Hyperproteinemia is a metabolic disorder associated with increased plasma protein concentration(PPC)and is often clinically complicated by malignant diseases or severe infections.At present,however,research on the mol...Hyperproteinemia is a metabolic disorder associated with increased plasma protein concentration(PPC)and is often clinically complicated by malignant diseases or severe infections.At present,however,research on the molecular mechanism underlying high PPC(HPPC)is scant.Here,an animal model of primary hyperproteinemia was constructed in an invertebrate(Bombyx mori)to investigate the effects of HPPC on circulating blood cells.Results showed that HPPC affected blood cell homeostasis,leading to increased reactive oxygen species levels,and induced programmed cell death dependent on the endoplasmic reticulum-calcium ion signaling pathway.HPPC induced the proliferation of blood cells,mainly granulocytes,by activating the Janus kinase/signal transducer and activator of transcription(JAK/STAT)signaling pathway.Supplementation with the endocrine hormone active substance 20 E significantly reduced the impact of HPPC on blood cell homeostasis.Thus,we identified a novel signaling pathway by which HPPC affects blood cell homeostasis,which differs from hyperglycemia,hyperlipidemia,and hypercholesterolemia.In addition,we showed that down-regulation of gene expression of the hematopoietic factor Gcm could be used as a potential early detection indicator for hyperproteinemia.展开更多
Gal alpha(1, 3) Gal (gal epitope) is a carbohydrate epitope and synthesized in large amount by alpha(1, 3) galactosyltransferase [alpha(1, 3) GT] enzyme on the cells of lower mammalian animals such as pigs and mice. H...Gal alpha(1, 3) Gal (gal epitope) is a carbohydrate epitope and synthesized in large amount by alpha(1, 3) galactosyltransferase [alpha(1, 3) GT] enzyme on the cells of lower mammalian animals such as pigs and mice. Human has no gal epitope due to the inactivation of alpha(1, 3) GT gene but produces a large amount of antibodies (anti-Gal) which recognize Gal alpha(1, 3) Gal structures specifically. In this study, a replication-deficient recombinant adenoviral vector Ad5sGT containing pig alpha(1, 3) GT cDNA was constructed and characterized. Adenoviral vector-mediated transfer of pig alpha(1, 3) GT gene into human tumor cells such as malignant melanoma A375, stomach cancer SGC-7901, and lung cancer SPC-A-1 was reported for the first time. Results showed that Gal epitope did not increase the sensitivity of human tumor cells to human complement-mediated lysis, although human complement activation and the binding of human IgG and IgM natural antibodies to human tumor cells were enhanced significantly after Ad5sGT transduction. Appearance of gal epitope on the human tumor cells changed the expression of cell surface carbohydrates reacting with Ulex europaeus I (UEA I) lectins, Vicia villosa agglutinin (VVA), Arachis hypogaea agglutinin (PNA), and Glycine max agglutinin (SBA) to different degrees. In addition, no effect of gal epitope on the growth in vitro of human tumor cells was observed in MTT assay.展开更多
INTRODUCTIONCrypt epithelial cells in normal small intestineproliferate at a high speed. But they are verydifficult to culture in vitro and passage stably. A lotof studies have been done[1-16]. Some domestic labsisola...INTRODUCTIONCrypt epithelial cells in normal small intestineproliferate at a high speed. But they are verydifficult to culture in vitro and passage stably. A lotof studies have been done[1-16]. Some domestic labsisolated and cultured crypt cells from embryonalintestines and aseptic animal intestine, but failed.We introduced normal rat epithelial cell line-IEC-6from the USA and its living condition for stablepassage was successfully established after trials. Thecell line was testified to be the small intestinalepithelial cell by electron microscopy,immunihistochemistry and enzymatic histoch-emistry. It has been applied to some relatedresearch work[17-21]. It was found that manyfactors were involved in the culture system. Ourpresent study focuses on the culture method and theinfluencing factors on IEC-6.展开更多
