Effects of lentivirus-mediated c-met RNAi on biological behaviors of human papillary thyroid cancer K1 cells

AIM: To evaluate the expression of c-Met in papillary thyroid cancer (PTC) by constructing lentiviral vectors for RNA interference (RNAi) of c-met gene and detecting its silencing effect on the biological behaviors of human papillary thyroid cancer cell line K1 cells. METHODS: Immunohistochemical assay was performed to detect the expression of c-Met protein in 35 cases of PTC and 25 cases of benign thyroid disease. Lentiviral vector for RNA of c-met gene was constructed and the silencing effect was detected by RT-PCR and Western blotting. The colony-forming ability, cell cycle, migration and invasion of K1 cells were measured by colony-forming assay, flow cytometry, wound-healing observation and Transwell experiment, respectively. In vivo tumorigenicity assay was performed to analyze in vivo proliferation of K1 cells in a xenograft model. RESULTS: The expression of c-Met in PTC was significantly higher than that in benign thyroid tissues. Lentiviral RNAi vectors targeting c-met gene were successfully constructed, and they efficiently inhibited the expression of c-met at mRNA and protein levels. Transfection of c-met lentiviral RNAi vectors inhibited the colony formation, cell cycle progression, migration, invasion and tumorigenicity of K1 cells. CONCLUSION: Lentivirus-mediated c-met RNAi efficiently inhibits colony formation, cell cycle progression, migration, invasion and tumorigenicity of K1 cells.     

梨ACC氧化酶基因(ACO)的片段克隆及其RNAi载体构建

以若光梨成熟果实为材料,提取总RNA,在ACC氧化酶基因(ACO)cDNA序列同源性较高区域设计一对特异引物,利用RT-PCR克隆了ACC氧化酶(ACO)基因片段。将ACO反义基因、与副球菌中类胡罗卜素合成有关的间隔基因YYT和ACO正义基因3个片段串联在一起,经鉴定后,插入到植物表达载体中,构建成能够表达ACC氧化酶基因的双链RNA的植物载体pYF028,为耐贮砂梨的遗传转化创造条件。     

Construction and expression of retroviral vector containing mIL-12 gene

AIM: To construct retroviral expression vector pLXmIL-12SN containing mouse IL-12 (mIL-12) gene and detect the gene expression in B16F10 cells. METHODS: Vector pGCp35-IRES-p40SN was digested to obtain p35-IRES-p40 (mIL-12 gene fragment) and retroviral vector pLXSN was digested by restriction enzyme, and then they were linked by ligase. The recombinant vector that mIL-12 gene fragment had correctly been inserted into pLXSN was identified by sequencing. The recombination vector with mIL-12 gene was transfected into B16F10 cell and was detected by RT-PCR. RESULTS: Expression vector pLXmIL-12SN was successfully constructed. mIL-12 gene was confirmed to express in B16F10 cells at the level of mRNA. CONCLUSION: Acquired vector pLXmIL-12SN and confirmed mIL-12 gene expression lays a foundation for mIL-12 gene therapy to eye diseases.     

Construction and identification of recombinant adenovirus vector containing CTLA4Ig gene

AIM:To construct a recombinant adenovirus expression vector containing CTLA4Ig gene.METHODS:The CTLA4Ig gene derived from the plasmid PCDNA3.0/CTLA4Ig by using polymerase chain reaction (PCR) was inserted into the backward position of cytomegalovirus (CMV) immediate early promoter of the shuttle plasmid (pAdTrack-CMV). After being identified by endonuclease, PCR and sequencing, the recombinant shuttle plasmid pAdTrack-CTLA4Ig was co-transformed into E.coli. BJ5183 cells with the adeoviral backbone plasmid pAdEasyl-1 to obtain the homologous recombination. The adenovirus was generated in 293 cells. A series methods such as PCR and fluorescence microscope was employed to identify the generated recombinant adenovirus.RESULTS:Recombinant CTLA4Ig adenoviruses were constructed and the titer of virus was generally up to 1.65×10¹² phaque forming units per liter (PFU/L).CONCLUSION:Success in constructing recombinant pAdTrack-CTLA4Ig will be the base of the further research on its expression in the mammalian cells, and be potenially used in the prevention of transplant rejection and autoimmunity diseases.     

