牛胎儿皮肤成纤维细胞的分离培养及转染Ipr1基因

为了为核移植提供供体细胞,本研究构建了真核表达载体PEGFP-SRA-Ipr1,并研究了牛胎儿皮肤成纤维细胞的体外分离培养,进行了Ipr1基因转染.克隆Ipr1基因和SR-A启动子,并构建了巨噬细胞特异性真核表达载体.用组织块贴壁法分离培养牛胎儿皮肤成纤维细胞,在体外经传代、纯化后,用电穿孔法将真核表达载体PEGFP-SRA-Ipr1转染至经体外纯化的第4～10代牛胎儿成纤维细胞,24 h后观察荧光表达,48 h后加入600μg·mL-1 G418,筛选1周,300 p.g·mL-1持续筛选,然后挑选单克隆,继续扩大培养.对稳定转染的牛胎儿成纤维细胞进行PCR检测和核型分析.结果,转染24 h后有绿色荧光蛋白表达,并经G418筛选获得稳定转染PEGFP-SRA-Ipr1的牛胎儿成纤维细胞株,经PCR检测在大约1500 bp处有目的片段,经流式细胞仪分析转染细胞染色体倍型未发生变化,仍是二倍体.说明目的基因已经成功整合,同时保持了遗传稳定性.可以作为核移植进行转基因克隆牛研究.     

脆壁克鲁维酵母基因置换技术的建立

以中性乳糖酶生产菌脆壁克鲁维酵母为材料,构建以kan基因为筛选标记、以脆壁克鲁维酵母乳糖酶基因上游调控区为同源臂的质粒pPkan。将质粒pPkan电击转化脆壁克鲁维酵母,对受体菌生长时期、电场强度、电击后培养时间等条件进行优化。结果表明,以OD600为0.8的脆壁克鲁维酵母为受体、电压1.5 kV、电场强度为7.5 kV.cm-1、电击后培养时间为90 min,可获得较高的转化率,最高转化效率为2.37×102转化子.μgDNA。在电击转化的同时加入限制性内切酶10μL,对电击转化的转化率无显著影响。经PCR筛选证实87.5%的转化子为同源重组,从而建立一套有效的脆壁克鲁维酵母基因置换技术。     

大鼠下丘脑室旁核在高张NaCl溶液抗失血性低血压中的作用

用大鼠股动脉放血致矢血性低血压的实验模型，以观察下丘脑室旁核（ＰＶＨ）在注射高张ＮａＣｌ溶液引起的升压反应中的作用。结果表明：（１）ＰＶＨ电损毁或假损毁后，小剂量高张ＮａＣｌ溶液静脉注射引起动脉血压（ＡＰ）有意义地迅速回升，但５ｍｉｎ后开始下降，损毁组下降速度快，３５ｍｉｎ后，平均动脉压（ＭＡＰ）回降至注射前水平。组间比较，２０ｍｉｎ后，损毁组ＭＡＰ回升值明显低于假损毁组（Ｐ＜０．０５）。（２）失血后５ｍｉｎ内假损毁组ＨＲ有意义地减慢；明显低于损毁组（Ｐ＜０．０５）。（３）ＰＶＨ微量注射高张ＮａＣｌ溶液（ＨＳ组）后，ＡＰ有意义地回升。对照组（ＮＳ组）则无明显回升。组间比较，ＨＳ组ＡＰ值显著高于ＮＳ组。结果提示：ＰＶＨ参与高张ＮａＣｌ溶液抗矢血性低血压时的升压反应，在血压恢复后的维持阶段起重要作用。     

Effects of chlorogenic acid (CGA) supplementation during in vitro maturation culture on the development and quality of porcine embryos with electroporation treatment after in vitro fertilization

Electroporation is the technique of choice to introduce an exogenous gene into embryos for transgenic animal production. Although this technique is practical and effective, embryonic damage caused by electroporation treatment remains a major problem. This study was conducted to evaluate the optimal culture system for electroporation‐treated porcine embryos by supplementation of chlorogenic acid (CGA), a potent antioxidant, during in vitro oocyte maturation. The oocytes were treated with various concentrations of CGA (0, 10, 50, and 100 μmol/L) through the duration of maturation for 44 hr. The treated oocytes were then fertilized, electroporated at 30 V/mm with five 1 msec unipolar pulses, and subsequently cultured in vitro until development into the blastocyst stage. Without electroporation, the treatment with 50 μmol/L CGA had useful effects on the maturation rate of oocytes, the total cell number, and the apoptotic nucleus indices of blastocysts. When the oocytes were electroporated after in vitro fertilization, the treatment with 50 μmol/L CGA supplementation significantly improved the rate of oocytes that developed into blastocysts and reduced the apoptotic nucleus indices (4.7% and 7.6, respectively) compared with those of the untreated group (1.4% and 13.0, respectively). These results suggested that supplementation with 50 μmol/L CGA during maturation improves porcine embryonic development and quality of electroporation‐treated embryos.     

