重组逆转录病毒浓缩方法研究

选择含有新霉素磷酸转移酶Ⅱ（Neor)的重组逆转录病毒载体,通过包装细胞进行包装,得到了含有重组逆转录病毒粒子的病毒溶液,该病毒溶液分别采用差速离心和滤膜截留两种方法进行浓缩,浓缩前,后的病毒溶液体外感染靶细胞NIH3T3以测定其滴度。结果显示,差速离心法的浓缩效率要优于滤膜截留法的浓缩效率,其浓缩效率能达到34．5％。     

人IL-12重组逆转录病毒载体在HeLa细胞中的表达

目的:包装携带人白细胞介素12(IL-12)的逆转录病毒,用于宫颈癌的治疗研究.方法:携带IL-12的逆转录病毒重组质粒pL35P40SN经PA317细胞包装,G418筛选.在NIH3T3细胞进行病毒滴度测定.然后用病毒感染人宫颈癌细胞HeLa.PCR、RT-PCR方法检测IL-12基因在HeLa中的整合和表达情况.结果:重组质粒pL35P40SN经PA317细胞包装后收获病毒上清,感染HeLa细胞,检测发现IL-12基因整合到细胞基因组DNA中,并且能有效的转录.结论:成功包装了携带IL-12基因的逆转录病毒,该病毒能有效感染HeLa细胞,并使携带的基因IL-12在细胞中表达,为今后IL-12基因治疗宫颈癌的研究奠定基础.     

重组逆转录病毒介导的转蚓激酶基因兔

目的通过重组逆转录病毒介导获得转蚓激酶基因家兔。方法将含有蚓激酶基因的表达盒插入到逆转录病毒载体pLNCL中获得了蚓激酶重组逆转录病毒载体,利用脂质体转染PA317包装细胞,G418进行筛选得到了阳性细胞克隆,克隆扩大培养后病毒上清直接注射雄性兔的睾丸组织转染精原干细胞。结果病毒上清经NIH3T3细胞进行滴度测定,最高滴度为1×104CFU/mL。病毒注射3月龄兔睾丸组织,过1.5个月取其睾丸、肾、脾、肝、肺进行组织切片观察,结果正常。提取交配所得F0、F1代仔兔基因组,经PCR、Southern检测,转基因阳性率F0代为6.3%(2/32)、F1代为15.3%(2/13)。结论通过重组逆转录病毒直接注射3月龄兔睾丸组织可获得转基因兔,病毒对兔的脏器无损害。     

逆转录病毒载体介导的LacZ基因在NIH-3T3细胞中的稳定表达

精原干细胞(SSCs)介导的转基因技术很可能成为制作转基因动物及治疗雄性不育的一条新途径。为了研究逆转录病毒载体介导法转染体外培养SSCs的可行性,用脂质体介导法将携带LacZ基因的重组逆转录病毒载体pLNCL导入包装细胞PA317,用含G418的培养液筛选得到5株稳定转染的产毒细胞。收集这些克隆的产毒上清,过滤后进行倍比稀释,用NIH-3T3细胞通过X-gal染色测定其浓缩前病毒滴度。结果显示,PA3173培养上清中病毒的浓缩前滴度最高,达1.1×103CFU/mL。再将筛选到的稳定转染的NIH-3T3细胞培养至单层,进行X-gal染色检测β-半乳糖苷酶的表达。结果显示,大多数稳定转染的NIH-3T3细胞均为X-gal ,表明这些细胞成功表达了目的基因LacZ。本研究结果为后期工作中用该载体感染体外培养SSCs奠定了基础。     

人转化生长因子β型基因在逆转录病毒载体的克隆及其细胞生长抑制作用

将人转化生长因子β型(HTGF-β)基因克隆到逆转录病毒载体pDO R-Neo中,构建一株重组质粒(pDOT)。然后将pDOT转染到逆转录病毒的包装细胞(ψ2和pA317细胞),组装成为一个携带HTGF-β基因的重组病毒。对重组病毒的滴度,所携带的HTGF-β基因的表达,对感染的NIH 3T3细胞生长的抑制作用进行了初步研究。     

人c-myc转基因细胞的建立

目的:建立人c-myc转基因细胞。方法:通过成功构建c-myc逆转录病毒表达载体,并经脂质体介导转染包装细胞293T,收集产重组病毒的293T培养上清,运用NIH3T3细胞测定了病毒滴度,用适当浓度的病毒感染L929细胞,经用Zeocin选择性培养基筛选细胞。结果:得到稳定高表达c-myc基因的L929转基因细胞。结论:运用逆转录病毒转染法可得到高表达的转基因细胞。     

