AIM:To investigate the effect of high mobility group A2(HMGA2) gene silencing on gastric cancer MKN-45 cells in vitro.METHODS:HMGA2 short hairpin RNA(shRNA) expression plasmids were constructed,including a pair of ran...AIM:To investigate the effect of high mobility group A2(HMGA2) gene silencing on gastric cancer MKN-45 cells in vitro.METHODS:HMGA2 short hairpin RNA(shRNA) expression plasmids were constructed,including a pair of random scrambled sequences.Human gastric cancer cell line MKN-45 cells were divided into three groups:blank control group(non-transfected cells),transfected group(cells transfected with HMGA2 shRNA recombinant plasmid) and scrambled sequence group(transfected with random scrambled plasmid).Cells were transfected with HMGA2 shRNA recombinant plasmids and scrambled plasmid in vitro,and the cells transfection efficiency was assayed by fluorescence microscopy.The HMGA2 messenger RNA(mRNA) expression was detected by reverse transcription polymerase chain reaction,gastric cancer cells apoptosis was detected by flow cytometry,cell proliferation was detected by methyl thiazol tetrazolium,and the protein expression of phosphatidylinositol 3-kinase(PI3K),protein kinase B(Akt),P27,caspase-9 and B-cell leukemia/lymphoma-2(Bcl-2) were analyzed by Western blotting.RESULTS:Compared with the blank control group and the scrambled sequence group,the levels of HMGA2 mRNA and protein expression in the transfected group were significantly reduced(P < 0.05).The relative HMGA2 mRNA expression levels of the blank control group,transfected group and scrambled sequence group were 0.674 ± 0.129,0.374 ± 0.048 and 0.689 ± 0.124,respectively.The relative HMGA2 protein expression levels of the blank control group,transfected group and scrambled sequence group were 0.554 ± 0.082,0.113 ± 0.032 and 0.484 ± 0.123,respectively.Moreover,transfection with the scrambled sequence had no effect on the expression of HMGA2.After being transfected with shRNA for 24,48 and 72 h,the cell apoptotic rates of the transfected group were 21.65% ± 0.28%,39.98% ± 1.82% and 24.51% ± 0.93%,respectively,which significantly higher than those of blank control group(4.72% ± 1.34%,5.83% ± 0.13% and 5.22% ± 1.07%) and scrambled sequence group(4.28% ± 1.33%,7.87% ± 1.43% and 6.71% ± 0.92%).After 24,48 and 72 h,the cell proliferation inhibition rates in the transfected group were 31.57% ± 1.17%,39.45% ± 2.07% and 37.56% ± 2.32%,respectively;the most obvious cell proliferation inhibition appeared at 48 h after transfection.Compared with the blank control group and scrambled sequence group,after transfection of shRNA for 72 h,the protein expression levels of PI3K(0.042 ± 0.005 vs 0.069 ± 0.003,0.067 ± 0.05),Akt(0.248 ± 0.004 vs 0.489 ± 0.006,0.496 ± 0.104) and Bcl-2(0.295 ± 0.084 vs 0.592 ± 0.072,0.594 ± 0.109) were significantly reduced.The protein expression levels of P27(0.151 ± 0.010 vs 0.068± 0.014,0.060 ± 0.013) and caspase-9(0.136 ± 0.042 vs 0.075 ± 0.010,0.073 ± 0.072) were significantly upregulated.CONCLUSION:HMGA2 shRNA gene silencing induces apoptosis and suppresses proliferation of MKN-45 cells.展开更多
The results from this study showed that the thresholds of brainstem auditory-evoked potentials peak following 10 successive days of intramuscular injection of Roman chickens with kanamycin, starting 3 days after birth...The results from this study showed that the thresholds of brainstem auditory-evoked potentials peak following 10 successive days of intramuscular injection of Roman chickens with kanamycin, starting 3 days after birth. Fluorescence immunohistochemistry analysis revealed few ganglion cells positively labeled for Ephrin A2 in the cochlea of experimental chickens from 2 days before until 7 days after the last kanamycin injection. The number of Ephrin A2-positive ganglion cell bodies was increased at 15 days after the last injection and was similar to that in normal chickens at 30 days following the cessation of kanamycin treatment. These experimental findings indicate that Ephrin A2 protein expression in the acoustic ganglia is synchronized with the connection damage and regeneration of cochlear hair cells after kanamycin exposure. Ephrin A2 may play an important role in the regeneration and plasticity of cochlear hair cells in the chick cochlea following kanamycin ototoxicity.展开更多
