[Objective] This study aimed to investigate the methylation levels of exogenous genes and promoters and the differences of protein expression in transgenic sheep obtained by different transgenic technologies. [Method]...[Objective] This study aimed to investigate the methylation levels of exogenous genes and promoters and the differences of protein expression in transgenic sheep obtained by different transgenic technologies. [Method] Exogenous genes eGFP （enhanced green fluorescent protein） and FGF5 （fibroblast growth factor 5） were separately transformed into sheep by somatic cell cloning, stem cell cloning and perivitelline injection to obtain transgenic sheep, with CMV as the promoter. Bisulfite sequencing method was adopted to detect the methylation status of the promoter region and coding region of exogenous genes in tail tissues of transgenic sheep. Western blot was adopted to detect the expression level of exogenous genes. [Result] The methylation level of the promoter region with stem cell cloning was the highest, followed by somatic cell cloning, while that with perivitelline injection was the lowest; the methylation level of the eGFP coding region with perivitelline injection was the highest, followed by stem cell cloning; the methylation level of the FGF5 coding region with somatic cell cloning was higher than that with perivitelline injection. The exogenous protein expression level was negatively correlated with the methylation level of the promoter region. [Conclusion] This study indicates that different transgenic methods may influence the methylation level of exogenous genes, thus affecting exogenous gene expression.展开更多
The rat chimera is an important animal model for the study of complex human diseases. In the present study we evaluated the chimeric potential of rat inner cell masses （ICMs） and fetal neural stem （FNS） cells. In ...The rat chimera is an important animal model for the study of complex human diseases. In the present study we evaluated the chimeric potential of rat inner cell masses （ICMs） and fetal neural stem （FNS） cells. In result, three rat chimeras were produced by day 5 （D5） Sprague-Dawley （SD） blastocysts injected with ICMs derived from day 6 （D6） and D5 Dark Agouti （DA） blastocysts; four rat chimeras had been generated by D5 DA blastocyst injected with D5 SD ICMs. For the requirement of gene modification, cultured rat inner cell mass cells were assessed to produce chimeras, but no chimeras were generated from injected embryos. The potential to generate chimeras from rFNS and transfected rFNS cells were tested, but no chimeric pups were produced. Only 2 of 41 fetuses derived from D5 DA blastocyst injection with SD LacZ transfected rFNS cells showed very low number of LacZ positive cells in the section. These results indicate that DA and SD rat ICMs arc able to contribute to chimeras, but their potential decreases significantly after culture in vitro （P〈0.05）, and rFNS cells only have the potential to contribute to early fetal development.展开更多
AIM: To compare the influence of different transplant sites in bone marrow mesenchymal stem cell (MSC)-based therapy for liver fibrosis. METHODS: MSCs isolated from Sprague Dawley (SD) rats were induced into hepatocyt...AIM: To compare the influence of different transplant sites in bone marrow mesenchymal stem cell (MSC)-based therapy for liver fibrosis. METHODS: MSCs isolated from Sprague Dawley (SD) rats were induced into hepatocyte-like cells. Liver fibrosis in SD rats was induced with carbon tetrachloride. Following hepatocyte induction in vitro, 4',6-diamidino- 2-phenylindole (DAPI)-labeled MSCs were transplanted by intravenous, intrahepatic, and intraperitoneal injection. Histopathological staining, immunohistochemistry, and biochemical analysis were used to compare the morphological and functional liver regeneration among different MSC injection modalities. The expression differences of interleukins, growth factor, extracellular matrix, matrix metalloproteinases, and tissue inhibitor of metalloproteinase were examined by real-time reverse transcription-polymerase chain reaction (RT-PCR) andenzyme linked immunosorbent assay (ELISA). RESULTS: Four days after exposure to hepatocyte differentiation medium, MSCs that did not express hepatocyte markers could express α-fetoprotein, albumin, and cytokeratin 18. The results of histopathological staining, immunohistochemistry, and biochemical analysis indicated that intravenous injection is more effective at rescuing liver failure than other injection modalities. DAPI-labeled cells were found around liver lobules in all three injection site groups, but the intravenous group had the highest number of cells. PCR and ELISA analysis indicated that interleukin-10 (IL-10) was highest in the intravenous group, whereas il1β, il6, tnfα and tgfβ, which can be regulated by IL10 and are promoters of liver fibrosis, were significantly lower than in the other groups. CONCLUSION: MSC administration is able to protect against liver fibrosis. Intravenous injection is the most favorable treatment modality through promotion of IL10 expression.展开更多
