1.Genistein inhibited hypoxia inducible factor-1? expression induced by cobalt chloride in human retinal pigment epithelium cells
Hua LI ; Hong YAN ; Jinshun PAN ; Bin WANG
Journal of Medical Postgraduates 2003;0(11):-
Objective:The time-dependent changes of hypoxia-inducible factor-1? (HIF-1?) expression induced by CoCl_(2 ) treatment and the effects of genistein on the level of HIF-1? expression in human retinal pigment epithelium cells were studied. Methods:Using a confocal scanning laser microscope coupled to a computer, HIF-1? expression was determined. Results:CoCl_(2 ) treatment could significantly elevate the level of HIF-1? expression. At 0.5 hour after CoCl_(2 ) treatment, the highest level was observed. Genistein 50 ?mol/L, 100 ?mol/L, 200 ?mol/L could suppress HIF-1? expression in a concentration-dependent manner. Conclusions:These results suggested that genistein could inhibit HIF-1? expression induced by CoCl_(2).
2.Killing K562 cells by RNA interference compared with imatinib
Xiangfang GU ; Xuemei SUN ; Junhao CHEN ; Yong LIU ; Jinshun PAN
Cancer Research and Clinic 2008;20(11):730-733
Objective To compare RNA interference (RNAi) with imatinib in killing K562 cells. Methods Design effective shRNA sequences special for bcr-abl silencing and insert them into the eukaryotic expression vector for RNAi by gene engineering. The recombinant plasmi(ts were then transfected into K562 cells. 48 hours later, the efficiency of transfection was identified by fluorescent microscope, bcr-abl mRNA level was detected by RT-PCR. Another group of K562 cells were treated respectively by imatinib with different concentration. All groups of K562 cells were finally analyzed in apoptosis, cell proliferation and phosphotyrosine-containing proteins. Results Both RNAi and imatinib induced apoptosis, decreased proliferation and reduced phosphotyrosine-containing proteins. Conclusion BNAi can kill K562 cells successfully as imatinib, and it may be a promising way to treat CML patients in clinic, especially for those who fail in imatinib or other chemotherapy.
3.Redution of false positivities of IgM antibodies against hepatitis E virus by a truncated immunodominant polypepfide of HEY open reading frame 2
Yongchun BI ; Jinshun PAN ; Hui ZHUANG ; Jing SUN ; Chao WU ; Qin TANG ; Yihua ZHOU
Chinese Journal of Laboratory Medicine 2009;32(7):821-824
Objective To exclude false positivities in detection of IgM antibodies against hepatitis E vires of genotype 4 (HEV-4) using a truncated immunodominant polypeptide of HEV open reading frames (ORF2). Methods The recombinant ORF2 immunodominant polypeptide corresponding to amino acids (AA) 459-607 and a truncated polypeptide corresponding to AA 472-607 were separately applied to coat ELISA plates. Anti-HEV IgM from 35 serum samples with HEV RNA positive, 69 serum samples from healthy individuals and 117 clinically suspicious HEV RNA positive serum samples was detected by an indirect ELISA and was confirmed by western blot in protein level and RT-PCR detecting in RNA level. Results Western blot analysis showed that the sera from HEV patients reacted with the dimmer of peptide 459-607, but they didn't react with the monomer and peptide 472-607. The ELISA showed that all 35 serum HEV RNA positive samples reacted with peptide 459-607 but not with peptide 472-607 and none of the 69 serum samples from healthy individuals reacted with either polypeptide. Among 117 chnically suspicious HEV RNA serum samples, 5 samples reacted simultaneously with both polypeptides. But the difference between 450 nm absorbance (A450) value was less than 0. 5. Western blot analysis demonstrated that all the 5 serum samples were anti-HEV IgM- negative. The 5 serum samples was detected negative by RT-PCR, indicating that the false pesitivities were caused by non-specific absorption. Conclusions ORF2 peptide 459-607 may be used to detect anti-HEV lgM efficiently. The false positivities caused by non-specific absorption can be largely excluded according to the difference between 45Ohm absorbance (A450) value when serum reacts with both polypeptides.
