Magnetic nanoparticle labeling of mesenchymal stem cells without transfection agent: cellular behavior and capability of detection with clinical 1.5 T magnetic resonance at the single cell level.

The purpose of this work was to evaluate the efficacy of labeling human mesenchymal stem cells (hMSCs) by ionic superparamagnetic iron oxide (SPIO) without a transfection agent and verifying its capability to be detected with clinical 1.5 T magnetic resonance (MR) at the single-cell level. Human hMSCs were incubated for 24 h with an ionic SPIO, Ferucarbotran. The labeling efficiency of hMSCs was determined by iron content measurement spectrophotometrically, and the influence of labeling on cell behavior was ascertained by examination of cell viability using the trypan blue exclusion method, cell proliferation analysis using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, mitochondrial membrane potential (MMP) change, differentiation capacity, and reactive oxygen species (ROS) production measured by dichlorofluorescein diacetate (DCFDA) fluorescent probe. Labeled hMSCs were scanned under 1.5 T MRI with three-dimensional (3D) and two-dimensional (2D) T(2)-weighted gradient echo (GRE) pulse sequences. Human hMSC labeling without transfection agent was efficient. The iron content in hMSCs was 23.4 pg Fe/cell. No significant change was found in viability, proliferation, MMP change, ROS production, or differentiation capacity. About 45.2% of the hMSCs could be detected using 1.5 T MRI at the single cell level with 3D GRE and four repetitions.     

Water- and fat-suppressed proton projection MRI (WASPI) of rat femur bone.

Investigators often study rats by microCT to investigate the pathogenesis and treatment of skeletal disorders in humans. However, microCT measurements provide information only on bone mineral content and not the solid matrix. CT scans are often carried out on cancellous bone, which contains a significant volume of marrow cells, stroma, water, and fat, and thus the apparent bone mineral density (BMD) does not reflect the mineral density within the matrix, where the mineral crystals are localized. Water- and fat-suppressed solid-state proton projection imaging (WASPI) was utilized in this study to image the solid matrix content (collagen, tightly bound water, and other immobile molecules) of rat femur specimens, and meet the challenges of small sample size and demanding submillimeter resolution. A method is introduced to recover the central region of k-space, which is always lost in the receiver dead time when free induction decays (FIDs) are acquired. With this approach, points near the k-space origin are sampled under a small number of radial projections at reduced gradient strength. The typical scan time for the current WASPI experiments was 2 hr. Proton solid-matrix images of rat femurs with 0.4-mm resolution and 12-mm field of view (FOV) were obtained. This method provides a noninvasive means of studying bone matrix in small animals.     

蛇毒抗高凝状态酶、氟尿嘧啶对BEL-7404细胞作用的实验研究

目的通过蛇毒抗高凝状态酶（AHCSE）、氟尿嘧啶（5-FU）对人肝癌细胞BEL-7404（简称7404细胞）、正常LO2肝细胞（简称LO2细胞）作用的比较，研究AHCSE对肝癌的作用以及探讨其可能的作用机制。方法应用光学显微镜、透射电镜、MTT法、TUNEL、FCM等方法观察7404细胞、LO2细胞经过AHCSE、5-FU处理后，细胞形态学、生物化学等方面的变化。结果7404细胞、LO2细胞经过AHCSE、52FU处理后，发生了形态学改变；小剂量AHCSE对7404细胞具有很强的抑制作用，但对LO2细胞几乎无影响；随着剂量的加大，AHCSE对7404细胞抑制率上升不明显，但是出现对LO2细胞的抑制作用。经AHCSE作用后，7404细胞发生了凋亡，凋亡率随AHCSE浓度的增加而增加；与5-FU对7404细胞作用相似，但5-FU对LO2细胞抑制作用明显增强。结论AHCSE、5-FU对BEL-7404细胞均有很强的抑制作用，诱导细胞凋亡是其作用机制之一，AHCSE可能成为一种新的肝癌细胞凋亡的诱导剂。     

支气管扩张症辨证模式初探

目的:探讨支气管扩张症的中医辨证分型规律及证候特点.方法:通过对563例支气管扩张症的临床流行病学调查,采集以症状、体征、舌、脉及相关理化检测为变量的基本信息,以频数分析、聚类分析、方差分析等方法,提炼支气管扩张症的证候分布规律及证候特点.结果:临床上支气管扩张症多见4种证候类型,分别为痰热壅肺证(45.65%)、肝火犯肺证(24.51%)、肺脾气虚证(22.38%)、气阴两虚证(7.46%).结论:较大样本的临床流行病学调查为研究支气管扩张症辨证分型规律提供了科学依据,并可以通过主症判别分析法建立证候识别模式,为临床实践提供依据.     

