目的制备并用绿色荧光标志骨髓间充质干细胞(BM⁃MSC),观察其对博来霉素所致大鼠肺纤维化损伤的影响。方法60只雄性SD大鼠随机分为5组,A组于气管内注入生理盐水,B、C、D、E组于气管内注入5 mg/kg博来霉素0.1 ml。建模后1 d,A、B组经尾静...目的制备并用绿色荧光标志骨髓间充质干细胞(BM⁃MSC),观察其对博来霉素所致大鼠肺纤维化损伤的影响。方法60只雄性SD大鼠随机分为5组,A组于气管内注入生理盐水,B、C、D、E组于气管内注入5 mg/kg博来霉素0.1 ml。建模后1 d,A、B组经尾静脉注射生理盐水1.0 ml,C组经尾静脉注射1.0 ml BM⁃MSC(1.0×106细胞),D组尾静脉注射1.0 ml BM⁃MSC(5.0×10^(5)细胞),建模后8 d,再给1.0 ml BM⁃MSC(5.0×10^(5)细胞)。E组尾静脉注射1.0 ml BM⁃MSC(1.0×107细胞)。建模后28、42 d处死各组一半大鼠,留取肺组织。行病理学检查,对HE染色切片评定肺组织肺泡炎程度,对Masson染色切片评定肺组织纤维化程度;测定肺组织的转化生长因子⁃β1(TGF⁃β1)、羟脯氨酸(HYP)、基质金属蛋白酶⁃2(MMP⁃2)和组织金属蛋白酶抑制剂⁃1(TIMP⁃1)的表达水平,分析TGF⁃β1、HYP、MMP⁃2/TIMP⁃1与肺泡炎程度及肺纤维化程度评分的相关性。结果A组肺泡结构正常,无明显的炎症或纤维化改变,B组博来霉素可致肺泡结构严重紊乱,大量胶原纤维沉积,与B组比较,C、D、E组注射BM⁃MSC后肺泡炎性和肺纤维化程度较轻。与A组比较,B组肺泡炎程度评分、肺纤维化程度评分、TGF⁃β1、HYP、MMP⁃2/TIMP⁃1水平增高(均P<0.05),MSC治疗后的C、D、E 3组可使上述指标下降(均P<0.05)。肺组织TGF⁃β1与肺泡炎程度评分、肺纤维化程度评分相关系数r=0.82、0.89,HYP与肺泡炎程度评分、肺纤维化程度评分相关系数r=0.86、0.92,MMP⁃2/TIMP⁃1与肺泡炎程度评分、肺纤维化程度评分相关系数r=0.83、0.92,均存在正相关(均P<0.05)。结论BM⁃MSC可减低博来霉素致肺纤维化损伤,较高剂量和分次移植效果较好,其机制与移植细胞长期存活无关,可能与下调TGF⁃β1水平、改善MMP/TIMP失衡有关。展开更多
Green fluorescent protein (GFP) fused to the F-actin binding domain of mouse talin labels the actin cytoskeleton in the living generative and sperm cells of a third generation transgenic rice (Oryza sativa L.) plant, ...Green fluorescent protein (GFP) fused to the F-actin binding domain of mouse talin labels the actin cytoskeleton in the living generative and sperm cells of a third generation transgenic rice (Oryza sativa L.) plant, A005-G-T-1-2. Observations were made on pollen at four major developmental stages, viz. I. uni-nucleate microspore stage; II. early bi-cellular pollen stage; III. late bi-cellular pollen stage; and IV. tri-cellular pollen stage. At each of these developmental stages vegetative nucleus, generative nucleus/ cell, and sperm cells were seen undergoing continuous and coordinated motion and migration. These movements seemed to be influenced by associated microfilament networks existing in the pollen. Based on these observations we propose that it is the interaction between the microfilament networks (usually one existing in the central cytoplasm and another in the cortex) that controls the dynamic movement of the vegetative nucleus, generative nucleus/cell and sperm cells. Furthermore, we have also observed that there is an array of microfilaments (oriented mostly parallel to the long axis of the cell) existing in the generative and sperm cells. As far as we are aware, this is the first report showing the existence of microfilaments in living generative and sperm cells of rice pollen. The implication and significance of the existence of microfilaments in generative and sperm cells in rendering self-propelled motion of these cells in relation to their passage and movement in the pollen tube and embryo sac for fertilization were discussed.展开更多
AIM:To investigate the role of bone marrow-derived endothelial progenitor cells(EPCs) in the angiogenesis of hepatocellular carcinoma(HCC).METHODS:The bone marrow of HCC mice was reconstructed by transplanting green f...AIM:To investigate the role of bone marrow-derived endothelial progenitor cells(EPCs) in the angiogenesis of hepatocellular carcinoma(HCC).METHODS:The bone marrow of HCC mice was reconstructed by transplanting green fluorescent protein(GFP) + bone marrow cells.The concentration of circulating EPCs was determined by colony-forming assays and fluorescence-activated cell sorting.Serum and tissue levels of vascular endothelial growth factor(VEGF) and colony-stimulating factor(CSF) were quantified by enzyme-linked immunosorbent assay.The distribution of EPCs in tumor and tumor-free tissues was detected by immunohistochemistry and real-time polymerase chain reaction.The incorporation of EPCs into hepatic vessels was examined by immunofluorescence and immunohistochemistry.The proportion of EPCs in vessels was then calculated.RESULTS:The HCC model was successful established.The flow cytometry analysis showed the mean percentage of CD133CD34 and CD133VEGFR2 double positive cells in HCC mice was 0.45% ± 0.16% and 0.20% ± 0.09% respectively.These values are