New 4-imino-4H-Chromeno[2,3-d]Pyrimidin-3(5H)-Amine: Synthesis, Cytotoxic Effects on Tumoral Cell Lines and in Silico ADMET Properties

The synthesis of new 4-imino-4H-chromeno[2,3-d]pyrimidin-3(5H)-amine in four steps including one step under microwave dielectric heating is reported. The structural identity of the synthesized compounds was established according to their spectroscopic analysis, such as FT-IR, NMR and mass spectroscopy. These new compounds were tested for their antiproliferative activities on seven representative human tumoral cell lines (Huh7 D12, Caco2, MDA-MB231, MDA-MB468, HCT116, PC3 and MCF7) and also on fibroblasts. Among them, only the compounds 6c showed micromolar cytotoxic activity on tumor cell lines (1.8 50 50 > 25 μM). Finally, in silico ADMET studies ware performed to investigate the possibility of using of the identified compound 6c as potential anti-tumor compound.

Synergistic anticancer properties of docosahexaenoic acid and 5-fluorouracil through interference with energy metabolism and cell cycle arrest in human gastric cancer cell line AGS cells

AIM: To explore the synergistic effect of docosahexaenoic acid(DHA)/5-fluorouracil(5-FU) on the human gastric cancer cell line AGS and examine the underlying mechanism.METHODS: AGS cells were cultured and treated with a series of concentrations of DHA and 5-FU alone or in combination for 24 and 48 h. To investigate the synergistic effect of DHA and 5-FU on AGS cells, the inhibition of cell proliferation was determined by MTT assay and cell morphology. Flow cytometric analysis was also used to assess cell cycle distribution, and the expression of mitochondrial electron transfer chain complexes(METCs)?Ⅰ, Ⅱ and Ⅴ in AGS cells was further determined by Western blot analysis. RESULTS: DHA and 5-FU alone or in combination could markedly suppress the proliferation of AGS cells in a significant time and dose-dependent manner. DHA markedly strengthened the antiproliferative effect of 5-FU, decreasing the IC50 by 3.56-2.15-fold in an apparent synergy. The morphological changes of the cells were characterized by shrinkage, cell membrane blebbing and decreased adherence. Cell cycle analysis showed a shift of cells into the G0/G1 phase from the S phase following treatment with DHA or 5-FU(G0/G1 phase: 30.04% ± 1.54% vs 49.05% ± 6.41% and 63.39% ± 6.83%, respectively, P < 0.05; S phase: 56.76% ± 3.14% vs 34.75% ± 2.35% and 25.63% ± 2.21%, respectively, P < 0.05). Combination treatment of DHA and 5-FU resulted in a significantly larger shift toward the G0/G1 phase and subsequent reduction in S phase(G0/G1 phase: 69.06% ± 2.63% vs 49.05% ± 6.41% and 63.39% ± 6.83%, respectively, P < 0.05; S phase: 19.80% ± 4.30% vs 34.75% ± 2.35% and 25.63% ± 2.21%, respectively, P < 0.05). This synergy was also reflected in the significant downregulation of the expression of METCs in AGS cells.CONCLUSION: Synergistic anticancer properties of DHA and 5-FU may involve interference with energy production of AGS cells via downregulation of METCs and cell cycle arrest.

Interaction between cisplatin,5-fluorouracil and vincristine on human hepatoma cell line (7721)

AIM To evaluate the killing effects of CDDP, 5-Fu and VCR on human hepaoma cell line (7721).METHODS The median-effect principle was used.RESULTS Killing effects of the individual drug were enhanced as the median concentration increased. Antagonism was produced when two drugs were used at a higher concentration (CI＞1), and synergism was achiened when CI＜1. Finally, the effect was influenced by both the ratios of drug concentration and the sequence of administration.CONCLUSION The drug administration order and drug concentrations are significant factors that need to be considered in clinical practice.INTRODUCTIONThe combined chemotherapy for malignant carcinoma is desired to produce efficacious synergism between each drug, alleviate side effects of drugs and delay drug resistance. Clinically, the interaction (namely synergism, summation and antagonism) of different anticancer drugs in combination is usually evaluated by Chou-Talalay′s combination index (i.e., median-effect principle)[1-9]. In this paper the combination effect between Cisplatin (Cis), 5-Fluorouracil (5-Flu) and Vincristine (VCR) on human hepatoma cell line 7721, was analyzed in vitro.

