小球藻类金属硫蛋白对酪氨酸酶活性的抑制作用研究

通过考察小球藻Zn-MT-like蛋白对酪氨酸酶单酚酶和二酚酶催化氧化酪氨酸活性的抑制作用,发现该蛋白具有延长单酚酶迟滞时间、抑制单酚酶稳定态酶和二酚酶活性的作用;随Zn-MT-like蛋白质量浓度的增大酶活性下降,1.2 mg/mL的小球藻Zn-MT-like蛋白可延长单酚酶迟滞时间至2 300 s,几乎能完全抑制单酚酶稳定态酶活性,且对二酚酶活性的抑制率高达87%。     

海参肽对成纤维细胞的作用及与其他抗皱成分的比较

皮肤老化问题越来越受到人们的重视,而伴随着蓝色经济的发展,海洋生物资源也得到了开发利用。在前期研究的基础上以混合酶解方法制备海参肽,通过MTT(噻唑蓝)方法研究海参肽对NIH/3T3细胞及胶原蛋白分泌量的作用,并与其他抗皱成分进行比较:其中海参肽质量浓度为200μg/m L时可以显著促进NIH/3T3细胞生长,增殖率为33.9%±13.8%;海参肽质量浓度为800μg/m L时,对NIH/3T3细胞胶原蛋白的分泌有显著的作用,羟脯氨酸含量为(1.96±0.16)μg/m L;燕麦多肽、蜗牛原液、多效亮肤混合液对NIH/3T3细胞的最大增殖率依次为14.2%±8.3%、26.7%±12.0%、26.2%±12.1%;表明海参肽较其他三种成分有更明显的促进NIH/3T3细胞增殖的作用。     

DNA甲基化对调控胰岛分化基因表达的影响

目的探讨DNA甲基化对调控胰岛分化基因表达的影响,为调控干细胞分化为胰岛细胞提供理论依据。方法采用甲基化DNA免疫共沉淀-实时定量PCR法(MeDIP-qPCR)检测129/J小鼠胚胎干细胞、NIT1细胞及NIH3T3细胞中Pdx-1、MafA、Nkx6.1和Oct4四种调控胰岛分化基因的DNA甲基化程度;同时采用实时定量RT-PCR检测上述3种细胞中4种基因mRNA表达水平,分析这些基因DNA甲基化水平差异与基因表达之间的关系。结果 Pdx-1、MafA和Nkx6.1基因在129/J小鼠胚胎干细胞和NIT1细胞中呈低甲基化,在NIH3T3细胞中则呈高甲基化,前两种细胞的甲基化程度明显低于后者(P<0.05);Oct4基因在129/J小鼠胚胎干细胞中未甲基化,在NIT1和NIH3T3细胞中呈低甲基化,NIT1细胞的甲基化程度明显低于NIH3T3细胞(P<0.05)。低甲基化的Pdx-1、MafA和Nkx6.1基因在NIT1细胞中可高效表达,而在NIH3T3和129/J小鼠胚胎干细胞中则未见表达(P<0.05);Oct4基因在129/J小鼠胚胎干细胞中可高效表达,在NIT1和NIH3T3细胞中则未见表达。结论转录起始区DNA甲基化程度可影响Pdx-1、MafA、Nkx6.1和Oct4基因的表达,参与β细胞的分化过程。     

