种子细胞双相接种技术促进组织工程骨体外成熟的研究

目的 比较观察双相接种法和静置接种法构建组织工程骨对种子细胞增殖、分化和成骨表达的影响。方法 取健康志愿者骨髓，密度梯度离心法分离骨髓间充质干细胞并进行体外扩增、诱导分化，然后应用双相接种法和静置接种法与脱钙骨基质（DBM）复合构建组织工程骨，体外培养3周，用倒置显微镜、扫描电镜观察细胞密度、形态及基质分泌情况，并在不同时相点测定组织块中DNA含量、碱性磷酸酶（ALP）活性及钙含量变化。结果 倒置显微镜下双相接种法构建的组织块可见大量具有一定折光性的梭形细胞存在于胶原基质中。扫描电镜见双相接种法构建的组织块切面较平坦，孔隙较小，细胞包埋在胶原中，而静置接种法构建的组织块可见细胞、细胞外基质较少。在体外培养过程中，双相接种法组织块中细胞的DNA含量、ALP活性均高于静置接种法，钙含量在培养2周后高于静置接种法。结论 双相接种法是一种高效的组织工程骨构建方法，有利于提高接种效率和促进组织工程骨的体外成熟。     

软骨细胞诱导骨髓基质干细胞构建带内支撑的组织工程化软骨

目的 探讨以聚羟基乙酸(PGA)包裹特定形态的医用假体材料--多孔高密度聚乙烯(HDPE,商品名为MEDPOR)为支架,应用软骨细胞诱导骨髓基质干细胞(BMSCs),共培养构建特定形态的带内支撑组织工程化软骨医用假体的可能性.方法 以直径3 mm、长5 mm的圆柱形HDPE,外裹 1 mm厚PGA为支架,将体外分别培养的新生猪BMSCs和耳郭软骨细胞按7∶3混合,以10×10 7/ml细胞浓度接种于支架上,同时以相同浓度的单纯软骨细胞和单纯BMSCs分别接种,作为阳性对照组(PC组)和阴性对照组(NC组).经体外培养2周及在裸鼠皮下移植4、8周后取材 ,行大体观察、组织学、组织化学及免疫组化检测.结果 各组细胞均与材料黏附良好.实验组和阳性对照组均形成了大体形态良好的HDPE-软骨复合体,内支撑的HDPE与外层软骨结合紧密.组织学可见成熟的软骨陷窝结构,软骨渗入HDPE孔隙内部、异染基质及Ⅱ型胶原呈强阳性表达.结论 以HDPE为内支撑,外裹PGA的支架,接种混合细胞,可于皮下构建特定形态、组织学良好的HDPE-软骨复合体.     

细胞移植的人嗅鞘细胞培养方法的研究

目的探讨适用于细胞移植的人嗅鞘细胞（OECs）分离与培养的方法。方法无菌条件下取自愿捐献孕5个月流产胎儿的完整嗅球进行嗅鞘细胞提取分离、纯化，并对嗅鞘细胞进行免疫组化染色及纯度检测，观察其形态学特点，检测其免疫组化染色的阳性率。结果本方法分离、培养的嗅鞘细胞大多呈三角形、梭形，有些突起较长，生长活跃，增殖迅速，P75、胶质纤维酸性蛋白（GFAP）免疫组化染色呈阳性，嗅鞘细胞阳性率为95．3％。结论该方法简便易行且不影响细胞功能，适用于细胞移植的研究。     

大鼠气管上皮细胞体内—体外转化模型的研究

本研究建立了大鼠气管上皮细胞体内－体外转化模型，大鼠气管内滴注苯并芘，三天后处死大鼠，消化气管上皮细胞，接种于无血清完全培养基。细胞形成集落后，换为选择培养基继续培养五周，统计转化率。结果显示，２５ｍｇ／ｋｇ和５０ｍｇ／ｋｇ的苯并芘可诱导大鼠气管上皮细胞转化及微核增加，用同样方法研究了煤焦沥青提取物，结果表明，剂量为８ｍｇ／ｋｇ和２５ｍｇ／ｋｇ的煤焦沥青提取物能明显诱导大鼠气管上皮细胞转化。     