目的探讨炎症条件的动物输注贮存红细胞对巨噬细胞(BMDMs)的调节作用以及贮存红细胞输注与细菌感染引发炎症反应的关系。方法将6~8周龄成年雄性C57BL/6小鼠[(18~22)g/只]40只随机均分为实验组和实验对照组(对照组),均通过动物尾静脉注...目的探讨炎症条件的动物输注贮存红细胞对巨噬细胞(BMDMs)的调节作用以及贮存红细胞输注与细菌感染引发炎症反应的关系。方法将6~8周龄成年雄性C57BL/6小鼠[(18~22)g/只]40只随机均分为实验组和实验对照组(对照组),均通过动物尾静脉注射铜绿假单胞菌200μL/只,并使用吸入式麻醉剂异氟烷(1%~3%)麻醉后,通过小鼠眼后静脉丛,实验组输注鼠源贮存悬浮红细胞(>14 d)400μL/只、对照组每只输注等量新鲜悬浮红细胞(贮存<24 h);于输注后2、4、8 h脱就猝死各结束2组小鼠生命5只,摘取鼠肝,体外培养铜绿假单胞菌感染(200μL/只)小鼠的股骨、胫骨骨髓来源的BMDMs,流式细胞术检测BMDMs中分化簇86(CD86)、分化簇197(CD197)[巨噬细胞1型(M1)基因特异性标志物]、分化簇209(CD209)[巨噬细胞2型(M2)基因特异性标记]表达水平,实时荧光定量PCR(qRT-PCR)法检测小鼠肝脏F4/80、M1、M2基因表达水平,并使用SPSS17.0统计学软件分析数据。结果实验组与对照组BMDMs中CD86和CD197的表达(%)分别为8688±1.01 vs 79.24±2.65、38.59±3.73 vs 25.95±0.86(P<0.05),CD209(%)为23.88±2.23 vs 21.91±3.58(P>0.05)。输注红细胞后2、4 h,小鼠肝F4/80基因表达水平实验组和对照组分别为1.83±0.11 vs 0.75±0.06、0.46±0.06 vs 0.33±0.06(P<0.05),8 h后分别为0.33±0.03 vs 0.35±0.05(P>0.05);输注红细胞2、4、8 h,小鼠肝M1基因中诱导型一氧化氮合酶(iNOS)基因表达水平实验组和对照组分别为3.44±0.20 vs 2.46±0.08、9.25±0.55 vs 2.67±0.12、2.80±0.08 vs 2.39±0.01,肿瘤坏死因子-α(TNF-α)分别为1.69±0.22 vs 1.13±0.03、1.44±0.24 vs 0.96±0.09、1.31±0.05 vs 0.96±0.06,单核细胞趋化蛋白1(MCP1)分别为4.96±0.08 vs 4.28±0.27、4.63±0.04 vs 2.07±0.09、2.28±0.19 vs 1.33±0.03(P<0.05);M2基因中精氨酸1(Arg1)基因表达水平实验组和对照组分别为0.81±0.21 vs 0.82±0.18、0.66±0.11 vs 0.58±0.09、0.39±0.17 vs 0.37±0.15,甘露糖受体C型2(Mrc2)分别为0.99±0.91 vs 0.97±0.08、0.98±0.12 vs 1.02±0.11、0.59±0.19 vs 0.57±0.08,重组蛋白163(CD163)分别为1.75±0.20 vs 1.69±0.18、0.22±0.02 vs 0.21±0.01、0.04±0.01 vs 0.03±0.01(P>0.05)。结论实验小鼠输注贮存红细胞明显促进其肝脏组织巨噬细胞朝向M1表型的极化。展开更多
OBJECTIVE: To study whether human umbilical cord blood CD34+ cells transduced with human aldehyde dehydrogenase class-1 (ALDH-1) and multidrug resistance gene (MDR1) have increases resistance to 4-Hydroperoxycyclo-pho...OBJECTIVE: To study whether human umbilical cord blood CD34+ cells transduced with human aldehyde dehydrogenase class-1 (ALDH-1) and multidrug resistance gene (MDR1) have increases resistance to 4-Hydroperoxycyclo-phosphamide (4-HC) and P-glycoprotein effluxed drugs. METHODS: A bicistronic retroviral vector G1Na-ALDH1-IRES-MDR1 was constructed and used to transfect the packaging cell lines GP + E86 and PA317 by LipofectAMINE method, using the medium containing VCR and 4-HC agents for cloning selection and ping-ponging supernatant infection between the ecotropic producer clone and the amphotropic producer clone, we obtained high titer amphotropic PA317 producing cells with high titers up to 5.6 x 10(5) CFU/ml. Cord blood CD34+ cells were transfected repeatedly with supernatant of retrovirus containing human ALDH-1 and MDR1cDNA under the stimulation of hemopoietic growth factors. RESULTS: Bicistronic retroviral vector construction was verified by restriction endonuclease analysis. Polymerase chain reaction (PCR), reverse transcription (RT)-PCR, Southern blot, Northern blot, fluorescenceactivated cell sorting (FACS) method and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analyses showed that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgenes recipient cells confered 4-fold stronger resistance to 4-HC and 5.5 to 7.2-fold P-glycoprotein effluxed drug than untransduced cells. CONCLUSION: The bicistronic retroviral vector-mediated transfer of two different types of drug resistance genes into human cord blood CD34+ cells and co-expression provided an experimental foundation for improving combination chemotherapy tolerance in tumor clinical trial.展开更多
基金supported by the National Natural Science Foundation of China(31972625)China Postdoctoral Science Foundation(2020M681718)+2 种基金Priority Academic Program Development(PAPD)of Jiangsu Higher Education InstitutionsPostgraduate Research&Practice Innovation Program of Jiangsu Province(KYCX21_2963)China Agriculture Research System(CARS)of Ministry of Finance and Ministry of Agriculture and Rural Areas。
