4.Lentivirus-mediated shRNA interference targeting ADAM17 down-regulates MMP-9 expression in airway epithelial cells via TNF-α/NF-κB signaling
Jianping YAN ; Yaqing LI ; Hui ZHONG ; Chun CHEN ; Chao GU
Chinese Pharmacological Bulletin 2014;(4):559-565
Aim To study the role of TNF-α/NF-κB signaling in matrix metalloproteinase ( MMP)-9 expres-sion induced by lipopolysaccharide ( LPS ) in airway epithelial cells, and to investigate the effects of lenti-virus mediated RNAi targeting a disintegrin and metal-loproteinase 17 ( ADAM17 ) gene on MMP-9 expression induced by LPS. Methods The ADAM17 siRNA ex-pression vector was constructed, and packaged to re-combinant lentivirus in 293T cells. The HBE4-E6/E7 cells were pretreated for 30 min by NF-κB inhibitor ( PDTC) and a recombinant human TNFR p75-Fc fu-sion protein ( Etanercept) , or infected by the recombi-nant lentivirus for 72 h, and then stimulated for 24 h by LPS or TNF-α. The release of TNF-α was detected by ELISA. The mRNA and protein levels of MMP-9 were analyzed respectively by RT-PCR and Western blot. NF-κB activity was detected by electrophoretic mobility shift assay. Results LPS and TNF-α signifi-cantly increased MMP-9 mRNA and protein expressions and the activation of NF-κB in HBE4-E6/E7 cells ( P<0. 05 ) . Etanercept and PDTC significantly inhibited MMP-9 expression and the activation of NF-κB induced by LPS ( P<0. 05 ) . Lentivirus mediated RNAi targe-ting ADAM17 significantly decreased TNF-α produc-tion, inhibited MMP-9 mRNA and protein expressions and the activation of NF-κB induced by LPS in HBE4-E6/E7 cells ( P <0. 05 ) . Lentivirus mediated RNAi targeting ADAM17 did not inhibit MMP-9 mRNA and protein expressions and the activation of NF-κB in-duced by TNF-α ( P>0. 05 ) . And PDTC significantly inhibited MMP-9 mRNA and protein expressions and the activation of NF-κB induced by TNF-α ( P <0. 05 ) . Conclusions TNF-α/NF-κB signaling partic-ipates in the regulation of MMP-9 expression induced by LPS in airway epithelial cells, and lentivirus-media-ted RNAi targeting ADAM17 plays an important role in that signaling pathway upstream by regulating TNF-αrelease.
5.Research and analysis on corneal endothelial cell morphology and corneal thickness in patients with diabetics
Wu-Ping, XU ; Chun-Hui, WEI ; Liu-Li, GU
International Eye Science 2014;(10):1860-1862
AIM: To assess the impact of diabetes on corneal endothelial cells through the quantitative analysis of corneal endothelial cell morphology for patients with diabetics.
METHODS: The corneal thickness and endothelial cell morphology of 360 eyes of 299 cases were detected using full automatic corneal endothelial cell analyzer. The normal control group included 175 eyes of 148 cases, and there were 185 eyes of 151 cases for the patients with diabetes, 110 eyes of 92 cases for the non-proliferating phase group and 75 eyes of 59 cases for the proliferating phase group. The average density of central corneal endothelial cells, proportion of hexagonal cells, coefficient of variation and corneal thickness were compared among groups, and then the statistical analysis was conducted.
RESULTS: Compared with the cornea of the normal group, in the diabetes group, the coefficient of variation of corneal endothelial cells and central corneal thickness increased, while the average density of central corneal endothelial cells and proportion of hexagonal cells decreased, showing a significant difference (P<0. 05). Compared with the cornea of non-proliferating phase group, in the proliferating phase group, the density of central corneal endothelial cells decreased, the coefficient of variation of corneal endothelial cells increased, while the proportion of hexagonal cells decreased with a significant difference (P<0. 05), and the central corneal thickness increased, showing no significant difference(P>0. 05).
CONCLUSION: Compared with the cornea of normal control group, in the diabetes group, the corneal endothelial cells show abnormal morphology, which aggravates with the severity of lesions, especially for the significant changes in the coefficient of variation and the proportion of hexagonal cells. As a result, the corneal resistance to damage in patients with diabetes will decrease.
6.Comparative analysis of abnormal thin-layer cytologic diagnosis, hybrid capture II HPV DNA testing results and histologic diagnosis in 2225 patients.