联氨基姜黄素脂质体纳米颗粒对小鼠乳腺癌4T1细胞生物学行为的影响

目的探讨联氨基姜黄素(Hydrazinocurcumin,HC)脂质体纳米颗粒(Liposome nanoparticles,NPs)对小鼠乳腺癌4T1细胞增殖、凋亡、侵袭及迁移的影响。方法采用薄膜分散-超声法制备HC-NPs,透射电镜检测纳米颗粒的粒径大小。将4T1细胞分为PBS组、NPs组和HC-NPs组,分别加入10%(v/v)PBS、10%(v/v)NP、10%(v/v)HC-NPs,培养24 h后,MTT法检测细胞的增殖活力;流式细胞术检测细胞周期和凋亡率的变化;刘氏染色法检测细胞的形态;Transwell试验检测4T1细胞侵袭和迁移能力;Western blot检测转导和转录激活因子STAT3及细胞增殖、凋亡、侵袭、迁移相关蛋白的表达水平。结果透射电镜下可见HC-NPs为150 nm左右的脂质体颗粒。HC-NPs对4T1细胞的增殖抑制率明显高于NPs组(P<0.01);与PBS组和NPs组相比,HC-NPs可使细胞形态不规则,诱导细胞周期发生G2/M期阻滞,细胞凋亡率明显增加(P<0.01),明显抑制细胞侵袭和迁移(P<0.01)及STAT3的激活,下调CyclinD1、c-Myc、Bcl-2、Survivin、MMP-9的表达,同时上调Bax的表达(P<0.05)。结论 HC-NPs可能通过抑制STAT3的激活,抑制4T1细胞增殖、侵袭和迁移能力,促进细胞凋亡。     

羧甲基葡甘聚糖-壳聚糖复合膜对小鼠成纤维细胞的相容性研究

利用溶液共混法制备了羧甲基葡甘聚糖-壳聚糖复合膜.以体外培养的小鼠成纤维细胞作为对象,通过复合膜浸渍液培养和膜材料直接培养法以及膜材料的溶血实验,评价材料的细胞相容性.采用MTT法检测膜材料浸渍液中细胞的增殖情况,光镜下观察膜材料浸渍液中细胞的生长和培养7天的NIH3T3-多孔膜复合物的组织切片,同时结合扫描电镜观察培养7天的NIH3T3-膜材料复合物表面的细胞生长状况.结果发现：复合膜浸渍液对细胞无毒性效应,NIH3T3细胞在复合膜上能很好地贴附,生长旺盛.比较了致密常规膜和多孔膜上细胞的生长状态,多孔膜上细胞能向材料内部迁移,呈立体生长.复合膜溶血率测定值＜5%.所以羧甲基葡甘聚糖-壳聚糖复合膜有良好的细胞相容性.     

基于发酵动力学模型的小球藻高密度发酵培养

构建了50 L发酵罐小球藻分批培养动力学模型,采用补料策略高密度发酵培养小球藻,考察了补料发酵过程中碳源的利用情况,采用实时荧光定量PCR技术分析了蛋白质合成关键酶二氨基庚二酸异构酶(dapF)、柠檬酸合成酶(CS)和葡萄糖?6-磷酸脱氢酶(G6PDH)的基因表达情况. 结果表明,小球藻经补料培养120 h,细胞生物量达106.65 g/L,平均生长速率为0.89 g/(L?h),葡萄糖的细胞得率为0.56 g/g,发酵过程中葡萄糖和尿素浓度对小球藻的dspF, CS和G6PDH基因表达量有重要影响.     

锌指蛋白转录抑制因子145′非翻译区内部核糖体进入位点的活性

目的检测锌指蛋白转录抑制因子14(positive regulatory domain zinc finger protein 14,PRDM14)5′非翻译区(5′untranslated region,5′UTR)内部核糖体进入位点(internal ribosome entry site,IRES)的活性。方法 PCR扩增PRDM14 5′UTR各截短序列的基因及全长基因,分别插入至双荧光素酶和红绿荧光报告基因中,构建重组质粒p RLPRDM14 S1-FL、p RL-PRDM14 S2-FL、p RL-PRDM14 S3-FL及p G-PRDM 14-R。将各重组质粒分别转染HEK293细胞,检测PRDM14 5′UTR IRES元件的活性、内部剪切位点和自身启动子的活性及影响IRES活性的序列。将重组质粒p RL-FL/5′UTR分别转染HCT-8/WT、Hep-2、Bel7402/WT和NIH3T3细胞,检测各细胞中PRDM14 5′UTR IRES元件的活性。结果 PRDM14 5′UTR具有内部核糖体进入位点元件活性;不具有内部剪切位点和自身启动子活性;PRDM14 5′UTR的活性激活中心位于25~60 nt,活性抑制中心位于60~95 nt;除NIH3T3细胞外,其他细胞均具有IRES活性。结论 PRDM14 5′UTR具有典型的IRES活性,为今后深入研究PRDM14表达的调控机制奠定了基础。     