Leucine-rich repeat-containing G protein-coupled receptor 5 marks different cancer stem cell compartments in human Caco-2 and LoVo colon cancer lines

BACKGROUND Colon cancer cell lines are widely used for research and for the screening of drugs that specifically target the stem cell compartment of colon cancers.It was reported that colon cancer carcinoma specimens contain a subset of leucine-rich repeatcontaining G protein-coupled receptor 5(LGR5)-expressing stem cells,these socalled“tumour-initiating”cells,reminiscent in their properties of the normal intestinal stem cells(ISCs),may explain the apparent heterogeneity of colon cancer cell lines.Also,colon cancer is initiated by aberrant Wnt signaling in ISCs known to express high levels of LGR5.Furthermore,in vivo reports demonstrate the clonal expansion of intestinal adenomas from a single LGR5-expressing cell.AIM To investigate whether colon cancer cell lines contain cancer stem cells and to characterize these putative cancer stem cells.METHODS A portable fluorescent reporter construct based on a conserved fragment of the LGR5 promoter was used to isolate the cell compartments expressing different levels of LGR5 in two widely used colon cancer cell lines(Caco-2 and LoVo).These cells were then characterized according to their proliferation capacity,gene expression signatures of ISC markers,and their tumorigenic properties in vivo and in vitro.RESULTS The data revealed that the LGR5 reporter can be used to identify and isolate a classical intestinal crypt stem cell-like population from the Caco-2,but not from the LoVo,cell lines,in which the cancer stem cell population is more akin to B lymphoma Moloney murine leukemia virus insertion region 1 homolog(+4 crypt)stem cells.This sub-population within Caco-2 cells exhibits an intestinal cancer stem cell gene expression signature and can both self-renew and generate differentiated LGR5 negative progeny.Our data also show that cells expressing high levels of LGR5/enhanced yellow fluorescent protein(EYFP)from this cell line exhibit tumorigenic-like properties in vivo and in vitro.In contrast,cell compartments of LoVo that are expressing high levels of LGR5/EYFP did not show these stem cell-like properties.Thus,cells that exhibit high levels of LGR5/EYFP expression represent the cancer stem cell compartment of Caco-2 colon cancer cells,but not LoVo cells.CONCLUSION Our findings highlight the presence of a spectrum of different ISC-like compartments in different colon cancer cell lines.Their existence is an important consideration for their screening applications and should be taken into account when interpreting drug screening data.We have generated a portable LGR5-reporter that serves as a valuable tool for the identification and isolation of different colon cancer stem cell populations in colon cancer lines.

Human embryonic stem cell lines with ccr5-del32 allele conferring resistance to HIV

A 32bp deletion in the chemokine receptor 5 (CCR5) gene (CMKBR5) was shown to be linked to HIV resistance. Bone marrow transplantation from the homozygous CCR5-del32 donor to a CDC Stage 2 HIV-positive recipient was demonstrated to confer a HIV resistance, resulting in discontinuation of antiretroviral therapy. In search for an unlimited source of CCR5-del32 cells for transplantation purposes, we tested 137 human embryonic stem cell (hESC) lines from the Reproductive Genetics Institute’s hESC lines collection, and report here the finding of 12 hESC lines with the CCR5-del32 allele, one of which represents a unique partenogenetic ESC line containing two copies of this deletion and may be studied for utility in stem cell transplantation treatment of HIV.

