Objective. To examine the effect of isobutyramide synthesized in our laboratory on human and murine globin gene expression and to test cell toxicity ofthe drug.Methods. MEL cells were transfected with the recombinant ...Objective. To examine the effect of isobutyramide synthesized in our laboratory on human and murine globin gene expression and to test cell toxicity ofthe drug.Methods. MEL cells were transfected with the recombinant construct μLCRAγψβδβand the stable transformants were cultured in the medium with different concentrations of isobutyramide. The experimental mice and rabbit were injected with different doses of isobutyramide. The globin mRNAs were analyzed by RNase protection assay. The hematological toxicity and electrolyte toxicity ofthe drug were tested.Results. An inducible and dose dependent expression of the human γ , β and mouse α globin gene was observed in the transfected MEL cells. The induction of the human γ globin gene is significant stronger than that of the β globin gene. With 2.5～5 mmol/L isobutyramide, the induction of the human γ globin gene is even more effective than that of mouse α globin gene. After a 15 day injection under the doses of 500～900mg·kg-1·d-1, the level of the mouse embryonic εy globin mRNA could be significantly induced up to 3～4 fold of that of uninjected controls. The changes of hemoglobin(Hb), RBC, hematocrit(HCT), WBC, derived from mice injected with different doses of isobutyramide at the interval of 24 hours for 2～4 weeks, were generally within the normal range. In rabbits injected with isobutyramide in the same regiment for 2 weeks, the concentration of blood K+, Na+, Cl-and CO2 were all within normal range and serum ionic osmotic pressure remained stable as well. Conclusion. Our results suggested that isobutyramide is a weak inducer ofcell differentiation, but it can selectively activate transcription of human γ globin gene at a certain degree, and it can act on early stages of erythroid progenitor differentiation in adult mice and activate transcription of embryonic εy-globin gene and have no hematological toxicity. Our results have further proved the potential value of isobutyramide in treatment of β-thalassemia and sickle cell disease.展开更多
We have confirmed efficient anti-tumor activities of the peripheral lymphocytes transduced with a p185HEH2-specific chimeric T-cell receptor gene both in murine and in human in our previous studies. To further test th...We have confirmed efficient anti-tumor activities of the peripheral lymphocytes transduced with a p185HEH2-specific chimeric T-cell receptor gene both in murine and in human in our previous studies. To further test the feasibility of chimeric T-cell receptor in a bone marrow transplantation model, we first, made two routine tumor cell lines： MT901 and MCA-205, to express human p185HER2 by retroviral gene transduction. Murine bone marrow cells were retrovirally transduced to express the chimeric T-cell receptor and gene-modified bone marrow cells were transplanted into lethally irradiated mouse. Six months post transplantation, p185HER2-positive tumor ceils： MT-901/HER2 or MCA-205/ HER2 was subcutaneously or intravenously injected to make mouse models simulating primary breast cancer or pulmonary metastasis. The in vivo anti-tumor effects were monitored by the size of the subcutaneous tumor or counting the tumor nodules in the lungs after India ink staining. The size of the subcutaneous tumor was significantly inhibited and the number of pulmonary nodules were significantly decreased in mouse recipients transplanted with chimeric T-cell receptor modified bone marrow cells compared with the control group. Our results suggest the efficient in vivo anti-tumor activities of chimeric T-cell receptor gene modified bone marrow cells.展开更多
AIM： To evaluate the expression of fibrinogenlike protein 2 （fgl2） and its correlation with disease progression in both mice and patients with severe viral hepatitis. METHODS： Balb/cJ or A/J mice were infected int...AIM： To evaluate the expression of fibrinogenlike protein 2 （fgl2） and its correlation with disease progression in both mice and patients with severe viral hepatitis. METHODS： Balb/cJ or A/J mice were infected intraperitoneally （ip） with 100 PFU of murine hepatitis virus type 3 （MHV-3）, liver and serum were harvested at 24, 48, and 72 h post infection for further use. Liver tissues were obtained from 23 patients with severe acute chronic （AOC） hepatitis B and 13 patients with mild chronic hepatitis B. Fourteen patients with mild chronic