much higher than in the sham-operation group(0.11% ± 0.13%,0.05% ± 0.11%,n = 9) at 14 d after modeling.At 21 d,the mean percentage of circulating CD133CD34 and CD133VEGFR2 cells is 0.23% ± 0.19%,0.25% ± 0.15% in HCC model vs 0.05% ± 0.04%,0.12% ± 0.11% in control.Compared to the transient increase observed in controls,the higher level of circulating EPCs were induced by HCC.In addition,the level of serum VEGF and CSF increased gradually in HCC,reaching its peak 14 d after modeling,then slowly decreased.Consecutive sections stained for the CD133 and CD34 antigens showed that the CD133+ and CD34+ VEGFR2 cells were mostly recruited to HCC tissue and concentrated in tumor microvessels.Under fluorescence microscopy,the bone-marrow(BM)-derived cells labeled with GFP were concentrated in the same area.The relative levels of CD133 and CD34 gene expression were elevated in tumors,around 5.0 and 3.8 times that of the tumor free area.In frozen liver sections from HCC mice,cells co-expressing CD133 and VEGFR2 were identified by immunohistochemical staining using anti-CD133 and VEGFR2 antibodies.In tumor tissue,the double-positive cells were incorporated into vessel walls.In immunofluorescent staining.These CD31 and GFP double positive cells are direct evidence that tumor vascular endothelial cells(VECs) come partly from BM-derived EPCs.The proportion of GFP CD31 double positive VECs(out of all VECs) on day 21 was around 35.3% ± 21.2%.This is much higher than the value recorded on day 7 group(17.1% ± 8.9%).The expression of intercellular adhesion molecule 1,vascular adhesion molecule 1,and VEGF was higher in tumor areas than in tumor-free tissues.CONCLUSION:Mobilized EPCs were found to participate in tumor vasculogenesis of HCC.Inhibiting EPC mobilization or recruitment to tumor tissue may be an efficient strategy for treating HCC.展开更多
Objective： To facilitate manipulation of gene expression in different host cells, we used pEGFP-N1 as backbone to construct a versatile vector that can drive foreign gene expression in prokaryotic and eukaryotic cell...Objective： To facilitate manipulation of gene expression in different host cells, we used pEGFP-N1 as backbone to construct a versatile vector that can drive foreign gene expression in prokaryotic and eukaryotic cells. Methods： A cloning and expression vector, pEGFP-NI-lac, was constructed by inserting the prokaryotic lac promoter of pUC 19 into the eukaryotic expression vector, pEGFP-N1, between the eukaryotic PCMV promoter and enhanced green fluorescent protein （EGFP） open reading frames. To assess the function of pEGFP-NI-lac, the nucleotide sequence encoding the hepatitis C virus （HCV） core protein was cloned into the multiple cloning sites. Western blotting analysis was used to detect the expression of the HCV core protein in Escherichia coli DH5a and HepG2 cells. Results： Restriction enzyme digestion and sequence analysis indicated that pEGFP-NI-lac was successfully constructed and the HCV core gene was cloned into this vector. The Western blotting results showed that pEGFP-NI-lac promoted expression of HCV core gene in prokaryotic E. coli DH5a and eukaryotic HepG2 cells. Conclusion： The pEGFP-NI-lac vector has been successfully constructed and functions in both prokaryotic and eukaryotic cells. The EGFP reporter can be used as an insert-inactivation marker for clone selection or as an expression tag. This vector can be used for cloning and expression of genes in both prokaryotic and eukaryotic cells, making gene cloning, expression and functional studies convenient as well as time- and labor-efficient展开更多
