人骨形态发生蛋白-2基因转染脂肪源性间充质干细胞的成骨研究

目的 探讨人骨形态发生蛋白-2（hBMP-2）基因修饰的人脂肪源性间充质干细胞（ADSCs）的成骨能力及基因转染对ADSCs成骨分化的生物学调控。方法 流式细胞技术鉴定从人脂肪组织中提取的细胞为间充质来源的干细胞。将ADSCs转染hBMP＋2基因，免疫沉淀＋Western blotting法和酶联免疫吸附试验（ELISA）检测hBMP-2表达，确定hBMP-2表达量及表达稳定性，碱性磷酸酶（ALP）染色、ALP定量测定、钙结节染色及RT—PCR分析hBMP-2基因转染对ADSCs向成骨细胞转化的调控，并将转基因细胞注入裸鼠股部肌内，通过X线及组织学检查观察体内成骨情况。结果 流式细胞术证实从脂肪中提取的细胞为间充质来源的干细胞。转基因后免疫沉淀＋Western blotting法和ELISA法显示转染hBMP-2的ADSCs具有较高且稳定的hBMP-2的表达，转染21d后表达量无明显降低。ALP染色、ALP定量测定、钙结节染色及RT—PCR发现转基因ADSCs向成骨细胞方向发生分化。裸鼠肌内注射转基因细胞后2周即显示有异位骨形成，4周明显增多，对照组无骨组织形成。结论 转染hBMP-2基因的ADSCs在持续稳定高表达目的蛋白的同时，自身向成骨细胞发生分化，并在裸鼠体内形成异位骨。因此，ADSCs有望作为新的基因工程种子细胞。     

复方肾华治疗大鼠改良慢性血清病肾炎的实验研究

目的　观察复方肾华胶囊对改良大鼠慢性血清病肾炎模型的治疗作用。方法　采取传统大鼠慢性血清病肾炎模型制作方法加上切除大鼠一侧肾脏、隔日饮饲牛血清白蛋白 (BSA)酸化水、腹腔注射大肠杆菌内毒素 (LPS)等措施 ,制作肾炎模型 ,与西药蒙诺作对照 ,观察中药肾华对肾炎大鼠尿蛋白、血生化、凝血指标、肾功能、病理、免疫荧光以及增殖细胞核抗原 (PCNA)表达的作用。结果　肾华胶囊能够显著减轻肾炎大鼠肾脏重量及肾重指数、降低肾炎大鼠尿蛋白、升高血清蛋白、降低胆固醇、甘油三酯、改善肾功能、纠正凝血功能紊乱、减轻肾脏病理改变、减轻免疫荧光变化、减弱PCNA的表达。结论　中药肾华胶囊对大鼠慢性血清病肾炎模型有较好的治疗作用     

Astrocyte and microglial motility in vitro is functionally dependent on the hyaluronan receptor RHAMM

RHAMM (Receptor for Hyaluronic Acid Mediated Motility) has been identified as a receptor for the extracellular matrix component hyaluronan (HA) and was recently shown to be essential for the locomotion of normal and transformed peripheral cells. Until now the potential role of RHAMM in the motility of neural-derived cells has not been investigated. Here, we report that cultured primary astrocytes, astrocyte cell lines, and microglia express this receptor and exhibit RHAMM-dependent motility. Immunocytochemical localization of RHAMM showed that it was often present as aggregates at the periphery of cells in contact with one another or concentrated on protruding processes of isolated cells. Glial cells contained 50 and 72 kDa forms of RHAMM, and both of these forms were found to have HA binding capacity. Time lapse imaging of cell locomotion revealed a significant inhibition of motility and process elongation by neutralizing anti-RHAMM antibodies and by peptides corresponding to the HA binding domains of RHAMM. These results demonstrate that RHAMM serves a role in glial cell locomotion in vitro and provide the basis for investigations of the motile behavior of glial cells in vivo after CNS injury.     

Cell mediated immunity (CMI) and post transplant viral infections — Role of a functional immune assay to titrate immunosuppression

Cell mediated immunity (CMI) was assessed by the ImmuKnow assay in 12 patients after kidney transplantation, who presented with viral infection. Treatment included lowering of immunosuppression in all cases and antiviral treatment if indicated. The assay was repeated during the follow up. The ImmuKnow assay at time of presentation of viral infections was 56.8 ± 58.2 (range 3–178; median 22) ATP ng/ml. With the clearance of viral infection and lowering of immunosuppression, the assay showed an increase in the level of CMI at 194.5 ± 118.9 (range 53–409; median 150) ATP ng/ml. There was viral clearance or stabilization in all cases and there was no incidence of allograft rejection. The ImmuKnow assay of CMI can be used to titrate initial immunosuppression reduction and its subsequent increase, in patients with viral infection after transplantation.     