甜瓜蔗糖磷酸合成酶基因全克隆及工程载体的构建

蔗糖磷酸合成酶在甜瓜果实蔗糖合成途径中起关键性的调节作用,克隆该基因并导入甜瓜低糖自交系,可改良甜瓜果实品质创新优良种质。从甜瓜幼苗叶片提取总RNA,利用RT-PCR与Southern blot方法相结合,重组子质粒经限制性内切酶酶切和PCR验证以及测序同源性比较,克隆到基因的5’(2859bp)和3’(852bp)。应用高保真Taq聚合酶PCR拼接蔗糖磷酸合成酶完整cDNA序列3692bp,该片段与GenBank中其它植物基因序列具有97%￣99%的同源性,登录号DQ364058。应用BamHⅠ、KpnⅠ、BglⅡ限制性酶切获得植物双元表达载体pROK2及基因的线性片段,通过T4连接酶反应,构建了以CaMV35S为启动子,以Tnos为终止子的工程载体pROK-SPS,该载体含有Npt-II选择标记基因。     

The construction of bicistronic eukaryotic vector carrying green fluorescent protein and hytk gene and its expression in bladder carcinoma

AIM: To facilitate the suicide gene delivery into neoplasm, a chimeric gene of HSV-tk and green fluorescent protein (gfp) was constructed. METHODS: Molecular cloning technique was used to construct this kind of eukaryotic vector. The internal ribosome entry site (IRES) of encephalomyocarditis virus (EMCV), which could coordinate expression of two genes in a single vector, was optioned. By using liposome-mediated transfection, eukaryotic expression vector tgCMV/hytk-IRES-gfp was transfected into human bladder carcinoma cells EJ. RESULTS: A bicistronic eukaryotic vector carrying gfp and hygromycin phosphotransferase-thymidine kinase fusion (hytk) gene was constructed. The results of PCR and microscopy detection show that the hytk-IRES-gfp gene was successfully transferred into EJ cells. There were no differences in the growth pattern or the morphology between EJ and EJ/hytk-GFP cells. In vitro experiments demonstrated dose- and time-dependent cell killing by transduction of the hytk-IRES-gfp gene followed by GCV treatment. The IC50 (the concentration required to elicit 50% growth inhibition) was 2.16 mg/L in treatment with GCV for 72 hours. CONCLUSION: These results suggest that this new kind of eukaryotic vector could serves as a new tool and method for neoplasm gene therapy.     

Effect of adenoviral vector containing AT1 receptor antisense cDNA on migration of vascular smooth muscle cells

AIM: To evaluate the role of human angiotensin II (AngII) type 1 receptor (AT1R) antisense cDNA (ahAT1) on migration of cultured artery smooth muscle cells (VSMCs). METHODS: Two recombinant adenoviral vectors, Ad/CMV.ahAT1 containing full length antisense cDNA targeting to human AT1R mRNA, and Ad/CMV.LacZ containing LacZ called report gene, were constructed by orientation clone technology and homologous recombination, and then were used to transfect VSMCs in vitro. AT1R expression detected by RT-PCR and immunohistochemistry, and migration of VSMCs measured by Boyden's Chamer methods, were compared between transfected and nontransfected VSMCs. RESULTS: Forty-eight hours after Ad/CMV. ahAT1 transfection, the level of AT1R mRNA decreased markedly (50% of control group), and AT1R protein expression was significantly less (P＜0.01 vs control-group and Ad/CMV.LacZ-group, respectively) in VSMCs. So it was migration distance of VSMCs among the three groups. CONCLUSION: These results indicate that antisense cDNA targeting to human AT1R transfer in vitro mediated by adenoviral vector has a powerful inhibitory effect on migration of VSMCs by attenuating AT1R expression.     

