Isolation and differentiation of embryonic stem cells from BALB/c mouse

Objective To invest the efficient method which can culture and induce embryonic stem cells to neurocyte in vitro. Methods Isolate the blastula of 3.5 d from BALB/c species mouse. Culture the cells from inner cell mass (inner cell mass, ICM) which were isolated by mechanical method on the mouse embryonic fibroblaste cell (MEF) feeder layer or 0.1% gelatin coated dishes. The stem cells were identified by characterized morphology, alkaline phosphatase stain, differential potency in vivo and immunochemistry stain. The isolated cells were differentiated by serial induction method that mimicking the intrinsic developmental process of the neural system. Results The isolated cells were positive for alkaline phosphatatse and SSEA-1 (stage specific embryonic antigen 1). Moreover they were identified pluripotent by differentiation in vivo. Therefore the isolated cells presented the characters of ESCs. Then the isolated cells were able to differentiate into neurocytes in vitro. Conclusion Mouse embryonic stem cells isolation, culture and differentiation system has been established.     

脂肪干细胞免疫学性状的初步实验观察

目的初步研究脂肪干细胞(Adiposederivedstemcells,ADSC)表面免疫分子的表达以及体外免疫调节功能,以期为组织工程提供同种异体种子细胞来源。方法体外培养人脂肪抽吸术中获取的脂肪干细胞,体外培养至第二代,流式细胞仪检测免疫分子HLA、HLA、B7-1、B7-2、CD40的表达。1×105个/孔ADSC细胞分别刺激单一异体淋巴细胞或混合双向淋巴细胞反应,观察淋巴细胞增殖情况。同时观察ADSC经IFN-γ作用后,免疫分子表达与淋巴细胞增殖的调节情况。结果ADSC表达HLA类分子,但未检测到HLA类分子阳性表达。B7-1(CD80)、B7-2(CD86)、CD28、CD40未见明显阳性表达。人IFN-γ刺激48h后,HLA类分子表达明显增高,HLAI表达未见明显增高。异体或经IFN-γ作用的ADSC均未能刺激异体淋巴细胞增殖。同样数量的ADSC可明显抑制双相混合淋巴细胞增殖,经IFN-γ作用后抑制作用未见明显减弱。结论ADSC具有一定的体外调节淋巴细胞反应的能力,有可能成为组织工程同种异体细胞来源。     

GM1 ganglioside partially rescues cultured dopaminergic neurons from MPP-induced damage: dependence on initial damage and time of treatment

GM1 ganglioside is believed to be important in promoting the recovery of neurons from injury. The present study assesses the ability of GM1 to repair or prevent the damage of dopamine neurons caused by the neurotoxin 1-methyl-4-phenylpyridinium (MPP⁺). Treatment of mesencephalic cell cultures with 2.5 μM MPP⁺ resulted in the loss of 30% of tyrosine hydoxylase (TH) immunoreactive neurons. In contrast, cultures administered 100 μM GM1 ganglioside for 3 days after toxin treatment contained nearly control numbers of TH⁺ neurons (97%). This reparative effect of GM1 was reflected in parallel increases in TH enzyme activity, dopamine and dopac levels. Cultures sustaining greater insult from higher doses of MPP⁺ (5.0–10.0 μM) did not benefit from ganglioside treatment, suggesting that rescue by GM1 depended on the degree of initial damage to cells. Moreover, the timing of ganglioside treatment was critical; pretreatment with GM1 alone did not prevent or attenuate the damage caused by subsequent incubation in 2.5 μM MPP⁺.     

EXAMINATION OF DEVELOPMENTAL NEUROTOXICITY BY THE USE OF TISSUE CULTURE MODEL SYSTEMS

1. The rotation-mediated three-dimensional reaggregate culture system is uniquely suited for studies on developmental neurotoxicity. In this system, it is possible to reconstruct central neuronal pathways and follow their development. 2. Exposure to drugs of abuse including methamphetamine and methylenedioxyamphetamine or the appetite suppressant, fenfluramine, reduces monoamines in the cultures in a dose-dependent manner and interrupts normal monoaminergic development. 3. While the monoaminergic neurones may attain normal rates of development following drug removal, the affected neurones are not capable of overcoming the drug-induced insults and a deficiency in monoamines persists throughout development. 4. In addition, the production of immortalized monoclonal hybrid cells obtained by fusion of fetal mesencephalic neurones with a neuroblastoma has yielded cell lines expressing a dopaminergic phenotype. 5. Such cells have been useful in establishing the relationship of neurotoxicity to cell lineage and can serve as models for the study of the cellular and molecular mechanisms of neurotoxicity.     

Hepatocyte isolation from pig livers after warm ischaemic injury

Abstract Hepatocyte cultures have been used extensively for a wide variety of physiological, pharmacological and experimental studies. The warm ischaemic period before isolation is kept to a minimum to achieve a high yield of cells isolated and a good viability for culture. We have recently introduced a new concept of liver resuscitation after warm ischaemia that is based on a 3-h reperfusion period with an improved perfusate and simultaneous dialysis. In this study, we applied the new technique for hepatocyte isolation from livers subjected to 80 min of complete ischaemia at 37 °C. Cell yield was improved by a resuscitating perfusion from 58% to 73% and viability from 39% to 76%.     

