东北地区部分行业增值税转型的效应分析

东北部分行业增值税转型试点不会造成财政收入的较大波动;它对宏观经济的影响取决于获得抵扣资金的企业对抵扣资金的使用途径;转型对内资企业有利,尤其是对新设内资企业有利;本次转型对企业结构和投资决策有一定的影响,会导致企业行为一定扭曲;它还会促使企业进行避税。因此,在局部地区推行消费型增值税的正面作用并不明显,实行具有中国特色的收入型增值税将更有利于解决现行增值税的问题。国家自然科学基金资助项目;; 教育部“十五”规划项目

Effects of transfection with acidic fibroblast growth factor by electroporation on skeletal muscle satellite cells

背景:课题组早期研究表明体外一定剂量酸性成纤维细胞生长因子对骨骼肌卫星细胞增殖有促进作用。目的:进一步验证电穿孔转染酸性成纤维细胞生长因子基因对骨骼肌卫星细胞生长、增殖及分化的影响。方法:原代培养、纯化骨骼肌卫星细胞,将带有酸性成纤维细胞生长因子基因的质粒P SECTAg-gfP-A fgf通过电转染的方法转染大鼠骨骼肌卫星细胞,荧光显微镜观察绿色荧光蛋白的表达情况并计算转染率,以流式细胞仪分析转染后细胞周期,绘制细胞生长曲线,观察转染后肌管形成情况,WESTErn blOTIng检测酸性成纤维细胞生长因子基因的表达。结果与结论:1免疫细胞化学检测:骨骼肌肌动蛋白呈阳性表达。2转染效率:P SECTAg-A fgf质粒电转染12 H后即可看见散在发绿色荧光的卫星细胞,72-96 H达高峰,阳性表达率约90%。3细胞周期检测:电转染后S期所占的百分比明显多于未转染对照组(P<0.05)。4细胞生长曲线检测:电转染细胞接种后第3天进入对数生长期,第5天后开始减少。5分化能力观察:电转染组肌管较未转染对照组明显减少,老化细胞较少。6WESTErn-blOT:酸性成纤维细胞生长因子基因在转染骨骼肌卫星细胞中表达。结果表明,通过电穿孔法可以将酸性成纤维细胞生长因子基因转染进骨骼肌卫星细胞并获得高效持久的表达,并有促进骨骼肌卫星细胞增殖及抑制分化为肌管的作用。BACKGROUND: Previous studies have shown that a certain dose of acidic fibroblast growth factor can promote skeletal muscle satellite cell proliferation in vitro.OBJECTIVE: To investigate the effects of transfection with acidic fibroblast growth factor by electroporation on growth, proliferation and differentiation of skeletal muscle satellite cells.METHODS: Skeletal muscle satellite cells were cultured and purified, and then transfected with plasmid p Sectag-GFP-a FGF by electroporation.The expression of green fluorescent protein was observed under fluorescence microscope, and the transfection efficiency was calculated.After transfection, cell cycle was analyzed by flow cytometry to draw the growth curve of skeletal muscle satellite cells.Western blot assay was employed to measure protein level of acidic fibroblast growth factor.RESULTS AND CONCLUSION:(1) Immunocytochemistry detection: The skeletal muscle satellite cells were positive for a-sarcomeric actin.(2) Transfection efficiency: At 12 hours after transfection with p Sectag-a FGF, several cells showed green fluorescence, and the green fluorescent expression reached the peak at 72-96 hours after transfection with a positive rate of about 90%.(3) Cell cycle: After electrotransfection, the proportion of cells at S phase in the electroporation group was higher than that in the control group(P < 0.05).(4) Cell growth curve: At 3 days after electrotransfection, the cells entered logarithmic growth phase but the proliferation slowed down at 5 days.(5) Differentiation capacity: There were fewer myotubes and aging cells in the electroporation group than the control group.(6) Western blot assay: Acidic fibroblast growth factor protein was highly expressed in the cells transfected with target gene detected by western blot assay.These findings indicate that by using electroporation method, acidic fibroblast growth factor can be transferred into skeletal muscle satellite cells and have a high-efficiency and long-term expression, which can promote the proliferation of skeletal muscle satellite cells and inhibit formation of myotubes