Objective： To construct sarvivin shRNA expression vector carting enhanced green fluorescent protein gene, transfect it into GBC-SDH cells via electroporation, and get GBC-SD cells which are stable expressing survivin...Objective： To construct sarvivin shRNA expression vector carting enhanced green fluorescent protein gene, transfect it into GBC-SDH cells via electroporation, and get GBC-SD cells which are stable expressing survivin shRNA. Methods： The siRNA sequence targeting survivin mRNA was synthesized and cloned into pEGFP-H1. The constructed plasmid and pEGFP-H1 were transfected into GBC-SI） cells respectively via liposome, and the transfecting effect was detected with Flow Cytometry. Then the transfected cells were selected with G418. Results： The recombinant plasmid was successfully constructed, named pEGFP-survivin. The gene transfection efficiencies in pEGFP-H1-transfected group and pEGFP-survivin- transfected group were the 80.29% ± 2.71% and 83.85% ±2.34%（P〉0.05）, which was successful to get the cells that are stable expressing shRNA, named GBC-SD/EGFP and GBC-SD/survivin. Conclusion： Survivin shRNA successfully and got GBC-SD cells which are stable expression vector was constructed expression shRNA.展开更多
Objective： To observe the survival and the number of olfactory ensheathing cells （OECs） transplanted in the contused spinal cord, so as to provide a basis for further studying the biological action of OECs.Methods...Objective： To observe the survival and the number of olfactory ensheathing cells （OECs） transplanted in the contused spinal cord, so as to provide a basis for further studying the biological action of OECs.Methods： The rat spinal cords were contused with NYU-impactor Ⅱ at T10 level by dropping a 10 g rod from a height of 25 mm. At the 1st week after injury, OECs isolated freshly from green fluorecense protein （GFP） of the rats were transplanted into the spinal cord at injured site and other two sites 1 mm apart from the caudal and rostral ends with the OECs number of 30000/μl×3 =90000. The survival and the number of OECs were qualitatively and semi-quantitatively observed under the fluorescense microscope from 1 week to 13 weeks after transplantation. The motor function of the cord was evaluated with BBB score.Results： GFP-OECs could survive at least for 13 weeks within the contused spinal cord. Their arrangement was from tight to loose and their number was decreased from 1 week to 13 weeks after injury. The average number of GFP-OECs was 536 at the 1st week, which was less than 1% of the number as compared with original transplantation. After then, the number of GFP-OECs was continually decreased,but the most obvious decrease was found during 1 week to 2 weeks. The extent of decrease at other time points was relatively mild. In contrast to the cell number, motor function of the cord was gradually recovered after transplantation.Conclusions： The survival and the number of GFPOECs are different between the animals and are affected by the pathological reaction of the host cord. Also it is related to the motor function recovery of the contused cord.展开更多
文摘目的制备并用绿色荧光标志骨髓间充质干细胞(BM⁃MSC),观察其对博来霉素所致大鼠肺纤维化损伤的影响。方法60只雄性SD大鼠随机分为5组,A组于气管内注入生理盐水,B、C、D、E组于气管内注入5 mg/kg博来霉素0.1 ml。建模后1 d,A、B组经尾静脉注射生理盐水1.0 ml,C组经尾静脉注射1.0 ml BM⁃MSC(1.0×106细胞),D组尾静脉注射1.0 ml BM⁃MSC(5.0×10^(5)细胞),建模后8 d,再给1.0 ml BM⁃MSC(5.0×10^(5)细胞)。E组尾静脉注射1.0 ml BM⁃MSC(1.0×107细胞)。建模后28、42 d处死各组一半大鼠,留取肺组织。行病理学检查,对HE染色切片评定肺组织肺泡炎程度,对Masson染色切片评定肺组织纤维化程度;测定肺组织的转化生长因子⁃β1(TGF⁃β1)、羟脯氨酸(HYP)、基质金属蛋白酶⁃2(MMP⁃2)和组织金属蛋白酶抑制剂⁃1(TIMP⁃1)的表达水平,分析TGF⁃β1、HYP、MMP⁃2/TIMP⁃1与肺泡炎程度及肺纤维化程度评分的相关性。结果A组肺泡结构正常,无明显的炎症或纤维化改变,B组博来霉素可致肺泡结构严重紊乱,大量胶原纤维沉积,与B组比较,C、D、E组注射BM⁃MSC后肺泡炎性和肺纤维化程度较轻。与A组比较,B组肺泡炎程度评分、肺纤维化程度评分、TGF⁃β1、HYP、MMP⁃2/TIMP⁃1水平增高(均P<0.05),MSC治疗后的C、D、E 3组可使上述指标下降(均P<0.05)。肺组织TGF⁃β1与肺泡炎程度评分、肺纤维化程度评分相关系数r=0.82、0.89,HYP与肺泡炎程度评分、肺纤维化程度评分相关系数r=0.86、0.92,MMP⁃2/TIMP⁃1与肺泡炎程度评分、肺纤维化程度评分相关系数r=0.83、0.92,均存在正相关(均P<0.05)。结论BM⁃MSC可减低博来霉素致肺纤维化损伤,较高剂量和分次移植效果较好,其机制与移植细胞长期存活无关,可能与下调TGF⁃β1水平、改善MMP/TIMP失衡有关。
