目的研究抗人神经纤毛蛋白-1(Neuropilin1,NRP-1)单克隆抗体对肝癌Hep G2细胞的生长抑制作用及其机制。方法小鼠腹水法制备抗NRP-1单克隆抗体(NRP-1 m Ab)并用r Protein A亲和柱纯化抗体,间接ELISA检测抗体的滴度水平。Western blot检测...目的研究抗人神经纤毛蛋白-1(Neuropilin1,NRP-1)单克隆抗体对肝癌Hep G2细胞的生长抑制作用及其机制。方法小鼠腹水法制备抗NRP-1单克隆抗体(NRP-1 m Ab)并用r Protein A亲和柱纯化抗体,间接ELISA检测抗体的滴度水平。Western blot检测NRP-1 m Ab对Hep G2细胞的特异性,细胞免疫荧光和流式细胞术检测NRP-1蛋白在肝癌细胞株Hep G2上的表达,MTT法检测NRP-1 m Ab对Hep G2的生长抑制作用,Western blot检测ERK1/2、P-ERK1/2、Akt、P-Akt蛋白的表达水平。结果 SDS-PAGE和间接ELISA检测纯化的NRP-1 m Ab纯度为95%以上,效价为1×10^(-6);Western blot检测结果显示NRP-1 m Ab可与Hep G2细胞膜上的NRP-1蛋白特异性结合。细胞免疫荧光染色结果显示NRP-1定位于Hep G2细胞膜,流式细胞术结果显示NRP-1蛋白在Hep G2细胞上表达水平较高;MTT法检测结果显示NRP-1 m Ab对Hep G2细胞有生长抑制作用。Western blot检测到在不同浓度NRP-1 m Ab作用下,Hep G2细胞裂解液P-ERK1/2、P-Akt蛋白的条带信号逐渐减弱。结论纯化的NRP-1m Ab能抑制Hep G2细胞的生长,其抑制作用是通过EGF和HGF信号通路实现的。展开更多
To explore how the intrinsic apoptosis pathway is controlled in the spontaneous fog （forebrain overgrowth） mutant mice with an Apafl splicing deficiency, we examined spleen and bone marrow cells from Apafl＋/＋ （＋...To explore how the intrinsic apoptosis pathway is controlled in the spontaneous fog （forebrain overgrowth） mutant mice with an Apafl splicing deficiency, we examined spleen and bone marrow cells from Apafl＋/＋ （＋/＋） and Apafl^fog/fog （fog/fog） mice for initiator caspase-9 activation by cellular stresses. When the mitochondrial inner membrane potential （△ψm） was disrupted by staurosporine, ＋/＋ cells but not fog/fog cells activated caspase-9 to cause apoptosis, indicating the lack of apoptosome （apoptosis protease activating factor 1 （Apaf-l）/cytochrome c/（d）ATP/procaspase-9） function in fog/fog cells. However, when a marginal （-20%） decrease in △ψm was caused by hydrogen peroxide （0.1 mM）, peroxynitrite donor 3-morpholinosydnonimine （0.1 mM） and UV-C irradiation （20 J/m^2）, both ＋/＋ and fog/fog cells triggered procaspase-9 auto-processing and its downstream cascade activation. Supporting our previous results, procaspase-9 pre-existing in the mitochondria induced its auto-processing before the cytosolic caspase activation regardless of the genotypes. Cellular ATP concentration significantly decreased under the hypoactive △ψm condition. Furthermore, we detected accumulation of citrate, a kosmotrope known to facilitate procaspase-9 dimerization, probably due to a feedback control of the Krebs cycle by the electron transfer system. Thus, mitochondrial in situ caspase-9 activation may be caused by the major metabolic reactions in response to physiological stresses, which may represent a mode of Apaf-l-independent apoptosis hypothesized from recent genetic studies.展开更多
To screen and evaluate protein biomarkers for the detection of gliomas (Astrocytoma grade Ⅰ-Ⅳ) from healthy individuals and gliomas from brain benign tumors by using surface enhanced laser desorption/ionization time...To screen and evaluate protein biomarkers for the detection of gliomas (Astrocytoma grade Ⅰ-Ⅳ) from healthy individuals and gliomas from brain benign tumors by using surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) coupled with an artificial neural network (ANN) algorithm. SELDI-TOF-MS protein fingerprinting of serum from 105 brain tumor patients and healthy individuals, included 28 patients with glioma (Astrocytoma Ⅰ-Ⅳ), 37 