富血小板血浆对体外培养条件下人真皮成纤维细胞增殖能力的影响

目的:探讨富血小板血浆(PRP)对人真皮成纤维细胞(hDFbs)在体外培养条件下增殖能力的影响,探讨PRP促进皮肤、黏膜伤口愈合的机制.方法:PRP和hDFbs来源于健康成年人,两次离心法制备PRP,倒置相差显微镜观察0、12.5%、25.0%、50.0%和100.0%PRP浓度作用下 hDFbs的增殖;免疫细胞化学检...     

苏拉明对体外培养视网膜色素上皮细胞增殖的影响

目的　研究苏拉明 (suramin)对培养人视网膜色素上皮细胞 (retinal pigm ent epithelium,RPE)增殖的影响 .方法　将不同质量浓度的苏拉明 (1.5 ,15和 15 0 mg· L- 1 )加入RPE细胞培养液 ,采用四甲基偶氮唑盐 (tetrazolium,MTT)比色法 ,细胞分裂指数计数和核仁组成区嗜银染色(Ag NORs)检测苏拉明对 RPE增殖活力的影响 .结果　含有10 0 m L· L- 1小牛血清的培养液可以显著刺激 RPE的增殖(P<0 .0 1) ,无血清组 A值为 0 .19± 0 .0 1、含血清组 A值为0 .30± 0 .0 1;苏拉明抑制了 10 0 m L· L- 1血清条件下 RPE的增殖 ,呈剂量依赖性 ,最大抑制率达 5 1% ,3种浓度组 A值分别为 0 .2 9± 0 .0 1,0 .2 4± 0 .0 1和 0 .14± 0 .0 1,对细胞的形态无明显影响 .结论　苏拉明对血清刺激 RPE增殖有显著非毒性抑制作用     

探讨不同实验条件对U87细胞体外增殖的影响

目的:探讨RNA干扰肝癌衍生生长因子(HDGF)后,U87细胞增殖抑制的最佳实验条件。方法:用LipofectamineTM2000将HDGF siRNA转染U87细胞后,将细胞接种于96孔板中,分别在无血清、含10%FBS和无血清Matrigel胶预处理细胞培养板条件下,采用MTS法检测细胞增殖能力。结果:HDGF表达水平下调后,U87细胞增殖能力受到抑制。在无血清、含10%FBS和无血清Matrigel胶预处理细胞培养板条件下,细胞增殖抑制率分别是18%、9%和28%。结论:在无血清Matrigel胶预先处理的培养条件下,能最大程度上反映出HDGFsiRNA对U87细胞增殖能力的抑制。     

血小板衍生生长因子D通过ERK信号通路对肺癌H1299细胞增殖、迁移和侵袭的影响及其机制

目的:探讨血小板衍生生长因子D (PDGF-D)对人肺癌H1299细胞增殖、迁移和侵袭的影响,并阐明其可能的作用机制。方法:选用人肺癌H1299细胞,分为对照组(转染空载体)和PDGF-D组(转染GV230-PDGF-D质粒),同时设空白组,采用实时荧光定量PCR (RT-qPCR)法和Western blotting法检测各组细胞中PDGF-D mRNA表达水平和蛋白表达量。H1299细胞分为对照组(转染空载体)、PDGF-D组(转染GV230-PDGF-D质粒)、PDGF-D+PD98059组(转染GV230-PDGF-D质粒+ERK抑制剂PD98059)和PD98059组,PD98059终浓度为10μmol·L^-1。MTT法检测各组细胞增殖活性,划痕实验检测各组细胞迁移率,Transwell实验检测各组细胞中侵袭细胞数,Western blotting法检测各组细胞中磷酸化细胞外信号调节基酶(p-ERK)、细胞外信号调节基酶(ERK)、锌指转录因子Snail、基质金属蛋白酶1 (MMP-1)和跨膜黏附糖蛋白CD44的蛋白表达水平。结果:RT-qPCR法检测,...     

人可溶性APRIL突变体的原核表达与纯化

目的 制备人可溶性增殖诱导配体(soluble a proliferation inducing ligand,sAPRIL)的两种突变体,为寻找APRIL 的竞争抑制剂创造条件.方法 采用一步反向PCR法,以人APRIL第186位甘氨酸(186G)和187位谷氨酰胺(187Q)残基为突变位点,构建如下两个突变体DNA,即sAPRIL突变体-1(mutant sAPRIL-1,msAPRIL-1)DNA(186G被赖氨酸残基K置换,187Q缺失)和msAPRIL-2 DNA(186G187Q置换为186K187G);测序后将两突变体DNA分别亚克隆于原核表达载体pQE-80L,继而在大肠杆菌DH5α中表达相应突变体蛋白,经SDS-PAGE和Westernblot鉴定表达产物,经Ni2 -NTA和Sephadex G-75柱层析纯化相应突变体蛋白.结果 一步反向PCR结合DNA测序,得到了与上述设计一致的两个sAPRIL突变体DNA.将两突变体DNA分别亚克隆于pQE-80L后,在大肠杆菌DH5α中成功表达了相应突变体蛋白.经Ni2 -NTA柱层析成功地纯化了两突变体蛋白,经Sephadex G-75柱层析成功获得了两突变体蛋白的三聚体分子.结论 成功制备了sAPRIL的两种突变体蛋白,为寻找基于sAPRIL突变体的抗肿瘤分子奠定了基础.     

