This study examined the effect of IL-10 on immunoglobulin-like transcript (ILT4) expression of human monocytic leukemic cell line THP-1, especially the role of the ILT4 promoter activity.ILT4 promoter area was amplifi...This study examined the effect of IL-10 on immunoglobulin-like transcript (ILT4) expression of human monocytic leukemic cell line THP-1, especially the role of the ILT4 promoter activity.ILT4 promoter area was amplified by PCR, and was cloned into the eukaryotic expressing vector pGL3-Basic.The pGL3-ILTP obtained was tested by double endonuclease digestion and sequencing.Then, the recombinant plasmid was transfected into THP-1 cells by using lipofectamine.After culture with IL-10 for 12 h, the mRNA extracted from THP-1 cells was detected by RT-PCR and the protein was detected by FACS.The dual-luciferase reporter assay system was employed to detect the activity of ILT4 promoter with or without IL-10.The results showed that the activity of pGL3-ILTP was significantly increased and was more than ten times that of pGL3-Basic cells.After culture with IL-10 for 12 h, the expression of ILT4 protein and its mean fluorescence intensity (MFI) were increased.Moreover, the mRNA was remarkably higher than that of the control group.Dual-luciferase reporter assay revealed that ILT4 promoter was much more activated after being treated with IL-10.We were led to conclude that pGL3-ILTP containing ILT4 promoter was constructed successfully.The expression of ILT4 could be up-regulated by IL-10 both at the transcriptional and translational level.Furthermore, ILT4 promoter could be much more active after addition of IL-10.This study suggests that IL-10 up-regulates ILT4 expression on monocytes via increasing ILT4 gene promoter activity, which may have implication for inducing transplantation tolerance in clinical practice.展开更多
AIM： The GFAP was traditionally considered to be a biomarker for neural gila （mainly astrocytes and nonmyelinating Schwann cells）. Genetically, a 2.2-kb human GFAP promoter has been successfully used to target astr...AIM： The GFAP was traditionally considered to be a biomarker for neural gila （mainly astrocytes and nonmyelinating Schwann cells）. Genetically, a 2.2-kb human GFAP promoter has been successfully used to target astrocytes in vitro and in vivo. More recently, GFAP was also established as one of the several makers for identifying hepatic stellate cells （HSC）. In this project, possible application of the same 2.2-kb human GFAP promoter for targeting HSC was investigated. METHODS： The GFAP-lacZ transgene was transfected into various cell lines （HSC, hepatocyte, and other nonHSC cell types）. The transgene expression specificity was determined by X-gal staining of the β-galactosidase activity. And the responsiveness of the transgene was tested with a typical pro-fibrotic cytokine TGF-β1. The expression of endogenous GFAP gene was assessed by real-time RT-PCR, providing a reference for the transgene expression. RESULTS： The results demonstrated for the first time that the 2.2 kb hGFAP promoter was not only capable of directing HSC-specific expression, but also responding to a known pro-fibrogenic cytokine TGF-β1 by upregulation in a doseand time-dependent manner, similar to the endogenous GFAP. CONCLUSION： In conclusion, these findings suggested novel utilities for using the GFAP promoter to specifically manipulate HSC for therapeutic purpose.展开更多
The p27Kip1 is a cell cycle repressor protein that regulates primarily the cell cycle transition from G1 to S phase and hence the DNA replication is in the S phase and cell division in the M phase. Expression of p27Ki...The p27Kip1 is a cell cycle repressor protein that regulates primarily the cell cycle transition from G1 to S phase and hence the DNA replication is in the S phase and cell division in the M phase. Expression of p27Kip1 protein has dual roles for both cancer prevention and promotion. For example, numerous nutritional and chemopreventive anti-cancer agents specifically increase the expression of p27Kip1 protein without directly affecting the expression of any other cell cycle regulatory proteins. On the other hand, pro-cancer agents (like glucose, insulin and other growth factors frequently seen in obesity and/or diabetes) specifically decrease the expression of p27Kip1 protein without directly affecting the expression of any other cell cycle regulatory proteins. Unlike expression of any other cell cycle regulatory proteins, expression of p27Kip1 protein is very unusual. The mRNA of p27Kip1 has a very long and unusual 5’-untranslated region (from -575 to -1 in human). It appears that the 5’-untranslated region of p27Kip1 mRNA forms two alternative secondary structures. One increases the expression of p27Kip1 protein when anti-cancer agents are added and another decrease the expression of p27K1p1 when pro-cancer agents are added. For this short concept proposal, Dr. Albert Einstein’s “visualized thought experiments (German: Gedanken experiment)” were used as a fundamental tool for understanding how either anti- or pro-cancer agents bring the primary structure of the 5’-untranslated region of p27Kip1 mRNA into two alternative secondary structures, thereby either increasing or decreasing, respectively, the translation initiation of p27Kip1 protein.展开更多
Background DNA analysis has shown a lack of significant compatibility between couples affected by unexplained recurrent spontaneous abortion (URSA) compared with normal fertile couples, 8 although one study that ma...Background DNA analysis has shown a lack of significant compatibility between couples affected by unexplained recurrent spontaneous abortion (URSA) compared with normal fertile couples, 8 although one study that made use of a PCR-sequence-specific oligonucleotide (SSO) method did observe evidence of significant compatibility in the HLA-DQA1 and DQB1 alleles between patients and aborted fetuses. 9 This study was designed to investigate whether URSA were associated with particular DQ alleles or promoter alleles.Methods Thirty-two patients with URSA and 54 women who had had at least one successful pregnancy were included in this study. HLA-DQ genotyping was performed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The HLA-DQB1 promoter was detected by the SSO and sequence-specific primer (SSP) methods. The DQA1, DQB1, and DQB1 promoter (QBP) gene frequencies in the patients were compared with the gene frequencies in normal controls. The data were analyzed statistically with the χ 2 and Fisher’s exact tests.Results The results showed that the frequency of DQB1 *0604/0605 was significantly higher and the frequency of DQB1 *0501/0502 was significantly lower in the patient group as compared with the normal controls. In addition, the frequencies of the DQA1 *01-DQB1 *0604/0605 and QBP6.2-DQB1 *0604/0605 haplotypes were overrepresented in the patients relative to the controls. Our results did not show any differences between URSA patients and the controls with regard to DQA1 and QBP allele frequencies. Conclusions Our data suggest that URSA is associated with the HLA-DQB1 coding region, and is not associated with its upstream regulatory region. The DQB1 *0604/0605, DQA1 *01-DQB1 *0604/0605, and QBP6.2-DQB1 *0604/0605 haplotypes may confer susceptibility to URSA, while the DQB1 *0501/0502 allele may protect women from URSA.展开更多
基金supported by grants from National Natural Sciences Foundation of China (No.30571755)Specialized Research Fund for the Doctoral Program of Higher Education (No.200804871175)
文摘This study examined the effect of IL-10 on immunoglobulin-like transcript (ILT4) expression of human monocytic leukemic cell line THP-1, especially the role of the ILT4 promoter activity.ILT4 promoter area was amplified by PCR, and was cloned into the eukaryotic expressing vector pGL3-Basic.The pGL3-ILTP obtained was tested by double endonuclease digestion and sequencing.Then, the recombinant plasmid was transfected into THP-1 cells by using lipofectamine.After culture with IL-10 for 12 h, the mRNA extracted from THP-1 cells was detected by RT-PCR and the protein was detected by FACS.The dual-luciferase reporter assay system was employed to detect the activity of ILT4 promoter with or without IL-10.The results showed that the activity of pGL3-ILTP was significantly increased and was more than ten times that of pGL3-Basic cells.After culture with IL-10 for 12 h, the expression of ILT4 protein and its mean fluorescence intensity (MFI) were increased.Moreover, the mRNA was remarkably higher than that of the control group.Dual-luciferase reporter assay revealed that ILT4 promoter was much more activated after being treated with IL-10.We were led to conclude that pGL3-ILTP containing ILT4 promoter was constructed successfully.The expression of ILT4 could be up-regulated by IL-10 both at the transcriptional and translational level.Furthermore, ILT4 promoter could be much more active after addition of IL-10.This study suggests that IL-10 up-regulates ILT4 expression on monocytes via increasing ILT4 gene promoter activity, which may have implication for inducing transplantation tolerance in clinical practice.
