Humans ; Nasal Cavity ; Paranasal Sinuses

Background Ultraviolet irradiation promotes cellular apoptosis by affecting the mitochondrial transmembrane potential,including human lens epithelial cells (LECs).Gene associated with retinoid-interferoninduced mortality-19 (GRIM-19),a cell death regulatory protein,is essential for the assembly and function of mitochondrial complex Ⅰ.However,whether LECs apoptosis induced by ultraviolet irradiation is related to GRIM-19 is still unclear.Objective The purpose of this study was to investigate the relationship between the apoptosis of human LECs caused by ultraviolet with GRIM-19 expression in vitro.Methods Human LEC line(SRA01/04)was cultured in α-MEM containing 10％ fetal bovine serum.The cells were exposed to ultraviolet ray at doses of 0,30,60,90,120 or 150 mJ/cm2 when cell growth reached the logarithmic phase and 80％ confluency.The rate of apoptosis of the cells was assayed using flow cytometry,and the level of expression and relative amount of GRIM-19 protein (GRIM-19/β-actin) were detected by Western blot.The relationship between apoptosis and the GRIM-19/β-actin value among the different treatment groups was compared using One-way ANOVA,and the correlation of LECs apoptosis rate and GRIM-19 expression level was assessed by Pearson linear analysis.Results A significant difference was found in the apoptosis rate among the different treatment groups(F=149.32,P＜0.01).Compared with the 0 mJ/cm2 ultraviolet irradiation group,the apoptosis rate of LECs was significantly increased in the 60,90,120 and 150 mJ/cm2 ultraviolet irradiation groups (q =17.02,-25.20,-29.41,-8.61,P ＜ 0.01).The expression of the GRIM-19 protein in the LECs suspension was enhanced by ultraviolet irradiation at 60,90,120 and 150 mJ/cm2.The relative expression of the GRIM-19 protein (GRIM-19/β-actin) was significantly different among the various groups (F=6.87,P＜0.05),and the GRIM-19/β-actin values in the 60,90,120,150 mJ/cm2 ultraviolet irradiation groups were elevated in comparison with the un-irradiated group(2.01±0.76,2.98± 1.80,3.97± 1.61,2.42± 1.28 vs.0.56±0.23),which showed statistically significant differences (q =4.12,-5.04,-7.09,-3.85,P ＜ 0.01).In addition,a positive correlation was seen between the rate of apoptosis and the expression of the GRIM-19 protein(r=0.71,P＜0.01).Conclusions GRIM-19 is expressed in normal human LECs.The apoptosis of human LECs accompanies the up-regulation of GRIM-19.The expression of GRIM-19 in LECs increases with ultraviolet irradiation in a doseindependent manner.