过表达溶质载体家族1成员5和敲低慢病毒载体构建及稳定转染RAW264.7细胞株

背景:溶质载体家族1成员5(solute carrier family 1 member 5,SLC1A5)在多种疾病中发挥了潜在作用,但确切作用机制尚不清楚。构建稳定的SLC1A5过表达和敲低细胞模型可为深入研究SLC1A5在疾病中的确切作用机制以及发现潜在治疗靶点提供有力的实验工具。目的:构建小鼠SLC1A5过表达和敲低的慢病毒载体,以建立稳定转染的RAW264.7细胞株,为深入探讨SLC1A5在炎症中的作用提供实验基础。方法:根据SLC1A5基因序列设计合成引物并使用聚合酶链反应扩增该基因片段。将目的基因定向接入经Age I/Nhe I酶切的载体质粒GV492中构建重组慢病毒质粒,对阳性克隆进一步筛选后测序比对结果;pHelper1.0质粒载体、pHelper2.0质粒载体、目的质粒载体与293T细胞共同培养并转染,获得慢病毒原液进行包装和滴度测定;在此基础上,通过体外培养RAW264.7细胞,确定嘌呤霉素工作质量浓度;不同滴度的慢病毒分别与RAW264.7细胞共同培养,根据荧光强度确定转染效率;用嘌呤霉素挑选出稳定转染细胞,实时荧光定量聚合酶链反应和蛋白免疫印迹方法检测稳定转染细胞株的SLC1A5基因和蛋白表达水平。结果与结论:(1)测序序列与目的序列一致提示重组慢病毒载体构建成功;(2)过表达SLC1A5慢病毒的滴度为1×10~9 TU/mL,敲低SLC1A5慢病毒的滴度为3×10~9 TU/mL;(3)确定RAW264.7细胞嘌呤霉素工作质量浓度为3μg/mL;(4)过表达/敲低SLC1A5慢病毒转染RAW264.7细胞的最佳条件皆为HiTransG P转染增强液且感染复数值等于50;(5)过表达SLC1A5稳转细胞株中SLC1A5基因和蛋白的表达量明显上调,而敲低SLC1A5稳转细胞株中SLC1A5基因和蛋白的表达量显著下调。结果表明,成功构建了小鼠SLC1A5过表达和敲低的慢病毒载体并获得稳定转染的RAW264.7细胞株。

转基因视网膜神经节细胞系RGC-5的研究进展

为研究青光眼性视网膜神经节细胞(RGCs)损害的细胞和分子机制,多种细胞培养模型已经建立,但是转基因的视网膜神经节细胞RGC-5细胞系在国内未见报道。RGC-5细胞系是应用鼠视网膜细胞通过转染技术建立的,除了其形态和电生理特性与RGCs有所不同外,细胞的生长条件以及细胞膜或者细胞内表达的物质与RGCs一致。应用RGC-5细胞系已经建立了多种凋亡模型,研究发现这些模型的细胞在凋亡时的基因变化以及对神经保护剂的反应都与RGCs一致。因此加强对RGC-5的研究和应用,对进一步阐明RGCs的损伤和保护机制将非常有意义。现就这个细胞系的建立及已做的相关研究做一综述。

Expression of interleukin 1β converting enzyme in 5-FU induced apoptosis in esophageal carcinoma cells

AIM To study the role of interleukin 1β converting enzyme (ICE) in antitumor drug induced apoptosis in tumor cells. METHODS Morphological changes in human esophageal carcinoma Eca 109 cells after treated with 5 fluorouracil (5 FU) were observed under light and electron microscope. Expression of ICE in the tumor cells exposed to 5 FU was examined by the immunocytochemical method. RESULTS The cells treated with 5 FU displayed disappearance of nucleoli, chromatin gathering under nuclear envelope, karyorrhexis, budding and the formation of apoptotic bodies. The expression of ICE was negative in control cells, and 5 FU could induce the ICE expression in Eca 109 cells undergoing apoptosis. The number and the staining intensity of positive cells increased with the extension of action time. CONCLUSION 5 FU may induce apoptosis in human esophageal carcinoma Eca 109 cells; ICE gene may be involved in the regulation of 5 FU induced apoptosis; and ICE protein may mediate apoptosis induced by 5 FU.

Inhibitory Effects of 5-Aza-2'-Deoxycytidine and Trichostatin A in Combination with p53-Expressing Adenovirus on Human Laryngocarcinoma Cells