hepatitis B with cirrhosis and 4 liver donors served as normal controls. In addition, peripheral blood mononuciear cells （PBMC） were isolated from 30 patients （unpaired） with severe AOC hepatitis B and 10 healthy volunteers as controls. Procoagulant activity representing functional prothrombinase activity in PBMC and white blood cells was also assayed. A polyclonal antibody against fgl2 was used to detect the expression of both mouse and human fgl2 protein in liver samples as well as in PBMC by immunohistochemistry staining in a separate set of studies. Alanine aminotransferase （ALT） and total bilirubin （TBil） in serum were measured to assess the severity of liver injury.RESULTS： Histological changes were found in liver sections 12-24 h post MHV-3 infection in Balb/cJ mice. In association with changes in liver histology, marked elevations in serum ALT and TBil were observed. House fgl2 （mfgl2） protein was detected in the endothelium of intrahepatic veins and hepatic sinusoids within the liver 24 h after MHV-3 infection. Liver tissues from the patients with severe AOC hepatitis B had classical pathological features of acute necroinflammation. Human fgl2 （hfgl2） was detected in 21 of 23 patients （91.30%） with severe AOC hepatitis B, while only 1 of 13 patients （7.69%） with mild chronic hepatitis B and cirrhosis had hfgl2 mRNA or protein expression. Twenty-eight of thirty patients （93.33%） with severe AOC hepatitis B and 1 of 10 with mild chronic hepatitis B had detectable hfgl2 expression in PBMC. No hfgl2 expression was found either in the liver tissue or in the PBMC from normal donors. There was a positive correlation between hfgl2 expression and the severity of the liver disease as indicated by the levels of TBil. PCA significantly increased in PBMC in patients with severe AOC hepatitis B. CONCLUSION： The molecular and cellular results reported here in both mice and patients with severe viral hepatitis suggest that virus-induced hfgl2 prothrombinase/fibroleukin expression and the coagulation activity associated with the encoded fgl2 protein play a pivotal role in initiating severe hepatitis. The measurement of hfgl2/fibroleukin expression in PBMC may serve as a useful marker to monitor the severity of AOC hepatitis B and a target for therapeutic intervention.展开更多
EKLF is an erythroid-specific, zinc finger-containing transcription factor essential for the activation of the mammalian beta globin gene in erythroid cells of definitive lineage. We have prepared a polyclonal anti-mo...EKLF is an erythroid-specific, zinc finger-containing transcription factor essential for the activation of the mammalian beta globin gene in erythroid cells of definitive lineage. We have prepared a polyclonal anti-mouse EKLF antibody suitable for Western blotting and immunoprecipitation （IP） qualities, and used it to define the expression patterns of the EKLF protein during mouse erythroid development. We have also used this antibody for the chromatin-immunoprecipitation （CHIP） assay. EKLF was found to bind in vivo at both the mouse beta-major-globin promoter and the HS2 site of beta-LCR in the mouse erythroleukemia cells （MEL） in a DMSO-inducible manner. The DMSO-induced bindings of EKLF as well as three other proteins, namely, RNA polymerase Ⅱ, acetylated histone H3, and methylated histone H3, were not abolished but significantly lowered in CB3, a MEL-derived cell line with null-expression of p45/NF-E2, an erythroid-enriched factor needed for activation of the mammalian globin loci. Interestingly, binding of EKLF in vivo was also detected in the mouse alpha-like globin locus, at the adult alpha globin promoter and its far upstream regulatory element alpha-MRE （HS26）. This study provides direct evidence for EKLF-binding in vivo at the major regulatory elements of the mouse beta-like globin gene clusters the data also have interesting implications with respect to the role of EKLF-chromatin interaction in mammalian globin gene regulation.展开更多
