Transplantation of human amnion epithelial cells improves learning and memory function in Alzheimer's disease-like pathology rat model

AIM: To observe the treatment effect and its immune regulation of human amnion epithelial cells (hAECs) on Alzheimer's disease (AD)-like pathology rat model. METHODS: The hAECs were isolated from amnion with trypsin digestion, and the phenotype of hAECs was analyzed by flow cytometry. SD rats (n=48) were randomly divided into sham control group, model group, medium group and hAECs group. AD-like pathology rat model was induced by bilateral intraventricular injection of lipopolysaccharide (LPS). hAECs (5×10⁵) were injected into the hippocampus of the AD-like pathology rats. At 2 weeks after transplantation, the animals were tested by Morris water maze to observe the function of learning and memory. The pathological change of the brain was observed by HE staining. The expression of amyloid β-protein 42(Aβ42) and Tau protein and the level of acetylcholine (ACh) in the injury brain were determined by immunohistochemistry. The survival and differentiation of hAECs in the hippocampus were measured by immunofluorescent technique. The percentages of lymphocyte subsets in the peripheral blood mononuclear cells were analyzed by flow cytometry. The contents of serum cytokines were detected by cytometric bead array. RESULTS: Compared with model group and medium group, hAECs group showed shortened escape latency (P<0.01), increased frequency of going through the platform (P<0.05), reduced loss of hippocampal neurons, decreased expression of Tau protein and Aβ42 in the hippocampus (P<0.05), increased ACh level in the hippocampus (P<0.05), decreased percentages of Th1 and Th17 subsets, increased percentages of Th2 and Treg cells (P<0.05), decreased concentrations of IFN-γ and IL-2 in the serum, and increased concentration of IL-4(P<0.05). CONCLUSION: hAECs improve the cognitive learning and memory function and alleviate pathologic damage of hippocampus through immune regulation in AD-like pathology rats.     

Effect of eleutheroside B/E on proliferation and expression of PPARγ and TGF-β1 in HBZY-1 cells cultured with high glucose

AIM: To explore the effects and mechanism of eleutheroside (ETS) B or E on the proliferation of HBZY-1 cells treated with high glucose. METHODS: The HBZY-1 cells were cultured under high glucose condition. The 4th generation of HBZY-1 cells was used for determining the optimal cell density, which was consistent with the growth regulation curve of the cells. The cells were divided into 6 groups: low glucose (LG) group, high glucose (HG) group, high glucose plus ETS-B/E (low dose, medium dose and high dose) groups, and high glucose plus losartan (LTG) group. After all cells were treated with the corresponding drugs at 24 h, 48 h and 72 h, the inhibitory rate of the proliferation was measured, and the expression of TGF-β1 and PPARγ was detected by immunocytochemistry and Western blotting. RESULTS: The best cell density was 2 000 cells/well, which was complied with the basic rules of the cell growth, and high glucose significantly promoted the HBZY-1 cell proliferation. At each time point, the inhibitory effects of ETS-B/E were significantly different between HG group and LTG group on the proliferation of the HBZY-1 cells (P<0.05). The expression of TGF-β1 was significantly inhibited, and the expression of PPARγ was significantly promoted by ETS-B/E (P<0.05). ETS-E showed stronger effect than ETS-B (P<0.05) in a concentration- and time-dependent manner. CONCLUSION: ETS-B/E significantly inhibits the proliferation of HBZY-1 cells under high glucose condition by decreasing TGF-β1 expression and promoting PPARγ expression.     

Ultrastructural impairment of islet microvascular endothelial cells in STZ-induced type 1 diabetic mice

AIM: To investigate the ultrastructural changes of islet microvascular endothelial cells in STZ-induced type 1 diabetic mice. METHODS: BALB/c mice were randomly divided into diabetic group and control group. The expression of insulin and platelet-endothelial cell adhesion molecule-1 (CD31) in islet microvessels was detected by immunohistochemical staining. The ultrastructural changes of islet β cells and islet microvessels were observed under transmission electron microscope. RESULTS: Compared with control group, the number of islet β cells, ratio of β cells/α cells, average number of secretory granules in β cells and insulin expression area per islet in diabetic group were significantly decreased (P<0.01). Besides, diabetic group had fewer microvessels with lower expression of CD31 (P<0.01). Mitochondria in islet microvascular endothelial cells and pericytes in diabetic group were swelling. The basement membrane of islet microvessels became thicker in diabetic group (P<0.01). CONCLUSION: Islet microvascular endothelial cells were impaired in type 1 diabetic mice.     

