Nanog is a newly identified homeodomain gene that functions to sustain the pluripotency of embryonic stem cells.However,the molecular mechanism through which nanog regulates stem cell pluripotency remains unknown.Mous...Nanog is a newly identified homeodomain gene that functions to sustain the pluripotency of embryonic stem cells.However,the molecular mechanism through which nanog regulates stem cell pluripotency remains unknown.Mouse nanog encodes a polypeptide of 305 residues with a divergent homeodomain similar to those in the NK-2 family.The rest ofnanog contains no apparent homology to any known proteins characterized so far.It is hypothesized that nanog encodes a transcription factor that regulates stem cell pluripotency by switching on or off target genes.To test this hypothesis,we constructed fusion proteins between nanog and DNA binding domains of the yeast transcription factor Gal4 and tested the transactivation potentials of these constructs.Our data demonstrate that both regions N- and C- terminal to the homeodomain have transcription activities.Despite the fact that it contains no apparent transactivation motifs,the C-terminal domain is about 7 times as active as the N-terminal one.This unique arrangement of dual transactivators may confer nanog the flexibility and specificity to regulate downstream genes critical for both pluripotency and differentiation of stem cells.展开更多
AIM： To investigate the ability of a genetically altered embryonic stem （ES） cell line to generate insulin-producing cells in vitro following transfer of the Nkx2.2 gene.METHODS： Hamster Nkx2.2 genes were transfer...AIM： To investigate the ability of a genetically altered embryonic stem （ES） cell line to generate insulin-producing cells in vitro following transfer of the Nkx2.2 gene.METHODS： Hamster Nkx2.2 genes were transferred into mouse ES cells. Parental and Nkx2.2-transfected ES cells were initiated toward differentiation in embryoid body （EB） culture for 5 d and the resulting EBs were transferred to an attached culture system. Dithizone （DTZ）, a zincchelating agent known to selectively stain pancreatic beta cells, was used to detect insulin-producing cells.The outgrowths were incubated in DTZ solution （final concentration, 100μg/mL） for 15 rain before being examined microscopically. Gene expression of the endocrine pancreatic markers was also analyzed by RT-PCR. In addition, insulin production was determined immunohistochemically and its secretion was examined using an ELISA.RESULTS： DTZ-stained cellular clusters appeared after approximately 14 d in the culture of Nkx2.2-transfected ES cells （Nkx-ES cells）, which was as much as 2 wk earlier, than those in the culture of parental ES cells （wt-ES）. The frequency of DTZ-positive cells among total cultured cells on day 28 accounted for approximately 1.0% and 0.1% of the Nkx-ES- and wt-ES-derived EB outgrowths, respectively. The DTZ-positive cellular clusters were found to be immunoreactive to insulin, while the gene expressions of pancreatic-duodenal homeobox 1 （PDX1）, proinsulin 1 and proinsulin 2 were observed in the cultures that contained DTZ-positive cellular clusters.Insulin secretion was also confirmed by ELISA, whereas glucose-dependent secretion was not demonstrated.CONCLUSION： Nkx2.2-transfected ES cells showed an ability to differentiate into insulin-producing cells.展开更多
The aim of this study was to develop a synthetic medium for the in vitro culture of bovine embryos, using various growth factors and cytokines （GF-CYK）： insulin-like growth factorl （IGF-Ⅰ）, insulin-like growth f...The aim of this study was to develop a synthetic medium for the in vitro culture of bovine embryos, using various growth factors and cytokines （GF-CYK）： insulin-like growth factorl （IGF-Ⅰ）, insulin-like growth factorⅡ （IGF-Ⅱ）, basic fibroblast growth factor （bFGF）, leukemia inhibitory factor （LIF）, granulocyte-macrophage colony stimulating factor （GM-CSF） and transforming growth factor beta Ⅰ （TGF-β1） ＋ hyaluronan （HA） ＋ recombinant albumin （RA）. The embryos were cultured in synthetic oviduct fluid （SOF） supplemented with： treatment 1 （T1）： bovine serum albumin （BSA） ＋ insulin, transferrin and selenium （ITS） （control）; or treatment 2 （T2）： GF-CYK ＋ HA ＋ RA. The blastocyst rates were not significantly different between TI and T2, at seven days post fertilization （dpf） （28.9% ± 2.4% and 31.8% ±2.2%）, and at 8 dpf （36.5% ±2.4% and 39.1% ±1.9%）, respectively （P 〉 0.05）. The total cell number （TCN） was significantly higher with T2 than that with T1 at 7 dpf（164.9 ±5.3 and 149.7 ±4.0） and 8 dpf （182.7 ±6.4 and 165.0 ±5.5） （P 〈 0.05）. The blastocyst diameter obtained with T2 was significantly greater （P 〈 0.05） than with T1 at 7 dpf （173.3 μm ±4.9 μm and 157.2μm ±4.1 μm, respectively）, however, no significant differences were observed at 8 dpf （190.3 μm 5.2 μm and 179.7 μm ± 5.3 μm, respectively）. In conclusion, the synthetic medium （T2） shows a comparable development rate to the control medium and improves the blastocyst diameter and the TCN.展开更多