Background Epithelial-myofibroblast transition (EMT) of human lens epithelial cells (LECs) induced by transforming growth factor-β2 (TGF-β2) is the main mechanism in the pathogenesis of posterior capsular opacification(PCO).Seeking an effective drug capable of inhibiting this process is important for the prevention and treatment of PCO.Objective The purpose of this study was to investigate the inhibitory effect of rapamycin (RAPA)on the proliferation of human LECs and TGF-β2-induced EMT.Methods Human LEC strain(SRA01/04)was cultured in DMEM with high glucose and 10％ fetal bovine serum.The cells were consequently cultured in serumfree DMEM with 5 mg/L TGF-β2,TGF-β2+10 mg/L RAPA,TGF-β2 + 100 mg/L RAPA,TGF-β2 + 1000 mg/L RAPA or TGF-β2 +10 000 mg/L RAPA for 72 hours,and SRA01/04 cultured in serum-free DMEM were used as control.The proliferation rate(A490)of SRA01/04 in the different groups was detected using the MTT assay and the rate of inhibition of RAPA was calculated.The expressions of the α-smooth muscle actin(α-SMA) and E-cadherin(E-cad)mRNA and protein were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot,respectively.The changes in the expression of α-SMA and E-cad in SRA01/04 were evaluated by Western blot 24,48 and 72 hours after TGF-β2 +400 mg/L RAPA treatment.Results The A490 value of SRA01/04 was 0.680±0.020,0.550±0.013,0.480±0.014,0.400±0.011 and 0.200±0.019 in the control group,TGF-β2 group,TGF-β2 + 10 mg/LRAPA group,TGF-β2 + 100 mg/L RAPA group,TGF-β2 + 1000 mg/L RAPA and TGF-β2 + 10 000 mg/L RAPA group,respectively,showing a gradually declining trend in SRA01/04 rate of proliferation with increasing RAPA concentrations (F =101.920,P =0.000).RT-PCR and Western blot assay showed that the relative expression levels of α-SMA mRNA (α-SMA mRNA/β-actin mRNA)and protein (α-SMA/β-actin)in the cells were significantly increased in the TGF-β2 treatment group.However,with exposure to RAPA,the relative expression levels of α-SMA mRNA and protein were significantly lowered with increasing RAPA concentrations,but the expression levels of E-cad mRNA and protein were raised (α-SMA mRNA:F =294.660,P =0.000 ; α-SMA protein:F =346.950,P =0.000 ; E-cad mRNA:F =264.250,P =0.000 ; E-cad protein:F =317.327,P =0.000).In addition,after exposure to 400 mg/L RAPA,the expression levels of α-SMA protein gradually reduced and those of E-cad protein gradually increased with increasing treatment durations,showing significant differences among the different time points (α-SMA:F =693.864,P =0.000 ;E-cad:F=369.286,P =0.000).Conclusions RAPA can inhibit the proliferation of SRA01/04 in vitro and arrest EMT of SRA01/04 induced by TGF-β2 in a dose-and time-dependent manner.

In the previous study, a high-throughput screening method was established to find the antagonists of CD36. In the present study, a new compound named IMB-1680 was found using this method. The anti-atherosclerotic activities of IMB-1680 were then evaluated. Dose-dependent activities of IMB-1680 were detected by using Sf9 [hCD36] and CHO [hCD36] models. Fluorescence microscopic photography and flow cytometry were used to analyze uptake of mLDL. Foam cell test with RAW264.7 macrophages was used to examine lipid accumulation. The results showed that IMB-1680 inhibited CD36 activity with IC50 of 2.80 and 8.79 micromol x L(-1) in Sf9[hCD36] and CHO [hCD36] cells, respectively. Fluorescence microscopic photography and flow cytometry revealed that IMB-1680 could significantly reduce DiI-AcLDL uptake. Meanwhile, IMB-1680 also could reduce lipids accumulation in RAW264.7 macrophages. In all, the data indicated that IMB-1680 might be a potent effective anti-atherosclerotic leading compound.
Animals ; CD36 Antigens ; antagonists & inhibitors ; genetics ; metabolism ; CHO Cells ; Cells, Cultured ; Cricetulus ; Dose-Response Relationship, Drug ; Foam Cells ; cytology ; High-Throughput Screening Assays ; Humans ; Lipoproteins, LDL ; metabolism ; Macrophages ; cytology ; metabolism ; Mice ; Molecular Structure ; Plasmids ; Receptors, Scavenger ; antagonists & inhibitors ; Sf9 Cells ; Spodoptera ; Transfection

Objective To establish the primary cat intestinal epithelial cells(IECs)culture methods and construct the cD-NA library for the following yeast two-hybrid experiment,so as to screen the virulence interaction factors among the final host. Methods The primary cat IECs were cultured by the tissue cultivation and combined digestion with collagenase XI and dispase I separately. Then the cat IECs cultured was identified with the morphological observation and cyto-keratin detection ,by using goat anti-cyto-keratin monoclonal antibodies. The mRNA of cat IECs was isolated and used as the template to synthesize the first strand cDNA by SMARTTM technology,and then the double-strand cDNAs were acquired by LD-PCR,which were subsequently cloned into the plasmid PGADT7-Rec to construct yeast two-hybrid cDNA library in the yeast strain Y187 by homologous recom-bination. Matchmaker?Insert Check PCR was used to detect the size distribution of cDNA fragments after the capacity calcula-tion of the cDNA library. Results The comparison of the two cultivation methods indicated that the combined digestion of colla-genase XI and dispase I was more effective than the tissue cultivation. The cat IECs system of continuous culture was established and the cat IECs with high purity were harvested for constructing the yeast two-hybrid cDNA library. The library contained 1.1× 106 independent clones. The titer was 2.8 × 109 cfu/ml. The size of inserted fragments was among 0.5-2.0 kb. Conclusion The yeast two-hybrid cDNA library of cat IECs meets the requirements of further screen research,and this study lays the foundation of screening the Toxoplasma gondii virulence interaction factors among the cDNA libraries of its final hosts.