Intratumoural interleukin 12 gene therapy stimulates the immune system and decreases angiogenesis in dogs with spontaneous cancer

Interleukin 12 (IL‐12) is a powerful immunostimulatory cytokine with a strong antitumoural activity. In this work, the immunological, anti‐angiogenic and clinical effects of three consecutive intratumoural IL‐12 electrogene therapy (EGT) treatments were evaluated in nine dogs with spontaneous cancer. In all the dogs, tumour biopsies and blood samples were taken prior, during and after the intratumoural IL‐12 EGT (on days 1, 8, 35 and 1, 3, 8, 15, 35, respectively). An initial decrease in immune cells was followed by an increase above baseline 1–3 weeks after treatment initiation. Interestingly, the decrease in peripheral leukocytes 2 days after the first intratumoural IL‐12 EGT coincided with erythema and tumour swelling. Transient increases of IL‐12 and interferon γ were measured in the serum and the tumour tissue, whereas IL‐10 transiently increased only in the serum. The effect of intratumoural IL‐12 EGT on the levels of IL‐24 and vascular endothelial growth factor in the sera and tumour biopsies differed per dog. Via contrast‐enhanced ultrasound (US) (on days 1, 8 and 35), we demonstrated that intratumoural IL‐12 EGT resulted in a significant decrease of the relative blood volume and blood flow speed in the tumour compared with baseline. Metastases were present in two dogs. In one of these dogs, IL‐12 EGT of the primary tumour caused a transient partial regression of the metastases, but not of the primary tumour. The second dog with metastases did not survive long enough to complete the entire treatment cycle. Despite encouraging immunostimulatory and anti‐angiogenic effects after intratumoural IL‐12 EGT, no clinically relevant outcomes were observed in this study, as persistent tumour regression could not be obtained. On the other hand, the laboratory and US results hold great promise for combinatorial strategies of intratumoural IL‐12 EGT with conventional antitumour (immuno)therapies.     

弓形虫SAG1基因在蜥蜴利什曼原虫中的表达

利用PCR扩增技术从弓形虫RH株全基因组中扩增出弓形虫抗原基因SAG1,将其克隆入真核表达载体质粒pLEXSY-neo2,构建真核表达穿梭质粒pLEXSY-neo2-SAG1。利用电穿孔转染技术将外源基因表达盒转入蜥蜴利什曼原虫,经G418筛选阳性克隆。表达的外源重组蛋白经SDS-PAGE和Western blot进行鉴定,证明SAG1重组蛋白在蜥蜴利什曼原虫中得到表达。     

电穿孔作用将外源基因导入中华蜜蜂卵的研究

将中华蜜蜂卵与带有绿色荧光蛋白基因的质粒DNA混合,经电穿孔作用后,人工孵化蜜蜂卵,并对孵化的幼虫进行检测.结果筛选出一组较理想的中华蜜蜂卵电穿孔参数(750 V·cm-1,100 μs,1);通过荧光检测,电击卵孵化的幼虫体内可以表达EGFP基因,阳性表达率最高可达1.05%,且外源DNA转染早期卵的效率显著高于晚期卵.结果表明电穿孔作用成功地将外源基因引入中华蜜蜂卵内,质粒DNA转染中华蜜蜂卵的效率与卵的生长时期有关.     

