殖和诱导分化作用.方法:采用免疫磁珠法(MACS)分离纯化再障患者骨髓CD34 细胞,造血细胞半固体和液体培养体系加入不同度补和解方制剂,检测对CD34 造血干/祖细胞增殖,形成祖细胞集落的提高率,并用流式细胞仪检测液体培养后细胞表面标记的变化.结果:补肾和解复方制剂(5～100μg.mL)能提高BFU-E、CFU-E、CFU-GEMM集落产率,补肾和解复方制剂50μg/mL是液体培养刺激CD34 细胞体外增殖的最佳浓度.补肾和解复方制剂诱导细胞14天后,细胞表面表达CD334 细胞随补肾和解复方制剂的浓度升高而增加,在补肾和解复方制剂的浓度升高而增加,在补肾和解复方制剂100μg/mL时以CD15 细胞数最高,CD71 细胞和G-A 细胞数仅在50μg/mL高于未加补肾和解复方制剂的对照组.结论:补肾和解复方制剂不但能促进再障患者CD34 造血干/祖细胞的增殖,并且能诱导定向分化,具有类生长因子和协同生长因子的作用.     

慢病毒介导的GFP转染对小鼠骨髓间充质干细胞表型、增殖和分化能力的影响

运用慢病毒(Lentivirus)载体构建绿色荧光蛋白(GFP)在小鼠骨髓间充质干细胞(MSCs)中的转染体系,检测转染后MSCs的表型和增殖能力,并诱导其向成肌细胞分化,检测此转染体系对MSCs细胞表型、增殖和分化能力的影响.骨髓细胞悬液破红后贴壁法培养MSCs,采用流式细胞术鉴定细胞表面标记,鉴定MSCs纯度;Lentivirus-GFP以1、10、50和100的感染复数(MOI)转染MSCs,作用48h后流式细胞术及免疫荧光法检测转染效率和表达的荧光强度;MTT法检测转染后MSCs的细胞活力.诱导MSCs-GFP向成肌细胞分化,蛋白印记法(WB)检测成肌细胞特异性蛋白desmin和α-SMA的表达.流式细胞术检测结果显示,培养的MSCs表达CD44、CD90和CD105,不表达CD34、CD45和CD188,符合干细胞特性.MOI为1、10、50和100的转染效率分别为23.45%、93.51%、95.44%和95.55%,MOI为10时,转染效率较高,且对MSCs活力无明显影响.MSCs-GFP体外经成肌细胞诱导分化后,表达特异性抗原desmin和α-SMA.表明本研究成功构建了小鼠骨髓来源MSCs的Lentivirus-GFP转染体系,有效标记了MSCs细胞,且此标记对MSCs的表型、增殖和分化能力等细胞生物学特性无明显影响.     

人胚胎脑神经干细胞分离纯化及体外培养的研究

为了探讨人胚神经干细胞体外培养条件下的生物学特性,为其应用于临床治疗奠定基础.取胎龄16周的人流产胚胎,胰酶消化结合机械法分离成单细胞悬液,以2×106个细胞/mL接种到含hEGF和h-bFGF的DMEM/F12、N2培养基进行体外培养;观察细胞生长情况,用10% FBS诱导神经干细胞球分化,免疫细胞化学鉴定. 结果显示从人胚大脑分离出的细胞经悬浮培养可以形成细胞球,表达Nestin蛋白.经诱导分化后具有表达神经元,神经胶质细胞的特异性抗原. 说明人胚神经干细胞在体外可以稳定生长,并能分化成为神经原及胶质细胞.     

全骨髓贴壁法离体培养大鼠骨髓间充质干细胞及其生物学特征鉴定

验证全骨髓贴壁法离体培养大鼠骨髓间充质干细胞(bone marrow mesenchymal stromal cells,BMSCs)的可行性,并对所获细胞进行生物学特征鉴定。采用全骨髓贴壁法离体培养大鼠BMSCs,通过倒置相差显微镜进行不同阶段的细胞形态学观察。通过流式细胞术进行BMSCs表型鉴定。通过成骨诱导分化实验检验其分化潜能。原代BMSCs接种48h后充分贴壁。72h后,呈集落式克隆,单体形态呈长梭形、三角形及多角形。细胞融合至80%~90%后呈"鱼群"或"旋涡"状排列。传至第四代的BMSCs失去其特征性形态,单体大而铺展,主要呈不规则形。传至第二代的BMSCs经流式细胞术检测后即呈现CD44、CD90阳性表达,而CD45阴性表达。第二代BMSCs成骨诱导3周后,碱性磷酸酶染色呈阳性,茜素红染色可见大量矿化结节形成。全骨髓贴壁法简便易行,可高效地体外分离、纯化及扩增大鼠BMSCs,所获细胞具备BMSCs的生物学特征。     