Matrix Metalloproteinase-2 and Tissue Inhibitor of Metalloproteinase-1 in the Retinal Pigment Epithelium and Interphotoreceptor Matrix: Vectorial Secretion and Regulation

Matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) play an essential role in both normal and pathological extracellular matrix degradation, and a TIMP has been associated with at least one type of retinal degeneration. We have studied expression of MMP-2 and TIMP-1 by zymography, immunocytochemistry, and immunoblotting in the retinal pigment epithelium (RPE) from normal, aged and diseased retinas. MMPs and TIMPs were found in the rat RPE, interphotoreceptor matrix (IPM), and in media conditioned by human and rat RPE in culture. In other polarized cells, MMPs and TIMP-2 are secreted vectorially towards the basal lamina. In the RPE, however, MMP-2 and TIMP-1 were secreted preferentially from the apical surface, the surface bordering the IPM. These findings provide new evidence that MMPs and TIMPs could play a role in the turnover of IPM components.Cell homogenates and conditioned media from RPE isolated from mutant Royal College of Surgeons (RCS) rats with inherited retinal dystrophy had similar amounts of MMP-2 and TIMP-1 as those from congenic control rats. The secretion of MMP-2 and TIMP-1 from RPE cell cultures isolated from young and aged human donors varied widely. However, with increasing cell passage number, secretion of MMPs and TIMPs from human RPE increased dramatically. Also, growing human RPE on bovine corneal endothelial cell-generated extracellular matrix instead of plastic reduced the secretion of both MMPs and TIMPs. These data suggest that the integrity of Bruch's membrane may serve to regulate RPE functions in MMP and TIMP secretion and that extracellular matrices contain signals that regulate MMP and TIMP synthesis and/or secretion by the RPE.     

Characteristics of rhabdomyosarcoma cell lines derived from uterine carcinosarcomas

 Rhabdomyosarcoma (RMS) is occasionally found in the female genital tract, and mostly appears as one of the heterologous mesenchymal components in uterine carcinosarcoma designated as malignant mixed müllerian tumour (MMMT). We examined the biological properties of a pure rhabdomyosarcoma (RMS) cell line designated FU-MMT-3, which was newly established from a surgical specimen taken from a patient with uterine MMMT. We also evaluated c-myc and MYCN gene amplification in three RMS cell lines (including FU-MMT-3) derived from three MMMTs by Southern blot analysis. FU-MMT-3 cells were propagated continuously for 57 serial passages over a 2-year period in vitro. FU-MMT-3 was able to produce tumours demonstrating pure RMS in athymic nude mice. Cytogenetically, FU-MMT-3 showed a triploidy pattern, with complex karyotypic abnormalities including trisomy of chromosome 8. All three RMS cell lines, including FU-MMT-3, showed amplification of the c-myc gene (approximately fourfold to eightfold), while no cell lines demonstrated MYCN gene amplification. FU-MMT-3 is considered to provide a useful system for the study of the biological behaviour of RMS in MMMTs. Extra copies of chromosome 8 and c-myc gene amplification may be associated with the rhabdomyoblastic differentiation in MMMT. Received: 7 January 1997 / Accepted: 2 May 1997     

Identification of cultured male epidermal allografts by detecting Y chromosome DNA fragments in female burn victims

ltiswellknownthattheskinofextensivelyburnedcasesthemselvesisinsufficienttoprovidecoverageforwounds.Thoughallogeneicskincancoverthewoundstemporari1y,itwillberejectedwithin2-3weeks.Thiscanthreatenthepatient'slifeifthereisnoenoughautogenousskintoreplacetherejectedoneontime.GreenetalL1]reportedthattheburnwoundsofpatientswithextensiveburnscouldbetreatedbytransplantingepidermalautograftsafterautologousepidermalcellsmulti-plyforthousandsoftimesinvltrowithinashortperiodoftime-Butittakesatleast2-3weeks…     