小鼠T-bet逆转录病毒载体的构建与功能验证

为在小鼠细胞中表达并研究T-bet功能,首先构建了含有报告基因Thy1.1的小鼠T-bet逆转录病毒载体,并将构建的载体质粒转染病毒包装细胞系包装成重组病毒,再利用重组病毒分别感染NIH-3T3和D9细胞系检测其感染能力。之后,使用该重组病毒感染T-bet敲除小鼠的CD4+T淋巴细胞,流式细胞术检测T-bet及其下游靶基因Ifng的表达情况。经验证,重组的逆转录病毒感染T-bet敲除小鼠T淋巴细胞后可以在细胞中表达T-bet,并进一步引起下游靶基因Ifng的表达上调,证明本研究中外源表达的转录因子T-bet具有正常功能。综上所述,本实验成功构建了含有报告基因的小鼠T-bet重组逆转录病毒载体,为进一步在小鼠细胞中研究T-bet功能奠定了基础。     

携带HSV-1TK基因的逆转录病毒载体构建及在HeLa细胞中的表达

目的:构建携带单纯疱疹病毒脱氧胸腺嘧啶激酶基因(herpes simplex virus thymidine kinase,HSV-TK)的逆转录病毒,用于宫颈癌的治疗研究。方法:用限制性内切酶从质粒pcDNA3.1/HA-myc-His(-)Z-TK切下HSV-1TK cDNA序列,亚克隆入逆转录病毒载体pLXSN得到重组质粒pLXSN-TK,鉴定正确的阳性重组质粒经PA317细胞包装,G418筛选,在NIH3T3细胞进行病毒滴度测定。然后用病毒感染人宫颈癌细胞HeLa。PCR、RT-PCR和Western blotting方法检测HSV-1TK基因在HeLa中的整合和表达情况。结果:重组质粒pLXSN-TK经PA317细胞包装后收获病毒上清,感染HeLa细胞,检测发现HSV-1TK基因整合到细胞基因组DNA中,并且能有效的转录和翻译。结论:成功构建了逆转录病毒pLXSN-TK,该病毒能有效感染HeLa细胞,并使携带的治疗基因HSV-1TK在细胞中表达,为今后HSV-1TK基因治疗宫颈癌的研究奠定基础。     

反义前C／C基因转移表达抗乙型肝炎病毒作用的研究

为了观察逆转录病毒载体包装细胞系统介导反义基因转移表达的抗乙型肝炎病毒（HBV）作用,将HBVayw前C／C基因（preC／C）片段反向插入逆转录病毒载体质粒。将重组体转染逆转录病毒包装细胞PA317后,获得重组逆转录病毒。用重组逆转录病毒感染2．2．15细胞后发现,感染后第3天,细胞培养上清HBV表面抗原（HBsAg）和e抗原（HBeAg）表达量即明显减少,抑制作用于感染后第5天达到高峰,其中对HBsAg表达的抑制率为27．0％,对HBeAg表达的抑制率为59.5％。反义基因重组逆转录病毒感染2.2．15细胞对HBV抗原表达的抑制作用至少可以持续到转导后的第11天。空载及正义基因重组逆转录病毒感染对2．215细胞HBV抗原表达无明显抑制作用。此外,反义基因重组逆转录病毒感染对2.2.15细胞HBVDNA复制也有抑制作用．无细胞毒性。     

Ca-Cd interaction in the prymnesiophyte Cricosphaera elongata

Abstract. ⁴⁵Ca and ¹⁰⁹Cd uptake were followed in Criscosphaera elongata Prymnesiophyceae. In both cases, after a rapid increase for the first 5 min, the incorporation rate slowed during the hour of observation. Verapamil, a blocker of voltage-dependent slow calcium channels, inhibited ⁴⁵Ca uptake except during the first rapid phase when adsorption should predominate. Cadmium also decreased ⁴⁵Ca labelling, suggesting that the two metals are antagonistic. However, verapamil was shown to augment ¹⁰⁹Cd incorporation, contrary to what occurs in animals cells; this effect is detectable in continuous labelling as well as in pulse experiments. The data support the presence of calcium channels in the alga and suggest several processes in Cd accumulation: adsorption on peripheral envelopes and diffusion of uncharged Cd.     