Objective:To explore the mechanism of acupoint injection of bone marrow mesenchymal stem cells(BM-MSCs) in improving blood flow in the rat with hind limb ischemia.Methods:Twenty-four SD rats were randomly divided into...Objective:To explore the mechanism of acupoint injection of bone marrow mesenchymal stem cells(BM-MSCs) in improving blood flow in the rat with hind limb ischemia.Methods:Twenty-four SD rats were randomly divided into 4 groups:normal control group(n=6),model group(n=6),BM-MSCs acupoint injection group(AI group,n=6) and BM-MSC intramuscular injection group(MI group,n=6).Sanyinjiao(SP 6),Housanli(ST 36),Zhaohai(KI 6),Huantiao(GB 30) and Yanglingquan(GB 34) were selected for the AI group,and five non-acupoints were selected on gastrocnemius and adductor of ischemic hind limbs in the MI group.BM-MSCs were injected to the latter two groups.The rat hind limb ischemia model was established with the method of blocking the femoral artery and its branches.Three weeks after injection of BM-MSCs,in each group,hindlimb adductor and gastrocnemius were taken from the ischemic side.Expressions of vascular endothelial growth factor(VEGF) and transfer growth factor-β1(TGF-β1) in the skeletal muscle were determined with immunohistochemical method,and the small arteries in the skeletal muscle were labeled with α-SMA immunohistochemical staining method,the density of small arteries(number of arterioles /number of muscle fibers) and the number of the blood vessel with VEGF positive expression were calculated.The serum levels of VEGF and nitric oxide(NO) were detected.Results:Compared with the model group,the expression of VEGF and TGF-β1,and the density of small arteries and the number of VEGF-positive blood vessels in the AI group and the MI group significantly increased(both P<0.01).Compared with the MI group,the density of small arteries and the number of VEGF-positive blood vessels in the AI group significantly increased(both P<0.01);Compared with the model group and the normal control group,the serum expression quantity of NO and VEGF in the AI group and the MI group were significantly increased(P<0.01).Conclusions:Acuppoint injection of BM-MSCs secrets more VEGF,TGF-β1 and NO to increase angiogenesis and arteriogenesis,so as to improve blood flow of the rats of hind limb ischemic.展开更多
基金Supported by National Natural Science Foundation of China (U1203381)Science and Technology Project of Xinjiang Uygur Autonomous Region (201111113)Science and Technology Support Project of Xinjiang Uygur Autonomous Region (201291147)~~
文摘[Objective] This study aimed to investigate the methylation levels of exogenous genes and promoters and the differences of protein expression in transgenic sheep obtained by different transgenic technologies. [Method] Exogenous genes eGFP （enhanced green fluorescent protein） and FGF5 （fibroblast growth factor 5） were separately transformed into sheep by somatic cell cloning, stem cell cloning and perivitelline injection to obtain transgenic sheep, with CMV as the promoter. Bisulfite sequencing method was adopted to detect the methylation status of the promoter region and coding region of exogenous genes in tail tissues of transgenic sheep. Western blot was adopted to detect the expression level of exogenous genes. [Result] The methylation level of the promoter region with stem cell cloning was the highest, followed by somatic cell cloning, while that with perivitelline injection was the lowest; the methylation level of the eGFP coding region with perivitelline injection was the highest, followed by stem cell cloning; the methylation level of the FGF5 coding region with somatic cell cloning was higher than that with perivitelline injection. The exogenous protein expression level was negatively correlated with the methylation level of the promoter region. [Conclusion] This study indicates that different transgenic methods may influence the methylation level of exogenous genes, thus affecting exogenous gene expression.
文摘The rat chimera is an important animal model for the study of complex human diseases. In the present study we evaluated the chimeric potential of rat inner cell masses （ICMs） and fetal neural stem （FNS） cells. In result, three rat chimeras were produced by day 5 （D5） Sprague-Dawley （SD） blastocysts injected with ICMs derived from day 6 （D6） and D5 Dark Agouti （DA） blastocysts; four rat chimeras had been generated by D5 DA blastocyst injected with D5 SD ICMs. For the requirement of gene modification, cultured rat inner cell mass cells were assessed to produce chimeras, but no chimeras were generated from injected embryos. The potential to generate chimeras from rFNS and transfected rFNS cells were tested, but no chimeric pups were produced. Only 2 of 41 fetuses derived from D5 DA blastocyst injection with SD LacZ transfected rFNS cells showed very low number of LacZ positive cells in the section. These results indicate that DA and SD rat ICMs arc able to contribute to chimeras, but their potential decreases significantly after culture in vitro （P〈0.05）, and rFNS cells only have the potential to contribute to early fetal development.
