Sucrose transporter is a functional protein family related to the sucrose transportation and signal transduction in plants. Using DNA recombination technology, it was prepared the specific antibody of rice sucrose tra...Sucrose transporter is a functional protein family related to the sucrose transportation and signal transduction in plants. Using DNA recombination technology, it was prepared the specific antibody of rice sucrose transporter (OsSUT2) based on its typical sequence of hydrophilic non-transmembrane to provide efficient molecular tool in the research on the physiological function of this protein. The cDNA sequences of the hydrophilic non-transmembrane region of central loop (CL) and TACL (Transmembrane and Apoplast regions and CL) of OsSUT2 were sub-cloned into the fusion expression vector pQE40 having dihydrofolate reductase (DHFR) to construct recombinant plasmid pQE40-OsSUT2-CL and pQE40-OsSUT2-TACL, respectively. The pQE40-OsSUT2-CL can be highly expressed in E. coli M15, especially in E. coli BL21 plysS. However, the pQE40-OsSUT2-TACL cannot be expressed in E. coli M15. DHFR-OsSUT2-CL purified with SDS-PAGE gel was injected into rabbit to obtain the produce antiserum. The specificity of obtained antibody was tested by Western blotting. It was indicated that the raised antibody can specifically identify OsSUT2, and it can be efficiently used in the research on the physiological function of OsSUT2.展开更多
目的:研究脑缺血后铁跨膜转运蛋白:转铁蛋白受体(Transferrin Receptor,TFR)、膜铁转运蛋白(Ferroportin,Fpn)、二价金属离子转运体(Divalent Metal Ion Transporter 1,DMT1)、人类猫白血病C亚类病毒受体(Feline Leukemia Virus Subgrou...目的:研究脑缺血后铁跨膜转运蛋白:转铁蛋白受体(Transferrin Receptor,TFR)、膜铁转运蛋白(Ferroportin,Fpn)、二价金属离子转运体(Divalent Metal Ion Transporter 1,DMT1)、人类猫白血病C亚类病毒受体(Feline Leukemia Virus Subgroup C Receptor,FLVCR)、胸腺癌抵抗蛋白(Breast Cancer Resistance Protein,BCRP)在益气活血法脑泰方(黄芪、川芎、地龙、僵蚕)干预后的表达变化。方法:随机将SD大鼠分为假手术组、模型组、脑泰方低、中、高剂量组(3、9、27 g/kg)。各组大鼠预处理灌胃给药连续3 d,大脑中动脉栓塞法(Middle Cerebral Artery Occlusion,MCAO)模型制备术后连续灌胃给药3d,1次/d。术后3 d取材,免疫组织化学及Western-blot检测TFR、Fpn、DMT1、FLVCR、BCRP的表达。结果:脑泰方高剂量组缺血海马CA2区TFR表达明显降低(P<0.05),DMT1量的表达减少(P<0.05),Fpn表达明显增高(P<0.05)。脑泰方各剂量组FLVCR表达明显增高(P<0.05),各脑泰方治疗组BCRP表达与模型组无差异。结论:脑泰方通过减少TFR、DMT1的表达,抑制铁的细胞内转运,增加Fpn、FLVCR的表达,促进胞内铁的外排,调节铁代谢,起到神经元的保护作用。展开更多