Objective： To investigate the effects of 5-Aza-2＇-deoxycytidine （5-Aza-Cdr） and trichostatin A （TSA） combined with p53-expressing adenovirus （Ad-p53） on Hep-2 cell line in vivo and in vitro, in order to explore its possibility in biological treatment of laryngocarcinoma. Methods： Effects of 5-Aza-Cdr and TSA in combination with Ad-p53 on Hep-2 cell line in vivo were determined by Cell Counting Kit-8 （CCK-8） assay. The effect of drug combination was calculated by Jin＇s formula. Effects on the cell line in vitro were investigated by establishing the nude mice model. Results： 5-Aza-Cdr and TSA showed inhibitory effects on the proliferation of Hep-2 cells in dose- and time-dependent manner. Ad-p53 can inhibit the growth of Hep-2 cells in vivo and in vitro. However, the combination of epigenetic reagents （5-Aza-Cdr/TSA） and Ad-p53 was less effective than individual use of Ad-p53. 5-Aza-Cdr and Ad-p53 inhibited the growth of transplanted tumors and reduced the volume of tumors, and the tumor volume of Ad-p53 group was significantly smaller than that of the control group （P0.05）. Conclusion： Both epigenetic reagents （5-Aza-Cdr/TSA） and Ad-p53 can suppress cell proliferation on Hep-2 in vivo and in vitro and there may be some antagonistic mechanism between Ad-p53 and epigenetic reagents （5-Aza-Cdr/ TSA）.

橘皮化合物5⁃ATAN诱导多发性骨髓瘤细胞凋亡及其机制研究

目的探讨橘皮提取物(5⁃ATAN)对多发性骨髓瘤细胞U266增殖和凋亡的影响及其机制。方法用6.25μmol/L、12.5μmol/L、25μmol/L、50μmol/L、100μmol/L的5⁃ATAN处理对数生长期U266细胞24h,四甲基偶氮唑蓝(MTT)法检测细胞增殖抑制率,流式细胞术检测细胞凋亡率和线粒体膜电位,Western blotting法检测细胞色素C、BCL⁃2、BAX、pro⁃caspase 3、pro⁃caspase 9及cleaved⁃caspase 3、cleaved⁃caspase 9、PARP、Bad、p⁃Bad的表达。结果5⁃ATAN对U266细胞的增殖有抑制作用,呈剂量依赖性(P<0.05);细胞凋亡率随药物浓度的增加显著增加(P<0.05),线粒体膜电位随5⁃ATAN浓度的增加显著降低(P<0.05)。抗凋亡蛋白Bcl⁃2、p⁃Bad及pro⁃caspase 3、pro⁃caspase 9及PARP的表达逐渐降低;细胞色素C及促凋亡蛋白Bax、cleaved⁃caspase 3、cleaved⁃caspase 9、PARP(89KDa)的表达逐渐升高(P<0.05)。结论5⁃ATAN抑制多发性骨髓瘤的增殖,促进其凋亡,可能与线粒体凋亡信号通路的激活有关。

Direct interaction between Rab5a and Rab4a enhanced epidermal growth factor-stimulated proliferation of gastric cancer cells

BACKGROUND Gastric cancer(GC)is one of the leading causes of cancer-related death worldwide.Although targeted therapies such as antibodies against human epidermal growth factor receptor 2 or vascular endothelial growth factor receptor 2 have been widely used in the treatment of metastatic cancer,the overall outcomes are poor.Therefore,elucidation of the mechanism underlying cancer progression is important to improve prognosis.Overexpression of the Rab5a gene has been confirmed to correlate with tumorigenesis of many cancers,but the mechanism underling,especially of GC,is still unclear.AIM To investigate the effects of Rab5a overexpression on the tumorigenesis of GC.METHODS First,the expression levels of Rab5a and Rab4a in primary tumorous tissues of GC patients diagnosed between 2015 and 2018 were analyzed.Then we constructed HGC-27 cell lines overexpressing green fluorescent protein-Rab5a or red fluorescent protein-Rab4a and investigated the interaction between Rab5a or Rab4a using Western blotting,co-immunoprecipitation,confocal microscopy,and colocalization analysis.Finally,epidermal growth factor-stimulated proliferation of these cell lines was analyzed using cell counting kit-8 cell viability assay.RESULTS Compared with normal gastric tissues,the expression levels of Rab5a and Rab4a increased progressively both in paracancerous tissues and in advanced cancerous tissues.Epidermal growth factor could promote the proliferation of HGC-27 cells,especially Rab5a-overexpressing HGC-27 cells.Notably,Rab5a and Rab4a cooverexpression promoted the proliferation of HGC-27 cells to the greatest extent.Further analysis identified a direct interaction between Rab5a and Rab4a in HGC-27 cells.CONCLUSION Co-overexpression of Rab5a and Rab4a in GC may promote the endosomal recycling of epidermal growth factor receptor,which in turn contributes to poor prognosis and tumor progression in GC patients.Inhibition of Rab5a or Rab4a expression might be a promising therapy for refractory GC.

