Objective: To investigate the effects of Shengmai Injection(生脉注射液, SMI) on the proliferation, apoptosis and N-myc downstream-regulated gene 2(NDRG2, a tumour suppressor gene) expression in varying densities of hu...Objective: To investigate the effects of Shengmai Injection(生脉注射液, SMI) on the proliferation, apoptosis and N-myc downstream-regulated gene 2(NDRG2, a tumour suppressor gene) expression in varying densities of human hepatic stellate cells LX-2. Methods: LX-2 cells were cultured in vitro. Then, cells were plated in 96-well plates at an approximate density of 2.5×10~4 cells/mL and cultured for 48, 72, 96 or 120 h followed by the application of different concentrations of SMI(0.6, 1.2, 2.4, 4.8 or 6 μL/mL). Cell proliferation was measured after an additional 24 or 48 h using the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay. The effects of SMI on different cell growth states(cultured for 48, 72, 96, or 120 h) were observed by light microscopy at 24 h after treatment. When the cells reached 80% confluence, apoptosis was detected by flow cytometry after 24 h. Lastly, LX-2 cells were treated with different concentrations of SMI and extracted with protein lysis buffer. The levels of NDRG2 were measured by Western blot. Results: When the LX-2 cells grew for 48, 72, 96 and 120 h, 4.8 and 6 μL/m L of SMI significantly inhibited cell proliferation at 24 and 48 h after treatment(P<0.05). And 2.4 μL/mL of SMI also inhibited cell proliferation at 24 h after treatment when cell growth for 48 h(P<0.05) and at 48 h after treatment when cell growth for 72, 96 and 120 h(P<0.05). The NDRG2 expression level in the LX-2 cell was significantly increased when treated with SMI at concentrations of 1.2, 2.4, 4.8 or 6 μL/mL(P<0.05). Conclusions: The inhibitory effects of SMI on the proliferation of LX-2 cells were related to not only concentration dependent but also cell density. In addition, SMI(2.4, 4.8 and 6 μL/mL) could accelerate apoptosis in LX-2 cells, and the mechanism might be associated with NDRG2 over-expression.展开更多
Gene deletion has been a valuable tool for unraveling the mysteries of molecular biology.Early approaches included gene trapping and gene targetting to disrupt or delete a gene randomly or at a specific location,respe...Gene deletion has been a valuable tool for unraveling the mysteries of molecular biology.Early approaches included gene trapping and gene targetting to disrupt or delete a gene randomly or at a specific location,respectively.Using these technologies in mouse embryos led to the generation of mouse knocko ut models and many scientific discoveries.The efficacy and specificity of these approaches have significantly increased with the advent of new technology such as cluste red regula rly inters paced short palindromic repeats for targetted gene deletion.However,several limitations including unwanted off-target gene deletion have hindered their widespread use in the field.Crerecombinase technology has provided additional capacity for cell-specific gene deletion.In this review,we provide a summary of currently available literature on the application of this system for targetted deletion of neuronal genes.This article has been constructed to provide some background info rmation for the new trainees on the mechanism and to provide necessary information for the design,and application of the Cre-recombinase system thro ugh reviewing the most f requent promoters that are currently available for genetic manipulation of neuro ns.We additionally will provide a summary of the latest technological developments that can be used for targeting neurons.This may also serve as a general guide for the selection of appropriate models for biomedical research.展开更多
Two experiments were conducted to investigate the effect of in ovo zinc （Zn） injection on the embryonic development, tissue Zn contents, antioxidation and related gene expressions of fertilized eggs of Arbor Acres b...Two experiments were conducted to investigate the effect of in ovo zinc （Zn） injection on the embryonic development, tissue Zn contents, antioxidation and related gene expressions of fertilized eggs of Arbor Acres broiler breeders. Experiment 1 was conducted to determine an optimal embryonic age for early in ovo injection. A total of 720 fertilized eggs with similar weights were randomly allotted to 4 treatments with 6 replicates per treatment and 30 eggs per replicate in a completely randomized design. The eggs were injected with 0.1 mL sterilized water at 3, 6 and 9 embryonic days of incubation （E3, E6 and E9） or non-injection （the control）, respectively. The results from experiment 1 showed that E3 and E6 injections increased （P〈0.05） the embryonic mortalities, and decreased （P〈0.05） hatchabilities compared to the non-injected control, but no differences （P〉0.05） between E9 injection and the non-injected control were observed in either embryonic mortality or hatchability. The findings suggest that the E9 is the optimal embryonic age for early in ovo injection. In experiment 2, a total of 672 fertilized eggs with similar weights were randomly allocated to 7 treatments with 6 replicates per treatment and 16 eggs per replicate in a completely randomized design. The eggs were injected with 0 （the negative control）, 50, 100, 150, 200, or 250 μg Zn/egg as reagent grade ZnSO4.7H20 in a 0.1-mL solution, or non-injection （the positive control）, respectively at E9-10. The results from the experiment 2 demonstrated that no differences （P〉0.05） among 50 and 100 μg Zn/egg groups and the negative control were observed in the embryonic mortality and hatchability, however, the injection of 200 μg Zn/egg increased （P〈0.05） the embryonic mortality, and injections of 150 and 200 μg Zn/egg decreased （P〈0.05） hatchabilities compared with the controls. The embryonic tibia Zn contents at E20 were increased （P〈0.05） by injections of 150, 200 and 250μg Zn/egg. Zinc inj展开更多