The objective of this study was to investigate the relationship between gene expression of nutrient（amino acid, peptide, sodium and proton） transporters in the small intestine and embryonic growth in domestic pigeon...The objective of this study was to investigate the relationship between gene expression of nutrient（amino acid, peptide, sodium and proton） transporters in the small intestine and embryonic growth in domestic pigeons（Columba livia）. One hundred and twenty-five fertilized eggs were randomly assigned into five groups and were incubated under optimal conditions（temperature of 38.1 °C and relative humidity of 55%）. Twenty embryos/birds from each group were sacrificed by cervical dislocation on embryonic day（E） 9, 11, 13, 15 and day of hatch（DOH）. The eggs, embryos（without yolk sac）, and organs（head, brain, heart, liver, lungs, kidney, gizzard, small intestine, legs, and thorax） were dissected, cleaned, and weighed. Small intestine samples were collected for RNA isolation. The m RNA abundance of intestinal nutrient transporters was evaluated by real-time reverse transcription-polymerase chain reaction（RT-PCR）. We classified these ten organs into four types according to the changes in relative weight during embryonic development. In addition, the gene expression of nutrient transporters was differentially regulated by embryonic day. The m RNA abundances of b^0,＋AT, EAAT3, y^＋LAT2, Pep T1, LAT4, NHE2, and NHE3 increased linearly with age, whereas m RNA abundances of CAT1, CAT2, LAT1, EAAT2, SNAT1, and SNAT2 were increased to higher levels on E9 or E11 and then decreased to lower levels until DOH. The results of correlation analysis showed that the gene expressions of b^0,＋AT, EAAT3, Pep T1, LAT4, NHE2, NHE3, and y^＋LAT2 had positive correlations with body weight（0.71〈correlation coefficient（CC）〈0.82, P〈0.0001）, while CAT1, CAT2, EAAT2, SNAT1, and SNAT2 had negative correlations with body weight（-0.86〈CC〈-0.64, P〈0.0001）. The gene expressions of b^0,＋AT, EAAT3, LAT4, Pep T1, NHE2, NHE3, and y^＋LAT2 showed positive correlations with intestinal weight（0.80〈CC〈0.91, P〈0.0001）, while CAT1, CAT2, and EAAT2 showed negative correlations with intestinal weight（-0.84〈CC〈-0.67, P〈0.0001）. It was concluded that the differences between growth trajectories of organs and gene expression of nutrient transporters in small intestine were due to their functional and physiological properties, which provided a comprehensive study of amino acid and peptide transporter m RNA in the small intestine during embryonic growth of pigeons.展开更多
OBJECTIVE:To investigate the effect of Soothing liver therapy on infertile women undergoing in vitro fertilization and embryo transfer(IVF-ET)and to explore its mechanism.METHODS:Fifty-eight women with tubal infertili...OBJECTIVE:To investigate the effect of Soothing liver therapy on infertile women undergoing in vitro fertilization and embryo transfer(IVF-ET)and to explore its mechanism.METHODS:Fifty-eight women with tubal infertility were randomized into two groups:30 in an experimental group treated with Xiaoyao powder(Shugan)plus gonadotropin-releasing hormone analog(GnRHa)/follicle-stimulating hormone(FSH)/human chorionic gonadotropin(hCG)and 28 in the control group who were treated with GnRHa/FSH/hCG only.The total gonadotropin(Gn)doses required,endometrial thickness,oocyte numbers,high quality embryo production rate and pregnancy rate of the two groups were compared.The concentration of growth differentiation factor-9(GDF-9)in follicular fluid was detected by western blotting and the expression of GDF-9 mRNA in granulosa cells was measured using reverse tran-scription-polymerase chain reaction amplification.RESULTS:In the experimental group,the Gn dose was significantly lower than that in the control group;the endometrial thickness,high quality embryo production and pregnancy rates were significantly higher and the expression of GDF-9 mRNA was also significantly higher than in the control group(all P<0.05).CONCLUSION:Shugan treatment can improve the pregnancy rate of women with tubal infertility;its mechanism is possibly related to the increased expression of GDF-9 in granulosa cells.展开更多
基金supported in part by the Tsinghua University BaiRen Scholar Program,NSFC 30270287the 973 Project--2001CB5101 from The Ministry of Science and Technology of China.