红肉苹果(Malus pumila var.niedzwetzkyana)MpMYB30基因的克隆及表达载体的构建

MYB转录因子是植物转录因子中最大的家族之一,在转录调节中起着多方面的重要作用。实验利用RACE技术从野生红肉苹果(Malus pumila var.niedzwetzkyana Schneid.)中获得了MpMYB30的全长序列,利用生物信息学的方法对其蛋白进行了分析和预测并将其连接到pCAMBIA-S1300+植物表达载体中。结果表明,该基因全长为1661bp,推导的氨基酸序列经系统发育分析与葡萄MYB30及蓖麻MYB基因相似性最高,命名为MpMYB30,其N端含有2个约55个氨基酸组成的MYB特征结构域。该基因推导的MpMYB30蛋白的分子式为C1790H2794N538O584S12,相对分子质量为41579.8ku,等电点为6.30,不存在信号肽和跨膜区域,蛋白质结构以α螺旋为主。利用XbalⅠ和SacⅠ对重组质粒进行酶切,并将其连接到pCAMBIA-S1300+载体上,通过抗性筛选和PCR酶切检测,证实了MpMYB30基因已经构建到植物表达载体pCAMBIA-S1300+上。     

Silencing of FoxM1 induces apoptosis of oral squamous-cell carcinoma cells by promoting mitochondrial release of cytochrome C

AIM: To study the effect of forkhead box protein M1 (FoxM1) silencing on apoptosis of oral squamous-cell carcinoma. METHODS: Oral squamous-cell carcinoma SCC9 cells were infected with FoxM1-shRNA lentivirus or negative control lentivirus. The silencing effect was measured by RT-qPCR and Western blot. The changes of cell viability effect was measured by MTT saay. The cell colony formation ability was measured by plate experiment. Flow cytometry was used to analyze the changes of apoptotic rate. Western blot was used to measure the protein levels of cleaved caspase-3 and cleaved caspase-9 in the cells. The changes of mitochondrial membrane potential were measured by JC-1 method. Western blot was used to measure the protein level of cytochrome C in the mitochondria and cytoplasm. RESULTS: Infection with FoxM1-shRNA lentivirus successfully reduced the expression of FoxM1 in oral squamous-cell carcinoma cells (P<0.05). Negative control lentivirus had no effect on the expression level of FoxM1 in the cells. The cell viability was reduced by FoxM1 silencing, and the ability of cell colony formation was also decreased. The apoptotic rate and the protein levels of cleaved caspase-3 and cleaved caspase-9 were all increased (P<0.05), and the mitochondrial membrane potential was decreased. The protein level of cytochrome C in the cytoplasm was increased, while the protein level of cytochrome C in the mitochondria was decreased (P<0.05). CONCLUSION: Silencing of FoxM1 induces the apoptosis of oral squamous-cell carcinoma cells by decreasing the mitochondrial membrane potential and promoting the release of cytochrome C from mitochondria.     

Effect of ischemic preconditioning on contents of cytochrome C and mitochondrial calcium in rats after focal cerebral ischemic-reperfusion

AIM: To observe the effect of ischemic preconditioning on contents of cytochrome C and mitochondrial calcium in rats after focal cerebral ischemic-reperfusion. METHODS: Focal cerebral ischemic model was made by occlusion of right middle artery in Wistar rats (ischemia for 2 h and reperfusion for 4 h). Rats were randomly divided into three groups: ischemia pretreatment, model and sham operation. Rats in ischemic pretreatment group were undergone transient ischemic preconditioning (30 min) and reperfusion (72 h). The contents of cytochrome C were measured according to Zhangjuntian's improved methods. The contents of mitochondrial calcium were detected by flame atom absorption. RESULTS: The contents of mitochondrial cytochrome C and calcium in model group were significantly lower than those in sham operation (P<0.05, P<0.01), whereas the contents of plasma cytochrome C were markedly higher (P<0.05, P<0.01). The change in ischemic pretreatment group was obviously difference as compared with the model group. CONCLUSION: Ischemic preconditioning reduces the release of mitochondrial cytochrome C and maintain mitochondrial calcium homeostasis.     