patients with brain benign tumor, and 40 age-matched healthy individuals. Two thirds of the total samples of every compared pair as training set were used to set up discriminating patterns, and one third of total samples of every compared pair as test set were used to cross-validate; simultaneously, discriminate-cluster analysis derived SPSS 10.0 software was used to compare Astrocytoma grade Ⅰ-Ⅱ with grade Ⅲ-Ⅳ ones. An accuracy of 95.7%, sensitivity of 88.9%, specificity of 100%, positive predictive value of 90% and negative predictive value of 100% were obtained in a blinded test set comparing gliomas patients with healthy individuals; an accuracy of 86.4%, sensitivity of 88.9%, specificity of 84.6%, positive predictive value of 90% and negative predictive value of 85.7% were obtained when patient's gliomas was compared with benign brain tumor. Total accuracy of 85.7%, accuracy of grade Ⅰ-Ⅱ Astrocytoma was 86.7%, accuracy ofⅢ-Ⅳ Astrocytoma was 84.6% were obtained when grade Ⅰ-Ⅱ Astrocytoma was compared with grade Ⅲ-Ⅳ ones (discriminant analysis). SELDI-TOF-MS combined with bioinformatics tools, could greatly facilitate the discovery of better biomarkers. The high sensitivity and specificity achieved by the use of selected biomarkers showed great potential application for the discrimination of gliomas patients from healthy individuals and glioma from brain benign tumors.展开更多
Objective: To explore the method to repair injured peripheral nerve using conduits made of human hair keratin (HHK). Methods: The tibial nerves of rabbits were transected leaving a gap 10 mm in length between the 2 se...Objective: To explore the method to repair injured peripheral nerve using conduits made of human hair keratin (HHK). Methods: The tibial nerves of rabbits were transected leaving a gap 10 mm in length between the 2 severed ends, which were either routinely sutured or bridged using HHK nerve conduits. Electro-physiological , anatomical and histological examinations were performed at different time postoperatively. Results: Electrophysiological study showed more obvious improvement in the neural function recovery in rabbits with HHK conduits bridging as compared with that in rabbits with routine suture. In the former group, HHK conduits were gradually degraded and absorbed with large amount of myelinated nerve fibers and Schwann cells regenerated around HHK conduits. In the latter group, however, the nerve tissues around the suture were degenerated and replaced by connective tissues. Conclusion: HHK may induce the regeneration of the nerve fibers and provides an ideal approach to repair nerve damages.展开更多
The newly emerged mosquito-borne Zika virus(ZIKV) strains pose a global challenge owing to its ability to cause microcephaly and neurological disorders. Several ZIKV vaccine candidates have been proposed, including in...The newly emerged mosquito-borne Zika virus(ZIKV) strains pose a global challenge owing to its ability to cause microcephaly and neurological disorders. Several ZIKV vaccine candidates have been proposed, including inactivated and live attenuated virus vaccines, vector-based vaccines, DNA and RNA vaccines. These have been shown to be efficacious in preclinical studies in mice