BACKGROUND:As the multipotent differentiation potential, bone marrow mesenchymal stem cells exert an important role in bone metabolism disorders, which is regulated by a variety of hormones and cytokines. Currently, the epigenetic regulatory mechanisms underlying osteogenic differentiation of bone marrow mesenchymal stem cells are unclear, and association between histone deacetylase (Hdac) and osteoporosis needs to be further explored. OBJECTIVE:To investigate the epigenetic mechanisms of bone formation by analyzing the expression of Hdac1, 3, 4 mRNA profile in bone marrow mesenchymal stem cells isolated from ovariectomized mice. METHODS:A total 30 female healthy Kunming mice were randomly divided into sham group and ovariectomy group. After 7 days of adaptive feeding, mice in the ovariectomized group (n=15) were subject to bilateral ovariectomy;mice in sham group (n=15) were sham-operated. RESULTS AND CONCLUSION:In the ovariectomy group, the trabecular bone of the femur was sparse or broken, the width of trabecular bone was narrowed, the trabecular separation was widened, and the trabecular bone volume was reduced. The level of Hdac3 mRNA was lower in bone marrow mesenchymal stem cells from ovariectomized mice compared with controls, but there was no significance between Hdac1, Hdac4 mRNA expression of the two groups. These findings demonstrate that Hdac might play an important role in bone remodeling in the model of estrogen deficiency-induced osteoporosis.

Objective To evaluate the role of vascular endothelial growth factor-D(VEGF-D)in lymphangiogenesis of breast cancer,and investigate its relationship with some clinicopathological parameters and prognosis. Methods VEGF-D expression was detected in 85 cases with primary breast carcinoma by immunohistochemistry,and VEGF-D mRNA was detected by RT-PCR.Podoplanin monoclonal antibody was used to mark lymphatic endothelial cell and lymphatic vessel density(LVD) was counted.The relationship among the aboved parameters,clinicopathological parameters and prognosis was analysed. Results It was revealed by immunohistochemistry that VEGF-D expression was ranked by 5 levels: negative,n=5(5.88%);"+",n=17(20%);"++",n=34(40%);"+++",n=22(25.88%),and "++++",n=7(8.24%).VEGF-D expression was associated with tumor lymph node metastasis and TNM clinical stage(P

Objective: To construct the lentiviral plenti6/V5-D-TOPO vector containing TβR Ⅱ Dnglytk and control vector by developing a lentivirus mediated gene transfer program incorporating a herpes simplex virus thymidine kinase(HSVtk) in the dominant negative TGF-β type Ⅱ receptor (TβR Ⅱ Dnglytk) expression vector. Methods: The PCR methods were used to amplify the genes TβR Ⅱ DN and HSV-tk from the respective plasmids. Then the genes were Linked by recombinant PCR technology to construct the fusion gene TβR Ⅱ Dnglytk and control vector TRANSglytk. According to the operation manual (from the Invitrogen company), ToPe cloning technology were used to construct the plasmids of plenti6/VS-D-TOPO(R)-TβRⅡ Dnglytk and plenti6/VS-D-TOPO -TRANSglytk. Both of the constructed plasmids were verified by sequencing.Results: The constructions of the plasmids of plenti6/V5-D-TOPO(R)-TβR Ⅱ Dnglytk and plenti6/V5-D-TOPO(R)-TRANSglytk were completed smoothly. The DNA sequencing results showed that both the plasmids were constructed correcdy and can be used in the production of infectious lentivirus vectors. Conclusion: Using TOPO cloning technology in the construction of the plasmids of plenti6/VS-D-TOPO -TβR Ⅱ Dnglytk and plenti6/VS-D-TOPO(R)-TRANSglytk and recombinant PCR in the fusion genes are feasible. The recombinant PCR combined with ToPe cloning technology can be the simple, highly efficient and rapid way to construct lentiviral vector and construction of plenti6/V5-D-TOPO(R)-TβR Ⅱ Dnglytk, which will lay a foundation for tumor immunotherapy.