Roles of Optineurin and Extracellular Vesicles in Glaucomatous Retinal Cell Loss

Objective To explore the role of extracellular vesicles(EVs)in the pathogenesis of glaucoma caused by E50K mutation.Methods A photoreceptor cell line,RGC-5,was transfected with empty plasmids and plasmids expressing wild-type(WT)optineurin(OPTN)or E50K OPTN to investigate the effects of OPTN glaucoma as well as to identify the role of EVs in glaucoma pathology.The RGC-5 cells were also stimulated with glutamate,and their viability was evaluated using flow cytometry or CCK-8 assay.EVs were extracted,labeled with PKH-26,and added into the medium for normal RGC-5 culture,and the status of the cells was observed thereafter.Results WT OPTN overexpression,E50K OPTN,and glutamate stimulation induced apoptosis of RGC-5 cells.However,when glutamate stimulation was used as an add-on treatment,the degree of apoptosis in WT OPTN-overexpressing RGC-5 cells was significantly lower than that in E50K OPTN-expressing and normal RGC-5 cells.The viability of normal RGC-5 cells was reduced when co-cultured with WT OPTN-overexpressing RGC-5 or E50K OPTN-overexpressing RGC-5.EVs released by the latter two transfected lines similarly reduced normal RGC-5 survival.Conclusion Our results indicate that WT OPTN overexpression may lead to photoreceptor apoptosis.However,overexpression also confers a degree of protection against high concentrations of extracellular glutamate.Additionally,EVs released by transfected RGC-5 cells may regulate the cell state.These findings may improve our understanding of the mechanisms of cell-cell interactions in pathological conditions,providing a basis for the use of EVs as novel targets for early diagnosis and treatment of glaucoma.

Effect of 5-Aza-2'-deoxycytidine on the expression of p16 in hepatocellular carcinoma cells in vitro

Objective: To study the effect of 5-Aza-2’ -deoxycytidine (5-Aza-cdR) on tumour suppressor gene p16 expres- sion in hepatocellular carcinoma cells. Method: Expression of pl6 mRNA and protein in hepatocellular carcinoma cell lines SMMC-7721 and HePG2 before and after treatment with 5-Aza-cdR were analyzed via reverse transcriptase polymerase chain reaction(RT-PCR) and immunohistochemistrty Results: The expression levels of p16 mRNA and protein were increased dramatically after treatment with 5-Aza-cdR. Conclusion: Our data show that, 5-Aza-2’ -deoxycytidine can increase the expression of pl6 gene both at transcription and translation. The findings suggested that 5-Aza-cdR may reactivate the pl6 gene by demethylation.

ESTABLISHMENT OF A HUMAN B CELL LINE THAT RESPONDS SPECIFICALLY TO B CELL GROWTH FACTOR

A human B cell line （3D5） that responds specifically to B cell growth factor （BCGF） hasbeen developed by a sequence of Staphylococcus aureus Cowen I activation,EB virus im-mortalization,and cloning.Proliferative response to PHA-stimulated T cell supernatant（PHA-T-Sup） and nonresponsiveness to rIL-2 stimulation were factors used to screen positivecells.Phenotype analysis with a flow cytometer indicated that:1) 3D5 is a B cell line:100% of the cells were positive for B1 marker and 59% were positive for sIg,while T3and Mo 1 were negative:2) 3D5 is an activated B cell line:both Tac and 4F2 markersof activated （but not of resting） B cells were 100% positive:3) 3D5 expresses high molecularweight BCGF （HMW-BCGF） receptor-associated epitope BA5.3D5 cells proliferated inresponse to cpBCGF stimulation in a dose-dependent manner.HMW-BCGF also induced3D5 cells to proliferate.Interestingly.no proliferation could be detected in the presenceof rIL-2,rIL-4,or rIFN-r.The data show that 3D5 cells are specifically BCGF-responsiveB cells.Using 3D5 cells as target,BCGF activity was detected in crude BCGF preparationsedimented by 85% （NH4）2SO4 and chromatographed in a DEAE-Sephadex A-25 column fromPHA-T-Sup.T24 cell supernatant with B cell differentiation factor （BCDF） activity couldnot induce 3D5 cells to differentiate into immunoglobulin-secreting cells.

