The nucleotide sequence deduced from the amino acid sequence of the scorpion insectotoxin AaIT was chemically synthesized and was expressed in Escherichia coli. The authenticity of this in vitro expressed peptide was ...The nucleotide sequence deduced from the amino acid sequence of the scorpion insectotoxin AaIT was chemically synthesized and was expressed in Escherichia coli. The authenticity of this in vitro expressed peptide was confirmed by N-terminal peptide sequencing. Two groups of bioassays, artificial diet incorporation assay and contact insecticidal effect assay, were carried out separately to verify the toxicity of this recombinant toxin. At the end of a 24 h experimental period, more than 60% of the testing diamondback moth (Plutella xylostella) larvae were killed in both groups with LC50 value of 18.4 microM and 0.70 microM respectively. Cytotoxicity assay using cultured Sf9 insect cells and MCF-7 human cells demonstrated that the toxin AaIT had specific toxicity against insect cells but not human cells. Only 0.13 microM recombinant toxin was needed to kill 50% of cultured insect cells while as much as 1.3 microM toxin had absolutely no effect on human cells. Insect cells produced obvious intrusions from their plasma membrane before broken up. We infer that toxin AaIT bind to a putative sodium channel in these insect cells and open the channel persistently, which would result in Na+ influx and finally cause destruction of insect cells.展开更多
药物的靶向性以及体内长效循环特性对于药物在病灶部位的累积和疗效发挥着至关重要的作用.本研究设计合成了长循环双靶向重组蛋白药物偶联体RPDC-L(long-circulated dualtargeted recombinant protein drug conjugate),用于抗肿瘤治疗.R...药物的靶向性以及体内长效循环特性对于药物在病灶部位的累积和疗效发挥着至关重要的作用.本研究设计合成了长循环双靶向重组蛋白药物偶联体RPDC-L(long-circulated dualtargeted recombinant protein drug conjugate),用于抗肿瘤治疗.RPDC-L包含4个部分,分别是靶向人表皮生长因子受体2(HER2)的anti-HER2纳米抗体、整合素受体αvβ_(3)的多肽配体RGD、白蛋白结合域以及小分子化疗药物阿霉素.其中,anti-HER2纳米抗体和RGD多肽提供肿瘤靶向功能;白蛋白结合域通过结合血液中的白蛋白,赋予药物较长的体内循环时间;化疗药物阿霉素通过酸响应聚乙二醇-腙键连接子与重组蛋白偶联,在微酸性的肿瘤微环境中通过pH响应性释放,进行肿瘤杀伤.与非长循环双靶向重组蛋白相比,融合表达白蛋白结合单元后,长循环双靶向重组蛋白在小鼠体内的半衰期时间提升到了1.51倍.与抗肿瘤药物阿霉素偶联后,药物在肿瘤组织中的累积提升到了1.6倍.体内抗肿瘤试验表明,RPDC-L实现安全高效的抗肿瘤效果,肿瘤抑制率达到78.4%,是非长循环双靶向重组蛋白药物偶联体的1.50倍.展开更多
目的:探讨在子宫内膜癌细胞中重组人程序性细胞死亡蛋白5(recombinant human programmed cell death protein 5,rhPDCD5)对紫杉醇化疗的促进作用。方法:子宫内膜癌KLE细胞培养完成后,通过重组人rh PDCD5(20μg/ml)处理KLE细胞,再分别以0...目的:探讨在子宫内膜癌细胞中重组人程序性细胞死亡蛋白5(recombinant human programmed cell death protein 5,rhPDCD5)对紫杉醇化疗的促进作用。方法:子宫内膜癌KLE细胞培养完成后,通过重组人rh PDCD5(20μg/ml)处理KLE细胞,再分别以0、1.0、5.0、10.0、50μmol/L紫杉醇(paclitaxel,PTX)处理24 h或以10μmol/L PTX处理0、12、24、48 h,提取细胞总RNA及蛋白后,CCK法检测KLE细胞的增殖情况,流式细胞术检测KLE细胞凋亡情况,实时定量PCR检测KLE细胞中PDCD5mRNA的表达量,实时定量PCR或Western blotting测定凋亡相关基因的Bax、Bcl2、caspase-3 mRNA或蛋白水平的变化。结果:PTX对PDCD5表达的促进作用具有剂量依赖性和时间依赖性;PTX的最佳作用浓度为10μmol/L,最佳作用时间为24 h。rh PDCD5明显增强紫杉醇对KLE细胞的抑制作用。CCK实验、流式细胞术及Western blotting检测显示:PTX+rhPDCD5联合处理组KLE细胞的增殖抑制率和凋亡率均较PTX组明显增加、pro-caspase 3的表达量明显增加(均P<0.01)。促进凋亡蛋白Bax和抑制凋亡蛋白Bcl2的比值亦较明显增加(P<0.01)。结论:rhPDCD5可协同PTX抑制子宫内膜癌KLE细胞的增殖、促进细胞的凋亡,可明显增强KLE细胞对PTX的药物敏感性。展开更多
OBJECTIVE: To investigate the direct effects of the Flt3 ligand (FL) on hematopoiesis, such as the stimulation of the formation of hematopoietic colonies and the proliferation of dendritic cells, as well as the indire...OBJECTIVE: To investigate the direct effects of the Flt3 ligand (FL) on hematopoiesis, such as the stimulation of the formation of hematopoietic colonies and the proliferation of dendritic cells, as well as the indirect stimulation of hematopoiesis, especially via the proliferation of endothelial cells. METHODS: Mononuclear cells from human cord blood were plated in methylcellulose medium containing different cytokines to induce hematopoietic colony formation. Dendritic cells (DCs) were induced from the mononuclear cells with a cytokine cocktail with or without recombinant human soluble FL (rhFL; 100 ng/ml). The Flt3 receptors on the surface of a human microvascular endothelial cell line (ECV) were analyzed by flow cytometry. The proliferation of ECV stimulated by rhFL was measured with the microculture tetrazolium assay. The levels of FL, IL-6, IL-8, G-CSF and GM-CSF in the supernatant of ECV cultures were measured by enzyme linked immunoabsorbent assay (ELISA). RESULTS: rhFL stimulates colony formation from cord blood when used as a sole stimulant. FL in combination with other cytokines increased colony formation significantly. The number of DCs was approximately 2.5 times higher when rhFL was used. rhFL stimulates the proliferation of ECV on which Flt3 receptors are expressed. Furthermore, ECV secretes FL, IL-6, IL-8, G-CSF and GM-CSF, which were augmented by tumor necrosis factor-alpha and rhFL. CONCLUSIONS: rhFL enhances hematopoietic colony formation and DC proliferation from human cord blood cells. FL not only stimulates the proliferation of ECV, but is also secreted by ECV. FL may exert direct and indirect effects on hematopoiesis.展开更多
