(-)-Epigallocatechin-3-gallate,reduces corneal damage secondary from experimental grade II alkali burns in mice

Background

Since recent reports have shown that (-)-Epigallocatechin-3-gallate (EGCG) could be used for treating proliferative and inflammatory disorders, we explored its use for the management of corneal chemical burns.

Investigation of the mechanisms of Genkwa Flos hepatotoxicity by a cell metabolomics strategy combined with serum pharmacology in HL-7702 liver cells

1. To investigate Genkwa Flos hepatotoxicity, a cell metabolomics strategy combined with serum pharmacology was performed on human HL-7702 liver cells in this study.

2. Firstly, cell viability and biochemical indicators were determined and the cell morphology was observed to confirm the cell injury and develop a cell hepatotoxicity model. Then, with the help of cell metabolomics based on UPLC-MS, the Genkwa Flos group samples were completely separated from the blank group samples in the score plots and seven upregulated as well as two down-regulated putative biomarkers in the loading plot were identified and confirmed. Besides, two signal molecules and four enzymes involved in biosynthesis pathway of lysophosphatidylcholine and the sphingosine kinase/sphingosine-1-phosphate pathway were determined to investigate the relationship between Genkwa Flos hepatotoxicity and these two classic pathways. Finally, the metabolic pathways related to specific biomarkers and two classic metabolic pathways were analyzed to explain the possible mechanism of Genkwa Flos hepatotoxicity.

3. Based on the results, lipid peroxidation and oxidative stress, phospholipase A₂/lysophosphatidylcholine pathway, the disturbance of sphingosine-1-phosphate metabolic profile centered on sphingosine kinase/sphingosine-1-phosphate pathway and fatty acid metabolism might be critical participators in the progression of liver injury induced by Genkwa Flos.     

PCNA、Survivin蛋白在人绒癌细胞株BeWo合体化过程中表达变化的研究

目的:通过检测人绒癌细胞株Be Wo合体化过程中增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、生存素(Survivin)蛋白表达的变化,探讨滋养细胞合体化后增殖性的变化,为恶性滋养细胞肿瘤,尤其是耐药恶性滋养细胞肿瘤的临床治疗提供新的思路和方法。方法:利用毛喉素(forskolin)诱导Be Wo细胞株融合;应用逆转录聚合酶链反应(RT-PCR)检测促融素(Syncytin)在forskolin作用不同时间的Be Wo细胞株中的表达;应用蛋白质印迹(Western blotting)检测PCNA、Survivin蛋白在forskolin作用不同时间的Be Wo细胞株中的表达;应用噻唑蓝比色分析实验(MTT)法检测forskolin作用不同时间的绒癌细胞株Be Wo的增殖能力。结果:1forskolin作用后的Be Wo细胞株Syncytin基因的表达增强,且随着forskolin作用时间的延长,Syncytin的表达更强,于48 h达到高峰。2forskolin作用后的Be Wo细胞株PCNA、Survivin蛋白的表达降低。3forskolin作用后的Be Wo细胞株的增殖能力下降,且不同作用时间的差异有统计学意义;forskolin作用的时间越长,Be Wo细胞株增殖能力下降越明显。结论:人绒癌细胞株Be Wo合体化后PCNA、Survivin蛋白的表达降低,说明人绒癌细胞株Be Wo合体化后增殖性降低,推测诱导滋养细胞合体化可能对临床治疗恶性滋养细胞肿瘤具有一定作用。     

