Human primary epithelial cells of renal pelvis was established to investigate the adherence of uropathogenic Escherichia coli (UPEC) to this cell line, in which the primary cell culture was performed by using cultivat...Human primary epithelial cells of renal pelvis was established to investigate the adherence of uropathogenic Escherichia coli (UPEC) to this cell line, in which the primary cell culture was performed by using cultivation of the normal epithelium of renal pelvis in keratinocyte serum free medium (K-SFM) with epidermal growth factor (EGF) and bovine pituitary extract (BPE). Both UPEC132 obtained from urine specimen of patients with pyelonephritis and the pilus-free representative strain E. coli K-12p678-54 were used to study the adherence of these strains on human primary epithelial cells of renal pelvis. The UPEC adherence was performed with observation on the morphological changes of the adhered cells, while the adhesion rates and indices were calculated in different times of experiment. In addition, the virulence genes hly and cnf1 of UPEC132 were detected by multiplex PCR assay. In this study, the human primary epithelial cells of renal pelvis was found to exhibit the character of the transitional epithelial cells. Compared with the control group, the adhesion rates and indices began to increase from 15 min of the experiment time and reached its peak in 120 min. The adhesion rate and index of UPEC132 to human primary epithelial cells of renal pelvis were 74.4% and 34.0 respectively. Many microscopic changes in the primary cells adhered with UPEC132 could be detected, such as rounding or irregularity in shape, unevenness in staining and the cytoplasmic and nuclear changes. It suggests that human primary epithelial cells of renal pelvis can be used for the experiment on UPEC adhesion, thus providing a basis for the further study on the pathogenesis of UPEC.展开更多
To investigate the role of connective tissue growth factor (CTGF) in transdifferentiation of human renal tubular epithelial cell (HKC), in vitro cultured HKC cells were divided into 3 groups: negtive control, low dose...To investigate the role of connective tissue growth factor (CTGF) in transdifferentiation of human renal tubular epithelial cell (HKC), in vitro cultured HKC cells were divided into 3 groups: negtive control, low dose CTGF-treated group (rh CTGF, 2.5 ng/ml) and high dose CTGF-treated (rhCTGF, 5.0 ng/ml). Then the expression of α-smooth muscle actin (α-SMA) were assessed by indirect immuno-fluorescence, and the percentage of α-SMA positive cells were assessed by flow cytometry. RT-PCR were also performed to examine the mRNA level of α-SMA. Upon the stimulation of different concentrations of rhCTGF, the expression of α-SMA were markedly stronger than that in negative controls. The percentages of α-SMA positive cells were significantly higher in the stimulated groups than that of negative controls (38.9 %, 65.5 % vs 2.4 %, P<0.01) .α-SMA mRNA levels were also up-regulated by the stimulation of rhCTGF (P<0.01). These results suggest that CTGF can promote the transdifferentiation of human renal tubular epithelial cells towards myofibroblast (Myo-F).展开更多
AIM: To enrich hepatic progenitors using epithelial cell adhesion molecule (EpCAM) as a marker from human fetal liver and investigate the expression of human leukocyte antigen (HLA) and their markers associated with h...AIM: To enrich hepatic progenitors using epithelial cell adhesion molecule (EpCAM) as a marker from human fetal liver and investigate the expression of human leukocyte antigen (HLA) and their markers associated with hepatic progenitor cells. METHODS: EpCAM +ve cells were isolated using magnetic cell sorting (MACS) from human fetuses (n = 10) at 15-25 wk gestation. Expression of markers for hepatic progenitors such as albumin, alpha-fetoprotein (AFP), CD29 (integrin β1), CD49f (integrin α6) and CD90 (Thy 1) was studied by using flow cytometry, immunocytochemistry and RT-PCR; HLA class Ⅰ (A, B, C) and class Ⅱ (DR) expression was studied by flow cytometry only. RESULTS: FACS analysis indicated that EpCAM +ve cells were positive for CD29, CD49f, CD90, CD34, HLA class Ⅰ, albumin and AFP but negative for HLA class Ⅱ (DR) and CD45. RT PCR showed that EpCAM +ve cells expressed liver epithelial markers (CK18), biliary specific marker (CK19) and hepatic markers (albumin, AFP). On immunocytochemical staining, EpCAM +ve cells were shown positive signals for CK18 and albumin. CONCLUSION: Our study suggests that these EpCAM +ve cells can be used as hepatic progenitors for cell transplantation with a minimum risk of alloreactivity and these cells may serve as a potential source for enrichment of hepatic progenitor.展开更多
