Objective To investigate the expression and change of the β3 integrin subunit and fibronectin in normal human oviductal tissue during various phases of the menstrual cycle and tubal ectopic pregnant tissueMethods Sam...Objective To investigate the expression and change of the β3 integrin subunit and fibronectin in normal human oviductal tissue during various phases of the menstrual cycle and tubal ectopic pregnant tissueMethods Samples of normal ( n=29 ) and pregnant fallopian tube ( n=22 ) tissues were obtained from women who had normal cycle and history of normal pregnancy. Normal oviductal tissue samples were divided into 4 groups based on their menstrual cycle. Both expression and distribution of the β3 subunit and fibronectin were determined with the immunohistochemical method and the image analysis.Results The β3 subunit was expressed in the cytoplasm of ciliated cells. The expression level of the β3 subunit was higher after ovulation than that before ovulation in isthmus epithelium (P<0.001), and declined significantly after ovulation in ampullae epithelium (P<0.001). In umbrella epithelium within 4 days after ovulation, the expression level of the β3 subunit was observed at rather higher level among other phases (P <0.001). The ciliated and secretory cells of the epithelium except for where the pregnancy occurred in tubal pregnancy expressed the β3 subunit, and no significant relationship was found between the normal tubal tissue of the secretory phase and tubal ectopic pregnant tissue (P>0.05). Fibronectin was expressed in the basement membrane of human oviductal epithelium and matrix. The expression level of fibronectin was higher in the hyperplastic phase than that in the secretory one (P<0.001). And it was lower in normal tubal tissue of the secretory phase than that in tubal ectopic pregnant tissue (P<0.001).Conclusion Theβ3 integrin subunit was expressed in the ciliated cells of human oviductal epithelium, and fibronectin was expressed in the basement membrane of human oviductal epithelium and matrix. Their expression and change in oviductal tissue is based on different phases of menstrual cycle. The β3 subunit could not related to the occurrence of tubal ectopic pregnancy. Fibronectin could be the potential molecular basis for the tubal ectopic pregnancy.展开更多
Objective To identify single nucleotide polymorphisms (SNPs) of human CatSper gene, themouse homologous gene product, which plays a crucial role in mouse male sterility.Methods We demonstrated a systematic screening o...Objective To identify single nucleotide polymorphisms (SNPs) of human CatSper gene, themouse homologous gene product, which plays a crucial role in mouse male sterility.Methods We demonstrated a systematic screening of SNPs in coding regions and flankingintronic regions of human CatSper gene in a sample subset from a total 210 male individuals byDNA sequencing. Then we used PCR single-strand conformation polymorphism (SSCP) analy-sis to determine the allele frequencies of the possible SNPs among the whole 210 Chinese Hanmale individuals.Results Three SNPs, including two novels, were identified and their allele frequencies weredetermined in the 210 Chinese Han male individuals. These SNPs were assembled into largeSNP database that promises to enable the dissection of the genetic basis of disease.展开更多
Objective To construct a single plasmid vector mediating doxycycline-inducible recombined human insulin gene expr-ession in myotube cell line. Methods An expression cassette of rtTAnls driven by promoter of human cyto...Objective To construct a single plasmid vector mediating doxycycline-inducible recombined human insulin gene expr-ession in myotube cell line. Methods An expression cassette of rtTAnls driven by promoter of human cytomegalovirus and a furin-cuttable recom-bined human insulin expression cassette driven by a reverse poly-tetO DNA motif were cloned into a single plasmid vector (prTR-tetO-mINS). The prTR-tetO-mINS and pLNCX were co-transfected into a myotube cell line (C2C12) and pLNCX vector were used as a control. After selection with G418, the transfected cells were induced with doxycycline at concentra-tions of 0, 2, and 10 μg/mL. RT-PCR was used to determine expression levels of recombinant insulin mRNA at the 5th day. Insulin production in cell cultures medium (at different incubation time) and cell extracts (at the 7th day) were analyzed with human pro/insulin RIA kits. Results Immune reactive insulin (IRI) level in cell medium was found increased at 24 hours of doxycycline incubation, and still increased at the 5th day. After withdrawn of doxycycline, IRI decreased sharply and was at baseline three days later. IRI and human insulin mRNA levels were positively related to different levels of doxycycline. A 25-fold increase in IRI was found against background expression at the 7th day. Conclusion Human insulin expression can be successfully regulated by doxycycline and the background was very low. This single tet-on insulin expression system may provide a new approach to a controlled insulin gene therapy in skeletal muscle.展开更多
基金This is a part of the project supported by Natural Science Foundation of Guangdong Province,China.