兔骨髓基质细胞诱导分化为胰岛素分泌细胞的方法

目的 探讨将骨髓基质细胞（MSCs）诱导成为胰岛素分泌细胞（IPCs）的方法。方法 体外培养新西兰兔骨髓及胰腺基质细胞和SD大鼠胰腺基质细胞，利用含有胰腺基质细胞培养基的混合培养基诱导培养MSCs。结果 以兔及SD大鼠胰腺基质细胞培养基均可诱导兔MSCs生成IPCs。结论 兔及SD大鼠的胰腺基质细胞培养基均可将兔MSCs诱导分化为IPCs。     

外伤性脾切除术后中医证候及免疫功能的研究

目的:研究外伤性脾切除术后中医辨证与免疫功能变化的关系.方法:选择15例脾切除和15例非脾切除患者作为观察对象,以其症状、舌苔、脉象及免疫功能的测定作为观察指标,观察其中医辨证与免疫功能变化.结果:脾切除组在术后早期可出现脾胃虚弱的证候群,同时免疫功能测定补体C3、免疫球蛋白IgG、免疫球蛋白IgM的水平明显降低,与非脾切除组有显著性差异.结论:脾切除容易造成患者术后出现的脾胃虚弱症候群,并降低患者的免疫功能.     

体外大鼠骨髓间充质干细胞多向分化潜能的研究

目的研究体外大鼠骨髓间充质干细胞向神经细胞等不同组织细胞多向分化的潜能。方法从SD大鼠股骨骨髓中获得间充质干细胞,原代培养后1∶2传代,传至第5代后分为普通传代培养组、神经细胞诱导组、成骨细胞诱导组和脂肪细胞诱导组。倒置相差显微镜下观察各组细胞生长情况、形态变化以及矿化结节和脂肪细胞的形成;流式细胞检测第5代大鼠骨髓间充质干细胞表面抗原CD29、CD44、CD90、CD31、CD34、CD45;免疫组织化学检测普通传代培养组和神经细胞诱导组细胞巢蛋白、微管相关蛋白-2、胶质纤维酸性蛋白和神经元特异性烯醇化酶等神经细胞相关蛋白的表达情况。结果大鼠骨髓间充质干细胞呈贴壁生长,细胞扩增至第5代时形态趋于一致,呈梭形。大鼠骨髓间充质干细胞表面抗原CD29(99.83%)、CD44(99.77%)、CD90(99.86%)均呈阳性表达,CD31(0.83%)、CD34(1.78%)、CD45(2.90%)无表达。在体外,普通传代培养细胞仅巢蛋白呈阳性表达;由神经细胞诱导的细胞巢蛋白、微管相关蛋白-2、胶质纤维酸性蛋白和神经元特异性烯醇化酶均呈阳性表达,且形态类似神经细胞;由成骨细胞诱导的细胞质内可见矿化结节形成;由脂肪细胞诱导的细胞质内出现多个猩红色呈簇状的脂肪滴。结论大鼠骨髓间充质干细胞易于提取、纯化和扩增,可于体外自发表达神经干细胞标志蛋白,并可通过诱导向神经细胞、成骨细胞及脂肪细胞分化。提示骨髓间充质干细胞不仅具有多向分化潜能,而且可能具有自发向神经干细胞分化的特性。     

体外细胞培养检验“方证对应”关系的方法学研究

目的通过在体外细胞培养中加入中药复方粗提制剂对具有典型的中医证型的肺癌患者的外周血淋巴细胞NK活性的影响，探索能否在体外实验中反映“方证对应”关系。方法以水煎醇沉法制备两种中药复方（益气养阴和健脾化痰）的粗提制剂，取具有典型的中医证型（气阴两虚或脾虚痰湿证型）的肺癌患者的外周血淋巴细胞，将同一个患者的标本分为四个体外实验组，即空白对照组，中药5mg／ml组及10mg／ml组，自细胞介素2（250^u/ml，阳性对照）组。按“辨证论治”原则加入相应的中药复方制剂。体外培养22h或94h，以MTT法检测各组的NK活性。结果无论培养22h或94h，中药组的NK活性与对照组比较皆无显著性差异（P〉0．05），而白细胞介素2（阳性对照）组的NK括性均明显升高（P〈0．05）。结论两种中药复方制剂都未能在体外反应出“方证对应”的效果，说明中药粗提制剂直接用于体外细胞培养，其结果的可靠性差。     

The end adjusts the means: Heterochromatin remodelling during terminal cell differentiation

All cells that constitute mature tissues in an eukaryotic organism undergo a multistep process of cell differentiation. At the terminal stage of this process, cells either cease to proliferate forever or rest for a very long period of time. During terminal differentiation, most of the genes that are required for cell ‘housekeeping’ functions, such as proto-oncogenes and other cell-cycle and cell proliferation genes, become stably repressed. At the same time, nuclear chromatin undergoes dramatic morphological and structural changes at the higher-order levels of chromatin organization. These changes involve both constitutively inactive chromosomal regions (constitutive heterochromatin) and the formerly active genes that become silenced and structurally modified to form facultative heterochromatin. Here we approach terminal cell differentiation as a unique system that allows us to combine biochemical, ultrastructural and molecular genetic techniques to study the relationship between the hierarchy of chromatin higher-order structures in the nucleus and its function(s) in dynamic packing of genetic material in a form that remains amenable to regulation of gene activity and other DNA-dependent cellular processes.