Vitamin C enhanced myocardial differentiation of dedifferentiated fat cells

AIM: In order to observe the myocardial differentiation capacity of the dedifferentiated fat (DFAT) cells treated with vitamin C in vitro. METHODS: DFAT cells were dedifferentiated from the mature rat adipocytes with ceiling adherent culture. The DFAT cells of passage 3 were used in the study. Vitamin C and/or neonatal rat heart tissue lysate were added into the culture medium to induce myocardial differentiation for 3 weeks. The cell morphology was observed under microscope. The myocardial-specific markers, such as cTnT, GATA-4 and NKx2.5, were examined by the methods of immunofluorescence, PCR and Western blot. RESULTS: Mature rat adipocytes dedifferentiated into fibroblast-like DFAT cells after ceiling adherent culture. The DFAT cells spontaneously differentiated into cardiomyocyte-like cells under normal culture condition with a low incidence. After treated with neonatal rat heart cell lysate, the DFAT cells became cardiomyocyte-like cells that had bigger size, longer shape and myotubule-structure. The expression of cTnT, GATA-4 and NKx2.5 was remarkably increased at both mRNA and protein levels as compared with the normal cultured DFAT cells. The expression of cTnT, GATA-4 and NKx2.5 was further increased in DFAT cells after treating with vitamin C. No spontaneous beating cell was observed. CONCLUSION: Vitamin C enhances the differentiation of DFAT cells into cardiomyocyte-like cells.     

过表达MyBPC1对牛骨骼肌卫星细胞增殖分化的影响

旨在探究肌球蛋白结合蛋白C1（myosin binding protein C1,MyBPC1）对牛骨骼肌卫星细胞增殖与成肌分化的影响,为进一步研究MyBPC1在细胞分化和肌肉发育过程中的调控作用提供依据。本研究利用西门塔尔胎牛原代牛骨骼肌卫星细胞体外诱导成肌分化模型模拟牛骨骼肌的生长发育过程。采用qRT-PCR和Western blot检测MyBPC1的细胞时序表达谱。试验分为两组。在RNA水平每组4个重复,每个重复20 μL;在蛋白水平每组3个重复,每个重复15 μg。采用qRT-PCR和Western blot检测牛骨骼肌卫星细胞转染MyBPC1的过表达效果,并进一步检测细胞增殖期标志因子Pax7、Ki67以及细胞分化期标志因子MyHC、MyOG的表达变化情况,观察牛骨骼肌卫星细胞肌管形成状态。结果,MyBPC1在牛骨骼肌卫星细胞分化前后表达水平存在极显著差异,牛骨骼肌卫星细胞诱导分化后MyBPC1的mRNA和蛋白表达量均极显著高于增殖期（P<0.01）。过表达MyBPC1后,细胞分化形成的肌管数量明显多于对照组,增殖标志因子Pax7的mRNA水平和蛋白表达水平无显著差异,分化标志因子MyHC的mRNA水平和蛋白表达水平极显著高于对照组（P<0.01）。过表达MyBPC1可以促进牛骨骼肌卫星细胞体外成肌分化,为进一步开展MyBPC1对牛骨骼肌卫星细胞的调控机制奠定基础。     

CD8+T细胞在不同年龄家兔胃肠道的分布规律

探讨不同年龄组家兔胃肠道各段CD8⁺T细胞的分布特征,为进一步研究CD8⁺T细胞的作用奠定形态学基础。采用免疫组织化学技术对不同年龄组(幼年组、青年组、老年组)家兔胃肠道内CD8⁺T细胞进行染色、观察及统计分析。结果显示,家兔的CD8⁺T细胞多呈圆形、椭圆形或呈不规则形。在不同年龄组家兔,胃肠不同部位的分布有如下特点:家兔的CD8⁺T细胞主要分布于胃肠道的固有层中;青年组CD8⁺T细胞的分布数量显著高于幼年组和老年组;各肠段之间CD8⁺T细胞分布的数量差异不显著,但其分布的总量按照小肠、大肠、胃的顺序依次降低。研究结果显示,CD8⁺T细胞在家兔小肠的分布最多,并且其数量随着年龄的增长呈现先上升后下降的趋势,青年家兔胃肠的分布最为丰富。     