文摘Objective To investigate the expression and change of the β3 integrin subunit and fibronectin in normal human oviductal tissue during various phases of the menstrual cycle and tubal ectopic pregnant tissueMethods Samples of normal ( n=29 ) and pregnant fallopian tube ( n=22 ) tissues were obtained from women who had normal cycle and history of normal pregnancy. Normal oviductal tissue samples were divided into 4 groups based on their menstrual cycle. Both expression and distribution of the β3 subunit and fibronectin were determined with the immunohistochemical method and the image analysis.Results The β3 subunit was expressed in the cytoplasm of ciliated cells. The expression level of the β3 subunit was higher after ovulation than that before ovulation in isthmus epithelium (P<0.001), and declined significantly after ovulation in ampullae epithelium (P<0.001). In umbrella epithelium within 4 days after ovulation, the expression level of the β3 subunit was observed at rather higher level among other phases (P <0.001). The ciliated and secretory cells of the epithelium except for where the pregnancy occurred in tubal pregnancy expressed the β3 subunit, and no significant relationship was found between the normal tubal tissue of the secretory phase and tubal ectopic pregnant tissue (P>0.05). Fibronectin was expressed in the basement membrane of human oviductal epithelium and matrix. The expression level of fibronectin was higher in the hyperplastic phase than that in the secretory one (P<0.001). And it was lower in normal tubal tissue of the secretory phase than that in tubal ectopic pregnant tissue (P<0.001).Conclusion Theβ3 integrin subunit was expressed in the ciliated cells of human oviductal epithelium, and fibronectin was expressed in the basement membrane of human oviductal epithelium and matrix. Their expression and change in oviductal tissue is based on different phases of menstrual cycle. The β3 subunit could not related to the occurrence of tubal ectopic pregnancy. Fibronectin could be the potential molecular basis for the tubal ectopic pregnancy.
文摘Objective To identify single nucleotide polymorphisms (SNPs) of human CatSper gene, themouse homologous gene product, which plays a crucial role in mouse male sterility.Methods We demonstrated a systematic screening of SNPs in coding regions and flankingintronic regions of human CatSper gene in a sample subset from a total 210 male individuals byDNA sequencing. Then we used PCR single-strand conformation polymorphism (SSCP) analy-sis to determine the allele frequencies of the possible SNPs among the whole 210 Chinese Hanmale individuals.Results Three SNPs, including two novels, were identified and their allele frequencies weredetermined in the 210 Chinese Han male individuals. These SNPs were assembled into largeSNP database that promises to enable the dissection of the genetic basis of disease.
基金Supported by a grant from the Educational Department of Liaoning Province (99022067).
文摘Objective To construct a single plasmid vector mediating doxycycline-inducible recombined human insulin gene expr-ession in myotube cell line. Methods An expression cassette of rtTAnls driven by promoter of human cytomegalovirus and a furin-cuttable recom-bined human insulin expression cassette driven by a reverse poly-tetO DNA motif were cloned into a single plasmid vector (prTR-tetO-mINS). The prTR-tetO-mINS and pLNCX were co-transfected into a myotube cell line (C2C12) and pLNCX vector were used as a control. After selection with G418, the transfected cells were induced with doxycycline at concentra-tions of 0, 2, and 10 μg/mL. RT-PCR was used to determine expression levels of recombinant insulin mRNA at the 5th day. Insulin production in cell cultures medium (at different incubation time) and cell extracts (at the 7th day) were analyzed with human pro/insulin RIA kits. Results Immune reactive insulin (IRI) level in cell medium was found increased at 24 hours of doxycycline incubation, and still increased at the 5th day. After withdrawn of doxycycline, IRI decreased sharply and was at baseline three days later. IRI and human insulin mRNA levels were positively related to different levels of doxycycline. A 25-fold increase in IRI was found against background expression at the 7th day. Conclusion Human insulin expression can be successfully regulated by doxycycline and the background was very low. This single tet-on insulin expression system may provide a new approach to a controlled insulin gene therapy in skeletal muscle.