Human immunodeficiency virus type-1 (HIV-1) chronic infection causes millions of deaths each year. We previously developed a novel HIV-1 Gag-spe cific exosome (EXO)-targeted T cell-based vaccine (Gag-Texo) using ConAs...Human immunodeficiency virus type-1 (HIV-1) chronic infection causes millions of deaths each year. We previously developed a novel HIV-1 Gag-spe cific exosome (EXO)-targeted T cell-based vaccine (Gag-Texo) using ConAstimulated polyclonal CD8+T (ConA-T) cells armed with Gag-specific dendritic cell (DC)-released EXOs, and showed that Gag-Texo stimulated more efficient cytotoxic T lymphocyte (CTL) responses than DCs. Tat HIV-1 early regulatory protein possesses immunomodulatory and adjuvant properties. To enhance Gag-Texo immunogenicity, we generated Tat-engineered OVA/Tat Texo and Gag/Tat-Texo vaccines using ConA-T cells armed with EXOs release by DCs infected with recombinant OVA/Tat- and Gag/Tat-expressing adenoviruses (AdVOVA/Tat and AdVGag/Tat). We then assessed vaccination-stimulated CTL responses in naive mice, and therapeutic immunity in transgenic HLA-A2 mice bearing Gag/HLA-A2-expressing BL6-10OVA/A2 melanoma lung metastases. We demonstrate that the OVA/Tat-Texo vaccine enhances functional OVA-specific CTL responses, compared to the OVA-Texo vaccine, and broadens CTL responses recognizing the cryptic OVA epitope in C57BL/6 mice. Furthermore, we determine that the Gag/Tat-Texo not only stimulates more efficient CTL responses than Gag-Texo, but also induces enhanced therapeutic immunity. We show that, 30% of Gag/Tat-Texo-immunized mice are free of tumor lung-metastases, compared to all Gag-Texo-immunized mice displaying lung-metastasis. In addition, the average number of tumor lung metastases colonies (32/lung) in the Gag/Tat-Texo-immunized mice was also significantly lower than that (78/lung) observed in Gag-Texo-immunized mice. Taken together, this indicates that HIV-1 Gag/Tat-Texo capable of stimulating enhanced Gag-specific CTL responses and therapeutic immunity may become a new immunotherapeutic vaccine candidate for controlling virus in HIV-1 patients.展开更多
基金Supported by The Natural Science Foundation of Guangxi,No. 2010GXNSFA013166the Key Project of Health Department of Guangxi,No.Zhong 2010021
文摘AIM:To investigate the effect of high mobility group A2(HMGA2) gene silencing on gastric cancer MKN-45 cells in vitro.METHODS:HMGA2 short hairpin RNA(shRNA) expression plasmids were constructed,including a pair of random scrambled sequences.Human gastric cancer cell line MKN-45 cells were divided into three groups:blank control group(non-transfected cells),transfected group(cells transfected with HMGA2 shRNA recombinant plasmid) and scrambled sequence group(transfected with random scrambled plasmid).Cells were transfected with HMGA2 shRNA recombinant plasmids and scrambled plasmid in vitro,and the cells transfection efficiency was assayed by fluorescence microscopy.The HMGA2 messenger RNA(mRNA) expression was detected by reverse transcription polymerase chain reaction,gastric cancer cells apoptosis was detected by flow cytometry,cell proliferation was detected by methyl thiazol tetrazolium,and the protein expression of phosphatidylinositol 3-kinase(PI3K),protein kinase B(Akt),P27,caspase-9 and B-cell leukemia/lymphoma-2(Bcl-2) were analyzed by Western blotting.RESULTS:Compared with the blank control group and the scrambled sequence group,the levels of HMGA2 mRNA and protein expression in the transfected group were significantly reduced(P < 0.05).The relative HMGA2 mRNA expression levels of the blank control group,transfected group and scrambled sequence group were 0.674 ± 0.129,0.374 ± 0.048 and 0.689 ± 0.124,respectively.The relative HMGA2 protein expression levels of the blank control group,transfected group and scrambled sequence group were 0.554 ± 0.082,0.113 ± 0.032 and 0.484 ± 0.123,respectively.Moreover,transfection with the scrambled sequence had no effect on the expression of HMGA2.After