Objective:To study the various processes involved in transcellular transport(TT) of huperzine A alone or in combination with ginkgolide B in Caco-2 and Madin-Darby canine renal(MDCK)cell monolayer.Methods:The transepi...Objective:To study the various processes involved in transcellular transport(TT) of huperzine A alone or in combination with ginkgolide B in Caco-2 and Madin-Darby canine renal(MDCK)cell monolayer.Methods:The transepithelial passage was assayed in the apical-to-basolateral(AP to BL) direction and opposite direction(BL to AP) in both cell lines.The determination of huperzine A and ginkgolide B were performed by high performance liquid chromatography(HPLC).The passage rates of huperzine A and ginkgolide B were calculated.Bi-directional TT(absorption and secretion) were taken in huperzine A and ginkgolide B in Caco-2 and MDCK cell monolayer.Results:TT absorption and secretion kinetics of huperzine A and ginkgolide B across two cells existed at the same time.The passage rates of huperzine A were increased significantly with adding different concentrations of ginkgolide B.Conclusions:The compound preparations of HA in combination with CB for dementia caused by cerebral ischemic have synergistic effects on the pharmacodynamics,and improve the bioavailability through BBB.展开更多
旨在研究犬腺病毒-2型(canine adenovirus type 2,CAV-2)作为溶瘤病毒的潜力并进一步探索精子黏附因子(SPAM1)协同CAV-2重塑肿瘤微环境在肿瘤免疫治疗中的应用价值。本研究以P15A-CAV-2反向遗传操作平台为基础,使用RED/ET同源重组系统...旨在研究犬腺病毒-2型(canine adenovirus type 2,CAV-2)作为溶瘤病毒的潜力并进一步探索精子黏附因子(SPAM1)协同CAV-2重塑肿瘤微环境在肿瘤免疫治疗中的应用价值。本研究以P15A-CAV-2反向遗传操作平台为基础,使用RED/ET同源重组系统构建携带SPAM1外源基因的中间载体,使用中间载体将CAV-2骨架载体E3区域缺失并替换SPAM1外源基因表达盒。再利用合成引物敲除kmccdB反向筛选表达盒构建P15A-CAV-2-mCMV-SPAM1-SV40 polyA感染性克隆质粒并在MDCK-E1A细胞系中拯救重组溶瘤病毒,对重组病毒体外溶瘤效应进行验证。根据测序与酶切验证结果证明,本研究成功构建并拯救出稳定表达外源基因SPAM1的重组溶瘤病毒1株;IFA试验证明,重组毒株能够高水平稳定表达外源基因且外源蛋白具有可弥散分布于细胞间的特点。缺失E3区域表达外源基因SPAM1的重组溶瘤病毒通过光镜观察和CCK8检测发现具有对犬癌细胞系A72强烈的杀伤效应。综上,本研究成功构建1株具有良好溶瘤效应的重组CAV-2,为后续应用于宠物肿瘤治疗奠定了基础。展开更多
[ Objective] To clone and express canine IL-2 gene and thus to provide theoretical support for the development of novel immune enhancers and genetic engineering vaccines. [ Method] Leukocytes separated from canine who...[ Objective] To clone and express canine IL-2 gene and thus to provide theoretical support for the development of novel immune enhancers and genetic engineering vaccines. [ Method] Leukocytes separated from canine whole blood were stimulated by concanavalin for 20 h, and then total RNA was extracted. According to the sequence of canine IL-2 gene published in the GenBank, a pair of primers was designed. After PCR am- plificetion, the target fragment was cloned into prokaryotic expression vector pET-28a. The recombinants were transformed into the host bacteria BL21. After IPTG induction, the expression products were analyzed by SDS-PAGE. [ Result] A 500 bp band with the expected size appeared in the RT-PCR products. After the pMD18-T-IL2 was identified by double digestion, an approximately 500 bp fragment was produced, which indicated successful cloning of the gene. After the pET-28a-lL2 was identified by restriction enzyme digestion and PCR, a 500 bp fragment was produced, which indicated successful construction of the expression vector. As revealed by the SDS-PAGE analysis, a protein band with molecular weight of about 20 kDa appeared. [ Conclusion] The canine IL-2 gene was cloned and expressed.展开更多
In this study, the full-length VP2 gene of canine parvovirus type 2 (CPV-2) was cloned into the pBacSC vector which possesses baculovirus transmembrane domain (gp64 TM) gene, baculovirus cytoplasmic domain (gp64 CTD) ...In this study, the full-length VP2 gene of canine parvovirus type 2 (CPV-2) was cloned into the pBacSC vector which possesses baculovirus transmembrane domain (gp64 TM) gene, baculovirus cytoplasmic domain (gp64 CTD) gene, and green florescence protein (GFP) gene. Baculovirus gp64 TM and gp64 CTD in the pBacSC vector were designed to display heterologous proteins on the baculovirus envelope. After