基金Supported by the Biomedical Research Councilthe Institute of Bioengineering and Nanotechnology,the Republic of Singapore
文摘AIM： The GFAP was traditionally considered to be a biomarker for neural gila （mainly astrocytes and nonmyelinating Schwann cells）. Genetically, a 2.2-kb human GFAP promoter has been successfully used to target astrocytes in vitro and in vivo. More recently, GFAP was also established as one of the several makers for identifying hepatic stellate cells （HSC）. In this project, possible application of the same 2.2-kb human GFAP promoter for targeting HSC was investigated. METHODS： The GFAP-lacZ transgene was transfected into various cell lines （HSC, hepatocyte, and other nonHSC cell types）. The transgene expression specificity was determined by X-gal staining of the β-galactosidase activity. And the responsiveness of the transgene was tested with a typical pro-fibrotic cytokine TGF-β1. The expression of endogenous GFAP gene was assessed by real-time RT-PCR, providing a reference for the transgene expression. RESULTS： The results demonstrated for the first time that the 2.2 kb hGFAP promoter was not only capable of directing HSC-specific expression, but also responding to a known pro-fibrogenic cytokine TGF-β1 by upregulation in a doseand time-dependent manner, similar to the endogenous GFAP. CONCLUSION： In conclusion, these findings suggested novel utilities for using the GFAP promoter to specifically manipulate HSC for therapeutic purpose.
文摘The p27Kip1 is a cell cycle repressor protein that regulates primarily the cell cycle transition from G1 to S phase and hence the DNA replication is in the S phase and cell division in the M phase. Expression of p27Kip1 protein has dual roles for both cancer prevention and promotion. For example, numerous nutritional and chemopreventive anti-cancer agents specifically increase the expression of p27Kip1 protein without directly affecting the expression of any other cell cycle regulatory proteins. On the other hand, pro-cancer agents (like glucose, insulin and other growth factors frequently seen in obesity and/or diabetes) specifically decrease the expression of p27Kip1 protein without directly affecting the expression of any other cell cycle regulatory proteins. Unlike expression of any other cell cycle regulatory proteins, expression of p27Kip1 protein is very unusual. The mRNA of p27Kip1 has a very long and unusual 5’-untranslated region (from -575 to -1 in human). It appears that the 5’-untranslated region of p27Kip1 mRNA forms two alternative secondary structures. One increases the expression of p27Kip1 protein when anti-cancer agents are added and another decrease the expression of p27K1p1 when pro-cancer agents are added. For this short concept proposal, Dr. Albert Einstein’s “visualized thought experiments (German: Gedanken experiment)” were used as a fundamental tool for understanding how either anti- or pro-cancer agents bring the primary structure of the 5’-untranslated region of p27Kip1 mRNA into two alternative secondary structures, thereby either increasing or decreasing, respectively, the translation initiation of p27Kip1 protein.
基金ThisstudywassupportedbytheNationalNaturalScienceFoundationofChina (No 3 9870 775 )
文摘Background DNA analysis has shown a lack of significant compatibility between couples affected by unexplained recurrent spontaneous abortion (URSA) compared with normal fertile couples, 8 although one study that made use of a PCR-sequence-specific oligonucleotide (SSO) method did observe evidence of significant compatibility in the HLA-DQA1 and DQB1 alleles between patients and aborted fetuses. 9 This study was designed to investigate whether URSA were associated with particular DQ alleles or promoter alleles.Methods Thirty-two patients with URSA and 54 women who had had at least one successful pregnancy were included in this study. HLA-DQ genotyping was performed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The HLA-DQB1 promoter was detected by the SSO and sequence-specific primer (SSP) methods. The DQA1, DQB1, and DQB1 promoter (QBP) gene frequencies in the patients were compared with the gene frequencies in normal controls. The data were analyzed statistically with the χ 2 and Fisher’s exact tests.Results The results showed that the frequency of DQB1 *0604/0605 was significantly higher and the frequency of DQB1 *0501/0502 was significantly lower in the patient group as compared with the normal controls. In addition, the frequencies of the DQA1 *01-DQB1 *0604/0605 and QBP6.2-DQB1 *0604/0605 haplotypes were overrepresented in the patients relative to the controls. Our results did not show any differences between URSA patients and the controls with regard to DQA1 and QBP allele frequencies. Conclusions Our data suggest that URSA is associated with the HLA-DQB1 coding region, and is not associated with its upstream regulatory region. The DQB1 *0604/0605, DQA1 *01-DQB1 *0604/0605, and QBP6.2-DQB1 *0604/0605 haplotypes may confer susceptibility to URSA, while the DQB1 *0501/0502 allele may protect women from URSA.