依维莫司联合全反式维甲酸逆转急性早幼粒细胞白血病细胞耐药的研究

目的探讨依维莫司联合全反式维甲酸(简称维甲酸)逆转急性早幼粒细胞白血病(APL)细胞株NB4-R1耐药的作用。方法应用CD11b染色流式细胞术及硝基四唑氮蓝(NBT)还原实验检测两药联合应用对细胞分化的影响, 流式细胞术检测细胞周期情况, Annexin V/PI双染色检测细胞凋亡情况, 蛋白质印迹法检测自噬相关蛋白微管结合蛋白轻链3(LC3)、Beclin 1及早幼粒白血病-维甲酸受体融合蛋白(PML-RARα)、磷酸化核糖体S6激酶(P-P70S6K)、磷酸化4E结合蛋白1(P-4E-BP1) 等表达水平。结果与维甲酸组比较, 联用组能诱导耐药细胞株NB4-R1细胞的分化, 并将细胞增殖阻止在G ₁期而对细胞凋亡无明显影响。100 nmol/L依维莫司组、1μmol/L维甲酸组、联用组、对照组NB4-R1细胞培养48 h后分化百分率分别为(2.29±0.57)%、(17.06±2.65)%、(54.47±4.91)%、(2.54±0.53)%; 处于G ₁期的细胞百分率分别为(35.20±11.97)%、(33.54±6.25)%、(53.70±8.73)%、(27.40±6.01)%; 四组细胞凋亡细胞百分率分别为(2.30±0.14)%、(2.25±0.21)%、(2.40±0.28)%、(1.95±0.07)%。与维甲酸组比较, 联用组mTOR信号通路下游的P70S6K、4E-BP1分子磷酸化水平下降, LC3-II和Beclin 1的表达上调, 且能部分降解融合蛋白PML-RARα。 结论依维莫司联合维甲酸能诱导NB4-R1细胞分化, 且能阻滞细胞周期而不致细胞凋亡, 其机制可能与依维莫司联合维甲酸抑制mTOR信号通路激活自噬作用从而降解PML-RARα蛋白有关。     

塞来昔布对大鼠心脏移植急性排斥反应的抑制作用

目的观察塞来昔布对大鼠心脏移植急性排斥反应的抑制作用。方法以SD大鼠为供者,Wistar大鼠为受者,进行40次腹部异位心脏移植。采用HE染色和原位末端标记(TUN EL)技术检测移植心切片,进行排斥反应的病理分级并计算移植心肌细胞的凋亡指数(AI)。结果移植心的细胞凋亡主要发生于心肌细胞;移植后第3、5d,塞来昔布治疗组心肌细胞凋亡指数分别为:1.03±0.42和3.28±2.42;对照组分别为2.35±1.51和11.35±3.46;两组比较,差异有统计学意义(P<0.001)。结论细胞凋亡是心脏移植急性排斥中组织损伤的重要机制;塞来昔布能明显抑制心肌细胞凋亡。     

Monolayer cultures of normal human bone cells contain multiple subpopulations of alkaline phosphatase positive cells

Summary Cytochemical staining of normal human bone cells in monolayer cultures for alkaline phosphatase (ALP) indicated that the cultures contained mixed-cell populations. Time course evaluations of the cytochemical staining revealed, in addition to the ALP-negative cell population, at least two subpopulations of ALP-positive human bone cells with different levels of ALP. A cytochemical method has been developed which separates the ALP-positive cells into high and intermediate ALP subpopulations. In this method, human bone cells were stained for ALP using an azo-dye method and incubating at 4°C for 10 and 30 minutes, respectively. We defined the cell population that stained positively for ALP at 10 minutes as strong ALP-positive cells, and both strong and intermediate cells were stained at 30 minutes. The intermediate cells were determined from the difference between the values at the two time points. The intra- and interassay variations of the assay, with the same investigator in blinded investigations, were both less than 10% and the interobserver variation was approximately 25%. Analysis of the distribution of ALP levels in cells with a laser densitometer confirmed the presence of at least three cell subpopulations. 1,25(OH)₂D₃ treatment increased the proportions of both ALP-positive cell populations, whereas TGF-beta treatment increased only the intermediate ALP-positive cell population. On the contrary, fluoride increased the proportion of the strong ALP cells, and IGF-1 had no effect on the proportions of either ALP-positive subpopulation. When the ALP-specific activity was compared with the percentage of each ALP-positive subpopulations for the cells treated with effectors, the ALP-specific activity correlated with the total ALP-positive and with the strong ALP-positive populations but not with the intermediate ALP-positive subpopulation. In summary, this study represents the first evidence that normal human bone cells in monolayer cultures contained at least two subpopulations of ALP-positive cells, and that bone cell effectors could have differential effects on each cell population.     