文摘Green fluorescent protein (GFP) fused to the F-actin binding domain of mouse talin labels the actin cytoskeleton in the living generative and sperm cells of a third generation transgenic rice (Oryza sativa L.) plant, A005-G-T-1-2. Observations were made on pollen at four major developmental stages, viz. I. uni-nucleate microspore stage; II. early bi-cellular pollen stage; III. late bi-cellular pollen stage; and IV. tri-cellular pollen stage. At each of these developmental stages vegetative nucleus, generative nucleus/ cell, and sperm cells were seen undergoing continuous and coordinated motion and migration. These movements seemed to be influenced by associated microfilament networks existing in the pollen. Based on these observations we propose that it is the interaction between the microfilament networks (usually one existing in the central cytoplasm and another in the cortex) that controls the dynamic movement of the vegetative nucleus, generative nucleus/cell and sperm cells. Furthermore, we have also observed that there is an array of microfilaments (oriented mostly parallel to the long axis of the cell) existing in the generative and sperm cells. As far as we are aware, this is the first report showing the existence of microfilaments in living generative and sperm cells of rice pollen. The implication and significance of the existence of microfilaments in generative and sperm cells in rendering self-propelled motion of these cells in relation to their passage and movement in the pollen tube and embryo sac for fertilization were discussed.
基金Supported by The National Natural Science Foundation of China,No. 30972904Jiangsu Provincial Key Medical Center for Hepatobiliary Disease,No. ZX200605
文摘AIM:To investigate the role of bone marrow-derived endothelial progenitor cells(EPCs) in the angiogenesis of hepatocellular carcinoma(HCC).METHODS:The bone marrow of HCC mice was reconstructed by transplanting green fluorescent protein(GFP) + bone marrow cells.The concentration of circulating EPCs was determined by colony-forming assays and fluorescence-activated cell sorting.Serum and tissue levels of vascular endothelial growth factor(VEGF) and colony-stimulating factor(CSF) were quantified by enzyme-linked immunosorbent assay.The distribution of EPCs in tumor and tumor-free tissues was detected by immunohistochemistry and real-time polymerase chain reaction.The incorporation of EPCs into hepatic vessels was examined by immunofluorescence and immunohistochemistry.The proportion of EPCs in vessels was then calculated.RESULTS:The HCC model was successful established.The flow cytometry analysis showed the mean percentage of CD133CD34 and CD133VEGFR2 double positive cells in HCC mice was 0.45% ± 0.16% and 0.20% ± 0.09% respectively.These values are much higher than in the sham-operation group(0.11% ± 0.13%,0.05% ± 0.11%,n = 9) at 14 d after modeling.At 21 d,the mean percentage of circulating CD133CD34 and CD133VEGFR2 cells is 0.23% ± 0.19%,0.25% ± 0.15% in HCC model vs 0.05% ± 0.04%,0.12% ± 0.11% in control.Compared to the transient increase observed in controls,the higher level of circulating EPCs were induced by HCC.In addition,the level of serum VEGF and CSF increased gradually in HCC,reaching its peak 14 d after modeling,then slowly decreased.Consecutive sections stained for the CD133 and CD34 antigens showed that the CD133+ and CD34+ VEGFR2 cells were mostly recruited to HCC tissue and concentrated in tumor microvessels.Under fluorescence microscopy,the bone-marrow(BM)-derived cells labeled with GFP were concentrated in the same area.The relative levels of CD133 and CD34 gene expression were elevated in tumors,around 5.0 and 3.8 times that of the tumor free area.In frozen liver sections from HCC mice,cells co-expressing CD133 and VEGFR2 were identified by immunohistochemical staining using anti-CD133 and VEGFR2 antibodies.In tumor tissue,the double-positive cells were incorporated into vessel walls.In immunofluorescent staining.These CD31 and GFP double positive cells are direct evidence that tumor vascular endothelial cells(VECs) come partly from BM-derived EPCs.The proportion of GFP CD31 double positive VECs(out of all VECs) on day 21 was around 35.3% ± 21.2%.This is much higher than the value recorded on day 7 group(17.1% ± 8.9%).The expression of intercellular adhesion molecule 1,vascular adhesion molecule 1,and VEGF was higher in tumor areas than in tumor-free tissues.CONCLUSION:Mobilized EPCs were found to participate in tumor vasculogenesis of HCC.Inhibiting EPC mobilization or recruitment to tumor tissue may be an efficient strategy for treating HCC.