芦根复方提取液体外3T3中性红摄取光毒性试验研究

通过乙醇提取方法,以芦根、甘草、当归、丹参、山茱萸及地骨皮为组方,制备芦根复方提取液,生药质量浓度为0.05 g/m L。采用体外3T3中性红摄取试验,评估制备的芦根复方提取液潜在的光毒性。比较3T3中性红摄取试验中受试样品和阳性对照盐酸氯丙嗪(CPZ)在无光照及光照条件下对Balb/c 3T3细胞的IC_(50)值、光刺激因子(PIF)和平均光效应(MPE),以此评价样品预期产生光毒性的可能。结果显示,阳性对照品CPZ存在明显光毒性,而芦根复方提取液样品预期无光毒性。     

不同碳源对小球藻(Chlorella vulgaris)生长及含油量的影响

为了研究不同碳源对小球藻(Chlorella vulgaris)生长及油脂积累的影响,选取了葡萄糖、麦芽糖、淀粉等共11种碳源分别作为唯一碳源对小球藻进行培养.研究小球藻的生长及油脂积累情况.结果表明,葡萄糖和麦芽糖都可作为唯一碳源,小球藻的最大细胞浓度分别为4.495×108 cell/L和2.725×108 cell/L,含油率分别为53.27％和38.54％.     

固定化小球藻去除氮磷的研究

使用海藻酸钠(SA)和聚乙烯醇(PVA)作为载体、以氯化钙(CaCl₂)和硼酸(H₃BO₃)为交联剂，制作固定化小球藻，在筛选最佳制备条件的基础上探讨固定化小球藻对人工废水和养殖用水的净化效果。结果表明，当SA和CaCl₂质量分数均为2%,PVA和H₃BO₃质量分数分别为1%和3%，交联时间为12 h时制备的固定化小球藻最佳。PVA质量分数为1%时固定化小球藻去除人工废水中的氨氮、亚硝态氮、硝态氮和总磷的效果最好，去除率分别为60.92%、77.04%、79.06%和83.38%。在草金鱼循环水实验中应用，固定化小球藻去除氮磷效果较对照组有显著优势。     

Ultraviolet C Irradiation Induces Different Expression of Cyclooxygenase 2 in NIH 3T3 Cells and A431 Cells: The Roles of COX-2 Are Different in Various Cell Lines

Ultraviolet C (UVC) is a DNA damage inducer, and 20 J/m(2) of UVC irradiation caused cell growth inhibition and induced cell death after exposure for 24-36 h. The growth of NIH 3T3 cells was significantly suppressed at 24 h after UVC irradiation whereas the proliferation of A431 cells was inhibited until 36 h after UVC irradiation. UVC irradiation increased COX-2 expression and such up-regulation reached a maximum during 3-6 h in NIH 3T3 cells. In contrast, UVC-induced COX-2 reached a maximum after 24-36 h in A431 cells. Measuring prostaglandin E2 (PGE2) level showed a biphasic profile that PGE2 release was rapidly elevated in 1-12 h after UVC irradiation and increased again at 24 h in both cell lines. Treatment with the selective COX-2 inhibitor, SC-791, during maximum expression of COX-2 induction, attenuated the UVC induced-growth inhibition in NIH 3T3 cells. In contrast, SC-791 treatment after UVC irradiation enhanced death of A431 cells. These data showed that the patterns of UVC-induced PGE2 secretion from NIH 3T3 cells and A431 cells were similar despite the differential profile in UVC-induced COX-2 up-regulation. Besides, COX-2 might play different roles in cellular response to UVC irradiation in various cell lines.     