and nonhuman primates, but their use will potentially be a threat to immunocompromised individuals and pregnant women. Virus-like particles(VLPs) are empty particles composed merely of viral proteins, which can serve as a safe and valuable tool for clinical prevention and treatment strategies. In this study, we used a new strategy to produce ZIKV VLPs based on the baculovirus expression system and demonstrated the feasibility of their use as a vaccine candidate. The pre-membrane(prM) and envelope(E) proteins were co-expressed in insect cells and selfassembled into particles similar to ZIKV. We found that the ZIKV VLPs could be quickly and easily prepared in large quantities using this system. The VLPs were shown to have good immunogenicity in immunized mice, as they stimulated high levels of virus neutralizing antibody titers, ZIKV-specific IgG titers and potent memory T cell responses. Thus, the baculovirus-based ZIKV VLP vaccine is a safe, effective and economical vaccine candidate for use against ZIKV.展开更多
文摘目的研究抗人神经纤毛蛋白-1(Neuropilin1,NRP-1)单克隆抗体对肝癌Hep G2细胞的生长抑制作用及其机制。方法小鼠腹水法制备抗NRP-1单克隆抗体(NRP-1 m Ab)并用r Protein A亲和柱纯化抗体,间接ELISA检测抗体的滴度水平。Western blot检测NRP-1 m Ab对Hep G2细胞的特异性,细胞免疫荧光和流式细胞术检测NRP-1蛋白在肝癌细胞株Hep G2上的表达,MTT法检测NRP-1 m Ab对Hep G2的生长抑制作用,Western blot检测ERK1/2、P-ERK1/2、Akt、P-Akt蛋白的表达水平。结果 SDS-PAGE和间接ELISA检测纯化的NRP-1 m Ab纯度为95%以上,效价为1×10^(-6);Western blot检测结果显示NRP-1 m Ab可与Hep G2细胞膜上的NRP-1蛋白特异性结合。细胞免疫荧光染色结果显示NRP-1定位于Hep G2细胞膜,流式细胞术结果显示NRP-1蛋白在Hep G2细胞上表达水平较高;MTT法检测结果显示NRP-1 m Ab对Hep G2细胞有生长抑制作用。Western blot检测到在不同浓度NRP-1 m Ab作用下,Hep G2细胞裂解液P-ERK1/2、P-Akt蛋白的条带信号逐渐减弱。结论纯化的NRP-1m Ab能抑制Hep G2细胞的生长,其抑制作用是通过EGF和HGF信号通路实现的。
文摘To explore how the intrinsic apoptosis pathway is controlled in the spontaneous fog （forebrain overgrowth） mutant mice with an Apafl splicing deficiency, we examined spleen and bone marrow cells from Apafl＋/＋ （＋/＋） and Apafl^fog/fog （fog/fog） mice for initiator caspase-9 activation by cellular stresses. When the mitochondrial inner membrane potential （△ψm） was disrupted by staurosporine, ＋/＋ cells but not fog/fog cells activated caspase-9 to cause apoptosis, indicating the lack of apoptosome （apoptosis protease activating factor 1 （Apaf-l）/cytochrome c/（d）ATP/procaspase-9） function in fog/fog cells. However, when a marginal （-20%） decrease in △ψm was caused by hydrogen peroxide （0.1 mM）, peroxynitrite donor 3-morpholinosydnonimine （0.1 mM） and UV-C irradiation （20 J/m^2）, both ＋/＋ and fog/fog cells triggered procaspase-9 auto-processing and its downstream cascade activation. Supporting our previous results, procaspase-9 pre-existing in the mitochondria induced its auto-processing before the cytosolic caspase activation regardless of the genotypes. Cellular ATP concentration significantly decreased under the hypoactive △ψm condition. Furthermore, we detected accumulation of citrate, a kosmotrope known to facilitate procaspase-9 dimerization, probably due to a feedback control of the Krebs cycle by the electron transfer system. Thus, mitochondrial in situ caspase-9 activation may be caused by the major metabolic reactions in response to physiological stresses, which may represent a mode of Apaf-l-independent apoptosis hypothesized from recent genetic studies.