文摘Hyperproteinemia is a metabolic disorder associated with increased plasma protein concentration(PPC)and is often clinically complicated by malignant diseases or severe infections.At present,however,research on the molecular mechanism underlying high PPC(HPPC)is scant.Here,an animal model of primary hyperproteinemia was constructed in an invertebrate(Bombyx mori)to investigate the effects of HPPC on circulating blood cells.Results showed that HPPC affected blood cell homeostasis,leading to increased reactive oxygen species levels,and induced programmed cell death dependent on the endoplasmic reticulum-calcium ion signaling pathway.HPPC induced the proliferation of blood cells,mainly granulocytes,by activating the Janus kinase/signal transducer and activator of transcription(JAK/STAT)signaling pathway.Supplementation with the endocrine hormone active substance 20 E significantly reduced the impact of HPPC on blood cell homeostasis.Thus,we identified a novel signaling pathway by which HPPC affects blood cell homeostasis,which differs from hyperglycemia,hyperlipidemia,and hypercholesterolemia.In addition,we showed that down-regulation of gene expression of the hematopoietic factor Gcm could be used as a potential early detection indicator for hyperproteinemia.
基金National..973" project, the Special Funds for Major State Bacsic Reseaxch of China (G1999053905) and NationalNatural Science Fou
文摘Gal alpha(1, 3) Gal (gal epitope) is a carbohydrate epitope and synthesized in large amount by alpha(1, 3) galactosyltransferase [alpha(1, 3) GT] enzyme on the cells of lower mammalian animals such as pigs and mice. Human has no gal epitope due to the inactivation of alpha(1, 3) GT gene but produces a large amount of antibodies (anti-Gal) which recognize Gal alpha(1, 3) Gal structures specifically. In this study, a replication-deficient recombinant adenoviral vector Ad5sGT containing pig alpha(1, 3) GT cDNA was constructed and characterized. Adenoviral vector-mediated transfer of pig alpha(1, 3) GT gene into human tumor cells such as malignant melanoma A375, stomach cancer SGC-7901, and lung cancer SPC-A-1 was reported for the first time. Results showed that Gal epitope did not increase the sensitivity of human tumor cells to human complement-mediated lysis, although human complement activation and the binding of human IgG and IgM natural antibodies to human tumor cells were enhanced significantly after Ad5sGT transduction. Appearance of gal epitope on the human tumor cells changed the expression of cell surface carbohydrates reacting with Ulex europaeus I (UEA I) lectins, Vicia villosa agglutinin (VVA), Arachis hypogaea agglutinin (PNA), and Glycine max agglutinin (SBA) to different degrees. In addition, no effect of gal epitope on the growth in vitro of human tumor cells was observed in MTT assay.
基金Supported by the National Natural Science Foundation of China, No.39100119
文摘INTRODUCTIONCrypt epithelial cells in normal small intestineproliferate at a high speed. But they are verydifficult to culture in vitro and passage stably. A lotof studies have been done[1-16]. Some domestic labsisolated and cultured crypt cells from embryonalintestines and aseptic animal intestine, but failed.We introduced normal rat epithelial cell line-IEC-6from the USA and its living condition for stablepassage was successfully established after trials. Thecell line was testified to be the small intestinalepithelial cell by electron microscopy,immunihistochemistry and enzymatic histoch-emistry. It has been applied to some relatedresearch work[17-21]. It was found that manyfactors were involved in the culture system. Ourpresent study focuses on the culture method and theinfluencing factors on IEC-6.