being transfected with shRNA for 24,48 and 72 h,the cell apoptotic rates of the transfected group were 21.65% ± 0.28%,39.98% ± 1.82% and 24.51% ± 0.93%,respectively,which significantly higher than those of blank control group(4.72% ± 1.34%,5.83% ± 0.13% and 5.22% ± 1.07%) and scrambled sequence group(4.28% ± 1.33%,7.87% ± 1.43% and 6.71% ± 0.92%).After 24,48 and 72 h,the cell proliferation inhibition rates in the transfected group were 31.57% ± 1.17%,39.45% ± 2.07% and 37.56% ± 2.32%,respectively;the most obvious cell proliferation inhibition appeared at 48 h after transfection.Compared with the blank control group and scrambled sequence group,after transfection of shRNA for 72 h,the protein expression levels of PI3K(0.042 ± 0.005 vs 0.069 ± 0.003,0.067 ± 0.05),Akt(0.248 ± 0.004 vs 0.489 ± 0.006,0.496 ± 0.104) and Bcl-2(0.295 ± 0.084 vs 0.592 ± 0.072,0.594 ± 0.109) were significantly reduced.The protein expression levels of P27(0.151 ± 0.010 vs 0.068± 0.014,0.060 ± 0.013) and caspase-9(0.136 ± 0.042 vs 0.075 ± 0.010,0.073 ± 0.072) were significantly upregulated.CONCLUSION:HMGA2 shRNA gene silencing induces apoptosis and suppresses proliferation of MKN-45 cells.
基金supported by the Natural Science Foundation of Shanghai,No.08ZR1414900 and 11ZR1423600
文摘The results from this study showed that the thresholds of brainstem auditory-evoked potentials peak following 10 successive days of intramuscular injection of Roman chickens with kanamycin, starting 3 days after birth. Fluorescence immunohistochemistry analysis revealed few ganglion cells positively labeled for Ephrin A2 in the cochlea of experimental chickens from 2 days before until 7 days after the last kanamycin injection. The number of Ephrin A2-positive ganglion cell bodies was increased at 15 days after the last injection and was similar to that in normal chickens at 30 days following the cessation of kanamycin treatment. These experimental findings indicate that Ephrin A2 protein expression in the acoustic ganglia is synchronized with the connection damage and regeneration of cochlear hair cells after kanamycin exposure. Ephrin A2 may play an important role in the regeneration and plasticity of cochlear hair cells in the chick cochlea following kanamycin ototoxicity.
文摘Human immunodeficiency virus type-1 (HIV-1) chronic infection causes millions of deaths each year. We previously developed a novel HIV-1 Gag-spe cific exosome (EXO)-targeted T cell-based vaccine (Gag-Texo) using ConAstimulated polyclonal CD8+T (ConA-T) cells armed with Gag-specific dendritic cell (DC)-released EXOs, and showed that Gag-Texo stimulated more efficient cytotoxic T lymphocyte (CTL) responses than DCs. Tat HIV-1 early regulatory protein possesses immunomodulatory and adjuvant properties. To enhance Gag-Texo immunogenicity, we generated Tat-engineered OVA/Tat Texo and Gag/Tat-Texo vaccines using ConA-T cells armed with EXOs release by DCs infected with recombinant OVA/Tat- and Gag/Tat-expressing adenoviruses (AdVOVA/Tat and AdVGag/Tat). We then assessed vaccination-stimulated CTL responses in naive mice, and therapeutic immunity in transgenic HLA-A2 mice bearing Gag/HLA-A2-expressing BL6-10OVA/A2 melanoma lung metastases. We demonstrate that the OVA/Tat-Texo vaccine enhances functional OVA-specific CTL responses, compared to the OVA-Texo vaccine, and broadens CTL responses recognizing the cryptic OVA epitope in C57BL/6 mice. Furthermore, we determine that the Gag/Tat-Texo not only stimulates more efficient CTL responses than Gag-Texo, but also induces enhanced therapeutic immunity. We show that, 30% of Gag/Tat-Texo-immunized mice are free of tumor lung-metastases, compared to all Gag-Texo-immunized mice displaying lung-metastasis. In addition, the average number of tumor lung metastases colonies (32/lung) in the Gag/Tat-Texo-immunized mice was also significantly lower than that (78/lung) observed in Gag-Texo-immunized mice. Taken together, this indicates that HIV-1 Gag/Tat-Texo capable of stimulating enhanced Gag-specific CTL responses and therapeutic immunity may become a new immunotherapeutic vaccine candidate for controlling virus in HIV-1 patients.