cloning the VP2 gene of CPV-2 into pBacSC vector, the recombinant plasmid pBacSC-VP2 was transformed into E. coli DH10Bac competent cells to form recombinant bacmid DNA. One recombinant baculovirus BacSC-VP2 that expresses the VP2 protein of CPV-2 was obtained. Confocal microscopy and immunogold electron microscopy were used to verify whether VP2 expressing on baculovirus envelope or cell membrane. Immunization of BALB/c mice with recombinant baculovirus BacSC-VP2, demonstrated that serum from the BacSC-VP2 treated models had higher levels of virus neutralization titers than the control groups. The results show that the recombinant baculovirus BacSC-VP2 can induce a strong immune response in a mouse model, suggesting that the pseudotyped baculovirus BacSC-VP2 can serve as a potential vaccine against CPV infections.展开更多
Background: Experimental studies have shown that cyclo-oxygenase-2 (Cox2) is related to the development and progression of tumors, since this enzyme is induced and expressed by cells such as macrophages, osteoblasts, ...Background: Experimental studies have shown that cyclo-oxygenase-2 (Cox2) is related to the development and progression of tumors, since this enzyme is induced and expressed by cells such as macrophages, osteoblasts, “activated” endothelial cells, and tumor cells. The activity in tumors includes proliferation, cell transformation, tumor growth, invasion and metastasis and may play an important role in carcinogenesis of the canine osteosarcoma, since it has high expression in tissue fragments. The combination of selective Cox2 inhibitors and other treatment modalities is the basis for a new anti-cancer therapy strategy. This in vitro study exposed primary cells of five different canine osteosarcoma cultures to selective Cox2 inhibitor at increasing concentrations and times. Results: For Cox2 negative cultures, despite the absence of differences, greater sensitivity of cells to treatment was observed. For Cox2 positive cultures, a higher number of necrotic cells were observed (P ≤ 0.05), when compared with negative cultures. For exposure times with Celecoxib doses, no difference (P > 0.05) was found between the three times analyzed for living, apoptotic and apoptotic/necrotic cells. There are similarities in the values of 24 h and 48 h, with slight reduction of living cells, increasing those undergoing apoptosis and apoptosis/necrosis. There was significance for necrosis (P ≤ 0.05). In 72 hours, a significant difference was observed between the other two previous values (P ≤ 0.05). It was found for the group of 100 μML-1, that there was a numerically greater signaling for apoptosis and lower (P = 0.08) for necrosis, and this point was the onset of the pharmacodynamic phenomenon, with drop in the values for living cells and increased number of necrotic cells, with a tendency (P = 0.08) for reducing the percentage of necrotic cells for the group of 100 μML-1 when compared to that of 10 μML-1. Conclusions: For Cox2 positive and negative cultures, there was difference for necrotic cells and there was no difference between Cox2 positive and Cox2 negative groups in relation to the percentage of living cells and apoptotic and apoptotic/necrotic cells. At time of 72 hours, higher percentage of living cells, lower percentage of apoptotic cells and increased percentage of necrotic cells in relation to groups of 24 and 48 hours was observed. A tendency for reducing the percentage of necrotic cells for the group of 100 μML-1 when compared to that of the group of 10 μML-1 was observed.展开更多