Objective Our hypothesis was if we rendered host' s cytotoxic tumor lymphocytes insensitive to TGF-β,these immune cells could be able to overcome the TGF-β mediated immunosuppression and reject the tumor.We aimed to develop a lentivirus mediated gene transfer program incorporating a herpes simplex virus thymidine kinase(HSV-tk)in our dominant negative TGF-β type Ⅱ receptor(TβRⅡDNglytk)expression vector.So we first need to construct the lentiviral pLenti6/V5-D-TOPO vector containing TβRⅡDNglytk and produce the recombinant lentivirus as the transfection vector.Methods PCR were used to amplify the genes TβRⅡDN and HSV-tk from the respective plasmids.Then the genes were linked by recombinant PCR technology to construct the fusion gene TβRⅡDNglytk and control vector TRANSglytk.According to the operation manual from the Invitrogen company,TOPO cloning technology was used to construct the plasmids of pLenti6/V5-D-TOPO(R)-TβRⅡDNglytk and pLenti6/V5-D-TOPO(R)-TRANSglytk.Both of the constructed plasmids were verified by sequencing.ViraPowerTM Lentiviral System and 293 FT cells provided by Invitrogen were used to produce the recombinant lentivirus vector,the tillers of the lentivirus were determined by 293 cells.Results The construction of the plasmids of pLenti6/V5-D-TOPO(R)-TβRⅡDNglytk and pLenti6/V5-D-TOPO(R)-TRANSglytk were completed succefully.The DNA sequencing results showed that both the plasmids were constructed correctly.Using them we successfully produced infectious lentivirus vectors with appropriate tilters.Conclusions Using TOPO cloning technology in the construction of the plasmids of pLenti6/V5-D-TOPO(R)-TβRⅡDNglytk and pLenti6/V5-D-TOPO(R)-TRANSglytk and recombinant PCR in the fusion genes is feasible.Recombinant PCR combine with TOPO cloning technology can be a simple,highly efficient and rapid way to construct lentiviral vector.The production of infectious lentivirus with appropriate tilters using 293FT is suitable and feasible.The construction of pLenti6/V5-D-TOPO(R)-TβRⅡDNglytk and production of the infectious lentivirus will lay a foundation for immunotherapy of prostate cancer.

Objective To examine the feasibility of using the serous-lined tunnel technique for orthotopic neobladder, continent cutaneous diversion and ureteral replacement by the intestinal segment. Methods In 31 patients of orthotopic ileal neobladder, the serous-lined tunnel techniques were used for antirefluxing ureteral implantation: In 13 patients of continent ileal pouch, the techniques were adopted for continent-valve construction and for uretersl implantation: In 3 patients (with lower ureteric cancer), the same techniques were applied for constructing the ileal ureters with a proximal antirefluxing mechanism. Results With a mean follow-up of 27 mon( 12-132 mon), 88 ureters implanted into ileal neobladders or continent pouches functioned well with neither obstruction nor reflux: 12 in 13 continent valves functioned well with no incontinence. 3 patients with ileal ureters showed no ileo-ureteric reflux and had reduced hydronephrosis comparing to that of before surgery.Conclusions Ureteral reimplantation and continent valve formation achieved by adopting the serouslined tunnel technique provide satisfactory results. The versatility of the technique is obvious in the present experience and the creative application of the serous-lined tunnel technique should be possible in urinary reconstruction.