文摘目的探讨炎症条件的动物输注贮存红细胞对巨噬细胞(BMDMs)的调节作用以及贮存红细胞输注与细菌感染引发炎症反应的关系。方法将6~8周龄成年雄性C57BL/6小鼠[(18~22)g/只]40只随机均分为实验组和实验对照组(对照组),均通过动物尾静脉注射铜绿假单胞菌200μL/只,并使用吸入式麻醉剂异氟烷(1%~3%)麻醉后,通过小鼠眼后静脉丛,实验组输注鼠源贮存悬浮红细胞(>14 d)400μL/只、对照组每只输注等量新鲜悬浮红细胞(贮存<24 h);于输注后2、4、8 h脱就猝死各结束2组小鼠生命5只,摘取鼠肝,体外培养铜绿假单胞菌感染(200μL/只)小鼠的股骨、胫骨骨髓来源的BMDMs,流式细胞术检测BMDMs中分化簇86(CD86)、分化簇197(CD197)[巨噬细胞1型(M1)基因特异性标志物]、分化簇209(CD209)[巨噬细胞2型(M2)基因特异性标记]表达水平,实时荧光定量PCR(qRT-PCR)法检测小鼠肝脏F4/80、M1、M2基因表达水平,并使用SPSS17.0统计学软件分析数据。结果实验组与对照组BMDMs中CD86和CD197的表达(%)分别为8688±1.01 vs 79.24±2.65、38.59±3.73 vs 25.95±0.86(P<0.05),CD209(%)为23.88±2.23 vs 21.91±3.58(P>0.05)。输注红细胞后2、4 h,小鼠肝F4/80基因表达水平实验组和对照组分别为1.83±0.11 vs 0.75±0.06、0.46±0.06 vs 0.33±0.06(P<0.05),8 h后分别为0.33±0.03 vs 0.35±0.05(P>0.05);输注红细胞2、4、8 h,小鼠肝M1基因中诱导型一氧化氮合酶(iNOS)基因表达水平实验组和对照组分别为3.44±0.20 vs 2.46±0.08、9.25±0.55 vs 2.67±0.12、2.80±0.08 vs 2.39±0.01,肿瘤坏死因子-α(TNF-α)分别为1.69±0.22 vs 1.13±0.03、1.44±0.24 vs 0.96±0.09、1.31±0.05 vs 0.96±0.06,单核细胞趋化蛋白1(MCP1)分别为4.96±0.08 vs 4.28±0.27、4.63±0.04 vs 2.07±0.09、2.28±0.19 vs 1.33±0.03(P<0.05);M2基因中精氨酸1(Arg1)基因表达水平实验组和对照组分别为0.81±0.21 vs 0.82±0.18、0.66±0.11 vs 0.58±0.09、0.39±0.17 vs 0.37±0.15,甘露糖受体C型2(Mrc2)分别为0.99±0.91 vs 0.97±0.08、0.98±0.12 vs 1.02±0.11、0.59±0.19 vs 0.57±0.08,重组蛋白163(CD163)分别为1.75±0.20 vs 1.69±0.18、0.22±0.02 vs 0.21±0.01、0.04±0.01 vs 0.03±0.01(P>0.05)。结论实验小鼠输注贮存红细胞明显促进其肝脏组织巨噬细胞朝向M1表型的极化。
基金theNationalNaturalScienceFoundationofChina (No 39770 331)
文摘OBJECTIVE: To study whether human umbilical cord blood CD34+ cells transduced with human aldehyde dehydrogenase class-1 (ALDH-1) and multidrug resistance gene (MDR1) have increases resistance to 4-Hydroperoxycyclo-phosphamide (4-HC) and P-glycoprotein effluxed drugs. METHODS: A bicistronic retroviral vector G1Na-ALDH1-IRES-MDR1 was constructed and used to transfect the packaging cell lines GP + E86 and PA317 by LipofectAMINE method, using the medium containing VCR and 4-HC agents for cloning selection and ping-ponging supernatant infection between the ecotropic producer clone and the amphotropic producer clone, we obtained high titer amphotropic PA317 producing cells with high titers up to 5.6 x 10(5) CFU/ml. Cord blood CD34+ cells were transfected repeatedly with supernatant of retrovirus containing human ALDH-1 and MDR1cDNA under the stimulation of hemopoietic growth factors. RESULTS: Bicistronic retroviral vector construction was verified by restriction endonuclease analysis. Polymerase chain reaction (PCR), reverse transcription (RT)-PCR, Southern blot, Northern blot, fluorescenceactivated cell sorting (FACS) method and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analyses showed that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgenes recipient cells confered 4-fold stronger resistance to 4-HC and 5.5 to 7.2-fold P-glycoprotein effluxed drug than untransduced cells. CONCLUSION: The bicistronic retroviral vector-mediated transfer of two different types of drug resistance genes into human cord blood CD34+ cells and co-expression provided an experimental foundation for improving combination chemotherapy tolerance in tumor clinical trial.