Comparative analysis of transient receptor potential channel 5 opposite strand-induced gene expression patterns and protein-protein interactions in triple-negative breast cancer

In patients with triple-negative breast cancer (TNBC), high tumour mutation burden and aberrant oncogene expression profiles are some of the causes of poor prognosis. Therefore, it is necessary to identify aberrantly expressed oncogenes, since they have the potential to serve as therapeutic targets. Transient receptor potential channel 5 opposite strand (TRPC5OS) has been previously shown to function as a novel tumour inducer. However, the underlying mechanism of TRPC5OS function in TNBC remain to be elucidated. Therefore, in the present study TRPC5OS expression was first measured in tissue samples of patients with TNBC and a panel of breast cancer cell lines (ZR-75-1, MDA-MB-453, SK-BR-3, JIMT-1, BT474 and HCC1937) by using qRT-PCR and Western blotting. Subsequently, the possible effects of TRPC5OS on MDA-MB-231 cells proliferation were determined using Cell Counting Kit-8 and 5-Ethynyl-2′-deoxyuridine assays after Lentiviral transfection of MDA-MB-231. In addition, potential interaction partners of TRPC5OS were explored using liquid chromatography-mass spectrometry (LC-MS)/MS. Gene expression patterns following TRPC5OS overexpression were also detected in MDA-MB-231 cells by using High-throughput sequencing. Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) analysis were then used to systematically verify the potential interactions among the TRPC5OS-regulated genes. The potential relationship between TRPC5OS-interacting proteins and gene expression patterns were studied using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) analysis. TRPC5OS expression was found to be significantly higher in TNBC tumour tissues and breast cancer cell lines compared with luminal tumour tissues and ZR-75-1. In addition, the overexpression of TRPC5OS significantly increased cell proliferation. High-throughput sequencing results revealed that 5,256 genes exhibited differential expression following TRPC5OS overexpression, including 3,269 upregulated genes and 1,987 downregulated genes. GO analysis results indicated that the functions of these differentially expressed genes were enriched in the categories of ‘cell division’ and ‘cell proliferation’ regulation. KEGG analysis showed that the TRPC5OS-regulated genes were associated with processes of ‘homologous recombination’ and ‘TNF signalling pathways’. Subsequently, 17 TRPC5OS-interacting proteins were found using LC-MS/MS and STRING analysis. The most important protein among interacting proteins was ENO1 which was associated with glycolysis and regulated proliferation of cancer. In summary, data from the present study suggest that TRPC5OS overexpression can increase TNBC cell proliferation and ENO1 may be a potential target protein mediated by TRPC5OS. Therefore, TRPC5OS may serve as a novel therapeutic target for TNBC.     

Hydroxyapatite from Natural Sources for Medical Applications

The aim of this work is to study the physical-chemical, mechanical, and biocompatible properties of hydroxyapatite obtained by hydrothermal synthesis, at relatively low temperatures and high pressures, starting from natural sources (Rapana whelk shells), knowing that these properties influence the behavior of nanostructured materials in cells or tissues. Thus, hydroxyapatite nanopowders were characterized by chemical analysis, Fourier-transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS), scanning electron microscopy (SEM), and X-ray diffraction (XRD). In vitro studies on osteoblast cell lines (cytotoxicity and cell proliferation), as well as preliminary mechanical tests, have been performed. The results showed that the obtained powders have a crystallite size below 50 nm and particle size less than 100 nm, demonstrating that hydrothermal synthesis led to hydroxyapatite nanocrystalline powders, with a Ca:P ratio close to the stoichiometric ratio and a controlled morphology (spherical particle aggregates). The tensile strength of HAp samples sintered at 1100 °C/90 min varies between 37.6–39.1 N/mm². HAp samples sintered at 1300 °C/120 min provide better results for the investigated mechanical properties. The coefficient of friction has an appropriate value for biomechanical applications. The results of cell viability showed that the cytotoxic effect is low for all tested samples. Better cell proliferation is observed for osteoblasts grown on square samples.     

Abnormal neurogenesis in the dentate gyrus of adult mice lacking 1,25-dihydroxy vitamin D3 (1,25-(OH)2 D3)

In this study, we employed 1α-hydroxylase knockout (1α-(OH)ase(-/-) ) mice to investigate the influence of 1,25-dihydroxy vitamin D(3) (1,25-(OH)(2) D(3) ) deficiency on the adult neurogenesis in the hippocampal dentate gyrus (DG). The numbers of both 24-hr-old BrdU(+) cells and proliferating cell nuclear antigen positive cells in 8-week-old 1α-(OH)ase(-/-) mice increased approximately twofold compared with wild-type littermates. In contrast, the numbers of 7- and 28-day-old BrdU(+) cells in 1α-(OH)ase(-/-) mice decreased by 50% compared with wild-type mice, while the proportion of BrdU(+) /NeuN(+) cells in BrdU(+) population showed no difference between 1α-(OH)ase(-/-) and wild-type mice. Apoptotic cells in the subgranular zone (SGZ) of DG markedly increased in 1α-(OH)ase(-/-) mice. Replenishment of 1,25-(OH)(2) D(3) , but not correction of serum calcium and phosphorus levels, completely prevented changes in the neurogenesis in 1α-(OH)ase(-/-) mice. The absence of 1,25-(OH)(2) D(3) led to an increase in the expression of L-type voltage-gated calcium channel (L-VGCC) and a decrease in the nerve growth factor (NGF) mRNA level. Treatment with the L-VGCC inhibitor nifedipine blocked the increased cell proliferations by 1,25-(OH)(2) D(3) deficiency. Administration of NGF significantly attenuated the loss of newborn neurons in 1α-(OH)ase(-/-) mice.