目的探讨半胱氨酸蛋白酶(caspase)在ω-3脂肪酸联合细胞外源性跨膜型TNF-α诱导MCF-7细胞凋亡过程中的作用。方法带有脂肪酸受体反应元件的跨膜型TNF-α表达载体转染MCF-7细胞,经ω-3脂肪酸处理后,MTT比色法检测细胞增殖能力,DNA梯形带...目的探讨半胱氨酸蛋白酶(caspase)在ω-3脂肪酸联合细胞外源性跨膜型TNF-α诱导MCF-7细胞凋亡过程中的作用。方法带有脂肪酸受体反应元件的跨膜型TNF-α表达载体转染MCF-7细胞,经ω-3脂肪酸处理后,MTT比色法检测细胞增殖能力,DNA梯形带分析ω-3脂肪酸对细胞凋亡的影响,RT-PCR和W estern b lot方法检测细胞caspase-1、8和9基因的表达变化,应用caspase特异性底物检测肿瘤细胞caspase-8活性变化,观察caspase特异性抑制剂Ac-IETD-CHO对ω-3脂肪酸联合细胞外源性跨膜型TNF-α抑制肿瘤细胞增殖、诱导凋亡的拮抗作用。结果与MCF-7细胞相比,MCF-7转染细胞经ω-3脂肪酸处理后,细胞增殖能力减弱,检测到明显的DNA梯形带,caspase-1、8、9基因表达增加,caspase-8活性增高,caspase抑制剂可以部分拮抗ω-3脂肪酸联合跨膜型TNF-α抑制肿瘤细胞增殖和诱导凋亡的发生。结论ω-3脂肪酸联合跨膜型TNF-α可以更有效地诱导MCF-7细胞凋亡,而上调caspase的表达和/或活性可能在此过程中发挥重要作用。展开更多
纳米载体体内吸收过程复杂,受生物吸收屏障影响,纳米制剂在促进活性分子吸收利用度方面受到质疑。本实验采用大豆分离蛋白(soy protein isolate,SPI)制备包埋β-胡萝卜素的植物基纳米颗粒(β-carotene loaded soy protein isolate nanop...纳米载体体内吸收过程复杂,受生物吸收屏障影响,纳米制剂在促进活性分子吸收利用度方面受到质疑。本实验采用大豆分离蛋白(soy protein isolate,SPI)制备包埋β-胡萝卜素的植物基纳米颗粒(β-carotene loaded soy protein isolate nanoparticles,BC-SPIs),并通过体外模拟消化模型研究BC-SPIs在消化过程中的结构特性变化。同时,通过Caco2细胞转运模型考察消化条件对消化后BC-SPIs跨膜转运的影响机制。此外,利用含黏液层的Caco2-HT29共培养模型考察消化前后BC-SPIs的黏液层渗透性。研究发现,在消化前,BC-SPIs可以直接通过网格蛋白和小窝蛋白依赖的内吞作用被Caco2单层细胞吸收;而在经过体外模拟消化后,BC-SPIs粒径增大,可以通过网格蛋白依赖的内吞作用、小窝蛋白依赖的内吞作用以及巨胞饮3种内吞形式被细胞直接吸收。消化后的BC-SPIs带有更高的负电荷,跨越黏液层屏障的能力提高了0.48倍,同时β-胡萝卜素的跨膜转运量提高了0.56倍。本研究明确了BC-SPIs在消化前和消化后的不同吸收途径,揭示了BC-SPIs在模拟消化条件下与胆盐互作及尺寸增大对其细胞转运吸收效率的促进作用。这些发现可为进一步提高纳米载体在生物利用度方面的应用潜力提供理论参考,有助于推动纳米技术在药物、保健品等领域的发展。展开更多
文摘Sucrose transporter is a functional protein family related to the sucrose transportation and signal transduction in plants. Using DNA recombination technology, it was prepared the specific antibody of rice sucrose transporter (OsSUT2) based on its typical sequence of hydrophilic non-transmembrane to provide efficient molecular tool in the research on the physiological function of this protein. The cDNA sequences of the hydrophilic non-transmembrane region of central loop (CL) and TACL (Transmembrane and Apoplast regions and CL) of OsSUT2 were sub-cloned into the fusion expression vector pQE40 having dihydrofolate reductase (DHFR) to construct recombinant plasmid pQE40-OsSUT2-CL and pQE40-OsSUT2-TACL, respectively. The pQE40-OsSUT2-CL can be highly expressed in E. coli M15, especially in E. coli BL21 plysS. However, the pQE40-OsSUT2-TACL cannot be expressed in E. coli M15. DHFR-OsSUT2-CL purified with SDS-PAGE gel was injected into rabbit to obtain the produce antiserum. The specificity of obtained antibody was tested by Western blotting. It was indicated that the raised antibody can specifically identify OsSUT2, and it can be efficiently used in the research on the physiological function of OsSUT2.