基金This work was supported by a grant from 863High Technology Program,Chinese Ministry of Sci-ence and Technology
文摘The nucleotide sequence deduced from the amino acid sequence of the scorpion insectotoxin AaIT was chemically synthesized and was expressed in Escherichia coli. The authenticity of this in vitro expressed peptide was confirmed by N-terminal peptide sequencing. Two groups of bioassays, artificial diet incorporation assay and contact insecticidal effect assay, were carried out separately to verify the toxicity of this recombinant toxin. At the end of a 24 h experimental period, more than 60% of the testing diamondback moth (Plutella xylostella) larvae were killed in both groups with LC50 value of 18.4 microM and 0.70 microM respectively. Cytotoxicity assay using cultured Sf9 insect cells and MCF-7 human cells demonstrated that the toxin AaIT had specific toxicity against insect cells but not human cells. Only 0.13 microM recombinant toxin was needed to kill 50% of cultured insect cells while as much as 1.3 microM toxin had absolutely no effect on human cells. Insect cells produced obvious intrusions from their plasma membrane before broken up. We infer that toxin AaIT bind to a putative sodium channel in these insect cells and open the channel persistently, which would result in Na+ influx and finally cause destruction of insect cells.
文摘药物的靶向性以及体内长效循环特性对于药物在病灶部位的累积和疗效发挥着至关重要的作用.本研究设计合成了长循环双靶向重组蛋白药物偶联体RPDC-L(long-circulated dualtargeted recombinant protein drug conjugate),用于抗肿瘤治疗.RPDC-L包含4个部分,分别是靶向人表皮生长因子受体2(HER2)的anti-HER2纳米抗体、整合素受体αvβ_(3)的多肽配体RGD、白蛋白结合域以及小分子化疗药物阿霉素.其中,anti-HER2纳米抗体和RGD多肽提供肿瘤靶向功能;白蛋白结合域通过结合血液中的白蛋白,赋予药物较长的体内循环时间;化疗药物阿霉素通过酸响应聚乙二醇-腙键连接子与重组蛋白偶联,在微酸性的肿瘤微环境中通过pH响应性释放,进行肿瘤杀伤.与非长循环双靶向重组蛋白相比,融合表达白蛋白结合单元后,长循环双靶向重组蛋白在小鼠体内的半衰期时间提升到了1.51倍.与抗肿瘤药物阿霉素偶联后,药物在肿瘤组织中的累积提升到了1.6倍.体内抗肿瘤试验表明,RPDC-L实现安全高效的抗肿瘤效果,肿瘤抑制率达到78.4%,是非长循环双靶向重组蛋白药物偶联体的1.50倍.
基金agrantfromtheIAEAFoundationfortheProtectionofAcuteIrradiationInjury (No CPR/9/0 2 5 )
文摘OBJECTIVE: To investigate the direct effects of the Flt3 ligand (FL) on hematopoiesis, such as the stimulation of the formation of hematopoietic colonies and the proliferation of dendritic cells, as well as the indirect stimulation of hematopoiesis, especially via the proliferation of endothelial cells. METHODS: Mononuclear cells from human cord blood were plated in methylcellulose medium containing different cytokines to induce hematopoietic colony formation. Dendritic cells (DCs) were induced from the mononuclear cells with a cytokine cocktail with or without recombinant human soluble FL (rhFL; 100 ng/ml). The Flt3 receptors on the surface of a human microvascular endothelial cell line (ECV) were analyzed by flow cytometry. The proliferation of ECV stimulated by rhFL was measured with the microculture tetrazolium assay. The levels of FL, IL-6, IL-8, G-CSF and GM-CSF in the supernatant of ECV cultures were measured by enzyme linked immunoabsorbent assay (ELISA). RESULTS: rhFL stimulates colony formation from cord blood when used as a sole stimulant. FL in combination with other cytokines increased colony formation significantly. The number of DCs was approximately 2.5 times higher when rhFL was used. rhFL stimulates the proliferation of ECV on which Flt3 receptors are expressed. Furthermore, ECV secretes FL, IL-6, IL-8, G-CSF and GM-CSF, which were augmented by tumor necrosis factor-alpha and rhFL. CONCLUSIONS: rhFL enhances hematopoietic colony formation and DC proliferation from human cord blood cells. FL not only stimulates the proliferation of ECV, but is also secreted by ECV. FL may exert direct and indirect effects on hematopoiesis.