二甲基亚砜诱导人肝癌细胞BEL-7402凋亡的研究

目的 :研究二甲基亚砜 (DMSO)对人肝癌细胞凋亡的诱导作用。方法 :用不同浓度的DMSO处理体外培养的人肝癌细胞BEL 74 0 2 ,应用普通光镜、荧光显微镜、MTT分析方法和流式细胞技术 (FCM )检测肝癌细胞凋亡的形态学变化、细胞存活率、凋亡百分率和细胞周期分布的变化。结果 :DMSO诱导BEL 74 0 2细胞核DNA凝缩和核片段化 ,最后形成凋亡小体 ;随着DMSO浓度的增加和处理时间的延长 ,细胞存活率明显下降 ,其IC50 为 1.9% ;2 %的DMSO处理细胞 12h ,凋亡率达 17.2 1% ,同时S期细胞明显增加 ,G2 M期细胞明显下降。结论 :DMSO可诱导人肝癌细胞凋亡 ,并使细胞受阻于S期而进入凋亡程序。     

支架及无支架条件下构建组织工程软骨的研究进展

支架材料的研究是组织工程的研究热点之一,软骨细胞复合培养给组织工程软骨的构建带来希望,单一的材料诸如胶原、透明质酸、壳聚糖、纤维蛋白凝胶等已经证明可以与软骨细胞复合培养,两种或者多种材料复合可以提高材料的性能,更好地用于组织工程软骨的构建,并满足软骨缺损修复的需要;同样,在无支架情况下应用软骨细胞聚集培养、沉淀培养的方法,可以构建组织工程软骨,并给软骨缺损的修复带来新希望,但目前的研究较少。两者是组织工程软骨构建的两个主要方向。     

Monoclonal antibody production to human and bovine 2′:3′-cyclic nucleotide 3′-phosphodiesterase (CNPase): high-specificity recognition in whole brain acetone powders and conservation of sequence between CNP1 and CNP2

Monoclonal antibodies against human and bovine 2′:3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) were generated by fusing FOX-NY myeloma cells with spleen cells from RBF/Dn mice previously immunized with the purified brain antigens. The enzyme isolated from bovine brain was quite basic, with an isoelectric point of 9.71 and both the bovine and human enzymes consisted of a closely spaced doublet at approximately 44 and 46 kDa on SDS-PAGE. Six monoclonals were identified as strongly recognizing the enzyme on both ELISA plates and on immunoblots of whole brain protein. Four monoclonals very weakly cross-reacted with guinea pig myelin basic protein. In contrast with two previous reports, some of our monoclonal antibodies did immunostain 2 or 3 protein bands in peripheral nerve, two bands closely corresponding to those immunostained in central nervous system (CNS) myelin, the Wolfgram protein fraction and in acetone powders of whole brain. Each of the 6 monoclonals reacting strongly on immunoblots recognized the enzyme in from 2 to 5 of the species examined (human, bovine, rat, mouse and rabbit). In addition, all 6 monoclonals that immunostained the enzyme in whole brain, myelin and Wolfgram protein immunoblots recognized both CNP1 (44 kDa) and CNP2 (46 kDa). The two closely spaced protein bands observed on SDS-PAGE and previously stained on immunoblots of CNS CNPase using polyvalent rabbit anti-bovine CNPase antisera, and now different monoclonal antibodies, appear to be immunologically related and to contain highly conserved sequences.     

Elemental composition and water content of myelinated axons and glial cells in rat central nervous system

The distribution of elements (e.g. Na, Cl, K) and water in CNS cells is unknown. Therefore, electron probe X-ray microanalysis (EPMA) was used to measure water content and concentrations (mmol/kg dry or wet weight) of Na, Mg, P, S, Cl, K and Ca in morphological compartments of myelinated axons and glial cells from rat optic nerve and cervical spinal cord white matter. Axons in both CNS regions exhibited similar water content ( 90%), and relatively high concentrations (wet and dry weight) of K with low Na and Ca levels. The K content of axons was related to diameter, i.e. small axons in spinal cord and optic nerve had significantly less (25–50%) K than larger diameter axons from the same CNS region. The elemental composition of spinal cord mitochondria was similar to corresponding axoplasm, whereas the water content (75%) of these organelles was substantially lower than that of axoplasm. In glial cell cytoplasm of both CNS areas, P and K (wet and dry weight) were the most abundant elements and water content was approximately 75%. CNS myelin had predominantly high P levels and the lowest water content (33–55%) of any compartment measured. The results of this study demonstrate that each morphological compartment of CNS axons and glia exhibits a characteristic elemental composition and water content which might be related to the structure and function of that neuronal region.