目的研究二甲双胍(metformin,Met)通过抑制NF-κBNLRP3通路改善草酸钙(Calcium Oxalate,CaOx)诱导人肾小管上皮细胞(HK-2)损伤。方法分别随机将人肾小管上皮细胞分成对照组、实验组(CaOx+HK-2)、高浓度Met组(CaOx+HK-2+1.2 mM Met)、低...目的研究二甲双胍(metformin,Met)通过抑制NF-κBNLRP3通路改善草酸钙(Calcium Oxalate,CaOx)诱导人肾小管上皮细胞(HK-2)损伤。方法分别随机将人肾小管上皮细胞分成对照组、实验组(CaOx+HK-2)、高浓度Met组(CaOx+HK-2+1.2 mM Met)、低浓度Met组(CaOx+HK-2+0.80 mM Met)、通路干预组(CaOx+HK-2+PDTC)五组。各组细胞培养24 h,采用酶联免疫吸附测定(Elisa)法检测细胞上清液中炎症因子(IL-6、IL-18、IL-1β),采用逆转录-聚合酶链反应(RT-PCR)法检测细胞中NF-κB、NLRP3、骨桥蛋白(OPN)mRNA。结果各组间细胞上清液IL-6、IL-18、IL-1β含量相比差异均有统计学意义(P均<0.05);各组间细胞中NF-κBmRNA表达量、NLRP3mRNA表达量、OPNmRNA表达量相比差异均有统计学意义(P均<0.05)。结论1.20 mmol/L或0.80 mmol/L Met可以通过抑制NF-κB/NLRP3通路调控下游的炎症相关蛋白及黏附蛋白表达,继而改善CaOx诱导人肾小管上皮细胞损伤及减少草酸钙肾结石形成。展开更多
[Objective] The aim of this study is to understand the effects of donor cell type,embryo stage,number and transfer position on the efficiency of goat transgenic clone.[Method] Using somatic cell nuclear transfer techn...[Objective] The aim of this study is to understand the effects of donor cell type,embryo stage,number and transfer position on the efficiency of goat transgenic clone.[Method] Using somatic cell nuclear transfer technology,the single goat fetal fibroblasts(GFF)and mammary gland epithelial cells(GMGE)harboring human lactoferrin(hLF)gene were transferred to the enucleated oocyte.Reconstructed karyoplast-cytoplast couplets were fused,activated,and cultured in vitro.Embryos at 2-8 cell stage were transferred into oviduct of synchronized recipients,and blastocysts were transferred into uterine horn.[Result] The pregnancy rate was similar between GFF and GMGE(oviduct transfer:26.47% vs.20.00%),and between oviduct transfer and uterine horn transfer(26.47% vs.25.00%)for GFF group;pregnancy rate in the group with the mean number of embryo transferred per recipient of 21.2 was significantly higher than in those the 5.93 group and 9.64 group(40.00% vs.26.67% and 21.43%).[Conclusion] These results indicate that pregnancy rate of goat transgenic clone couldn't be affected by donor cell type,embryo stage and transfer position but be done by the number of embryo transferred per recipient.In addition,the study also suggests the feasibility of making transgenic goat using GMGE as donor cells.展开更多
基金This work was supported by the grants from the National Natural Science Foundation of China （30470096）.
文摘Human primary epithelial cells of renal pelvis was established to investigate the adherence of uropathogenic Escherichia coli (UPEC) to this cell line, in which the primary cell culture was performed by using cultivation of the normal epithelium of renal pelvis in keratinocyte serum free medium (K-SFM) with epidermal growth factor (EGF) and bovine pituitary extract (BPE). Both UPEC132 obtained from urine specimen of patients with pyelonephritis and the pilus-free representative strain E. coli K-12p678-54 were used to study the adherence of these strains on human primary epithelial cells of renal pelvis. The UPEC adherence was performed with observation on the morphological changes of the adhered cells, while the adhesion rates and indices were calculated in different times of experiment. In addition, the virulence genes hly and cnf1 of UPEC132 were detected by multiplex PCR assay. In this study, the human primary epithelial cells of renal pelvis was found to exhibit the character of the transitional epithelial cells. Compared with the control group, the adhesion rates and indices began to increase from 15 min of the experiment time and reached its peak in 120 min. The adhesion rate and index of UPEC132 to human primary epithelial cells of renal pelvis were 74.4% and 34.0 respectively. Many microscopic changes in the primary cells adhered with UPEC132 could be detected, such as rounding or irregularity in shape, unevenness in staining and the cytoplasmic and nuclear changes. It suggests that human primary epithelial cells of renal pelvis can be used for the experiment on UPEC adhesion, thus providing a basis for the further study on the pathogenesis of UPEC.
