BACKGROUND： Previous studies have shown that p75 neurotrophin receptor plays an important role in peripheral nerve injury. However, the role of p75 neurotrophin receptor in the regeneration of peripheral nerves remai...BACKGROUND： Previous studies have shown that p75 neurotrophin receptor plays an important role in peripheral nerve injury. However, the role of p75 neurotrophin receptor in the regeneration of peripheral nerves remains poorly understood. OBJECTIVE： To study the effect of p75 neurotrophin receptor on facial nerve regeneration. DESIGN, TIME AND SETTING： A randomized controlled experiment was performed in the Regeneration Laboratory of Flinders University, Australia and the Biomedical Laboratory of Dentistry School, Shandong University from March 2005 to February 2006. MATERIALS： Cholera toxin B subunit, fast blue, and biotin rabbit-anti goat IgG were provided by Sigma, USA; goat-anti choleratoxin B subunit ant/body was provided by List Biologicals, USA. METHODS： In p75 neurotrophin receptor knockout and wild type 129/sv mice, the facial nerves on one side were crushed. At days 2 and 4 following injury, regenerating motor neurons in the facial nuclei were labeled by fast blue, and the regenerating axon was labeled by the anterograde tracer choleratoxin B subunit. MAIN OUTCOME MEASURES： Axonal regenerative velocity and number were detected by immunohistochemical staining of choleratoxin B subunit, growth-associated protein, protein gene product 9.5, and calcitonin-gene-related peptide; survival of motor neurons in the facial nuclei was detected by retrograde fast blue. RESULTS： Axonal growth in the facial nerve of p75 neurotrophin receptor knockout mice was significantly less than in wild type mice. At day 7 after injury, the number of regenerating motor neurons in p75 neurotrophin receptor knockout mice remained significantly less than in wild type mice （P 〈 0.05）. The number of positively stained fibers for growth-associated protein-43, protein gene product 9.5, and calcitonin-gene-related peptide in p75 neurotrophin receptor knockout mice was significantly less than in wild type mice （P 〈 0.01）. CONCLUSION： p75 neurotrophin receptor promoted axonal regeneration and enhanced the survival rate of motor neurons following facial nerve injury.展开更多