文摘目的:研究脑缺血后铁跨膜转运蛋白:转铁蛋白受体(Transferrin Receptor,TFR)、膜铁转运蛋白(Ferroportin,Fpn)、二价金属离子转运体(Divalent Metal Ion Transporter 1,DMT1)、人类猫白血病C亚类病毒受体(Feline Leukemia Virus Subgroup C Receptor,FLVCR)、胸腺癌抵抗蛋白(Breast Cancer Resistance Protein,BCRP)在益气活血法脑泰方(黄芪、川芎、地龙、僵蚕)干预后的表达变化。方法:随机将SD大鼠分为假手术组、模型组、脑泰方低、中、高剂量组(3、9、27 g/kg)。各组大鼠预处理灌胃给药连续3 d,大脑中动脉栓塞法(Middle Cerebral Artery Occlusion,MCAO)模型制备术后连续灌胃给药3d,1次/d。术后3 d取材,免疫组织化学及Western-blot检测TFR、Fpn、DMT1、FLVCR、BCRP的表达。结果:脑泰方高剂量组缺血海马CA2区TFR表达明显降低(P<0.05),DMT1量的表达减少(P<0.05),Fpn表达明显增高(P<0.05)。脑泰方各剂量组FLVCR表达明显增高(P<0.05),各脑泰方治疗组BCRP表达与模型组无差异。结论:脑泰方通过减少TFR、DMT1的表达,抑制铁的细胞内转运,增加Fpn、FLVCR的表达,促进胞内铁的外排,调节铁代谢,起到神经元的保护作用。
文摘目的探讨半胱氨酸蛋白酶(caspase)在ω-3脂肪酸联合细胞外源性跨膜型TNF-α诱导MCF-7细胞凋亡过程中的作用。方法带有脂肪酸受体反应元件的跨膜型TNF-α表达载体转染MCF-7细胞,经ω-3脂肪酸处理后,MTT比色法检测细胞增殖能力,DNA梯形带分析ω-3脂肪酸对细胞凋亡的影响,RT-PCR和W estern b lot方法检测细胞caspase-1、8和9基因的表达变化,应用caspase特异性底物检测肿瘤细胞caspase-8活性变化,观察caspase特异性抑制剂Ac-IETD-CHO对ω-3脂肪酸联合细胞外源性跨膜型TNF-α抑制肿瘤细胞增殖、诱导凋亡的拮抗作用。结果与MCF-7细胞相比,MCF-7转染细胞经ω-3脂肪酸处理后,细胞增殖能力减弱,检测到明显的DNA梯形带,caspase-1、8、9基因表达增加,caspase-8活性增高,caspase抑制剂可以部分拮抗ω-3脂肪酸联合跨膜型TNF-α抑制肿瘤细胞增殖和诱导凋亡的发生。结论ω-3脂肪酸联合跨膜型TNF-α可以更有效地诱导MCF-7细胞凋亡,而上调caspase的表达和/或活性可能在此过程中发挥重要作用。
文摘纳米载体体内吸收过程复杂,受生物吸收屏障影响,纳米制剂在促进活性分子吸收利用度方面受到质疑。本实验采用大豆分离蛋白(soy protein isolate,SPI)制备包埋β-胡萝卜素的植物基纳米颗粒(β-carotene loaded soy protein isolate nanoparticles,BC-SPIs),并通过体外模拟消化模型研究BC-SPIs在消化过程中的结构特性变化。同时,通过Caco2细胞转运模型考察消化条件对消化后BC-SPIs跨膜转运的影响机制。此外,利用含黏液层的Caco2-HT29共培养模型考察消化前后BC-SPIs的黏液层渗透性。研究发现,在消化前,BC-SPIs可以直接通过网格蛋白和小窝蛋白依赖的内吞作用被Caco2单层细胞吸收;而在经过体外模拟消化后,BC-SPIs粒径增大,可以通过网格蛋白依赖的内吞作用、小窝蛋白依赖的内吞作用以及巨胞饮3种内吞形式被细胞直接吸收。消化后的BC-SPIs带有更高的负电荷,跨越黏液层屏障的能力提高了0.48倍,同时β-胡萝卜素的跨膜转运量提高了0.56倍。本研究明确了BC-SPIs在消化前和消化后的不同吸收途径,揭示了BC-SPIs在模拟消化条件下与胆盐互作及尺寸增大对其细胞转运吸收效率的促进作用。这些发现可为进一步提高纳米载体在生物利用度方面的应用潜力提供理论参考,有助于推动纳米技术在药物、保健品等领域的发展。