Ai-chun WANG ; Yi-qun GU ; Jun WANG ; Qiu-li ZHOU ; Li WANG ; Li-juan LU ; Hui ZHANG
Chinese Journal of Pathology 2011;40(1):46-47
Adult
;
Aged
;
Carcinoma, Squamous Cell
;
pathology
;
virology
;
Cervical Intraepithelial Neoplasia
;
pathology
;
virology
;
Colposcopy
;
Cytodiagnosis
;
DNA Probes, HPV
;
DNA, Viral
;
isolation & purification
;
Female
;
Humans
;
Middle Aged
;
Nucleic Acid Hybridization
;
Papillomaviridae
;
genetics
;
isolation & purification
;
Papillomavirus Infections
;
Uterine Cervical Neoplasms
;
pathology
;
virology
;
Vaginal Smears
;
Young Adult
7.Construction of fusion gene between IgGHV and IL-2 as IgHV nucleic acid vaccine against lymphoma.
Hui LIU ; Nai-Bai CHANG ; Xi-Chun GU ; Ping ZHU
Journal of Experimental Hematology 2006;14(6):1160-1162
The purpose of this study was to construct the IgHV and IL-2 coexpressed vector. The IgHV gene fragments were obtained from the peripheral blood of patients with lymphoma, and were cloned into eukaryotic expression vector. Meanwhile, the gene fragments of IgHV linked with gene of IL-2 were inserted into pcDNA3.0 to form a fusion gene of IgHV-IL-2. Then fusion genes were transfected into COS cells by Lipofectin and the expression of IL-2 was detected by ELISA. The results showed that the IgHV/pcDNA3.0 expression vector was successfully constructed. The 3' end of IgHV was linked to IL-2 gene, and IL-2 could be correctly expressed. In conclusion, the expression vector of IgHV-IL-2 can express IL-2 correctly in COS cells.
Cancer Vaccines
;
immunology
;
Eukaryotic Cells
;
metabolism
;
Genes, Immunoglobulin
;
Genetic Vectors
;
Humans
;
Immunoglobulin Heavy Chains
;
genetics
;
Immunoglobulin Variable Region
;
genetics
;
Interleukin-2
;
biosynthesis
;
genetics
;
Lymphoma
;
immunology
;
Recombinant Fusion Proteins
;
biosynthesis
;
genetics
;
immunology
;
Vaccines, DNA
;
biosynthesis
;
genetics
;
immunology
8.The clinical significance of CT angiography in the diagnosis of arterioportal shunts associated with hepatocellular carcinoma
Xiao-Chun MENG ; Hong SHAN ; Kang-Shun ZHU ; Jian-Sheng ZHANG ; Xiao-Hong WANG ; Jie QIN ; Hui-Zhen GU ;
Chinese Journal of Radiology 2001;0(09):-
Objective To evaluate the clinical significance of CT angiography(CTA)in the diagnosis of arterioportal shunts(APS)associated with hepatocellular carcinoma(HCC).Methods One hundred and twenty-seven consecutive HCC patients accepted both dynamic enhancement CT and DSA examinations.The interval between CT and DSA exam was from 3 to 15 days.Based on transverse CT images in hepatic artery phase,CTA was performed for all the patients.By contrast with DSA results,the capabilities of transverse CT and transverse images combined with CTA in APS diagnosis were analyzed. Results In all 127 HCC cases,52 cases with APS were confirmed by DSA(40.94%),33 with central type of APS and 19 with peripheral type.Diagnostic sensitivity of APS based on transverse CT and combined CTA with transverse CT images were both 94.23%(49/52).However,specificity was 84.00%(63/75) and 97.33%(73/75),respectively,accuracy was 88.19%(112/127)and 96.06%(122/127),the predictive value of positive cases was 80.33%(49/61)and 96.08%(49/51),and the predictive value of negative cases was 95.45%(63/66)and 96.05%(73/76).Combined with CTA,false positive cases of 4 central type of APS and 6 peripheral type of APS were excluded which were demonstrated by transverse CT images.By contrast with DSA,the coincidence rate of the type of APS diagnosed by transverse images combined with CTA was 88.46%(46/52),including 90.91%(30/33)of central type of APS and 84.21%(16/19)of peripheral type.The supplying arteries of central type of APS were intuitively displayed by CTA in 23 cases,19 from proper hepatic artery and 4 from gastro-duodenal artery.Conclusion CTA techniques based on the dynamic enhancement CT exams could effectively promote the specificity and the accuracy of APS diagnosis.
9.Effects of SOD2 and its C47T mutation on oxidative injury in cochlea hair cell.