Daily insulin injection is necessary for the treatment of the insulin-dependent diabetes. However, the process is painful and inconvenient. Accordingly, we have made exploratory efforts to establish an alternative met...Daily insulin injection is necessary for the treatment of the insulin-dependent diabetes. However, the process is painful and inconvenient. Accordingly, we have made exploratory efforts to establish an alternative method for continuous insulin supply via intramuscular injection of a designed plasmid encoding the single-strand insulin analogue (SIA), which provides safe, effective and prolonged control of insulin-dependent diabetes. To generate a SIA, a short flexible peptide was alternatively introduced into the natural proinsulin to replace its original long and rigid C-peptide. Then, the synthetic promoter SP301 was used to drive potent and specific expression of SIA in skeletal muscle cells. By combining the Pluronic L64 and low-voltage electropulse (L/E), the specialized gene delivery technique was applied to efficiently transfer the constructed plasmid into skeletal muscle cells via intramuscular injection. Through these efforts, a plasmid-based intramuscular gene expression system was established and improved, making it applicable for gene therapy. The plasmid-expressed SIA showed biological functions that were similar to that of natural insulin. A single L/E-pSP301-SIA administration provided sustained SIA expression in vivo for about 1.5 months. In addition, the diabetic mice treated with L/E-pSP301-SIA were much healthier than those with other treatments. This plasmid-based system was safe for the treatment of diabetes and did not cause immune responses or pathological damage. The results confirmed that, in a mouse model, long-term positive effects were achieved by a single intramuscular L/E-pSP301-SIA injection, which consequently provided reliable experimental basis for its clinical application for the treatment of diabetes mellitus with promising prospects.展开更多
It is reported here that Bt toxin gene has been successfully transferred into maize inbred line by ovary injection for the first time both at home and abroad. One transgenic plant (To) has been confirmed by Southern b...It is reported here that Bt toxin gene has been successfully transferred into maize inbred line by ovary injection for the first time both at home and abroad. One transgenic plant (To) has been confirmed by Southern blotting and PCR test, and 71 progenies (T1) from T0 have been obtained through self-pollination. Of these 71 progenies, seven plants demonstrated positive results in the PCR test; four were used to feed Asian corn borer, and certain effect of insect-resistance was observed. The experiments on the ovary injection in Hainan Province have also been repeated, thus providing new chance to the application of genetic engineering to the maize improvement.展开更多
We previously found that the K141N mutation in heat shock protein B8 （HSPB8） was responsible for Charcot-Marie-Tooth disease type 2L in a large Chinese family. The objective of the present study was to generate a tr...We previously found that the K141N mutation in heat shock protein B8 （HSPB8） was responsible for Charcot-Marie-Tooth disease type 2L in a large Chinese family. The objective of the present study was to generate a transgenic mouse model bearing the K141N mutation in the human HSPB8 gene, and to determine whether this K141NHSPB8 transgenic mouse model would manifest the clinical phenotype of Charcot-Marie-Tooth disease type 2L, and consequently be suitable for use in studies of disease pathogenesis. Transgenic mice overexpressing K141N HSPB8 were generated using K141N mutant HSPB8 cDNA cloned into a pCAGGS plasmid driven by a human cytomegalovirus expression system. PCR and western blot analysis confirmed integration of the KI41NHSPB8 gene and widespread expression in tissues of the transgenic mice. The K141N HSPB8 transgenic mice exhibited decreased muscle strength in the hind limbs and impaired motor coordination, but no obvious sensory disturbance at 6 months of age by behavioral assessment. Electrophysiological analysis showed that the compound motor action potential amplitude in the sciatic nerve was significantly decreased, but motor nerve conduction velocity remained normal at 6 months of age. Pathological analysis of the sciatic nerve showed reduced myelinated fiber density, notable axonal edema and vacuolar degeneration in K141N HSPB8 transgenic mice, suggesting axonal involvement in the peripheral nerve damage in these animals. These findings indicate that the KI4mHSPB8 transgenic mouse successfully models Charcot-Marie-Tooth disease type 2L and can be used to study the pathogenesis of the disease.展开更多
基金Supported by the National Natural Sciences Foundation of China(No.81072973)