Determination of Estrogenic Compounds in Water of Jiulong River Using Polar Organic Chemical Integrative Sampler

极性有机物一体化采样器(POCIS)作为富集水体中有机物的新型采样技术,可以反映目标物在被测水体中的时间权重浓度,因此在环境监测中是对主动采样方式的重要补充.本研究采用POCIS进行了4种固醇类雌激素化合物(雌酮、雌二醇、雌三醇及雌炔醇)的富集动力学,以及温度和目标物浓度对POCIS富集雌激素影响的实验室模拟研究.结果表明,POCIS对目标化合物的富集放置时间7 d内呈现显著的线性关系(r2≥0.988 1,P12%),随着温度的升高,目标化合物在POCIS上的采样速率减小.而目标物浓度对POCIS富集没有显著的影响(rSd 12%).As for different concentrations of target compounds,the sampling rate was not affected by the concentrations( RSD < 5%).After laboratory experiments,an environmental field study was performed in Jiulong River.The results showed that there was a relatively good correlation between the measured and calculated values( r2= 0.720 9,P < 0.001).It demonstrated that POCIS can be used in aquatic field systems.近海海洋环境科学国家重点实验室访问学者基金项目; 海洋公益性行业科研专项(201105013

X射线双星系统的探测

搜寻X射线双星并建立大规模样本是解决众多科学问题(如\"银河系中有多少黑洞X射线双星?黑洞和中子星之间有无质量间隙?球状星团中有无黑洞?\"等)的基础.爱因斯坦探针卫星拥有更灵敏的探测能力,将在运行的几年之中,预计可以发现一批新的中子星和黑洞X射线双星,研究其空间和动力学质量分布成为解决上述问题的关键.国家重点研发计划(编号:2016YFA040080X);;国家自然科学基金(编号:1133305);;中国科学院战略性先导科技专项(编号:XDA15052100,XDB23040000)资助项

抗HEV嵌合抗体的构建及在CHO细胞中的表达

通过RT-PCR方法从分泌戊型肝炎(戊肝)病毒中和性鼠源单克隆抗体(单抗)8C11的杂交瘤细胞中克隆出抗体基因的重链可变区(VH)、轻链可变区(VK)序列,并分别克隆到含有人gamma1重链和kappa轻链恒定区序列的pcDNA3 1/Hygro和pcDNA3 1(+)质粒中,共转染中华仓鼠卵巢癌细胞(CHO)细胞。RT-PCR结果表明,转染的CHO细胞转录了嵌合重链及轻链基因,间接ELISA及Westernblot结果表明:翻译出的两种多肽在细胞内正确组装成嵌合抗体分子,并可分泌至细胞外,表达的嵌合抗体保留了原鼠单抗的抗原结合特异性及对8H3结合抗原的增强作用。8C11嵌合抗体的成功表达可降低鼠源性,为探讨戊肝抗体治疗的可能性奠定了基础

多样性人源天然噬菌体抗体库的构建及初步应用

目的:构建多样性良好的人源天然噬菌体抗体库。方法:从正常人外周血中分离淋巴细胞,以RT-PCR和半巢式PCR扩增重链可变区VH基因和轻链可变区VL基因,以重叠延伸PCR将VH、VL组装成scFv基因,并将其克隆入噬菌粒载体pCANTAB-5E中。以pCANTAB-5E电转化大肠杆菌TG1,构建人源天然噬菌体抗体库,测序分析抗体基因的家族信息和多样性,并用多种抗原对其进行筛选。结果:获得了库容为2×108的人源天然噬菌体抗体库。分别用5种抗原对其进行筛选,均可获得特异性噬菌体抗体的富集。结论:成功地构建了一个多样性良好的人源天然噬菌体抗体库,可用于制备具有应用前景的人源抗体