Distribution of glutathione and glutathione-related enzyme systems in mitochondria and cytosol of cultured cerebellar astrocytes and granule cells

The cellular and regional distribution of glutathione (GSH) and GSH-related enzyme systems involved in cellular defense against reactive oxygen species and electrophilic xenobiotics in the nervous system has been extensively studied. However, little is known about the subcellular distribution of GSH systems in brain tissue and cultured neural cells. The present study investigates the distribution of mitochondrial and cytosolic GSH and GSH-related enzymes in cultured cerebellar astrocytes and granule cells, and compares them with levels in the adult rat cerebellum. Cytosolic GSH levels and cytosolic activities of glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) in astrocytes were 57, 153, 245, and 92% higher than those found in granule cells, respectively. In contrast, granule cells contained significantly higher mitochondrial GSH levels than astrocytes. Granule cells also demonstrated comparable mitochondria/cytosolic concentrations of GSH and GR, GPX and GST activities to those observed in the cerebellar tissue, whereas ratios in astrocytes were markedly lower. Although in vitro treatments with 100 μM ethacrynic acid depleted both cytosolic and mitochondrial GSH in cultured astrocytes and granule cells in a time-dependent fashion, cellular GSH in granule cells was more resistant to the GSH-depleting agent than astrocytes. These results suggest that although GSH and GSH-related enzymes are abundant in cytosolic compartments of astrocytes, mitochondrial pools are relatively small. Since brain mitochondria are sites of significant hydrogen peroxide generation, the mitochondrial localization of GSH and its associated enzymes in neural cells provide important defenses against toxic oxygen species in the nervous system. Differences in subcellular distribution of GSH systems in individual neural cell types may provide a basis for selective cellular and/or subcellular expression of neurotoxicity.     

Chemical traumatization of adult mouse olfactory epithelium in situ stimulates growth and differentiation of olfactory neurons in vitro

This study demonstrates that ZnSO₄ induced chemical trauma results in an in situ regeneration of the olfactory epithelium which, when maintained in vitro, provides an enriched population of olfactory neurons. Therefore, the ability of the olfactory epithelium to respond to chemical trauma with increased mitotic activity can be used to increase growth of neurons in culture. Tissue obtained from normal or vehicle-treated adult mice produced few olfactory neurons, when maintained in culture, compared to cultures established from tissue following an in situ ZnSO₄ trauma. Maximal neuronal yields were obtained in cultures established from tissue that was removed 4–6 days following chemical trauma. The morphological appearance and the presence of cell specific intermediate filament proteins were used to classify the cell types in these olfactory epithelial cultures. Single cells and aggregates of cells which were immunopositive for keratin, but immunonegative for neurofilament protein and GFAP, were identified as epithelioid. Flattened polygonal cells immunopositive for GFAP were identified as glia. A small population of flattened cells was immunonegative for all of the antibodies used in this study. Cells that had processes were immunonegative for GFAP and keratin. Some were immunopositive for 200 kDa and 160 kDa neurofilament proteins but immunonegative for the 68 kDa neurofilament protein. A few of these cells showed positive immunoreactivity with the olfactory marker protein (OMP) antibody and most likely represented the most mature olfactory neurons in the cultures. This trauma-induced culture model using olfactory tissue from adult mice can serve as a source of CNS neurons for comparison with cultured embryonic neurons.     

ITP患者T细胞对巨核细胞生成作用的影响

本文应用改良的血浆凝块技术和共培养系统研究特发性血小板减少性紫癜(ITP)患者T细胞在体外对异体骨髓巨核细胞生成作用的影响,以探讨ITP的发病机制。实验结果表明,从总体来看,ITP患者的T细胞当加到培养物中和靶细胞共培养时,对巨核系祖细胞集落形成单位(CFU-MK)的集落效应无明显的影响。其中三例PAIgG升高患者的T细胞对CFU-MK有不同程度的抑制作用。以上结果提示ITP的发病机制相当复杂。除体液免疫机制外,也有细胞免疫机制参与,T细胞或其亚群对巨核系祖细胞生成抑制在ITP的发病机制中可能起着一定的作用。