水分胁迫对黄瓜种子萌发及幼苗生长的影响

采用PEG高渗溶液法研究了黄瓜种子的萌发特性,同时采用盆栽控水法研究了黄瓜幼苗在4个水分梯度下的生长发育习性。结果表明：随着水分胁迫程度的加剧,黄瓜发芽率、发芽势、胚根长、胚芽长及黄瓜苗高呈下降趋势;脯氨酸含量呈上升趋势,且水分胁迫越严重,趋势越明显;而黄瓜幼苗叶绿素含量和过氧化物酶活性在水分胁迫初期呈上升趋势,后期呈下降趋势,但与对照组相比,都有所增加,在中度水分胁迫下达到最高值;黄瓜幼苗叶片数和总叶面积与对照组相比,均有所下降,中度水分胁迫下降趋势最低,与对照组相比差异不显著。     

大载体转染猪胎儿成纤维细胞的电转条件优化

背景 随着生物技术发展,研究的生理机制和生物功能日益复杂,提高大载体的转染效率对多基因共表达系统、基因编辑技术、转基因育种等具有重要的意义。在转基因育种中,使用的转基因载体相对较大,而且转基因动物的制备效率也与供体细胞猪胎儿成纤维（porcine Fetal Fibroblasts,PFFs）细胞的转染效率有关。目的 研究主要从转染参数、质粒用量和拓扑结构三方面,比较3种电转仪ECM ^?830/NEPA 21/Nucleofector ^TM2b的大载体转染效率,以探索大载体转染PFFs的最佳条件。方法 使用3种不同电转仪将长达26 kb的携带增强型绿荧光蛋白基因的pPXAT-EGFP质粒转染1×10 ⁶个PFFs,48 h后使用流式细胞仪测定荧光细胞比例,从电转参数、质粒用量和拓扑结构三方面分别比较瞬时转染效率。结果 首先比较电转仪不同参数的转染效率,结果显示当电转参数为脉冲电压300 V,脉冲长度1 ms,脉冲间隔50 ms,脉冲次数3次,NEPA 21转染PFFs的效率最高,为13.24%±1.63%,而Nucleofector ^TM 2b的最佳电转参数为U-023,其转染效率高达46.36%±3.95%。然后在最佳电转参数下分别比较6、8、10和12 μg的26 kb超螺旋质粒的转染效率,ECM ^?830和Nucleofector ^TM 2b转染PFFs的最佳质粒用量为12 μg,其转染效率分别为8.44%±0.90%（电转参数：脉冲电压300 V,脉冲长度1 ms,脉冲次数3 次）和14.63%±3.21%(电转参数：U-023),而NEPA 21使用10 μg质粒转染PFFs时效率达到最高（6.09%±0.72%）。最后比较不同质粒拓扑结构的转染效率,结果显示线性化质粒的转染效率较低,仅为超螺旋质粒转染效率的34.96%—48.39%。结论 因此Nucleofector ^TM 2b转染PFFs的最佳条件为：U-023程序、12 μg超螺旋质粒;NEPA 21为：脉冲电压200 V,脉冲长度3 ms,脉冲间隔50 ms,脉冲次数3次、10 μg超螺旋质粒;ECM ^?830则在脉冲电压300 V,脉冲长度1 ms,脉冲次数3 次条件下转染12 μg超螺旋质粒可获得最佳转染效率。综合比较上述3种电转仪,26 kb大载体转染PFFs的最佳电转仪是Nucleofector ^TM 2b。     

水稻干尖线虫Ab-lea基因在高渗透压下的表达

【目的】水稻干尖线虫(Aphelenchoides besseyi)在侵染寄主以前,能够以变渗隐生状态在土壤和种子中的高渗透压环境下长期存活,使防治该线虫极为困难。通常,非生物胁迫下,胚胎发育晚期丰富蛋白(late embryogenesis abundant protein,LEA)的基因表达量会显著上调。作为逆境表达蛋白,LEA能在逆境下保护生物。为探究水稻干尖线虫LEA编码基因(Ab-lea)在高渗透压胁迫下的表达模式及功能,【方法】根据转录组数据克隆到Ab-lea。以无机溶剂饱和Mg SO4·7H2O溶液为高渗透压胁迫剂,对水稻干尖线虫进行高渗透压胁迫,240 min后该线虫进入变渗隐生。通过RT-PCR验证Ab-lea在线虫进入变渗隐生及维持变渗隐生过程中的表达特性。使用RNAi技术,探究该线虫Ab-lea沉默后,高渗透压胁迫下存活率变化。【结果】RT-PCR结果表明,Ab-lea在进入变渗隐生前期及维持变渗隐生过程中表达量上调。Ab-lea沉默后,水稻干尖线虫在进入变渗隐生后存活率显著下降。【结论】表明该基因可能参与调控水稻干尖线虫变渗隐生。本研究为进一步探究该线虫抗高渗透压胁迫的生理机制奠定基础,为防治该线虫提供新思路。