文摘Nanog is a newly identified homeodomain gene that functions to sustain the pluripotency of embryonic stem cells.However,the molecular mechanism through which nanog regulates stem cell pluripotency remains unknown.Mouse nanog encodes a polypeptide of 305 residues with a divergent homeodomain similar to those in the NK-2 family.The rest ofnanog contains no apparent homology to any known proteins characterized so far.It is hypothesized that nanog encodes a transcription factor that regulates stem cell pluripotency by switching on or off target genes.To test this hypothesis,we constructed fusion proteins between nanog and DNA binding domains of the yeast transcription factor Gal4 and tested the transactivation potentials of these constructs.Our data demonstrate that both regions N- and C- terminal to the homeodomain have transcription activities.Despite the fact that it contains no apparent transactivation motifs,the C-terminal domain is about 7 times as active as the N-terminal one.This unique arrangement of dual transactivators may confer nanog the flexibility and specificity to regulate downstream genes critical for both pluripotency and differentiation of stem cells.
文摘AIM： To investigate the ability of a genetically altered embryonic stem （ES） cell line to generate insulin-producing cells in vitro following transfer of the Nkx2.2 gene.METHODS： Hamster Nkx2.2 genes were transferred into mouse ES cells. Parental and Nkx2.2-transfected ES cells were initiated toward differentiation in embryoid body （EB） culture for 5 d and the resulting EBs were transferred to an attached culture system. Dithizone （DTZ）, a zincchelating agent known to selectively stain pancreatic beta cells, was used to detect insulin-producing cells.The outgrowths were incubated in DTZ solution （final concentration, 100μg/mL） for 15 rain before being examined microscopically. Gene expression of the endocrine pancreatic markers was also analyzed by RT-PCR. In addition, insulin production was determined immunohistochemically and its secretion was examined using an ELISA.RESULTS： DTZ-stained cellular clusters appeared after approximately 14 d in the culture of Nkx2.2-transfected ES cells （Nkx-ES cells）, which was as much as 2 wk earlier, than those in the culture of parental ES cells （wt-ES）. The frequency of DTZ-positive cells among total cultured cells on day 28 accounted for approximately 1.0% and 0.1% of the Nkx-ES- and wt-ES-derived EB outgrowths, respectively. The DTZ-positive cellular clusters were found to be immunoreactive to insulin, while the gene expressions of pancreatic-duodenal homeobox 1 （PDX1）, proinsulin 1 and proinsulin 2 were observed in the cultures that contained DTZ-positive cellular clusters.Insulin secretion was also confirmed by ELISA, whereas glucose-dependent secretion was not demonstrated.CONCLUSION： Nkx2.2-transfected ES cells showed an ability to differentiate into insulin-producing cells.
文摘The aim of this study was to develop a synthetic medium for the in vitro culture of bovine embryos, using various growth factors and cytokines （GF-CYK）： insulin-like growth factorl （IGF-Ⅰ）, insulin-like growth factorⅡ （IGF-Ⅱ）, basic fibroblast growth factor （bFGF）, leukemia inhibitory factor （LIF）, granulocyte-macrophage colony stimulating factor （GM-CSF） and transforming growth factor beta Ⅰ （TGF-β1） ＋ hyaluronan （HA） ＋ recombinant albumin （RA）. The embryos were cultured in synthetic oviduct fluid （SOF） supplemented with： treatment 1 （T1）： bovine serum albumin （BSA） ＋ insulin, transferrin and selenium （ITS） （control）; or treatment 2 （T2）： GF-CYK ＋ HA ＋ RA. The blastocyst rates were not significantly different between TI and T2, at seven days post fertilization （dpf） （28.9% ± 2.4% and 31.8% ±2.2%）, and at 8 dpf （36.5% ±2.4% and 39.1% ±1.9%）, respectively （P 〉 0.05）. The total cell number （TCN） was significantly higher with T2 than that with T1 at 7 dpf（164.9 ±5.3 and 149.7 ±4.0） and 8 dpf （182.7 ±6.4 and 165.0 ±5.5） （P 〈 0.05）. The blastocyst diameter obtained with T2 was significantly greater （P 〈 0.05） than with T1 at 7 dpf （173.3 μm ±4.9 μm and 157.2μm ±4.1 μm, respectively）, however, no significant differences were observed at 8 dpf （190.3 μm 5.2 μm and 179.7 μm ± 5.3 μm, respectively）. In conclusion, the synthetic medium （T2） shows a comparable development rate to the control medium and improves the blastocyst diameter and the TCN.