Self-Targeting of Carbon Dots into the Cell Nucleus: Diverse Mechanisms of Toxicity in NIH/3T3 and L929 Cells

It is important to understand the nanomaterials intracellular trafficking and distribution and investigate their targeting into the nuclear area in the living cells. In our previous study, we firstly observed penetration of nonmodified positively charged carbon dots decorated with quaternary ammonium groups (QCDs) into the nucleus of mouse NIH/3T3 fibroblasts. Thus, in this work, we focused on deeper study of QCDs distribution inside two healthy mouse NIH/3T3 and L929 cell lines by fluorescence microspectroscopy and performed a comprehensive cytotoxic and DNA damage measurements. Real-time penetration of QCDs across the plasma cell membrane was recorded, concentration dependent uptake was determined and endocytic pathways were characterized. We found out that the QCDs concentration of 200 µg/mL is close to saturation and subsequently, NIH/3T3 had a different cell cycle profile, however, no significant changes in viability (not even in the case with QCDs in the nuclei) and DNA damage. In the case of L929, the presence of QCDs in the nucleus evoked a cellular death. Intranuclear environment of NIH/3T3 cells affected fluorescent properties of QCDs and evoked fluorescence blue shifts. Studying the intracellular interactions with CDs is essential for development of future applications such as DNA sensing, because CDs as DNA probes have not yet been developed.     

The Protective Effects of EMF-LTE against DNA Double-Strand Break Damage In Vitro and In Vivo

With the rapid growth of the wireless communication industry, humans are extensively exposed to electromagnetic fields (EMF) comprised of radiofrequency (RF). The skin is considered the primary target of EMFs given its outermost location. Recent evidence suggests that extremely low frequency (ELF)-EMF can improve the efficacy of DNA repair in human cell-lines. However, the effects of EMF-RF on DNA damage remain unknown. Here, we investigated the impact of EMF-long term evolution (LTE, 1.762 GHz, 8 W/kg) irradiation on DNA double-strand break (DSB) using the murine melanoma cell line B16 and the human keratinocyte cell line HaCaT. EMF-LTE exposure alone did not affect cell viability or induce apoptosis or necrosis. In addition, DNA DSB damage, as determined by the neutral comet assay, was not induced by EMF-LTE irradiation. Of note, EMF-LTE exposure can attenuate the DNA DSB damage induced by physical and chemical DNA damaging agents (such as ionizing radiation (IR, 10 Gy) in HaCaT and B16 cells and bleomycin (BLM, 3 μM) in HaCaT cells and a human melanoma cell line MNT-1), suggesting that EMF-LTE promotes the repair of DNA DSB damage. The protective effect of EMF-LTE against DNA damage was further confirmed by attenuation of the DNA damage marker γ-H2AX after exposure to EMF-LTE in HaCaT and B16 cells. Most importantly, irradiation of EMF-LTE (1.76 GHz, 6 W/kg, 8 h/day) on mice in vivo for 4 weeks reduced the γ-H2AX level in the skin tissue, further supporting the protective effects of EMF-LTE against DNA DSB damage. Furthermore, p53, the master tumor-suppressor gene, was commonly upregulated by EMF-LTE irradiation in B16 and HaCaT cells. This finding suggests that p53 plays a role in the protective effect of EMF-LTE against DNA DSBs. Collectively, these results demonstrated that EMF-LTE might have a protective effect against DNA DSB damage in the skin, although further studies are necessary to understand its impact on human health.     

Docosahexaenoic acid modulates phorbol ester-induced activation of extracellular signal-regulated kinases 1 and 2 in NIH/3T3 cells