甲醇利用型细菌发酵生产吡咯喹啉醌的培养条件

以甲醇为唯一碳源,对生丝微菌TH205进行培养,研究了甲醇流加速度、Fe^2 浓度对细胞生长及吡咯喹啉醌(PQQ)表达量的影响。结果表明：培养基中添加浓度为7μg／mL的Fe^2 ,并控制甲醇的流加速度使培养基中甲醇浓度为1mL／L时,细胞的生长情况较好,且此时PQQ的发酵产量为26．6μg／mL。     

新疆红景天中红景天苷含量的测定

用反相高效液相色谱法，流动相为甲醇-0．1mol／L磷酸二氢钠(15：85)，紫外检测波长为280nm，流速1．0mL／min，测定新疆产红景天中红景天苷含量，结果表明，红景天苷对照品在10～320μg/mL内线性关系良好，r=0．999。平均加样回收率为96．5％；RSD为3．4％。稳定性良好。该法简便，分离完全，结果可靠，适用于红景天苷的含量测定。     

兔骨髓间充质干细胞复合脱细胞牛松质骨体外相容性研究

探讨脱细胞牛松质骨（Acellular Bovince Cancellous Bone，ABcB）生物衍生材料与骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs）的生物相容性，为组织工程方法修复骨缺损提供依据。将兔骨髓间充质干细胞与ABCB复合并体外培养，然后进行形态学、细胞增殖、蛋白质含量测定。骨髓间充质干细胞能够贴附在ABCB孔隙内生长，细胞生长及蛋白合成功能不受ABCB的影响。ABCB生物相容性良好，可作为组织工程的支架材料应用于骨缺损的修复；BMSCs可以作为骨组织工程种子细胞。     

SD大鼠骨髓间充质干细胞的分离培养和鉴定

目的:探讨体外分离培养SD大鼠骨髓间充质干细胞rMSCs的方法,检测传代细胞的细胞周期,并分析部分细胞表型,对rMSCs进行初步的鉴定。方法:密度梯度离心结合贴壁培养法分离培养rMSCs,传代扩增,倒置显微镜进行细胞形态学观察,免疫组化检测细胞表面抗原,流式细胞仪检测细胞周期。结果:密度梯度离心结合贴壁培养法能有效分离纯化rMSCs,细胞呈均一的成纤维细胞样,细胞周期显示90.16%P1代细胞处于G0/G1期,免疫组化结果为:CD44、CD29阳性、CD34阴性,说明培养的细胞即非造血干细胞,也非成纤维细胞。结论:本实验分离培养的细胞群与间充质干细胞的生物学特性吻合,说明密度梯度离心结合贴壁培养法能有效分离纯化rMSCs,细胞稳定表达CD44、CD29,是实用、可行的方法,所培养的细胞可用于细胞移植等研究。     

生物力学作用对骨髓间充质干细胞增殖和成骨分化影响研究进展

骨髓间充质干细胞在体内所处的力学环境比较复杂,为了探索生物力学与骨髓间充质干细胞增殖分化之间的关系,对近年来生物力学微环境对骨髓间充质干细胞增殖分化影响的研究现状和最新进展进行了综述。认为牵张力与流体剪切力对骨髓间充质干细胞产生的刺激大部分会使其向成骨方向分化,而较大的压缩力和静水压力对骨髓间充质干细胞产生的刺激大部分会使其偏向软骨方向分化,小部分向成骨方向分化,每种分化方向都有其最适的分化条件。通过综述生物力学对骨髓间充质干细胞增殖及成骨分化的影响,为骨髓间充质干细胞的骨组织工程与再生医学研究及更好地应用于临床治疗提供思路和参考依据。     