基金ThisworkwassupportedbyagrantfromtheScience&TechnologyFoundationofHubeiProvince (No .2 0 0 3AA30 1C14 )
文摘To investigate the role of connective tissue growth factor (CTGF) in transdifferentiation of human renal tubular epithelial cell (HKC), in vitro cultured HKC cells were divided into 3 groups: negtive control, low dose CTGF-treated group (rh CTGF, 2.5 ng/ml) and high dose CTGF-treated (rhCTGF, 5.0 ng/ml). Then the expression of α-smooth muscle actin (α-SMA) were assessed by indirect immuno-fluorescence, and the percentage of α-SMA positive cells were assessed by flow cytometry. RT-PCR were also performed to examine the mRNA level of α-SMA. Upon the stimulation of different concentrations of rhCTGF, the expression of α-SMA were markedly stronger than that in negative controls. The percentages of α-SMA positive cells were significantly higher in the stimulated groups than that of negative controls (38.9 %, 65.5 % vs 2.4 %, P<0.01) .α-SMA mRNA levels were also up-regulated by the stimulation of rhCTGF (P<0.01). These results suggest that CTGF can promote the transdifferentiation of human renal tubular epithelial cells towards myofibroblast (Myo-F).
基金Council of Scientific and Industrial Research Network Grant CMM002ICMR Grant (GAP 0215)
文摘AIM: To enrich hepatic progenitors using epithelial cell adhesion molecule (EpCAM) as a marker from human fetal liver and investigate the expression of human leukocyte antigen (HLA) and their markers associated with hepatic progenitor cells. METHODS: EpCAM +ve cells were isolated using magnetic cell sorting (MACS) from human fetuses (n = 10) at 15-25 wk gestation. Expression of markers for hepatic progenitors such as albumin, alpha-fetoprotein (AFP), CD29 (integrin β1), CD49f (integrin α6) and CD90 (Thy 1) was studied by using flow cytometry, immunocytochemistry and RT-PCR; HLA class Ⅰ (A, B, C) and class Ⅱ (DR) expression was studied by flow cytometry only. RESULTS: FACS analysis indicated that EpCAM +ve cells were positive for CD29, CD49f, CD90, CD34, HLA class Ⅰ, albumin and AFP but negative for HLA class Ⅱ (DR) and CD45. RT PCR showed that EpCAM +ve cells expressed liver epithelial markers (CK18), biliary specific marker (CK19) and hepatic markers (albumin, AFP). On immunocytochemical staining, EpCAM +ve cells were shown positive signals for CK18 and albumin. CONCLUSION: Our study suggests that these EpCAM +ve cells can be used as hepatic progenitors for cell transplantation with a minimum risk of alloreactivity and these cells may serve as a potential source for enrichment of hepatic progenitor.
文摘目的研究二甲双胍(metformin,Met)通过抑制NF-κBNLRP3通路改善草酸钙(Calcium Oxalate,CaOx)诱导人肾小管上皮细胞(HK-2)损伤。方法分别随机将人肾小管上皮细胞分成对照组、实验组(CaOx+HK-2)、高浓度Met组(CaOx+HK-2+1.2 mM Met)、低浓度Met组(CaOx+HK-2+0.80 mM Met)、通路干预组(CaOx+HK-2+PDTC)五组。各组细胞培养24 h,采用酶联免疫吸附测定(Elisa)法检测细胞上清液中炎症因子(IL-6、IL-18、IL-1β),采用逆转录-聚合酶链反应(RT-PCR)法检测细胞中NF-κB、NLRP3、骨桥蛋白(OPN)mRNA。结果各组间细胞上清液IL-6、IL-18、IL-1β含量相比差异均有统计学意义(P均<0.05);各组间细胞中NF-κBmRNA表达量、NLRP3mRNA表达量、OPNmRNA表达量相比差异均有统计学意义(P均<0.05)。结论1.20 mmol/L或0.80 mmol/L Met可以通过抑制NF-κB/NLRP3通路调控下游的炎症相关蛋白及黏附蛋白表达,继而改善CaOx诱导人肾小管上皮细胞损伤及减少草酸钙肾结石形成。
基金Supported by the National High-tech R&D Program(2004AA213072)the Doctor Fund of Henan University of Science and Technology~~
文摘[Objective] The aim of this study is to understand the effects of donor cell type,embryo stage,number and transfer position on the efficiency of goat transgenic clone.[Method] Using somatic cell nuclear transfer technology,the single goat fetal fibroblasts(GFF)and mammary gland epithelial cells(GMGE)harboring human lactoferrin(hLF)gene were transferred to the enucleated oocyte.Reconstructed karyoplast-cytoplast couplets were fused,activated,and cultured in vitro.Embryos at 2-8 cell stage were transferred into oviduct of synchronized recipients,and blastocysts were transferred into uterine horn.[Result] The pregnancy rate was similar between GFF and GMGE(oviduct transfer:26.47% vs.20.00%),and between oviduct transfer and uterine horn transfer(26.47% vs.25.00%)for GFF group;pregnancy rate in the group with the mean number of embryo transferred per recipient of 21.2 was significantly higher than in those the 5.93 group and 9.64 group(40.00% vs.26.67% and 21.43%).[Conclusion] These results indicate that pregnancy rate of goat transgenic clone couldn't be affected by donor cell type,embryo stage and transfer position but be done by the number of embryo transferred per recipient.In addition,the study also suggests the feasibility of making transgenic goat using GMGE as donor cells.