基金Supported by the National High Technology Research and Development Program of China (863 Program, 2009AA02Z111)the National Natural Science Foundation of China (30872223)the Funds of the State Key Laboratory of Pathogen and Biosecurity
文摘Objective： To facilitate manipulation of gene expression in different host cells, we used pEGFP-N1 as backbone to construct a versatile vector that can drive foreign gene expression in prokaryotic and eukaryotic cells. Methods： A cloning and expression vector, pEGFP-NI-lac, was constructed by inserting the prokaryotic lac promoter of pUC 19 into the eukaryotic expression vector, pEGFP-N1, between the eukaryotic PCMV promoter and enhanced green fluorescent protein （EGFP） open reading frames. To assess the function of pEGFP-NI-lac, the nucleotide sequence encoding the hepatitis C virus （HCV） core protein was cloned into the multiple cloning sites. Western blotting analysis was used to detect the expression of the HCV core protein in Escherichia coli DH5a and HepG2 cells. Results： Restriction enzyme digestion and sequence analysis indicated that pEGFP-NI-lac was successfully constructed and the HCV core gene was cloned into this vector. The Western blotting results showed that pEGFP-NI-lac promoted expression of HCV core gene in prokaryotic E. coli DH5a and eukaryotic HepG2 cells. Conclusion： The pEGFP-NI-lac vector has been successfully constructed and functions in both prokaryotic and eukaryotic cells. The EGFP reporter can be used as an insert-inactivation marker for clone selection or as an expression tag. This vector can be used for cloning and expression of genes in both prokaryotic and eukaryotic cells, making gene cloning, expression and functional studies convenient as well as time- and labor-efficient
文摘Objective： To construct sarvivin shRNA expression vector carting enhanced green fluorescent protein gene, transfect it into GBC-SDH cells via electroporation, and get GBC-SD cells which are stable expressing survivin shRNA. Methods： The siRNA sequence targeting survivin mRNA was synthesized and cloned into pEGFP-H1. The constructed plasmid and pEGFP-H1 were transfected into GBC-SI） cells respectively via liposome, and the transfecting effect was detected with Flow Cytometry. Then the transfected cells were selected with G418. Results： The recombinant plasmid was successfully constructed, named pEGFP-survivin. The gene transfection efficiencies in pEGFP-H1-transfected group and pEGFP-survivin- transfected group were the 80.29% ± 2.71% and 83.85% ±2.34%（P〉0.05）, which was successful to get the cells that are stable expressing shRNA, named GBC-SD/EGFP and GBC-SD/survivin. Conclusion： Survivin shRNA successfully and got GBC-SD cells which are stable expression vector was constructed expression shRNA.
文摘Objective： To observe the survival and the number of olfactory ensheathing cells （OECs） transplanted in the contused spinal cord, so as to provide a basis for further studying the biological action of OECs.Methods： The rat spinal cords were contused with NYU-impactor Ⅱ at T10 level by dropping a 10 g rod from a height of 25 mm. At the 1st week after injury, OECs isolated freshly from green fluorecense protein （GFP） of the rats were transplanted into the spinal cord at injured site and other two sites 1 mm apart from the caudal and rostral ends with the OECs number of 30000/μl×3 =90000. The survival and the number of OECs were qualitatively and semi-quantitatively observed under the fluorescense microscope from 1 week to 13 weeks after transplantation. The motor function of the cord was evaluated with BBB score.Results： GFP-OECs could survive at least for 13 weeks within the contused spinal cord. Their arrangement was from tight to loose and their number was decreased from 1 week to 13 weeks after injury. The average number of GFP-OECs was 536 at the 1st week, which was less than 1% of the number as compared with original transplantation. After then, the number of GFP-OECs was continually decreased,but the most obvious decrease was found during 1 week to 2 weeks. The extent of decrease at other time points was relatively mild. In contrast to the cell number, motor function of the cord was gradually recovered after transplantation.Conclusions： The survival and the number of GFPOECs are different between the animals and are affected by the pathological reaction of the host cord. Also it is related to the motor function recovery of the contused cord.