悬浮培养型 BHK -21 细胞的生长规律和不同接种密度培养效果的研究

为探索悬浮培养型BHK-21细胞的生长规律和最佳接种密度,以3．0×10^5个／mL、4．0×10^5个／mL、5．0×10^5个／mL、6．0×10^5个/mL和7．0×10^5个／mL等5个接种密度培养了悬浮培养型BHK-21细胞,绘制了细胞生长曲线和活力变化曲线．结果显示,悬浮培养型BHK-21细胞生长曲线呈“S”型,接种培养前24h细胞处于适应期,生长曲线上密度增长不大,期后细胞进入对数增长期,生长密度呈指数增长．以3．0×10^5个／mL和4．0×10^5个／mL接种培养的在120h达到最大增殖密度,分别为4．65×100个／mL和5．59×10^6个／mL,倍增时间分别为26．9h和26．7h;以5．0×10^6个／mL、6．0×10^6个/mL和7．0×10^6个／mL接种培养的在96h就达到最大增殖密度,分别为5．14×10^6个／mL、5．36×10^6个／mL和5．1×10^6个／mL,倍增时间分别为22．4h、24．1h和25．2h．各组在培养的96h以前细胞活力均在90％以上且变化较小,120h和144h时细胞活力开始下降,且接种密度越高活力下降越快．该细胞以4．0×10^5个／mL、5．0×10^5个／mL、6．0×10^5个／mL接种培养能达到较高培养密度,细胞生长快,且细胞峰值持续时间长．     

Electrospun Nanofibers of Hydroxyapatite/Collagen/Chitosan Promote Osteogenic Differentiation of BMSCs

Bone tissue engineering, aiming at developing bone substitutes for repair and regeneration of bone defects instead of using autologous bone grafts, has attracted wide attention in the field of tissue engineering and regenerative medicine. Developing biomimetic biomaterial scaffolds able to regulate osteogenic differentiation of stem cells could be a promising strategy to improve the therapeutic efficacy. In this study, clectrospun composite nanofibers of hydroxyapatite/collagen/chitosan （ HAp/Col/CTS ） resembling the fibrous nanostructure and constituents of the hierarchically organized natural bone, were prepared to investigate their capacity for promoting bone mesenchymal stem cells （BMSCs） to differentiate into the osteogenic lineage in the absence and presence of the osteogenlc supplementation, respectively. Call morphology, proliferation and quantified specific osteogenic protein expression on the electrospun HAp/Coi/CTS scaffolds were evaluated in comparison with different controls including dectrospun nanofibrous CTS, HAp/CTS and tissue culture plate. Our remits showed that the nanofibrous HAp/Col/CTS scaffolds supported better spreading and proliferation of the BMSCs than other substrates （ P 〈 0.01 ）. Expressions of osteogenesis protein markers, alkaline phosphatase （ALP） and Col, were significantly upregulated on the HAp/Col/CTS than those on the CTS （P 〈0.01） and HAp/ CTS （P 〈 0. 05 ） scaffolds in the absence of the osteogeulc supplementation. Moreover, presence of osteogeulc supplementation also proved to enhance osteogeule differentiation of BMSCs on HAp/ Col/CTS scaffolds, indicative of a synergistic effect. This study highlights the potential of BMSCs/HAp/Col/CTS cell-scaffold system for functional bone repair and regeneration applications.     

人胎儿骨髓间充质干细胞的分离及生物学鉴定

通过原代细胞培养,从引产胎儿骨髓组织中分离干细胞,然后进行生物学鉴定,旨在体外建立培养胎儿骨髓干细胞的有效方法,为进一步研究干细胞奠定基础．本研究对四个月的引产胎儿骨髓组织进行原代细胞培养,采用贴壁筛选法,在含有15％胎牛血清的L-DMEM／IMDM(1:1)混和培养液中培养,7 d后细胞可长满瓶底．显微镜下观察,细胞形态均一,呈长梭形．传代后,在8代以内的细胞贴壁能力较强,生长速度较快．将其命名为BMMS-03．在第3代时,对培养的细胞进行了于细胞标志物的生物学鉴定,采用流式细胞仪对经免疫荧光染色的细胞进行检测．结果显示:97．2％的细胞呈CD105阳性反应,66．0％的细胞呈CD106阳性反应, 9．2％的细胞呈CD34阳性反应．阴性对照组阳性反应为0．5％．生物学鉴定的初步结果提示,从胎儿骨髓组织中分离培养成功的细胞为骨髓间充质干细胞,其细胞形态学特征、CD105 、CD106 和CD34-的检测结果均符合间充质干细胞的特征．本研究成功地建立了体外培养胎儿骨髓间充质干细胞的有效方法,所获得的间充质干细胞纯度较高,增殖较快,适用于干细胞生物学和组织工程学的研究．     