景观工程的施工细节处理控制

如何才能造就一项令人满意的园林景观,施工过程中控制不可或缺。施工细节处理得当对于提升景观过程的质量与品质具有现实的意义。本文对景观过程中的硬质铺装是和景观植物施工的细节处理进行可简要梳理,供大家参考。     

高尔夫球场景观区的设计与营造

在高尔夫球场中,景观区受重视的程度越来越高,在球场中所占的比重也越来越大。从原先的林克斯风格一统天下的局面,逐渐分化为林克斯球场、森林球场、湿地球场、园林球场等等。本文从几个方面阐述了高尔夫球场中景观区所发挥的作用、景观区设计原则和景观区主要组成部分的设计方法。在景观区设计及营造过程中,将中国优秀的园林设计理念融入其中,也许能给中国的高尔夫球场带来新的发展契机。     

Glutamate-induced apoptosis is related with the damage of mitochondria that results in cytochrome C release into cytosol in cultured hippocampal neurons

AIM: To set up a glutamate-induced cell damage model in cultured hippocampal neurons, and to determine whether glutamate-induced neuronal apoptosis changes and whether this process is mediated by mitochondrial signal transduction pathways involving the release of cytochrome C. METHODS: Hippocampal neurons, isolated and cultured from new born Wistar rats, were exposed to various concentrations of glutamate. Extent of cell death was assessed by measuring the release of lactate dehydrogenase (LDH) in the culture media. Based on these data, an appropriate concentration of glutamate was selected, and all subsequent experiments were carried out under the concentration. Kinetics of glutamate-induced both apoptotic and necrotic cell death after exposure to glutamate for various times(3-24 h) were determined by flow cytometry and LDH release. The caspase-3 protein levels and cytochrome C release from mitochondria into cytosol in hippocampal neurons were determined by Western blotting. RESULTS: Glutamate treatment induced hippocampal neurons death in dose-dependent and time-dependent manners. A significant increase in LDH release (18.4%) was induced in the cells treated with 50 μmol/L glutamate, compared to control untreated cells(P<0.05). A significant increases in LDH release and apoptosis were observed at 6 h after glutamate treatment (P<0.05). The significant increase in the level of caspase-3 protein occurred at 3 h after glutamate exposure, which preceded neuronal cell apoptosis, and reached maximum levels at 6 h(62.4%). Treatment with glutamate induced a rapid release of cytochrome C into cytosol. Cytosolic cytochrome C showed a significant increase (P<0.05) as early as 30 min after glutamate treatment, which preceded the increase in caspase-3 level, and after 3 h, the level of cytochrome C was higher in the cytosol compared to the mitochondria. Concomitant with these changes in cytosol, the mitochondrial levels of cytochrome C decreased significantly. CONCLUSION: Exposure to 50 μmol/L glutamate induces apoptosis in cultured hippocampal neurons. The glutamate-induced apoptosis may be via the damage of mitochondria that results in cytochrome C release into cytosol, which activates caspase cascade and induces apoptotic cell death.     

城乡绿道建设探析——以广东珠三角绿道建设为例

绿道是源自欧美发达国家的先进理念,绿道建设基本不占用建设用地,符合建设低碳城市的发展要求。近几年绿道建设率先在我国广东珠三角地区开展,随后在全国各地快速发展起来,全面提升了城乡居民的生活质量,完善了城市功能,强化了地方风貌特征。文章在综合分析国内外绿道建设与研究相关文献的基础上,以广东珠三角绿道建设为例,着重对绿道建设的生态功能、游憩功能进行了探析,并针对当前绿道建设存在的问题提出建议。