将麦芽糖结合蛋白(maltose-binding protein,MBP)、霍乱毒素B亚基(cholera toxin B subunit,CTB)以及增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因重组构建MBP-CTB-EGFP并原核表达纯化,利用HEK293T细胞模型探究MBP...将麦芽糖结合蛋白(maltose-binding protein,MBP)、霍乱毒素B亚基(cholera toxin B subunit,CTB)以及增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因重组构建MBP-CTB-EGFP并原核表达纯化,利用HEK293T细胞模型探究MBP-CTB-EGFP穿透细胞膜的能力,从而开发新型黏膜佐剂.进一步地,利用原肌球蛋白(tropomyosin,TM)重组融合蛋白MBP-CTB-TM,将其应用于Balb/c小鼠致敏实验,探究融合蛋白的致敏性及其对小鼠食物过敏相关免疫反应的影响,以达到降低过敏原剂量提高建模效率的效果.结果表明:MBP-CTB-EGFP具有作为黏膜佐剂的能力,从而促进外源蛋白进入HEK293T细胞内,且与转染方式相比,携带外源蛋白进入细胞的效率更高.另一方面,利用融合蛋白MBP-CTB-TM免疫小鼠,TM特异性IgE的OD450 nm达到0.4,而天然TM致敏组仅为0.05,此外融合蛋白致敏还导致高水平的TM特异性IgG1、IgG2a产生.本研究表明,融合蛋白MBP-CTB-TM致敏效果更好,有可能达到降低过敏原用量的效果,为后续食物过敏研究提供了有力工具.展开更多
】目的 :构建小鼠蛋白激酶 CK2 β亚基 c DNA重组表达质粒 ,以便深入进行 CK2结构与功能的研究。方法 :通过反转录 PCR从 NIH3T3小鼠成纤维细胞中获得小鼠蛋白激酶 CK2 β亚基编码区 c DNA,PCR扩增酶为高保真 DNA聚合酶。将 N de I/ H i...】目的 :构建小鼠蛋白激酶 CK2 β亚基 c DNA重组表达质粒 ,以便深入进行 CK2结构与功能的研究。方法 :通过反转录 PCR从 NIH3T3小鼠成纤维细胞中获得小鼠蛋白激酶 CK2 β亚基编码区 c DNA,PCR扩增酶为高保真 DNA聚合酶。将 N de I/ H ind 彻底双酶切 PCR产物定向连接到牛小肠碱性磷酸酶去 5′端磷酸的同样彻底 Nde I/ H ind 双酶切的表达载体 p T7- 7中 ,转化感受态大肠杆菌 DH5 α获得转化子 ,用 0 .8%琼脂糖凝胶电泳初步筛选转化子 ,将阳性克隆进行单和双酶切分析鉴定。随机挑选两个阳性克隆进行 DNA测序。结果 :转化子的阳性筛选率为 10 0 % ,限制性酶切分析结果表明插入片段和重组质粒的大小与理论推测值相符。 DNA测序结果表明 :两个重组小鼠 CK2 β亚基克隆中的插入片段序列均与两种已报道的小鼠 CK2 β亚基 c DNA编码区序列分别存在一个碱基差异 ,但我们报道的这 2个差异碱基与大鼠、兔、猪和人 CK2 β亚基 c DNA的编码区相应碱基序列一致。结论 :本实验克隆的小鼠蛋白激酶 CK2 β亚基 c展开更多
目的:探讨20S蛋白酶体β5亚单位(PSMB5)对小鼠神经干细胞(NSCs)增殖和分化能力的影响.方法:体外分离培养新生BALB/c小鼠(P0)的NSCs,应用免疫荧光染色观察PSMB5在NSCs中的表达与分布;利用real time RT-PCR方法检测PSMB5 mRNA的表达;荧光...目的:探讨20S蛋白酶体β5亚单位(PSMB5)对小鼠神经干细胞(NSCs)增殖和分化能力的影响.方法:体外分离培养新生BALB/c小鼠(P0)的NSCs,应用免疫荧光染色观察PSMB5在NSCs中的表达与分布;利用real time RT-PCR方法检测PSMB5 mRNA的表达;荧光分光光度法测定PSMB5相关的糜蛋白酶活性的变化;应用PSMB5-siRNA抑制P0 NSCs中PSMB5基因的表达;使用含有PSMB5基因的重组慢病毒((lenti-PSMB5))感染成年小鼠(P90)NSCs.利用CCK-8试验检测PSMB5对NSCs增殖能力的影响,利用BrdU和Tuj1染色观察PSMB5基因敲低和过表达对NSCs增殖及分化能力的影响.结果:PSMB5主要分布于NSCs的胞浆,P0的NSCs分化后,PSMB5 mRNA的表达下调.成年和老年小鼠NSCs中糜蛋白酶体活性较新生小鼠显著降低.PSMB5敲减后NSCs增殖能力和向神经元分化能力减弱,PSMB5过表达后NSCs增殖能力和向神经元分化能力增强.结论:PSMB5可能参与小鼠NSCs增殖及分化的调控.展开更多
基金the Natural Science Foundation of Shandong Province,No. Y2008C54
文摘BACKGROUND： Previous studies have shown that p75 neurotrophin receptor plays an important role in peripheral nerve injury. However, the role of p75 neurotrophin receptor in the regeneration of peripheral nerves remains poorly understood. OBJECTIVE： To study the effect of p75 neurotrophin receptor on facial nerve regeneration. DESIGN, TIME AND SETTING： A randomized controlled experiment was performed in the Regeneration Laboratory of Flinders University, Australia and the Biomedical Laboratory of Dentistry School, Shandong University from March 2005 to February 2006. MATERIALS： Cholera toxin B subunit, fast blue, and