Effects of brain-derived neurotrophic factor on induced differentiation of SH-SY5Y cells in vitro

BACKGROUND： Previous studies have demonstrated that brain-derived neurotrophic factor （BDNF） promotes neural differentiation. However, the mechanisms involved in cell cycle-related protein regulation, which highly correlates to neural proliferation and apoptosis, remain poorly understood. OBJECTIVE： To investigate the effects of various concentrations of BDNF on cycle-related protein mRNA expression in induce-differentiated SH-SY5Y cells in vitro prior to and following G2 phase, and to analyze the neuroprotective effects of BDNF. DESIGN, TIME AND SETTING： A comparison, observational study, based on cell biology, was performed at the Department of Biochemistry, Medical College of Tongji University, from March 2005 to October 2006. MATERIALS： SH-SY5Y cells were provided by Shanghai Institute of Cytology, Chinese Academy of Science; BDNF by Alomone Labs, Israel; all-trans retinoic acid （ATRA） by Sigma-Aldrich, USA. METHODS： SH-SY5Y cells were randomly divided into three groups： blank control [cells were treated in Insulin-Transferrin-Selenium （ITS） solution for 7 days], ATRA （cells were treated with ITS solution containing 10 μmol/L ATRA for 7 days）, and BDNF （cells were treated identical to the ATRA group for 5 days, and then respectively treated in ITS solution containing 1, 10, and 100 μg/L BDNF for 2 days）. The experiment was repeated three times for each group. MAIN OUTCOME MEASURES： mRNA expression levels of cyclin A1, B1, B2, cyclin-dependent kinase 1, and 5 were detected using quantitative real-time RT-PCR; percentage of cells in G1, S, and G2 phases were detected using fluorescence-activated cell sorting. RESULTS： mRNA expression levels of cyclin A1 in the high-dose BDNF group was significantly less than the ATRA group （P 〈 0.05）.mRNA expression levels of cyclin B1 was significantly less in the different BDNF concentration groups compared with the control and ATRA groups （P 〈 0.05 or P 〈 0.01）. mRNA expression levels of cyclin B2 and cyclin-dependent kinase 1 were significantly decreased in the high-dose BDNF group （P 〈 0.05 or P 〈 0.01）. Cyclin-dependent kinase 5 mRNA expression was significantly greater in the low-dose and moderate-dose BDNF groups compared with the ATRA group （P 〈 0.05）. The percentage of cells in G1 phase was significantly greater in the different BDNF concentration groups compared with the ATRA and control groups （P 〈 0.01）. Moreover, the percentage of cells in S phase was significantly less in the three BDNF groups compared with the ATRA group （P 〈 0.01）. However, the percentage of cells in S phase was significantly less in the low-dose and high-dose BDNF groups compared with the control group （P 〈 0.01）. CONCLUSION： BDNF enhanced the percentage of cells in G1 phase, but did not alter mRNA expression of cell cycle-related proteins prior to or following G2 phase. These results suggested that BDNF was not a risk factor for inducing apoptosis.

The Expression Levels of KAI1 Gene and Its Mechanism in Human Lung Cancer Cell Lines

Background and Objective Lung cancer is one of the most malignant cancers which is hazarding the people’s health and life in the world. In the past half century, the incidence and mortality