四种灵长类肘关节的生物力学研究

对懒猴、猕猴、叶猴和长臂猿的肘关节形态与功能和前臂伸、屈肌肌肉电生理的研究结果表明,随着上肢运动功能的加强,肘关节的灵活性亦相应增大。猕猴各肌的肌电活动相对较弱,肘关节的结构明显不同于其它3个种,表现出对四足型运动和维持关节稳定性的适应特点。叶猴肘关节的形态结构和前臂各肌的肌电活动类似于长臂猿,表现出与臂摆荡有关的活动特点。对前臂伸、屈肌有关指数的判别分析表明,与骨骼相比,4个种的前臂各肌的形态差异明显较大。肱肌的近侧起点指数。肱桡肌的止点指数和肱三头肌内侧头的起点指数可作为4个种的鉴定特征。     

High-pressure effect on the equilibrium and kinetics of cyanide binding to chloroperoxidase

The kinetics of cyanide binding to chloroperoxidase were studied using a high-pressure stopped-flow technique at 25 degrees C and pH 4.7 in a pressure range from 1 to 1000 bar. The activation volume change for the association reaction is delta V not equal to + = -2.5 +/- 0.5 ml/mol. The total reaction volume change, determined from the pressure dependence of the equilibrium constant, is delta V degrees = -17.8 +/- 1.3 ml/mol. The effect of temperature was studied at 1 bar yielding delta H not equal to + = 29 +/- 1 kJ/mol, delta S not equal to + = -58 +/- 4 J/mol per K. Equilibrium studies give delta H degrees = -41 +/- 3 kJ/mol and delta S degrees = -59 +/- 10 J/mol per K. Possible contributions to the binding process are discussed: changes in spin state, bond formation and conformation changes in the protein. An activation volume analog of the Hammond postulate is considered.     

Mass-spectrometric investigations on hydration of nucleic acid components in vacuum. II. N-methylated adenines

Association reactions between water and N-methylated adenines (M), occurring under field-ionization conditions in a mass spectrometer at the tungsten point emitter surface, were studied at a number of temperatures. The origin of the peaks observed in the mass spectra at m/e ratios corresponding to MH+ was assigned to M X H2O monohydrates , of those corresponding to (MH X H2O)+ and (MH2)2+ to double hydrates M(H2O)3. Measurements of the relative peak intensities allowed calculation of apparent equilibrium constants, Kass, for successive hydration steps, and hence the respective van't Hoff enthalpies of hydration. Both the equilibrium and the energy parameters obtained support the available quantum-mechanical single-layer hydration schemes of adenine and of its N-methylated derivatives.     

THE ROLE OF UNICELLS IN THE POLYMORPHIC SCENEDESMUS ARMATUS (CHLOROPHYCEAE)1

The UTEX 2193 strain of Scenedesmus armatus (Chod.) Chod, when cultured in any of several media (whether natural or artificial, concentrated or dilute) produced a variety of colonial morphologies as well as a unicell population. Morphological expression was related to culture ape. When the initial cell density was just a feu1 hundred cells per mL. the culture first produced a unicell population, then spiny colonies, and as stationary phase was approached, spine-less colonies. Two classes of spiny colonies were detected. Type I colonies had elongate cells with the terminal cells shorter than median cells. Spines were longer than cell length. The wider, oval, grainy cells of Type II colonies were uniform m length. Spines were shorter and thicker than those on Type I colonies. Only Type I colonies produced unicells: the latter appeared as two morphs. The smaller unicell was obovoid with four delicate spines: the larger had ovate cells bearing four thicker spines. Control of unicell development in all media was achieved by carefully monitoring colony type and cell number used for the inoculum. A unicellular population developed in batch culture in defined media, both concentrated and dilute, when the initial cell density (either Type I or Type II colonies) was low (below 1000 cells-mL^?1), as well as in synchronous cultures. With higher initial cell densities, e.g. 2 × 10⁴ cells·mL^?1, the inoculum had to contain Type I colonies to produce unicells. Unicells were also produced in water from Agronomy Pond, where the strain originated. We discuss the role of unicell populations in the distribution of Scenedesmus.     

棉花751与多父本杂交受精细胞学研究及性状分析

本文报导了以棉花７５１为母本与鸡脚叶棉、乌干达棉、红叶棉、汉寿大桃、草棉等５个父本的混合花粉进行杂交。授粉后,观察了７５１大小孢子和雌雄配子体的发生、发育及其受精过程。切片观察表明在大小孢子的发生、发育与花蕾大小成正相关。大小孢子的发生在减数分裂前是不同步的。小孢子的发生早于大孢子发生１￣２个时期。减数分裂后它们的发育逐渐趋向同步。开花时达到同步成熟。用多父本的混合花粉授粉,其受精过程仍为单精入卵