基金Project supported by the Spark Program of Guangdong,China(No.2012A020603012)
文摘The objective of this study was to investigate the relationship between gene expression of nutrient（amino acid, peptide, sodium and proton） transporters in the small intestine and embryonic growth in domestic pigeons（Columba livia）. One hundred and twenty-five fertilized eggs were randomly assigned into five groups and were incubated under optimal conditions（temperature of 38.1 °C and relative humidity of 55%）. Twenty embryos/birds from each group were sacrificed by cervical dislocation on embryonic day（E） 9, 11, 13, 15 and day of hatch（DOH）. The eggs, embryos（without yolk sac）, and organs（head, brain, heart, liver, lungs, kidney, gizzard, small intestine, legs, and thorax） were dissected, cleaned, and weighed. Small intestine samples were collected for RNA isolation. The m RNA abundance of intestinal nutrient transporters was evaluated by real-time reverse transcription-polymerase chain reaction（RT-PCR）. We classified these ten organs into four types according to the changes in relative weight during embryonic development. In addition, the gene expression of nutrient transporters was differentially regulated by embryonic day. The m RNA abundances of b^0,＋AT, EAAT3, y^＋LAT2, Pep T1, LAT4, NHE2, and NHE3 increased linearly with age, whereas m RNA abundances of CAT1, CAT2, LAT1, EAAT2, SNAT1, and SNAT2 were increased to higher levels on E9 or E11 and then decreased to lower levels until DOH. The results of correlation analysis showed that the gene expressions of b^0,＋AT, EAAT3, Pep T1, LAT4, NHE2, NHE3, and y^＋LAT2 had positive correlations with body weight（0.71〈correlation coefficient（CC）〈0.82, P〈0.0001）, while CAT1, CAT2, EAAT2, SNAT1, and SNAT2 had negative correlations with body weight（-0.86〈CC〈-0.64, P〈0.0001）. The gene expressions of b^0,＋AT, EAAT3, LAT4, Pep T1, NHE2, NHE3, and y^＋LAT2 showed positive correlations with intestinal weight（0.80〈CC〈0.91, P〈0.0001）, while CAT1, CAT2, and EAAT2 showed negative correlations with intestinal weight（-0.84〈CC〈-0.67, P〈0.0001）. It was concluded that the differences between growth trajectories of organs and gene expression of nutrient transporters in small intestine were due to their functional and physiological properties, which provided a comprehensive study of amino acid and peptide transporter m RNA in the small intestine during embryonic growth of pigeons.
基金Supported by the National Natural Science Foundation of China(No.81173294)
文摘OBJECTIVE:To investigate the effect of Soothing liver therapy on infertile women undergoing in vitro fertilization and embryo transfer(IVF-ET)and to explore its mechanism.METHODS:Fifty-eight women with tubal infertility were randomized into two groups:30 in an experimental group treated with Xiaoyao powder(Shugan)plus gonadotropin-releasing hormone analog(GnRHa)/follicle-stimulating hormone(FSH)/human chorionic gonadotropin(hCG)and 28 in the control group who were treated with GnRHa/FSH/hCG only.The total gonadotropin(Gn)doses required,endometrial thickness,oocyte numbers,high quality embryo production rate and pregnancy rate of the two groups were compared.The concentration of growth differentiation factor-9(GDF-9)in follicular fluid was detected by western blotting and the expression of GDF-9 mRNA in granulosa cells was measured using reverse tran-scription-polymerase chain reaction amplification.RESULTS:In the experimental group,the Gn dose was significantly lower than that in the control group;the endometrial thickness,high quality embryo production and pregnancy rates were significantly higher and the expression of GDF-9 mRNA was also significantly higher than in the control group(all P<0.05).CONCLUSION:Shugan treatment can improve the pregnancy rate of women with tubal infertility;its mechanism is possibly related to the increased expression of GDF-9 in granulosa cells.