Phosphorylation of extracellular signal-regulated kinases (ERK1/ERK2) has been implicated in cell proliferation of mammalian cells. In the present study, we investigated the role of docosahexaenoic acid (DHA) in the modulation of ERK1/ERK2 phosphorylation, stimulated either with phorbol 12-myristate 13-acetate (PMA) or transforming growth factor-alpha (TGFα) in NIH/3T3 cells. We observed that both PMA and TGFα induced ERK1/ERK2 phosphorylation within 5 min of stimulation. PMA acts upstream of MEK and via activation of protein kinase C (PKC), as GF109203X, a potent PKC inhibitor, and U0126, a MEK inhibitor, abolished its actions on ERK1/ERK2 phosphorylation. TGFα did not act via PKC because GF109203X failed to curtail the degree of ERK1/ERK2 phosphorylation in these cells. DHA alone failed to induce the phosphorylation of these mitogen-activated protein (MAP) kinases; however, this fatty acid significantly curtailed the PMA-but not TGFα-induced MAP kinase enzyme activity and phosphorylation in NIH/3T3 cells. Furthermore, we observed that DHA significantly inhibited PMA-induced translocation of two PKC isoforms, PKCα and PKCε, from cytosol to plasma membrane. Interestingly, DHA failed to inhibit the PMA-induced translocation PKCδ isoform in these cells. Furthermore, DHA decreased PMA-induced proliferation of NIH/3T3 cells. In this study, we show for the first time that DHA inhibits MAP kinase (ERK1/ERK2) activation and proliferation of NIH/3T3 cells via its inhibitory action on PKCα and ε isoforms.     

Lactoferrin Directly Scavenges Hydroxyl Radicals and Undergoes Oxidative Self-Degradation: A Possible Role in Protection against Oxidative DNA Damage

In this study, we examined the protective effect of lactoferrin against DNA damage induced by various hydroxyl radical generation systems. Lactoferrin (LF) was examined with regard to its potential role as a scavenger against radical oxygen species using bovine milk LF. Native LF, iron-saturated LF (holo-LF), and apolactoferrin (apo-LF) effectively suppressed strand breaks in plasmid DNA due to hydroxyl radicals produced by the Fenton reaction. In addition, both native LF and holo-LF clearly protected calf thymus DNA from fragmentation due to ultraviolet irradiation in the presence of H₂O₂. We also demonstrated a protective effect of all three LF molecules against 8-hydroxydeoxyguanosine (8-OHdG) formation in calf thymus DNA following ultraviolet (UV) irradiation with H₂O₂. Our results clearly indicate that native LF has reactive oxygen species-scavenging ability, independent of its nature as a masking component for transient metals. We also demonstrated that the protective effect of LF against oxidative DNA damage is due to degradation of LF itself, which is more susceptible to degradation than other bovine milk proteins.     

Identification of Spiro-Fused [3-azabicyclo[3.1.0]hexane]oxindoles as Potential Antitumor Agents: Initial In Vitro Evaluation of Anti-Proliferative Effect and Actin Cytoskeleton Transformation in 3T3 and 3T3-SV40 Fibroblast

Novel heterocyclic compounds containing 3-spiro[3-azabicyclo[3.1.0]hexane]oxindole framework (4a, 4b and 4c) have been studied as potential antitumor agents. The in silico ADMET (adsorption, distribution, metabolism, excretion and toxicity) analysis was performed on 4a–c compounds with promising antiproliferative activity, previously synthetized and screened against human erythroleukemic cell line K562 tumor cell line. Cytotoxicity of 4a–c against murine fibroblast 3T3 and SV-40 transformed murine fibroblast 3T3-SV40 cell lines were evaluated. The 4a and 4c compounds were cytotoxic against 3T3-SV40 cells in comparison with those of 3T3. In agreement with the DNA cytometry studies, the tested compounds have achieved significant cell-cycle perturbation with higher accumulation of cells in G0/G1 phase. Using confocal microscopy, we found that with 4a and 4c treatment of 3T3 cells, actin filaments disappeared, and granular actin was distributed diffusely in the cytoplasm in 82–97% of cells. The number of 3T3-SV40 cells with stress fibers increased to 7–30% against 2% in control. We discovered that transformed 3T3-SV40 cells after treatment with compounds 4a and 4c significantly reduced the number of cells with filopodium-like membrane protrusions (from 86 % in control cells to 6–18% after treatment), which indirectly suggests a decrease in cell motility. We can conclude that the studied compounds 4a and 4c have a cytostatic effect, which can lead to a decrease in the number of filopodium-like membrane protrusions.     