Canine parvovirus(CPV)is a high morbidity and lethality virus which causes severe enteric disease in dogs.In an attempt to gain canine-origin neutralizing antibodies against CPV,single chain variable fragment(scFv)bac...Canine parvovirus(CPV)is a high morbidity and lethality virus which causes severe enteric disease in dogs.In an attempt to gain canine-origin neutralizing antibodies against CPV,single chain variable fragment(scFv)bacteria display libraries against the protective antigen VP2 were constructed and screened.VP2 specific scFvs were selected following three rounds of screening procedures.Selected scFvs were characterized by FCM and ELISA.Seven scFvs showed high affinity and specific binding to CPV.Moreover,the neutralizing activity of the antibody was preliminarily identified by CPV(100 TCID_(50))in vitro and scFv-2 showed the neutralizing ability with a titer of 32768.This study generated the first canine-origin neutralizing scFv against CPV.The neutralizing scFv would be constructed into full-length antibody in the future.This study laid the foundation for the generation of an effective therapeutic reagent with long half-life and no immunological rejection for the prevention and treatment of CPV infection.展开更多
Objectives To evaluate the changes in atrial effective refractory period (AERP) proprieties and in ionic currents in PVs myocytes from dogs subjected to rapid atrial pacing in PVs and right atrial appendage (RAA) ...Objectives To evaluate the changes in atrial effective refractory period (AERP) proprieties and in ionic currents in PVs myocytes from dogs subjected to rapid atrial pacing in PVs and right atrial appendage (RAA) and to relate these changes to the ability to induce AF. Methods Twelve mongrel dogs in normal sinus rhythm were paced from the superior left PVs or RAA at 500 bpm for 4 hours. Electrophysiologic studies conducted to determine changes in AERP, dispersion and rhythm. Ionic currents were studies with the patch clamp technique in single PVs myocytes in sham operated dogs and compared with those from PVs pacing and RAA pacing groups. Results The presence of rapid atrial pacing was associated with a marked shortening in AERP in both PVs and RAA pacing group with a marked increase of AERP dispersion in PVs pacing. Both L-type calcium current (Ica L ) and the transient outward current ( Ito ) were reduced in both groups with an increased significance in PVs pacing group. The density of ICa-L was decreased significantly from ( - 6. 03 ± 0. 63 ) pA./pF in the control group to ( -3.21 ±0. 34) pA/pF in PVs pacing group and ( - 4. 75 ± 0. 41 ) pA./pF in RAA pacing group ( n = 6, P 〈 0. 05 ) while the density of Ito was decreased significantly from (8.45 ± 0. 71 ) pA./pF in the control group to ( 5.21 ± 0. 763 ) pA./pF in PVs pacing group and (6. 84 ±0. 69 ) pA./pF in RAA pacing group ( n = 6, P 〈 0. 05 ). Conclusions Our findings provide likely ionic mechanisms of shortened repolarization in induced atrial tachycardia with a decrease in Ica L and /tocurrent densities which is the likely mechanism for a decrease in Action potential duration (APD) rate adaptation in the canine rapid pacing model more pronounced in PVs pacing group underlying the crucial role of PVs in initiating AF.展开更多
基金supported by Clinical Special Funds of China University Medical Journals(11321611)
文摘Objective:To study the various processes involved in transcellular transport(TT) of huperzine A alone or in combination with ginkgolide B in Caco-2 and Madin-Darby canine renal(MDCK)cell monolayer.Methods:The transepithelial passage was assayed in the apical-to-basolateral(AP to BL) direction and opposite direction(BL to AP) in both cell lines.The determination of huperzine A and ginkgolide B were performed by high performance liquid chromatography(HPLC).The passage rates of huperzine A and ginkgolide B were calculated.Bi-directional TT(absorption and secretion) were taken in huperzine A and ginkgolide B in Caco-2 and MDCK cell monolayer.Results:TT absorption and secretion kinetics of huperzine A and ginkgolide B across two cells existed at the same time.The passage rates of huperzine A were increased significantly with adding different concentrations of ginkgolide B.Conclusions:The compound preparations of HA in combination with CB for dementia caused by cerebral ischemic have synergistic effects on the pharmacodynamics,and improve the bioavailability through BBB.