In the world,hepatocellular carcinoma(HCC)is among the top 10 most prevalent malignancies.HCC formation has indeed been linked to numerous etiological factors,including alcohol usage,hepatitis viruses and liver cirrho...In the world,hepatocellular carcinoma(HCC)is among the top 10 most prevalent malignancies.HCC formation has indeed been linked to numerous etiological factors,including alcohol usage,hepatitis viruses and liver cirrhosis.Among the most prevalent defects in a wide range of tumours,notably HCC,is the silencing of the p53 tumour suppressor gene.The control of the cell cycle and the preservation of gene function are both critically important functions of p53.In order to pinpoint the core mechanisms of HCC and find more efficient treatments,molecular research employing HCC tissues has been the main focus.Stimulated p53 triggers necessary reactions that achieve cell cycle arrest,genetic stability,DNA repair and the elimination of DNA-damaged cells’responses to biological stressors(like oncogenes or DNA damage).To the contrary hand,the oncogene protein of the murine double minute 2(MDM2)is a significant biological inhibitor of p53.MDM2 causes p53 protein degradation,which in turn adversely controls p53 function.Despite carrying wt-p53,the majority of HCCs show abnormalities in the p53-expressed apoptotic pathway.High p53 in-vivo expression might have two clinical impacts on HCC:(1)Increased levels of exogenous p53 protein cause tumour cells to undergo apoptosis by preventing cell growth through a number of biological pathways;and(2)Exogenous p53 makes HCC susceptible to various anticancer drugs.This review describes the functions and primary mechanisms of p53 in pathological mechanism,chemoresistance and therapeutic mechanisms of HCC.展开更多
文摘Objective. To examine the effect of isobutyramide synthesized in our laboratory on human and murine globin gene expression and to test cell toxicity ofthe drug.Methods. MEL cells were transfected with the recombinant construct μLCRAγψβδβand the stable transformants were cultured in the medium with different concentrations of isobutyramide. The experimental mice and rabbit were injected with different doses of isobutyramide. The globin mRNAs were analyzed by RNase protection assay. The hematological toxicity and electrolyte toxicity ofthe drug were tested.Results. An inducible and dose dependent expression of the human γ , β and mouse α globin gene was observed in the transfected MEL cells. The induction of the human γ globin gene is significant stronger than that of the β globin gene. With 2.5～5 mmol/L isobutyramide, the induction of the human γ globin gene is even more effective than that of mouse α globin gene. After a 15 day injection under the doses of 500～900mg·kg-1·d-1, the level of the mouse embryonic εy globin mRNA could be significantly induced up to 3～4 fold of that of uninjected controls. The changes of hemoglobin(Hb), RBC, hematocrit(HCT), WBC, derived from mice injected with different doses of isobutyramide at the interval of 24 hours for 2～4 weeks, were generally within the normal range. In rabbits injected with isobutyramide in the same regiment for 2 weeks, the concentration of blood K+, Na+, Cl-and CO2 were all within normal range and serum ionic osmotic pressure remained stable as well. Conclusion. Our results suggested that isobutyramide is a weak inducer ofcell differentiation, but it can selectively activate transcription of human γ globin gene at a certain degree, and it can act on early stages of erythroid progenitor differentiation in adult mice and activate transcription of embryonic εy-globin gene and have no hematological toxicity. Our results have further proved the potential value of isobutyramide in treatment of β-thalassemia and sickle cell disease.
文摘We have confirmed efficient anti-tumor activities of the peripheral lymphocytes transduced with a p185HEH2-specific chimeric T-cell receptor gene both in murine and in human in our previous studies. To further test the feasibility of chimeric T-cell receptor in a bone marrow transplantation model, we first, made two routine tumor cell lines： MT901 and MCA-205, to express human p185HER2 by retroviral gene transduction. Murine bone marrow cells were retrovirally transduced to express the chimeric T-cell receptor and gene-modified bone marrow cells were transplanted into lethally irradiated mouse. Six months post transplantation, p185HER2-positive tumor ceils： MT-901/HER2 or MCA-205/ HER2 was subcutaneously or intravenously injected to make mouse models simulating primary breast cancer or pulmonary metastasis. The in vivo anti-tumor effects were monitored by the size of the subcutaneous tumor or counting the tumor nodules in the lungs after India ink staining. The size of the subcutaneous tumor was significantly inhibited and the number of pulmonary nodules were significantly decreased in mouse recipients transplanted with chimeric T-cell receptor modified bone marrow cells compared with the control group. Our results suggest the efficient in vivo anti-tumor activities of chimeric T-cell receptor gene modified bone marrow cells.