Chun-hui GU ; Yi-min LIU ; Xiao GUO ; Xu-dong LI ; Zhi WANG ; Li-mei CHEN
Chinese Journal of Industrial Hygiene and Occupational Diseases 2011;29(2):116-120
OBJECTIVETo study the effect of SOD2 and its C47T mutation on oxidative injury in cochlea hair cells.
METHODSHEI-OC1 cells were transfected with the SOD2 of Ala16 and Vla16. Cells' proliferation ability was determined by MTT assay. The intracellular SOD2 activities were detected by xanthine oxidase method. Intracellular ROS were determined by DCFH-DA after exposure to 100 µmol/L t-BHP and the early apoptotic and necrotic rate or late apoptotic rate were quantified by flow cytometry (FCM) using Annexin V/PI double staining.
RESULTSMTT method showed the transfection of SOD2 gene and empty plasmid did not affect the proliferation capacity. SOD2 vitality in Ala(16) and Val(16) SOD2 transfected cells increased 2.51 and 2.71 times respectively (P < 0.01), but the difference between the two transfection groups was not statistically significant (P > 0.05). After exposed to t-BHP, the majority of the untransfected and empty plasmid transfected cells sent '++' class bright fluorescence, while in Ala(16) and Val(16) SOD2 transfected groups, only about half cells sent '±' ∼ '+' level fuzzy fluorescence. determination of FCM suggested the early apoptotic and necrotic rate or late apoptotic rate decreased after SOD2 transfection (P < 0.01), but the difference between the two genotypes of SOD2 was not statistically significant (P > 0.05).
CONCLUSIONHigh expression of SOD2 below 3.71 times can reduce intracellular ROS level in HEI-OC1 cells, while SOD2 C47T mutation had no effect on them. SOD2 can be considered as NIHL susceptibility gene and its rs4880 SNP may be not directly related to NIHL genetic susceptibility.
Cell Line ; Cochlea ; metabolism ; Hair Cells, Auditory ; metabolism ; Humans ; Mutation ; Oxidative Stress ; Polymorphism, Single Nucleotide ; Reactive Oxygen Species ; metabolism ; Superoxide Dismutase ; genetics
10.Effect of phosphatidylinositol 3-kinase/serine-threonine kinase signalling pathway during the proliferation process of HL-60 cells exposed to benzoquinone.
Yi-min LIU ; Yong-sheng LI ; Zhi WANG ; Xu-dong LI ; Xiao GUO ; Chun-hui GU
Chinese Journal of Industrial Hygiene and Occupational Diseases 2011;29(5):334-337
OBJECTIVETo explore the effects of phosphatidylinositol 3-kinase/Serine-threonine kinase (PI3K/Akt) signal pathway on the proliferation of HL-60 cells exposed to benzoquinone (BQ).
METHODSHL60 cells were divided into 3 groups: control group (treated with PBS), BQ group (treated with 3 micromol/L BQ) and LY294002 plus BQ group (treated with 20 micromol/L LY294002 plus 3 micromol/L BQ). The cell proliferation was measured with alamar blue dye assay. Western blot assay was used to detect the expression of p-Akt and Akt proteins and flow cytometer was used to observe the cell cycle.
RESULTSThe cell proliferation rate and the cell proportion in the S, G2 phase of BQ group were 185.00% +/- 30.00%, 48.23% +/- 1.37% and 15.40% +/- 1.21%, respectively, which were significantly higher than those (100.00% +/- 0.00%, 42.47% +/- 0.45% and 5.40% +/- 0.40%) of control group (P<0.05). But the cell proportion rate (36.37% +/- 0.40%) in the G1 phase in BQ group was significantly lower than that (52.13 +/- 0.75%) in control group (P<0.05). The expression level of p-Akt protein in BQ group was significantly higher than that in control group (P<0.05). The cell proliferation rate and the cell proportion in the S, G2 phase of LY294002 plus BQ group were 82.59% +/- 15.00%, 42.03% +/- 0.50% and 3.87% +/- 0.47%, respectively, which were significantly lower than those of BQ group (P<0.05). But the cell proportion rate (54.43% +/- 0.40%) in the G1 phase in LY294002 plus BQ group was significantly higher than that in BQ group (P<0.05).
CONCLUSIONThe PI3K/Akt signal pathway may play an important role in the proliferation of HL-60 cells exposed to BQ.
Benzoquinones ; toxicity ; Cell Proliferation ; drug effects ; HL-60 Cells ; Humans ; Phosphatidylinositol 3-Kinase ; metabolism ; Protein-Serine-Threonine Kinases ; metabolism ; Signal Transduction