抗HEV中和抗体13D8的单链抗体的构建及表达

目的 :降低HEV中和性单抗 (mAb) 13D8的鼠源性 ,表达其单链抗体 (scFv)。方法 :从分泌 13D8鼠mAb的杂交瘤细胞中 ,通过RT PCR克隆mAb的VL、VH 基因 ,并进一步组装成VH linker VL 型的scFv片段。将scFv片段克隆到pTO T7载体中 ,在大肠杆菌中进行表达。用ELISA、Westernblot检测scFv的活性。结果 :SDS PAGE表明 ,13D8的scFv在E .coli中得到高效表达 ,表达量达菌体总蛋白的 2 6 .8%左右 ,表达产物主要以包涵体的形式存在。间接ELISA和Westernblot检测表明 ,表达的 13D8的scFv能与HEVOFR2区中一段重组蛋白(NE2 )特异结合。竞争ELISA表明 ,scFv与原鼠mAb识别的为同一表位。结论 :成功地表达出具有免疫学活性的 13D8的scFv

Uptake and Metabolism Kinetics of TBT in Whelk(Thais clavigera)Exposed to Dietary

将牡蛎消化腺分别暴露在1000ng.l-1和100ng.l-1TbT水溶液中4周,然后将染毒的牡蛎消化腺分别投喂疣荔枝螺(THAIS ClAVIgErA)。经过45d的暴露和30d的净化,我们发现雌雄疣荔枝螺的消化和生殖系统能较快地吸收TbT(吸收速率ku=0.004-0.022.d-1),并且其代谢(生物代谢系数bdI=5.59-23.30)和排出速率(净化速率kE=0.024-0.053.d-1)也相对较快,各器官中TbT的代谢产物MbT占了相对较高的比例,因此TbT在食物链传递过程中没有出现生物放大的现象。此外,TbT有逐渐从雌螺消化系统向生殖系统转移的趋势,并且雌螺生殖系统对TbT的吸收和富集能力(ku=0.006-0.022.d-1,生物放大系数bMf=0.181-0.664)要显著强于雄螺(ku=0.004-0.014.d-1,生物放大系数bMf=0.142-0.376),但其代谢和净化速率(bdI=5.59-10.50,kE=0.024-0.025.d-1)却显著低于雄螺(bdI=11.5-12.4,kE=0.031-0.050.d-1),雌螺的生殖系统被认为是TbT转移和富集的潜在靶器官,这对我们今后开展TbT污染的环境监测和评价具有重要的参考价值。Oysters were respectively exposed to 1000 ng·L-1 and 100 ng·L-1 tributyltin(TBT) aqueous for 4 weeks,which was as dietary to feed the female and male Thais clavigera whelks for 45 days.Then these Thais clavigera were depurated for 30 days.The results show that TBT rapidly accumulated in their digestive and reproductive organs(ku=0.004-0.022 d-1).Moreover,elimination and biotransformation of TBT were also rapid(BDI=5.6-23.30,ke=0.024-0.053 d-1).MBT was the dominant metabolite in each tissue.Therefore,bio-magnification of TBT did not occur during the trophic transfer process.Additionally,to females,the mobilization of TBT from digestive to reproductive organs and bioaccumulation of TBT(ku=0.006-0.022 d-1,BMF=0.181-0.664) were more obvious than that of the males.However,lower metabolism and elimination of TBT(BDI=5.6-10.5,ke=0.024-0.025 d-1) in female reproductive organs,which indicated that the reproductive organs of females were the main targets of TBT accumulation.The results are important to the risk assessment of TBT contamination in coastal environments.国家“863”项目No.2007AA09Z126;国家自然科学基金项目No.40476048;20777060;海洋公益性行业科研专项经费;No.20080509

亲和层析法用于噬菌体抗体库的筛选

本研究报道一种基于固定化金属亲和层析(IMAC)的噬菌体抗体库液相筛选方法。将纯化的带有His标签的抗原与噬菌体抗体库混合,噬菌体抗体与抗原充分结合后再加入亲和介质,使噬菌体抗体抗原复合物通过His标签与介质结合,然后通过充分洗涤去除非特异性噬菌体抗体,最后将特异性噬菌体抗体洗脱下来,感染TG1,进行下一轮筛选。整个筛选过程中抗原与抗体的结合在液相中完成,不仅消除了固相介质对抗原表位的影响,也更有利于噬菌体抗体与抗原的充分作用。将此方法应用于HEV NE2蛋白特异性人源噬菌体抗体的筛选,抗原竞争ELISA,阳性血清阻断,可溶性单链抗体表达检测及测序结果表明,最终获得2个特异性人源抗体

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