文摘旨在研究犬腺病毒-2型(canine adenovirus type 2,CAV-2)作为溶瘤病毒的潜力并进一步探索精子黏附因子(SPAM1)协同CAV-2重塑肿瘤微环境在肿瘤免疫治疗中的应用价值。本研究以P15A-CAV-2反向遗传操作平台为基础,使用RED/ET同源重组系统构建携带SPAM1外源基因的中间载体,使用中间载体将CAV-2骨架载体E3区域缺失并替换SPAM1外源基因表达盒。再利用合成引物敲除kmccdB反向筛选表达盒构建P15A-CAV-2-mCMV-SPAM1-SV40 polyA感染性克隆质粒并在MDCK-E1A细胞系中拯救重组溶瘤病毒,对重组病毒体外溶瘤效应进行验证。根据测序与酶切验证结果证明,本研究成功构建并拯救出稳定表达外源基因SPAM1的重组溶瘤病毒1株;IFA试验证明,重组毒株能够高水平稳定表达外源基因且外源蛋白具有可弥散分布于细胞间的特点。缺失E3区域表达外源基因SPAM1的重组溶瘤病毒通过光镜观察和CCK8检测发现具有对犬癌细胞系A72强烈的杀伤效应。综上,本研究成功构建1株具有良好溶瘤效应的重组CAV-2,为后续应用于宠物肿瘤治疗奠定了基础。
基金funded by the Project of Liaoning Education Department (L2010263)
文摘[ Objective] To clone and express canine IL-2 gene and thus to provide theoretical support for the development of novel immune enhancers and genetic engineering vaccines. [ Method] Leukocytes separated from canine whole blood were stimulated by concanavalin for 20 h, and then total RNA was extracted. According to the sequence of canine IL-2 gene published in the GenBank, a pair of primers was designed. After PCR am- plificetion, the target fragment was cloned into prokaryotic expression vector pET-28a. The recombinants were transformed into the host bacteria BL21. After IPTG induction, the expression products were analyzed by SDS-PAGE. [ Result] A 500 bp band with the expected size appeared in the RT-PCR products. After the pMD18-T-IL2 was identified by double digestion, an approximately 500 bp fragment was produced, which indicated successful cloning of the gene. After the pET-28a-lL2 was identified by restriction enzyme digestion and PCR, a 500 bp fragment was produced, which indicated successful construction of the expression vector. As revealed by the SDS-PAGE analysis, a protein band with molecular weight of about 20 kDa appeared. [ Conclusion] The canine IL-2 gene was cloned and expressed.
文摘In this study, the full-length VP2 gene of canine parvovirus type 2 (CPV-2) was cloned into the pBacSC vector which possesses baculovirus transmembrane domain (gp64 TM) gene, baculovirus cytoplasmic domain (gp64 CTD) gene, and green florescence protein (GFP) gene. Baculovirus gp64 TM and gp64 CTD in the pBacSC vector were designed to display heterologous proteins on the baculovirus envelope. After cloning the VP2 gene of CPV-2 into pBacSC vector, the recombinant plasmid pBacSC-VP2 was transformed into E. coli DH10Bac competent cells to form recombinant bacmid DNA. One recombinant baculovirus BacSC-VP2 that expresses the VP2 protein of CPV-2 was obtained. Confocal microscopy and immunogold electron microscopy were used to verify whether VP2 expressing on baculovirus envelope or cell membrane. Immunization of BALB/c mice with recombinant baculovirus BacSC-VP2, demonstrated that serum from the BacSC-VP2 treated models had higher levels of virus neutralization titers than the control groups. The results show that the recombinant baculovirus BacSC-VP2 can induce a strong immune response in a mouse model, suggesting that the pseudotyped baculovirus BacSC-VP2 can serve as a potential vaccine against CPV infections.