Differentiation of bone mar-row derived Thy-1~+β_2M~- cells into hepatocytes induced by coculture with transgenic CFSCs

Studies of transplantation in vivo indicted thatbone marrow derived stem cells had a potential to differenti-ate into mature hepatocytes. However, there are lots ofdoubts and uncertainties in the influencing factors and con-trol agents of effectively inducing stem cell differentiation invitro, the efficiency of stem cells‘ differentiation into hepato-cytes and differentiated cells‘ life-span and functional state,etc. In this study, rat bone marrow derived Thy-l^ β2M^- cells(BDTCs) were induced to differentiate into hepatocytes byco-culturing with CFSC/HGF feeder layers which expressedhHGF efficiently and stably. RT-PCR and immunofluores-cent texts proved induced BDTCs expressed infant and adulthepatocyte specific genes. Further more, these cells displayedfunctions of indocyanine green (ICG) uptake, ammoniummetabolism and albumin production. It was shown thatgrowth factors together with hepatic nonparenchyma cellsprovided a feasible microenvironment for differentiation ofbone marrow stem cells into hepatocytes. The studies notonly provided a significant biological model for going deepinto the mechanism of stem cell plasticity, but also offered atheoretical and technical foundation of gene and stem cellengineering-based regenerative medicine for end-stage liverdiseases.     

Nanofibrous Scaffold Containing Osteoblast-Derived Extracellular Matrix for the Proliferation of Bone Marrow Mesenchymal Stem Cells

Extracellular matrix( ECM) plays a prominent role in establishing and maintaining an appropriate microenvironment for tissue regeneration. The aims of this study were to construct a tissue engineered scaffold by reconstituting osteoblast cell-derived ECM( O-ECM) on the electrospun nanofibrous scaffold,and further to evaluate its subsequent application for promoting the proliferation of bone marrow mesenchymal stem cells( BMSCs). To engineer a biomimetic scaffold, calvarial osteoblasts and electrospun poly-llactic acid( PLLA) nanofibers were prepared and subjected to decellularize for O-ECM deposition. To evaluate and characterize the O-ECM/PLLA scaffold, the morphology was examined and several specific mark proteins of osteoblasts matrix were evaluated.Furthermore,the cell counting kit-8( CCK-8) assay was used to detect the proliferation of the BMSCs cultivated on the O-ECM/PLLA scaffold. The results indicated O-ECM/PLLA scaffold was loaded with Collagen I, Fibronectin, and Laminin, as the composition of the marrow ECM. After decellularization,O-ECM deposition was observed in O-ECM/PLLA scaffold. Moreover,the O-ECM/PLLA scaffold could significantly enhance the proliferation of BMSCs,suggesting better cytocompatibility compared to the other groups tested. Taken together,a biomimetic scaffold based on the joint use of O-ECM and PLLA biomaterials,which represents a promising approach to bone tissue engineering, facilitates the expansion of BMSCs in vitro.     

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)     

一种基于Propidium Monoazide与PCR结合的选择性检测细菌活细胞的方法

建立了一种PMA与PCR结合的细菌活细胞检测的方法,可有效抑制微生物死细胞DNA的PCR扩增,从而排除死细胞对微生物活细胞检测的影响．结果表明,PMA浓度大于3 μg/ml时可抑制死细胞DNA的PCR扩增,PMA浓度高达50 μg/ml时仍不影响活细胞DNA的PCR扩增;并且得出曝光时间大于3 min可使PMA与死细胞DNA分子共价交联,抑制其PCR扩增;浊度影响PMA交联的结果表明浊度小于10 NTU时,PMA仍能有效地抑制死细胞DNA的PCR扩增,而当浊度大于100 NTU时,PMA失去效果．