4.Characterization of the homodimer and antigenicity of ORF2 polypeptides of genotype 4 hepatitis Evirus
Jinshun PAN ; Hui ZHUANG ; Chao WU ; Jinyong ZHOU ; Qin TANG ; Yihua ZHOU
Chinese Journal of Microbiology and Immunology 2008;28(11):993-997
Objective To characterize the dimerization and the antigenicity of the ORF2 polypep-tide of hepatitis E virus (HEV, genotype 4). Methods HEV ORF2 gene was cloned from the serum of a patient with hepatitis E. The genotype was determined by sequencing. Three ORF2 polypeptides differing in size and other polypeptides with point mutations were produced in E. coli. The recombinant polypeptides were purified and analyzed by SDS-PAGE and Western blot. Results The ORF2 polypeptide containing 459-607 amino acid formed homedimer even in 8 mol/L urea. The truncated polypeptides containing amino acid 472-607 or 459-594 formed monomer only. The mutations at amino acid 562 or 595 disrupted the ho-modimer, whereas the mutations at amino acid 476 or 580 did not. Anti-HEV from hepatitis E patients only reacted with the homodimer form of the polypeptide 459-607 and did not react with monomer or tnmcated pol-ypeptides. Conclusion The amino acid 459-607 of HEV ORF2 is essential for dimerization of the ORF2 polypeptide. Residues at amino acid 562 and 595 are critical for the dimerization. The antigenicity of the polypeptide 459-607 mainly depends on its homodimer form.
5.The inhibitory effect of Withaferin A on the growth of orthotopic xenograft tumor of hepatocellular carcinoma in nude mice and the mechanism
Xianmin MU ; Wei SHI ; Yue XU ; Shi HU ; Jing YANG ; Che XU ; Chen ZHANG ; Jinshun PAN ; Biao GENG ; Qiang YOU
Journal of Chinese Physician 2017;19(12):1800-1803,1806
Objective To investigate the inhibitory effect of Withaferin A ( WFA) on the growth of orthotopic xenograft tumor of hepatocellular carcinoma in nude mice and the mechanism of its antitumoral effect. Methods For in vivo model, anti-tumor efficacy of Withaferin A was evaluated in nude mice mod-els of human liver cancer orthotopic xenograft. The nude mice were randomly divided into model group, Sunitinib group,and Withaferin A groups [6, 3 mg/(kg·d)]. All mice were given intraperitoneal injec-tion for 14 days. Tumor volume and tumor weight were observed. Antiangiogenic effects were assessed in vi-vo by the tumor inhibition rate and microvessel density. Quantitative polymerase chain reaction ( QPCR) as-say was used to detect the mRNA expression of vascular endothelial growth factor ( VEGF) , basic fibroblast growth factor (bFGF), angiopoietin-2 (Ang-2), vascular endothelial growth factor receptor 2 (VEGFR2) from tumor tissues. For in vitro experiments, the cell count kit 8 ( CCK8 ) assay was used to detect the effect of Withaferin A on HepG2 cells proliferation. QPCR assay and enzyme-linked immunosorbent assay ( ELISA) were used to detect the mRNA expression of VEGF. Results Compared to the model group, the high-dose Withaferin A group and the Sunitinib group had a significantly lower tumor weight (P<0. 05). The tumor inhibition rate was 42. 69% in the high-dose Withaferin A group, 20. 22% in the low-dose With-aferin A group, and 49. 43% in the Sunitinib group. The growth of HepG2 cells was significantly inhibited by different concentrations of Withaferin A,and the 50% concentration of inhibition ( IC50 ) of Withaferin A were (2. 64 ± 0. 18)μmol/L at 24 h. Withaferin A (6,3 μmol/L) could inhibit the protein and mRNA ex-pression of VEGF ( P<0. 05 ) . Conclusions Withaferin A significantly reduces the growth of orthotopic xenograft tumor of hepatocellular carcinoma in nude mice via antiangiogenic effect. Downregulation of the protein and mRNA expression of VEGF by WFA may be one mechanism of its anti-liver cancer effect.