基金Supported by Millitary Medicine and Health Foundation of China, No. 08Z030
文摘AIM: To compare the influence of different transplant sites in bone marrow mesenchymal stem cell (MSC)-based therapy for liver fibrosis. METHODS: MSCs isolated from Sprague Dawley (SD) rats were induced into hepatocyte-like cells. Liver fibrosis in SD rats was induced with carbon tetrachloride. Following hepatocyte induction in vitro, 4',6-diamidino- 2-phenylindole (DAPI)-labeled MSCs were transplanted by intravenous, intrahepatic, and intraperitoneal injection. Histopathological staining, immunohistochemistry, and biochemical analysis were used to compare the morphological and functional liver regeneration among different MSC injection modalities. The expression differences of interleukins, growth factor, extracellular matrix, matrix metalloproteinases, and tissue inhibitor of metalloproteinase were examined by real-time reverse transcription-polymerase chain reaction (RT-PCR) andenzyme linked immunosorbent assay (ELISA). RESULTS: Four days after exposure to hepatocyte differentiation medium, MSCs that did not express hepatocyte markers could express α-fetoprotein, albumin, and cytokeratin 18. The results of histopathological staining, immunohistochemistry, and biochemical analysis indicated that intravenous injection is more effective at rescuing liver failure than other injection modalities. DAPI-labeled cells were found around liver lobules in all three injection site groups, but the intravenous group had the highest number of cells. PCR and ELISA analysis indicated that interleukin-10 (IL-10) was highest in the intravenous group, whereas il1β, il6, tnfα and tgfβ, which can be regulated by IL10 and are promoters of liver fibrosis, were significantly lower than in the other groups. CONCLUSION: MSC administration is able to protect against liver fibrosis. Intravenous injection is the most favorable treatment modality through promotion of IL10 expression.
基金supported by the TCM Technology Project of Beijing (No.JJ2007-026)
文摘Objective:To explore the mechanism of acupoint injection of bone marrow mesenchymal stem cells(BM-MSCs) in improving blood flow in the rat with hind limb ischemia.Methods:Twenty-four SD rats were randomly divided into 4 groups:normal control group(n=6),model group(n=6),BM-MSCs acupoint injection group(AI group,n=6) and BM-MSC intramuscular injection group(MI group,n=6).Sanyinjiao(SP 6),Housanli(ST 36),Zhaohai(KI 6),Huantiao(GB 30) and Yanglingquan(GB 34) were selected for the AI group,and five non-acupoints were selected on gastrocnemius and adductor of ischemic hind limbs in the MI group.BM-MSCs were injected to the latter two groups.The rat hind limb ischemia model was established with the method of blocking the femoral artery and its branches.Three weeks after injection of BM-MSCs,in each group,hindlimb adductor and gastrocnemius were taken from the ischemic side.Expressions of vascular endothelial growth factor(VEGF) and transfer growth factor-β1(TGF-β1) in the skeletal muscle were determined with immunohistochemical method,and the small arteries in the skeletal muscle were labeled with α-SMA immunohistochemical staining method,the density of small arteries(number of arterioles /number of muscle fibers) and the number of the blood vessel with VEGF positive expression were calculated.The serum levels of VEGF and nitric oxide(NO) were detected.Results:Compared with the model group,the expression of VEGF and TGF-β1,and the density of small arteries and the number of VEGF-positive blood vessels in the AI group and the MI group significantly increased(both P<0.01).Compared with the MI group,the density of small arteries and the number of VEGF-positive blood vessels in the AI group significantly increased(both P<0.01);Compared with the model group and the normal control group,the serum expression quantity of NO and VEGF in the AI group and the MI group were significantly increased(P<0.01).Conclusions:Acuppoint injection of BM-MSCs secrets more VEGF,TGF-β1 and NO to increase angiogenesis and arteriogenesis,so as to improve blood flow of the rats of hind limb ischemic.