槲皮素通过生长抑制特异性基因5调节微小RNA-23a-3p和磷脂酰肌醇3激酶/蛋白激酶B信号通路抑制垂体神经内分泌肿瘤的增殖机制

目的:探讨槲皮素对垂体神经内分泌肿瘤(PitNETs)生长的抑制作用及其可能机制。方法:通过Starbase工具预测生长抑制特异性基因5(GAS5)与微小RNA-23a-3p(miR-23a-3p)结合情况,双荧光素酶报告实验和RNA免疫沉淀测定GAS5和miR-23a-3p之间的结合关系。体外培养人垂体瘤细胞系RC-4B/C,分为对照组(Control)、槲皮素组(Quercetin)、GAS5抑制组(Si-GAS5)和槲皮素+si-GAS5组(Quercetin+si-GAS5),对比分析槲皮素对GAS5、miR-23a-3p及磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(AKT)信号通路的影响,以及人垂体瘤复苏细胞(RC-4B/C)生长和侵袭能力的影响。结果:Starbase工具预测GAS5与miR-23a-3p结合,双荧光素酶报告实验和RNA免疫沉淀测定揭示了GAS5和miR-23a-3p之间的直接结合。槲皮素促进GAS5的表达,抑制miR-23a-3p和PI3K/AKT的表达,抑制RC-4B/C细胞的增殖和侵袭(均P<0.05);下调GAS5、miR-23a-3p表达增多,促进RC-4B/C细胞的增殖和侵袭(均P<0.05);槲皮素可以逆转GAS5下调引起的miR-23a-3p、PI3K/AKT表达增加,抑制RC-4B/C细胞的增殖和侵袭(均P<0.05)。结论:槲皮素可能通过促进GAS5表达竞争性抑制miR-23a-3p,降低了PI3K/AKT,抑制垂体神经内分泌肿瘤的生长。

瘦素对5-氟尿嘧啶损伤结肠癌细胞的影响

目的探讨不同浓度的瘦素对5-氟尿嘧啶(5-FU)损伤结肠癌细胞的影响。方法同时用5-FU及不同浓度的瘦素进行体外干预结肠癌HT-29细胞株。MTT法检测5-FU50%细胞生长抑制率(IC50)的变化。流式细胞仪、原位末端转移酶标记法(TUNEL)进行周期及凋亡分析。以RT-PCR方法检测caspase-9,caspase-3mRNA的表达。结果瘦素抑制5-FU对HT-29的杀伤作用。抑制5-FU诱导的细胞周期阻滞及细胞凋亡。RT-PCR表明加入瘦素后caspase-9,caspase-3mRNA的表达均下降,且呈剂量依赖性。结论瘦素通过下调caspase-9,caspase-3的表达促进结肠癌细胞产生凋亡抵抗,抑制5-FU的损伤作用。

细粒棘球蚴细胞系13G-5培育

从１８例棘球蚴病人的包囊分离棘球蚴细胞，经３６批次体外培养获得１株细粒棘球蚴细胞系（１３Ｇ－５细胞系），已培养１４０ｄ，传２１代，并继续扩大增殖培养。测定了该细胞系第１３代细胞生长曲线和集落形成率（４％）。１３Ｇ－５细胞系５代以前为多形性细胞，后逐渐以成纤维型细胞占优势。探讨了棘球蚴细胞培养的制约因素。

5-Aza-CdR对胶质瘤细胞生长及LRRC4基因异常甲基化的影响

LRRC4是一个新发现的胶质瘤抑瘤基因,它在多种胶质瘤细胞系和胶质瘤组织表达缺失或下调,前期研究结果表明胶质瘤细胞和组织中LRRC4的编码区未发生突变、缺失或重排.为了获得LRRC4作为胶质瘤抑瘤基因的进一步证据,采用去甲基化制剂5-Aza-CdR处理LRRC4表达缺失的SF126和SF767胶质瘤细胞,MSP和RT-PCR检测表明,LRRC4的启动子在表达缺失的SF126和SF767细胞存在完全的甲基化,而5-Aza-CdR能逆转LRRC4启动子的甲基化状态,恢复LRRC4的表达.MTT法测定显示,5-Aza-CdR使SF126和SF767胶质瘤细胞增殖受到明显抑制,并呈时间和剂量的依赖性.同时流式细胞仪检测显示,5-Aza-CdR使SF126和SF767胶质瘤细胞周期阻滞于G0/G1期.因此,5-Aza-CdR能抑制胶质瘤细胞SF126和SF767增殖并干扰其细胞周期,LRRC4启动子异常甲基化是其在胶质瘤细胞中表达缺失的重要机制,5-Aza-CdR能逆转LRRC4基因的甲基化,恢复LRRC4的表达,为LRRC4作为胶质瘤去甲基化治疗的靶标提供了科学依据.