A Novel Prodrug of a γ‐Glutamylcyclotransferase Inhibitor Suppresses Cancer Cell Proliferation in vitro and Inhibits Tumor Growth in a Xenograft Mouse Model of Prostate Cancer

γ‐Glutamylcyclotransferase (GGCT) depletion inhibits cancer cell proliferation. However, whether the enzymatic activity of GGCT is critical for the regulation of cancer cell growth remains unclear. In this study, a novel diester‐type cell‐permeable prodrug, pro‐GA, was developed based on the structure of N‐glutaryl‐l ‐alanine (GA), by structure optimization using temporary fluorophore‐tagged prodrug candidates. The antiproliferative activity of pro‐GA was demonstrated using GGCT‐overexpressing NIH‐3T3 cells and human cancer cells including MCF7, HL‐60, and PC3 cells. By contrast, normal cells were not significantly affected by pro‐GA treatment. Moreover, pro‐GA administration exhibited anticancer effects in a xenograft model using immunocompromised mice inoculated with PC3 cells. These results indicate that the enzymatic activity of GGCT accelerates tumor growth and that GGCT inhibition is a promising therapeutic strategy for the treatment of GGCT‐overexpressing tumors.     

Biological Evaluation of Photodynamic Effect Mediated by Nanoparticles with Embedded Porphyrin Photosensitizer

Clinically approved photodynamic therapy (PDT) is a minimally invasive treatment procedure that uses three key components: photosensitization, a light source, and tissue oxygen. However, the photodynamic effect is limited by both the photophysical properties of photosensitizers as well as their low selectivity, leading to damage to adjacent normal tissue and/or inadequate biodistribution. Nanoparticles (NPs) represent a new option for PDT that can overcome most of the limitations of conventional photosensitizers and can also promote photosensitizer accumulation in target cells through enhanced permeation and retention effects. In this in vitro study, the photodynamic effect of TPP photosensitizers embedded in polystyrene nanoparticles was observed on the non-tumor NIH3T3 cell line and HeLa and G361 tumor cell lines. The efficacy was evaluated by viability assay, while reactive oxygen species production, changes in membrane mitochondrial potential, and morphological changes before and after treatment were imaged by atomic force microscopy. The tested nanoparticles with embedded TPP were found to become cytotoxic only after activation by blue light (414 nm) due to the production of reactive oxygen species. The photodynamic effect observed in this evaluation was significantly higher in both tumor lines than the effect observed in the non-tumor line, and the resulting phototoxicity depended on the concentration of photosensitizer and irradiation time.     

Photo-Polymerization Damage Protection by Hydrogen Sulfide Donors for 3D-Cell Culture Systems Optimization

Photo-polymerized hydrogels are ideally suited for stem-cell based tissue regeneration and three dimensional (3D) bioprinting because they can be highly biocompatible, injectable, easy to use, and their mechanical and physical properties can be controlled. However, photo-polymerization involves the use of potentially toxic photo-initiators, exposure to ultraviolet light radiation, formation of free radicals that trigger the cross-linking reaction, and other events whose effects on cells are not yet fully understood. The purpose of this study was to examine the effects of hydrogen sulfide (H₂S) in mitigating cellular toxicity of photo-polymerization caused to resident cells during the process of hydrogel formation. H₂S, which is the latest discovered member of the gasotransmitter family of gaseous signalling molecules, has a number of established beneficial properties, including cell protection from oxidative damage both directly (by acting as a scavenger molecule) and indirectly (by inducing the expression of anti-oxidant proteins in the cell). Cells were exposed to slow release H₂S treatment using pre-conditioning with glutathione-conjugated-garlic extract in order to mitigate toxicity during the photo-polymerization process of hydrogel formation. The protective effects of the H₂S treatment were evaluated in both an enzymatic model and a 3D cell culture system using cell viability as a quantitative indicator. The protective effect of H₂S treatment of cells is a promising approach to enhance cell survival in tissue engineering applications requiring photo-polymerized hydrogel scaffolds.