MSTN基因对牛肌动蛋白细胞骨架调节通路的影响

为探究肌肉生长抑制素（myostatin,MSTN）对牛骨骼肌生长发育的作用机制,本研究前期利用定量蛋白质组学与磷酸化蛋白质组学分析野生型蒙古牛（MG.WT）和MSTN^+/-蒙古牛（MG.MSTN^+/-）腿臀肌肌肉组织中蛋白质水平和磷酸化修饰水平的差异变化,使用已建立的牛骨骼肌卫星细胞体外诱导成肌分化模型,检测设计合成的MSTN siRNA （si-MSTN）干扰效果;采用实时荧光定量PCR和Western blotting方法检测转染si-MSTN的增殖期（GM）和分化第3天（DM3）牛骨骼肌卫星细胞中肌动蛋白细胞骨架调节通路相关基因的mRNA和蛋白水平的表达变化,研究敲低MSTN表达对肌动蛋白细胞骨架调节通路的影响。结果显示,在MSTN^+/-蒙古牛肌肉组织中共鉴定到16个肌动蛋白细胞骨架调节通路相关基因表达丰度上调;转染si-MSTN细胞中的MSTN表达水平极显著降低（P<0.01）;在转染si-MSTN的GM期牛骨骼肌卫星细胞中,肌动蛋白细胞骨架调节通路相关基因ENAH、ACTN4和Cdc42的mRNA水平均显著升高（P<0.05）,PFN1、RhoA和ACTN4的蛋白水平均显著或极显著升高（P<0.05;P<0.01）;在转染si-MSTN的DM3牛骨骼肌卫星细胞中,ENAH、CFL1、SCIN和Cdc42基因mRNA水平均显著升高（P<0.05）,RhoA基因mRNA水平极显著升高（P<0.01）,PFN1和ACTN4的蛋白水平均显著升高（P<0.05）。结果表明,干扰MSTN可以促进肌动蛋白细胞骨架调节通路相关基因的表达,探明了MSTN可能通过介导肌动蛋白细胞骨架调节通路影响牛骨骼肌卫星细胞增殖和成肌分化的分子机制,为进一步研究MSTN对牛成肌分化的调控机制提供参考。     

Production and Identification of Clone Sheep Using Bone Marrow Mesenchymal Stem Cells as Donor Cell

In order to obtain the cloned sheep using bone marrow mesenchymal stem cells（BMSCs）as the donor cells,the BMSCs of sheep were chosen and reconstructed embryos were built to transfer.10 published microsatellite markers were chosen,and the DNA samples from clone sheep,donor cells and surrogate ewes were amplified,and the relationship of father-son（RCP）was analyzed using the Quantity One for genotyping.The results showed that the reconstructed embryos were successfully built for electric fusion using sheep BMSCs as nuclear donor,and making nuclear transplantation into enucleated mature oocytes of which the fusion rate was 80.62%.20 surrogate ewe were chosen to be implanted with the reconstructed embryos at morula stage by implant surgery,and 5 lambs were born and only 3 were survived.The genotype of cloned sheep was in line with the dornor cell and the RCP were more than 99.999%.In conclusion,the first clone sheep were obtained successfully by using BMSCs as a nuclear donor in this experiment.     

Biodegradation of Linear Alkyl Benzene Sulphonate

The paper researches on the degradation of LAS (Linear Alkyl benzene Sulphonate) by combining AS1.860 immobilized with low intensity ultrasonic irradiation. The paper also discusses the influence of pH, rotary velocity, LAS concentration and the condition of low intensity ultrasonic irradiation on the degradation of LAS, the degradation rate of LAS is the main index of our experiment, the results of orthogonal test shows that low ultrasonic irradiation can increase the metabolizing of microorganism cells and facilitate the biodegradation of immobilized cells to LAS, here we research the degradation condition of 50mg/L LAS simulation wastewater with low ultrasonic irradiation, and the results show that the influence is obvious, the optimization degradation rate is about 83%, nine point five percent higher than that of the immobilized cells without ultrasonic irradiation.     

内毒素对肠黏膜微血管内皮细胞内分泌一氧化氮和内皮素的影响

目的是研究内毒素（lipopolysaccharide; LPS）导致肠黏膜微血管内皮细胞损伤的作用机制。将所培养的肠黏膜微血管内皮细胞分为空白对照组和LPS组，每组按时间分为4个亚组：3h、6h、9h、12h。空白对照组加维持培养液，LPS组加1μg/ml LPS培养液，在CO2培养箱内静置培养。结果表明，一氧化氮（NO）分泌明显升高，3h后达到高峰，随后逐渐降低，而内皮素（endothelin; ET）分泌急剧升高，9h后达到高峰，随后又开始逐渐降低，但一直保持在较高的水平。所以引起了NO和ET的升高可能是LPS导致肠黏膜微血管内皮细胞的损伤机制。