基金Supported by the National Natural Science Foundation of China for Distinguished Young Scholars, No. 30225040 for Dr Ning Q,No. 30123019 for Dr Luo XP
文摘AIM： To evaluate the expression of fibrinogenlike protein 2 （fgl2） and its correlation with disease progression in both mice and patients with severe viral hepatitis. METHODS： Balb/cJ or A/J mice were infected intraperitoneally （ip） with 100 PFU of murine hepatitis virus type 3 （MHV-3）, liver and serum were harvested at 24, 48, and 72 h post infection for further use. Liver tissues were obtained from 23 patients with severe acute chronic （AOC） hepatitis B and 13 patients with mild chronic hepatitis B. Fourteen patients with mild chronic hepatitis B with cirrhosis and 4 liver donors served as normal controls. In addition, peripheral blood mononuciear cells （PBMC） were isolated from 30 patients （unpaired） with severe AOC hepatitis B and 10 healthy volunteers as controls. Procoagulant activity representing functional prothrombinase activity in PBMC and white blood cells was also assayed. A polyclonal antibody against fgl2 was used to detect the expression of both mouse and human fgl2 protein in liver samples as well as in PBMC by immunohistochemistry staining in a separate set of studies. Alanine aminotransferase （ALT） and total bilirubin （TBil） in serum were measured to assess the severity of liver injury.RESULTS： Histological changes were found in liver sections 12-24 h post MHV-3 infection in Balb/cJ mice. In association with changes in liver histology, marked elevations in serum ALT and TBil were observed. House fgl2 （mfgl2） protein was detected in the endothelium of intrahepatic veins and hepatic sinusoids within the liver 24 h after MHV-3 infection. Liver tissues from the patients with severe AOC hepatitis B had classical pathological features of acute necroinflammation. Human fgl2 （hfgl2） was detected in 21 of 23 patients （91.30%） with severe AOC hepatitis B, while only 1 of 13 patients （7.69%） with mild chronic hepatitis B and cirrhosis had hfgl2 mRNA or protein expression. Twenty-eight of thirty patients （93.33%） with severe AOC hepatitis B and 1 of 10 with mild chronic hepatitis B had detectable hfgl2 expression in PBMC. No hfgl2 expression was found either in the liver tissue or in the PBMC from normal donors. There was a positive correlation between hfgl2 expression and the severity of the liver disease as indicated by the levels of TBil. PCA significantly increased in PBMC in patients with severe AOC hepatitis B. CONCLUSION： The molecular and cellular results reported here in both mice and patients with severe viral hepatitis suggest that virus-induced hfgl2 prothrombinase/fibroleukin expression and the coagulation activity associated with the encoded fgl2 protein play a pivotal role in initiating severe hepatitis. The measurement of hfgl2/fibroleukin expression in PBMC may serve as a useful marker to monitor the severity of AOC hepatitis B and a target for therapeutic intervention.
文摘EKLF is an erythroid-specific, zinc finger-containing transcription factor essential for the activation of the mammalian beta globin gene in erythroid cells of definitive lineage. We have prepared a polyclonal anti-mouse EKLF antibody suitable for Western blotting and immunoprecipitation （IP） qualities, and used it to define the expression patterns of the EKLF protein during mouse erythroid development. We have also used this antibody for the chromatin-immunoprecipitation （CHIP） assay. EKLF was found to bind in vivo at both the mouse beta-major-globin promoter and the HS2 site of beta-LCR in the mouse erythroleukemia cells （MEL） in a DMSO-inducible manner. The DMSO-induced bindings of EKLF as well as three other proteins, namely, RNA polymerase Ⅱ, acetylated histone H3, and methylated histone H3, were not abolished but significantly lowered in CB3, a MEL-derived cell line with null-expression of p45/NF-E2, an erythroid-enriched factor needed for activation of the mammalian globin loci. Interestingly, binding of EKLF in vivo was also detected in the mouse alpha-like globin locus, at the adult alpha globin promoter and its far upstream regulatory element alpha-MRE （HS26）. This study provides direct evidence for EKLF-binding in vivo at the major regulatory elements of the mouse beta-like globin gene clusters the data also have interesting implications with respect to the role of EKLF-chromatin interaction in mammalian globin gene regulation.
文摘In the world,hepatocellular carcinoma(HCC)is among the top 10 most prevalent malignancies.HCC formation has indeed been linked to numerous etiological factors,including alcohol usage,hepatitis viruses and liver cirrhosis.Among the most prevalent defects in a wide range of tumours,notably HCC,is the silencing of the p53 tumour suppressor gene.The control of the cell cycle and the preservation of gene function are both critically important functions of p53.In order to pinpoint the core mechanisms of HCC and find more efficient treatments,molecular research employing HCC tissues has been the main focus.Stimulated p53 triggers necessary reactions that achieve cell cycle arrest,genetic stability,DNA repair and the elimination of DNA-damaged cells’responses to biological stressors(like oncogenes or DNA damage).To the contrary hand,the oncogene protein of the murine double minute 2(MDM2)is a significant biological inhibitor of p53.MDM2 causes p53 protein degradation,which in turn adversely controls p53 function.Despite carrying wt-p53,the majority of HCCs show abnormalities in the p53-expressed apoptotic pathway.High p53 in-vivo expression might have two clinical impacts on HCC:(1)Increased levels of exogenous p53 protein cause tumour cells to undergo apoptosis by preventing cell growth through a number of biological pathways;and(2)Exogenous p53 makes HCC susceptible to various anticancer drugs.This review describes the functions and primary mechanisms of p53 in pathological mechanism,chemoresistance and therapeutic mechanisms of HCC.