文摘To screen and evaluate protein biomarkers for the detection of gliomas (Astrocytoma grade Ⅰ-Ⅳ) from healthy individuals and gliomas from brain benign tumors by using surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) coupled with an artificial neural network (ANN) algorithm. SELDI-TOF-MS protein fingerprinting of serum from 105 brain tumor patients and healthy individuals, included 28 patients with glioma (Astrocytoma Ⅰ-Ⅳ), 37 patients with brain benign tumor, and 40 age-matched healthy individuals. Two thirds of the total samples of every compared pair as training set were used to set up discriminating patterns, and one third of total samples of every compared pair as test set were used to cross-validate; simultaneously, discriminate-cluster analysis derived SPSS 10.0 software was used to compare Astrocytoma grade Ⅰ-Ⅱ with grade Ⅲ-Ⅳ ones. An accuracy of 95.7%, sensitivity of 88.9%, specificity of 100%, positive predictive value of 90% and negative predictive value of 100% were obtained in a blinded test set comparing gliomas patients with healthy individuals; an accuracy of 86.4%, sensitivity of 88.9%, specificity of 84.6%, positive predictive value of 90% and negative predictive value of 85.7% were obtained when patient's gliomas was compared with benign brain tumor. Total accuracy of 85.7%, accuracy of grade Ⅰ-Ⅱ Astrocytoma was 86.7%, accuracy ofⅢ-Ⅳ Astrocytoma was 84.6% were obtained when grade Ⅰ-Ⅱ Astrocytoma was compared with grade Ⅲ-Ⅳ ones (discriminant analysis). SELDI-TOF-MS combined with bioinformatics tools, could greatly facilitate the discovery of better biomarkers. The high sensitivity and specificity achieved by the use of selected biomarkers showed great potential application for the discrimination of gliomas patients from healthy individuals and glioma from brain benign tumors.
基金Supported by National 863 Project of China (No. 102090503)
文摘Objective: To explore the method to repair injured peripheral nerve using conduits made of human hair keratin (HHK). Methods: The tibial nerves of rabbits were transected leaving a gap 10 mm in length between the 2 severed ends, which were either routinely sutured or bridged using HHK nerve conduits. Electro-physiological , anatomical and histological examinations were performed at different time postoperatively. Results: Electrophysiological study showed more obvious improvement in the neural function recovery in rabbits with HHK conduits bridging as compared with that in rabbits with routine suture. In the former group, HHK conduits were gradually degraded and absorbed with large amount of myelinated nerve fibers and Schwann cells regenerated around HHK conduits. In the latter group, however, the nerve tissues around the suture were degenerated and replaced by connective tissues. Conclusion: HHK may induce the regeneration of the nerve fibers and provides an ideal approach to repair nerve damages.
基金supported by the Science and Technology Basic Work Program (2013FY113500) from the Ministry of Science and Technology of Chinathe strategic priority research program of the Chinese Academy of Sciences (ZDRW-ZS2016-4)
文摘The newly emerged mosquito-borne Zika virus(ZIKV) strains pose a global challenge owing to its ability to cause microcephaly and neurological disorders. Several ZIKV vaccine candidates have been proposed, including inactivated and live attenuated virus vaccines, vector-based vaccines, DNA and RNA vaccines. These have been shown to be efficacious in preclinical studies in mice and nonhuman primates, but their use will potentially be a threat to immunocompromised individuals and pregnant women. Virus-like particles(VLPs) are empty particles composed merely of viral proteins, which can serve as a safe and valuable tool for clinical prevention and treatment strategies. In this study, we used a new strategy to produce ZIKV VLPs based on the baculovirus expression system and demonstrated the feasibility of their use as a vaccine candidate. The pre-membrane(prM) and envelope(E) proteins were co-expressed in insect cells and selfassembled into particles similar to ZIKV. We found that the ZIKV VLPs could be quickly and easily prepared in large quantities using this system. The VLPs were shown to have good immunogenicity in immunized mice, as they stimulated high levels of virus neutralizing antibody titers, ZIKV-specific IgG titers and potent memory T cell responses. Thus, the baculovirus-based ZIKV VLP vaccine is a safe, effective and economical vaccine candidate for use against ZIKV.