文摘Objective: To investigate the effects of Shengmai Injection(生脉注射液, SMI) on the proliferation, apoptosis and N-myc downstream-regulated gene 2(NDRG2, a tumour suppressor gene) expression in varying densities of human hepatic stellate cells LX-2. Methods: LX-2 cells were cultured in vitro. Then, cells were plated in 96-well plates at an approximate density of 2.5×10~4 cells/mL and cultured for 48, 72, 96 or 120 h followed by the application of different concentrations of SMI(0.6, 1.2, 2.4, 4.8 or 6 μL/mL). Cell proliferation was measured after an additional 24 or 48 h using the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay. The effects of SMI on different cell growth states(cultured for 48, 72, 96, or 120 h) were observed by light microscopy at 24 h after treatment. When the cells reached 80% confluence, apoptosis was detected by flow cytometry after 24 h. Lastly, LX-2 cells were treated with different concentrations of SMI and extracted with protein lysis buffer. The levels of NDRG2 were measured by Western blot. Results: When the LX-2 cells grew for 48, 72, 96 and 120 h, 4.8 and 6 μL/m L of SMI significantly inhibited cell proliferation at 24 and 48 h after treatment(P<0.05). And 2.4 μL/mL of SMI also inhibited cell proliferation at 24 h after treatment when cell growth for 48 h(P<0.05) and at 48 h after treatment when cell growth for 72, 96 and 120 h(P<0.05). The NDRG2 expression level in the LX-2 cell was significantly increased when treated with SMI at concentrations of 1.2, 2.4, 4.8 or 6 μL/mL(P<0.05). Conclusions: The inhibitory effects of SMI on the proliferation of LX-2 cells were related to not only concentration dependent but also cell density. In addition, SMI(2.4, 4.8 and 6 μL/mL) could accelerate apoptosis in LX-2 cells, and the mechanism might be associated with NDRG2 over-expression.
文摘Gene deletion has been a valuable tool for unraveling the mysteries of molecular biology.Early approaches included gene trapping and gene targetting to disrupt or delete a gene randomly or at a specific location,respectively.Using these technologies in mouse embryos led to the generation of mouse knocko ut models and many scientific discoveries.The efficacy and specificity of these approaches have significantly increased with the advent of new technology such as cluste red regula rly inters paced short palindromic repeats for targetted gene deletion.However,several limitations including unwanted off-target gene deletion have hindered their widespread use in the field.Crerecombinase technology has provided additional capacity for cell-specific gene deletion.In this review,we provide a summary of currently available literature on the application of this system for targetted deletion of neuronal genes.This article has been constructed to provide some background info rmation for the new trainees on the mechanism and to provide necessary information for the design,and application of the Cre-recombinase system thro ugh reviewing the most f requent promoters that are currently available for genetic manipulation of neuro ns.We additionally will provide a summary of the latest technological developments that can be used for targeting neurons.This may also serve as a general guide for the selection of appropriate models for biomedical research.
基金supported by the Key International Cooperation Program of the National Natural Science Foundation of China (31110103916)the Agricultural Science and Technology Innovation Program, China (ASTIPIAS08)the earmaked fund for the China Agriculture Research System (CARS-42)
文摘Two experiments were conducted to investigate the effect of in ovo zinc （Zn） injection on the embryonic development, tissue Zn contents, antioxidation and related gene expressions of fertilized eggs of Arbor Acres broiler breeders. Experiment 1 was conducted to determine an optimal embryonic age for early in ovo injection. A total of 720 fertilized eggs with similar weights were randomly allotted to 4 treatments with 6 replicates per treatment and 30 eggs per replicate in a completely randomized design. The eggs were injected with 0.1 mL sterilized water at 3, 6 and 9 embryonic days of incubation （E3, E6 and E9） or non-injection （the control）, respectively. The results from experiment 1 showed that E3 and E6 injections increased （P〈0.05） the embryonic mortalities, and decreased （P〈0.05） hatchabilities compared to the non-injected control, but no differences （P〉0.05） between E9 injection and the non-injected control were observed in either embryonic mortality or hatchability. The findings suggest that the E9 is the optimal embryonic age for early in ovo injection. In experiment 2, a total of 672 fertilized eggs with similar weights were randomly allocated to 7 treatments with 6 replicates per treatment and 16 eggs per replicate in a completely randomized design. The eggs were injected with 0 （the negative control）, 50, 100, 150, 200, or 250 μg Zn/egg as reagent grade ZnSO4.7H20 in a 0.1-mL solution, or non-injection （the positive control）, respectively at E9-10. The results from the experiment 2 demonstrated that no differences （P〉0.05） among 50 and 100 μg Zn/egg groups and the negative control were observed in the embryonic mortality and hatchability, however, the injection of 200 μg Zn/egg increased （P〈0.05） the embryonic mortality, and injections of 150 and 200 μg Zn/egg decreased （P〈0.05） hatchabilities compared with the controls. The embryonic tibia Zn contents at E20 were increased （P〈0.05） by injections of 150, 200 and 250μg Zn/egg. Zinc inj
基金supported by the National Natural Science Foundation of China(No.31971390)the International Cooperative Project of Sichuan Province on Science and Technology Innovation(China)(No.2021YFH0142).