biotin rabbit-anti goat IgG were provided by Sigma, USA; goat-anti choleratoxin B subunit ant/body was provided by List Biologicals, USA. METHODS： In p75 neurotrophin receptor knockout and wild type 129/sv mice, the facial nerves on one side were crushed. At days 2 and 4 following injury, regenerating motor neurons in the facial nuclei were labeled by fast blue, and the regenerating axon was labeled by the anterograde tracer choleratoxin B subunit. MAIN OUTCOME MEASURES： Axonal regenerative velocity and number were detected by immunohistochemical staining of choleratoxin B subunit, growth-associated protein, protein gene product 9.5, and calcitonin-gene-related peptide; survival of motor neurons in the facial nuclei was detected by retrograde fast blue. RESULTS： Axonal growth in the facial nerve of p75 neurotrophin receptor knockout mice was significantly less than in wild type mice. At day 7 after injury, the number of regenerating motor neurons in p75 neurotrophin receptor knockout mice remained significantly less than in wild type mice （P 〈 0.05）. The number of positively stained fibers for growth-associated protein-43, protein gene product 9.5, and calcitonin-gene-related peptide in p75 neurotrophin receptor knockout mice was significantly less than in wild type mice （P 〈 0.01）. CONCLUSION： p75 neurotrophin receptor promoted axonal regeneration and enhanced the survival rate of motor neurons following facial nerve injury.
文摘将麦芽糖结合蛋白(maltose-binding protein,MBP)、霍乱毒素B亚基(cholera toxin B subunit,CTB)以及增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因重组构建MBP-CTB-EGFP并原核表达纯化,利用HEK293T细胞模型探究MBP-CTB-EGFP穿透细胞膜的能力,从而开发新型黏膜佐剂.进一步地,利用原肌球蛋白(tropomyosin,TM)重组融合蛋白MBP-CTB-TM,将其应用于Balb/c小鼠致敏实验,探究融合蛋白的致敏性及其对小鼠食物过敏相关免疫反应的影响,以达到降低过敏原剂量提高建模效率的效果.结果表明:MBP-CTB-EGFP具有作为黏膜佐剂的能力,从而促进外源蛋白进入HEK293T细胞内,且与转染方式相比,携带外源蛋白进入细胞的效率更高.另一方面,利用融合蛋白MBP-CTB-TM免疫小鼠,TM特异性IgE的OD450 nm达到0.4,而天然TM致敏组仅为0.05,此外融合蛋白致敏还导致高水平的TM特异性IgG1、IgG2a产生.本研究表明,融合蛋白MBP-CTB-TM致敏效果更好,有可能达到降低过敏原用量的效果,为后续食物过敏研究提供了有力工具.
文摘】目的 :构建小鼠蛋白激酶 CK2 β亚基 c DNA重组表达质粒 ,以便深入进行 CK2结构与功能的研究。方法 :通过反转录 PCR从 NIH3T3小鼠成纤维细胞中获得小鼠蛋白激酶 CK2 β亚基编码区 c DNA,PCR扩增酶为高保真 DNA聚合酶。将 N de I/ H ind 彻底双酶切 PCR产物定向连接到牛小肠碱性磷酸酶去 5′端磷酸的同样彻底 Nde I/ H ind 双酶切的表达载体 p T7- 7中 ,转化感受态大肠杆菌 DH5 α获得转化子 ,用 0 .8%琼脂糖凝胶电泳初步筛选转化子 ,将阳性克隆进行单和双酶切分析鉴定。随机挑选两个阳性克隆进行 DNA测序。结果 :转化子的阳性筛选率为 10 0 % ,限制性酶切分析结果表明插入片段和重组质粒的大小与理论推测值相符。 DNA测序结果表明 :两个重组小鼠 CK2 β亚基克隆中的插入片段序列均与两种已报道的小鼠 CK2 β亚基 c DNA编码区序列分别存在一个碱基差异 ,但我们报道的这 2个差异碱基与大鼠、兔、猪和人 CK2 β亚基 c DNA的编码区相应碱基序列一致。结论 :本实验克隆的小鼠蛋白激酶 CK2 β亚基 c