重庆市正常人群肠道双歧杆菌的定量调查

本文介绍了一种双歧杆菌的选择鉴别培养基,并用该培养基对重庆市正常人群肠道双歧杆菌进行了定量分析,进而确定了正常值,为进一步研究双歧杆菌与人类的健康和疾病以及生态学防治提供了科学依据.同时也显示了正常人类肠道中双收杆菌的含量与年龄及性别无显著差异.     

In vitro preimplantation mouse embryo development with incubation temperatures of 37 and 39°C

Embryos from two strains of mice were used to assess the effect of incubation temperature on pronuclear and twocell development to the morula/blastocyst (M/B) stage. Embryos from B6D2F2 and B6SJLF1 strains were cultured in medium M16 at either 37 or 39°C until 120 hr post human chorionic gonadotropin (hCG) or 0, 24, or 48 hr at 37°C and the remaining time at 39°C. Overall M/B development for pronuclear embryos was 0.6, 0, 32.3, and 52.4% for 0—96, 24—72, 48—48, and 96—0 hr at 37 and 39°C, respectively. Only 0—96 and 24—72 hr at 37 and 39°C were not different (P >0.10). Overall M/B development for two-cell embryos was 48.1, 78.1, and 98.0% for 0—72, 24—48, and 72—0 hr at 37 and 39°C, respectively. Percentage development at each time was different (P <.01) for each category. Additionally, the number of nuclei for morulae and blastocysts tended to be higher for embryos initiating culture at the two-cell stage compared to pronuclear embryos. The first cell cycle was most dramatically affected by a 2°C increase in incubator temperature. More advanced embryos can tolerate slight increases in incubator temperature more readily than pronuclear embryos.     

Regulation of peripheral-type benzodiazepine receptors in cultured astrocytes by monoamine and amino acid neurotransmitters

Exposure of primary cultured astrocytes for 3 days to 1 μM of either dopamine, serotonin or norepinephrine resulted in upregulation (25–34% increase in B_max) of the peripheral-type benzodiazepine receptors (PBRs) labeled with [³H]Ro5-4864. A similar treatment with γ-aminobutyric acid [GABA] caused a 2-fold increase in the affinity (K_d) of [³H]Ro5-4864. The monoamines tested and GABA had no effect on the binding parameters of [³H]PK 11195, another selective PBR ligand. The present study indicates that Ro5-4864 binding sites are susceptible to regulation by specific neurotransmitters and provides further evidence for the distinction between Ro5-4864 and PK 11195 binding sites of the PBRs in cultured astrocytes.     

Pulsed electromagnetic fields alter phenotypic expression in chondroblasts in tissue culture

We hypothesize that pulsed electromagnetic fields (PEMF) alter phenotypic expression of chondroblasts by promoting the production of alkaline phosphatase (AP) and altering the structure of proteoglycans. Chondroblasts from the hypertrophic zone of tibial epiphyses (HC), sternum (SC), and skin fibroblasts (F) were cultured from 16 day chick embryos. Cultures were randomly designated control (C) or experimental (E). E received PEMF for 24 h in a 6 h on, 6 h rest sequence. The controls were in the same incubator shielded by Mu metal. Assays for AP activity were performed and normalized to protein content. Proteoglycan synthesis assay involved labeling with 35S fractionating in a 5% to 20% surcrose gradient determining total protein and chondroitin sulfate content. PEMF showed no change of AP activity on F. A high AP basal activity was found in HC, but was not increased above the control. PEMF increased AP in the SC samples (E/C ratio). The sucrose gradient data showed a shift in peaks for SC only altering the ratio of carbohydrate to protein for the SC. Analysis of carbohydrate and protein indicated that the effect was decreased synthesis or degradation of protein. We conclude that PEMF alters the phenotypic expression of sternal chondroblasts in our in vitro system.     

人胎儿雪旺氏细胞的体外培养及其纯化研究

本实验用酶消化法从孕１４～１９周人胎儿周围神经中分离培养雪旺氏细胞，结合差速贴壁和阿糖胞苷处理进行纯化，根据Ｓ－１００蛋白免疫细胞化学染色和形态学特征，分析不同时期雪旺氏细胞的纯化程度。实验结果显示：培养４ｄ，倒置相差显微镜下观察，雪旺氏细胞呈典型的双极或三极形，端对端或旋涡状排列，Ｓ－１００蛋白免疫细胞化学染色呈阳性反应，其比例约占９８％；在２～３周内，雪旺氏细胞的纯度维持在８５％～９０％；培养３５ｄ，雪旺氏细胞约占８０％；单纯采用阿糖胞苷或差速贴壁处理，培养３５ｄ，雪旺氏细胞约占７０％；原代培养９２ｄ，传代１１代细胞生长良好。实验结果表明，多种方法联合使用较单一方法所得的纯度高。本方法快速、简便，能为雪旺氏细胞作为移植材料提供足够的来源。