文摘Daily insulin injection is necessary for the treatment of the insulin-dependent diabetes. However, the process is painful and inconvenient. Accordingly, we have made exploratory efforts to establish an alternative method for continuous insulin supply via intramuscular injection of a designed plasmid encoding the single-strand insulin analogue (SIA), which provides safe, effective and prolonged control of insulin-dependent diabetes. To generate a SIA, a short flexible peptide was alternatively introduced into the natural proinsulin to replace its original long and rigid C-peptide. Then, the synthetic promoter SP301 was used to drive potent and specific expression of SIA in skeletal muscle cells. By combining the Pluronic L64 and low-voltage electropulse (L/E), the specialized gene delivery technique was applied to efficiently transfer the constructed plasmid into skeletal muscle cells via intramuscular injection. Through these efforts, a plasmid-based intramuscular gene expression system was established and improved, making it applicable for gene therapy. The plasmid-expressed SIA showed biological functions that were similar to that of natural insulin. A single L/E-pSP301-SIA administration provided sustained SIA expression in vivo for about 1.5 months. In addition, the diabetic mice treated with L/E-pSP301-SIA were much healthier than those with other treatments. This plasmid-based system was safe for the treatment of diabetes and did not cause immune responses or pathological damage. The results confirmed that, in a mouse model, long-term positive effects were achieved by a single intramuscular L/E-pSP301-SIA injection, which consequently provided reliable experimental basis for its clinical application for the treatment of diabetes mellitus with promising prospects.
文摘It is reported here that Bt toxin gene has been successfully transferred into maize inbred line by ovary injection for the first time both at home and abroad. One transgenic plant (To) has been confirmed by Southern blotting and PCR test, and 71 progenies (T1) from T0 have been obtained through self-pollination. Of these 71 progenies, seven plants demonstrated positive results in the PCR test; four were used to feed Asian corn borer, and certain effect of insect-resistance was observed. The experiments on the ovary injection in Hainan Province have also been repeated, thus providing new chance to the application of genetic engineering to the maize improvement.
基金funded by the National Natural Science Foundation of China,No.81071001,30900805
文摘We previously found that the K141N mutation in heat shock protein B8 （HSPB8） was responsible for Charcot-Marie-Tooth disease type 2L in a large Chinese family. The objective of the present study was to generate a transgenic mouse model bearing the K141N mutation in the human HSPB8 gene, and to determine whether this K141NHSPB8 transgenic mouse model would manifest the clinical phenotype of Charcot-Marie-Tooth disease type 2L, and consequently be suitable for use in studies of disease pathogenesis. Transgenic mice overexpressing K141N HSPB8 were generated using K141N mutant HSPB8 cDNA cloned into a pCAGGS plasmid driven by a human cytomegalovirus expression system. PCR and western blot analysis confirmed integration of the KI41NHSPB8 gene and widespread expression in tissues of the transgenic mice. The K141N HSPB8 transgenic mice exhibited decreased muscle strength in the hind limbs and impaired motor coordination, but no obvious sensory disturbance at 6 months of age by behavioral assessment. Electrophysiological analysis showed that the compound motor action potential amplitude in the sciatic nerve was significantly decreased, but motor nerve conduction velocity remained normal at 6 months of age. Pathological analysis of the sciatic nerve showed reduced myelinated fiber density, notable axonal edema and vacuolar degeneration in K141N HSPB8 transgenic mice, suggesting axonal involvement in the peripheral nerve damage in these animals. These findings indicate that the KI4mHSPB8 transgenic mouse successfully models Charcot-Marie-Tooth disease type 2L and can be used to study the pathogenesis of the disease.