基金FAPESP(2009/53493-9)and(2009/53777-7)for the financial support
文摘Background: Experimental studies have shown that cyclo-oxygenase-2 (Cox2) is related to the development and progression of tumors, since this enzyme is induced and expressed by cells such as macrophages, osteoblasts, “activated” endothelial cells, and tumor cells. The activity in tumors includes proliferation, cell transformation, tumor growth, invasion and metastasis and may play an important role in carcinogenesis of the canine osteosarcoma, since it has high expression in tissue fragments. The combination of selective Cox2 inhibitors and other treatment modalities is the basis for a new anti-cancer therapy strategy. This in vitro study exposed primary cells of five different canine osteosarcoma cultures to selective Cox2 inhibitor at increasing concentrations and times. Results: For Cox2 negative cultures, despite the absence of differences, greater sensitivity of cells to treatment was observed. For Cox2 positive cultures, a higher number of necrotic cells were observed (P ≤ 0.05), when compared with negative cultures. For exposure times with Celecoxib doses, no difference (P > 0.05) was found between the three times analyzed for living, apoptotic and apoptotic/necrotic cells. There are similarities in the values of 24 h and 48 h, with slight reduction of living cells, increasing those undergoing apoptosis and apoptosis/necrosis. There was significance for necrosis (P ≤ 0.05). In 72 hours, a significant difference was observed between the other two previous values (P ≤ 0.05). It was found for the group of 100 μML-1, that there was a numerically greater signaling for apoptosis and lower (P = 0.08) for necrosis, and this point was the onset of the pharmacodynamic phenomenon, with drop in the values for living cells and increased number of necrotic cells, with a tendency (P = 0.08) for reducing the percentage of necrotic cells for the group of 100 μML-1 when compared to that of 10 μML-1. Conclusions: For Cox2 positive and negative cultures, there was difference for necrotic cells and there was no difference between Cox2 positive and Cox2 negative groups in relation to the percentage of living cells and apoptotic and apoptotic/necrotic cells. At time of 72 hours, higher percentage of living cells, lower percentage of apoptotic cells and increased percentage of necrotic cells in relation to groups of 24 and 48 hours was observed. A tendency for reducing the percentage of necrotic cells for the group of 100 μML-1 when compared to that of the group of 10 μML-1 was observed.
文摘本研究旨在探究己糖激酶2(hexokinase 2,HK2)在犬乳腺肿瘤(canine mammary tumor,CMT)中的表达情况及与患犬预后的相关性。将临床收集的121例犬乳腺肿块样本制作石蜡切片,经组织病理学分析判读后随机抽取恶性乳腺肿瘤与正常乳腺组织样本各3份进行高通量转录组测序,筛选差异表达基因。使用实时荧光定量PCR(quantitative real time PCR,qPCR)和免疫组织化学法检测HK2在犬全身组织及乳腺肿瘤组织中的表达情况,对术犬长期随访调查并分析HK2与乳腺肿瘤患犬临床特征及预后的相关性。结果表明:1)本研究中收集到的121例犬乳腺肿块临床样本中良性乳腺肿瘤33例,恶性乳腺肿瘤78例;2)经高通量转录组测序筛选出HK2、MEST、GDF5等36个显著差异表达基因,其中,HK2经qPCR验证以及基于其在细胞代谢中的关键作用且在人类某些肿瘤中异常表达,故将HK2作为研究对象;3)HK2 mRNA与蛋白表达水平在犬正常乳腺组织中均表现为低表达,而CMT中HK2 mRNA与蛋白表达水平均显著升高(P<0.01);4)同时HK2的表达水平与肿瘤组织类型、肿瘤大小、脉管浸润、Ki-67指数以及预后显著相关(P<0.01)。HK2在CMT组织中呈高表达,并与肿瘤的临床特征及预后显著相关。HK2具有作为CMT的诊断与预后评估因子的潜力。
基金Supported by the National Key R&D Program of China(2017YFD0501102,2016YFD0501003)Science and Technology Department of Heilongjiang Province(GX18B018)Education Department of Heilongjiang Province(TSTAU-R2018017)。
文摘Canine parvovirus(CPV)is a high morbidity and lethality virus which causes severe enteric disease in dogs.In an attempt to gain canine-origin neutralizing antibodies against CPV,single chain variable fragment(scFv)bacteria display libraries against the protective antigen VP2 were constructed and screened.VP2 specific scFvs were selected following three rounds of screening procedures.Selected scFvs were characterized by FCM and ELISA.Seven scFvs showed high affinity and specific binding to CPV.Moreover,the neutralizing activity of the antibody was preliminarily identified by CPV(100 TCID_(50))in vitro and scFv-2 showed the neutralizing ability with a titer of 32768.This study generated the first canine-origin neutralizing scFv against CPV.The neutralizing scFv would be constructed into full-length antibody in the future.This study laid the foundation for the generation of an effective therapeutic reagent with long half-life and no immunological rejection for the prevention and treatment of CPV infection.