Role of the cell recognition molecule, cognin, in GABAergic differentiation in chick retina

Previous work showed that GABAergic differentiation in developing chick retina depends on insulin and cell interactions. Here, we investigated whether it depended on cell signaling mediated by retina cognin, a 50 kDa cell recognition molecule. Cognin mediates cell adhesion in vitro and occurs on retinal neurons that become both GABAergic and cholinergic. We investigated two markers of GABAergic differentiation: glutamate decarboxylase (GAD) activity and high-affinity GABA uptake. Both increase during differentiation of retinal neurons in culture and can be easily measured. We blocked cognin-mediated cell signaling with cognin antibody and found a reduction of the developmental increase in GAD activity in cultures of retinal neurons from 7 and 11 day chick embryos. There was no reduction of high-affinity GABA uptake. This suggested that cognin-mediated signaling was necessary for the normal developmental increase in GAD but not for high-affinity GABA uptake. These results contrasted with our previous observations on cholinergic differentiation in cultured retinal neurons. We found that cognin antibody blocked the normal developmental increase in choline acetyltransferase (ChAT) only if the cells were exposed before embryonic day 7. Thus, while both GAD and ChAT activity appear to be controlled by cell signaling involving cognin, the periods of developmental sensitivity for the two differentiation markers are different. Antibodies to other adhesion molecules, Ng-CAM, and N-cadherin, did not similarly affect GAD activity. Antibodies to laminin at a 10-fold higher concentration inhibited GAD activity only in early embryonic retina. Tests for protein synthesis and “housekeeping” enzyme activity demonstrated that the cognin antibody effect was selective for neuronal differentiation pathways. Thus, GABAergic differentiation in developing retina is sensitive to cell signaling mediated in part by cognin.     

流式细胞光度术在鼻咽癌上皮样细胞株细胞周期动力学研究中的应用

本实验应用流式细胞光度术研究鼻咽癌上皮样细胞株（ＣＮＥ）的细胞周期动力学。结果发现ＣＮＥ细胞仍保持原代细胞的ＤＮＡ含量水平（超三倍体和亚四倍体），随着细胞接种时间的延长细胞周期中Ｇ０／Ｇ１时相细胞的百分数逐渐升高，而Ｓ％和Ｇ２＋Ｍ％则逐渐降低。该项研究还发现随着孵育时间的延长，细胞周期中各时相细胞的运动均逐渐延缓，其中以Ｇ０／Ｇ１和Ｇ２＋Ｍ时相细胞的运动减慢较为明显。     

重组人肿瘤坏死因子诱导HL－60白血病细胞分化及调控原癌基因表达的作用

采用２０～２００Ｕ／ｍｌ的重组人肿瘤坏死因子（ｒｈＴＮＦ－α）处理体外液相培养的人早幼粒白血病细胞珠ＨＬ－６０，观察到５０～２００Ｕ／ｍｌ的小剂量ＴＮＦ具有诱导其向单核－巨噬细胞途径分化及抑制其增殖的效应。采用细胞总ＲＮＡ打点杂交技术检测１～１００Ｕ／ｍｌＴＮＦ诱导ＨＬ－６０细胞早期（１２小时内）对其ｃ－ｍｙｃ，ｃ－ｆｏｓ原癌基因表达的影响，发现ＴＮＦ可抑制ｃ－ｍｙｃｍＲＮＡ水平及ｃ－ｆｏｓ短暂性表达增高。结果表明此小剂量ｒｈＴＮＦ－α具有诱导白血病细胞分化的效应及调控多癌基因表达的作用。     

应用胶原凝胶构建组织工程化皮肤的研究

目的探讨以胶原凝胶为支架材料构建组织工程化皮肤的可行性。方法体外分离、培养人皮肤表皮细胞和成纤维细胞;利用自制的胶原蛋白制成胶原凝胶作为组织工程支架材料;在成功构建人工真皮的基础上种植表皮细胞,构建复合人工皮肤;采用HE染色与免疫组织化学的方法对复合人工皮肤进行组织学检测。结果HE染色可见,构建的复合人工皮肤具有表皮和真皮双层结构;免疫组织化学染色显示,Ⅳ型胶原、纤维连接蛋白和层粘连蛋白阳性,在形态结构上与正常皮肤相似。结论培养的人表皮细胞和成纤维细胞种植于胶原凝胶支架上,气-液界面培养可构建出具有类似正常皮肤结构的组织工程化皮肤。     

突触蛋白-Ⅰ在胚胎干细胞体外神经分化过程中的表达变化

目的探讨突触蛋白-I（synapsin-I）在胚胎干细胞（ESCs）体外神经分化过程中的表达变化及作用。方法采用“五步法”和维甲酸（RA）法两种途径体外诱导ESCs向神经细胞分化，并以另一种可向神经细胞分化的肿瘤细胞-PC12细胞的诱导过程作参照，从不同的途径、不同的细胞进行比较．通过免疫组织化学、RT-PCR、Western blot方法观察这一过程中synapsin-I的表达变化．找出synapsin-I在ESCs向神经细胞分化过程中表达变化的共同规律。结果结合形态学和其它神经特异性指标的变化，synapsin-I在ESCs和PC12细胞向神经细胞分化的过程中具有早期即有表达，后逐渐升高，至分化成熟阶段达最高，后期又逐渐下降的变化规律。结论在ESCs的分化过程中，synapsin-I的表达存在特定的时空规律，并与ESCs的形态学改变相关，提示synapsin-I可能对ESCs在神经分化过程中的形态分化起着重要的作用。