文摘Objectives To evaluate the changes in atrial effective refractory period (AERP) proprieties and in ionic currents in PVs myocytes from dogs subjected to rapid atrial pacing in PVs and right atrial appendage (RAA) and to relate these changes to the ability to induce AF. Methods Twelve mongrel dogs in normal sinus rhythm were paced from the superior left PVs or RAA at 500 bpm for 4 hours. Electrophysiologic studies conducted to determine changes in AERP, dispersion and rhythm. Ionic currents were studies with the patch clamp technique in single PVs myocytes in sham operated dogs and compared with those from PVs pacing and RAA pacing groups. Results The presence of rapid atrial pacing was associated with a marked shortening in AERP in both PVs and RAA pacing group with a marked increase of AERP dispersion in PVs pacing. Both L-type calcium current (Ica L ) and the transient outward current ( Ito ) were reduced in both groups with an increased significance in PVs pacing group. The density of ICa-L was decreased significantly from ( - 6. 03 ± 0. 63 ) pA./pF in the control group to ( -3.21 ±0. 34) pA/pF in PVs pacing group and ( - 4. 75 ± 0. 41 ) pA./pF in RAA pacing group ( n = 6, P 〈 0. 05 ) while the density of Ito was decreased significantly from (8.45 ± 0. 71 ) pA./pF in the control group to ( 5.21 ± 0. 763 ) pA./pF in PVs pacing group and (6. 84 ±0. 69 ) pA./pF in RAA pacing group ( n = 6, P 〈 0. 05 ). Conclusions Our findings provide likely ionic mechanisms of shortened repolarization in induced atrial tachycardia with a decrease in Ica L and /tocurrent densities which is the likely mechanism for a decrease in Action potential duration (APD) rate adaptation in the canine rapid pacing model more pronounced in PVs pacing group underlying the crucial role of PVs in initiating AF.
文摘为研制一种预防犬科动物狂犬病的新型疫苗,将含有狂犬病毒ERA株糖蛋白基因(Rabies glycoprotein,Rgp)表达盒的穿梭质粒pVAXΔE3Rgp中的Rgp表达盒克隆入犬2型腺病毒(Canine adenovirus type2,CAV2)骨架质粒pPoly2-CAV2中,获得重组质粒pPoly2-CAV2-ΔE3-Rgp,释放其基因组,转染MDCK细胞系,获得E3缺失区(Deletion of early protein3,ΔE3)含有Rgp表达盒的重组病毒CAV2-ΔE3-Rgp。该重组病毒能在MDCK细胞上产生典型的腺病毒细胞病变。通过酶切、PCR、基因测序,表明该重组病毒含有完整的Rgp表达盒。通过RT-PCR、Western blot等检测,表明该重组病毒能够表达Rgp抗原。用该重组病毒免疫犬,3次免疫后,可以诱导犬产生特异的抗CAV2HI抗体,其效价超过1∶256和抗狂犬病病毒(Rabies virus,RV)中和抗体,其效价超过0.50IU/mL。试验结果表明,获得的重组病毒免疫犬后,能够产生抗狂犬病毒和腺病毒的高效价保护性抗体,是一种有潜力的犬科动物狂犬病毒腺病毒二联疫苗候选株。