Objective: To determine if fusion genes of HSV-tk gene and cytokine gene have synergy on the cell killing of the Hep-2 human laryngeal carcinoma cell line in vitro. Methods: Different fusion genes expressing vectors PL(TI) SN, PL(TT)SN and PL(TK)SN were generated by recombinant DNA technology. Hep-2 was infected by the recombinant retrovirus. The positive clones were obtained after G418 selection and were termed Hep/TI, Hep/TT and Hep/TK respectively. The integration and expression of fusion genes in Hep-2 cells were identified by RT-PCR and Southern blot. The growth state and GCV killing effect of fusion genes modified cells were used to investigate the expression of fusion genes and antitumour effect on Hep-2 cells. Results: RT-PCR and Southern blot analysis confirmed the integration and expression of fusion genes in Hep-2 cells. There was no significant difference in cell proliferation between the Hep/TI and Hep/ TK.but the growth of Hep/TT was restrained. After the treatment of GCV the Hep/TI, Hep/TT and Hep/TK all showed high sensitivity to GCV. The killing effect of GCV on Hep/TT was the most siginificant and bystander effects were observed siginificantly in vitro. Conclusion: The fusion genes of HSV-tk and cytokine gene have synergistic effects on killing Hep-2 cell after treatment of GCV in vitro,which might have therapeutic potentials for laryngocarcinoma.

Objective: To construct the eukaryotic expression vector pSurp-EGFP regulated by the survivin gene promoter and to detect the specific expression of the promoter in human laryngeal cancer Hep-2 cells by green fluorescent protein assay.Methods: Thesurvivin gene promoter was generated by polymerase chain reaction(PCR) and the CMV promoter of the pShuttle vector replaced by the survivin gene promoter to generate the plasmid pSurp.The three plasmids pShuttle,pSurp and pEGFP-C1 were respectively double-enzyme digested so as to produce the plasmids pCMV-EGFP and pSurp-EGFP carrying the CMV or survivin promoter.The purified pCMV-EGFP and pSurp-EGFP were transfected into Hep-2 cell and vascular endothelial cell ECV304 using liposome transfection reagent and the expressions of EGFP detected by the fluorescent microscope.Results: Thesurvivin gene promoter was successfully cloned by PCR,and thesurvivin gene promoter-regulated pSurp-EGFP was constructed.Green fluorescence was observed in Hep-2 cells but not in ECV304. Conclusion: The high specific activity of the survivin gene promoter in Hep-2 cells that we successfully constructed attributes to the studies of tumor specific gene therapy.

ObjectiveTo detect the value of ultrasound-guided steroids injection for the treatment of plantar fasciitis.MethodsThirty-eight physical therapy ineffective plantar fasciitis patients were enrolled in this study, and randomly divided into ultrasound-guided and palpation-guided groups. Pain intensity was measured using a visual analog scale (VAS) and plantar fasciitis were evaluated by high frequency ultrasound including assessment of the thickness before injection and at 4 weeks, 12 weeks post injection.ResultsThirty-eight patients who received either ultrasound guided or palpation-guided injection had significantly lower visual analog scale scores and lower plantar fascia thickness (bothP<0.01) at 4 weeks, 12 weeks post injection. The differences in plantar fascia thickness, VAS score between the two groups before, and at the 4 weeks follow-up were not statistically significant [2.52±0.77vs 2.68±0.82,P>0.05; (4.56±0.25) mmvs (4.72±0.38) mm, P>0.05]. However, the ultrasound guided group had lower mean visual analog scale score (1.47±0.77vs 2.37±0.68,P<0.01) and lower mean plantar fascia thickness [(4.02±0.24) mmvs (4.53±0.35) mm,P<0.01] than the palpation-guided group at 12 weeks post injection. The calcification of the plantar fascia at 12 weeks post injection in ultrasound guided group was completely disappeared or significantly decreased.ConclusionUltrasound-guided injection for treating plantar fasciitis is more accurate and effective than palpation-guided injection, and is of great clinical value for both patients and doctors.

Background Fungal corneal ulcer is a visual-threatening eye disease,and drug therapy has a limiting efficacy.Corneal transplantation or eye enucleation sometimes is necessary to the severe patients.Corneal collagen cross-linking (CXL) is an effective method for some corneal diseases,but the study on CXL for fungal corneal ulcer is lack.Objective This study was to evaluate the clinical effectiveness and safety CXL for fungal corneal ulcer.Methods Fifteen 8-week-old healthy New Zealand white rabbits were used in this study and other 5 rabbits served as normal controls.Fungal corneal ulcer models were established in the right eyes of other 10 rabbits by infecting sickle bacteria liquid after corneal scratching and removing corneal epithelium,then decellularized ostrich corneal patch covered the defected cornea.The models were randomly divided into the non-treatment group and the CXL treatment group.Corneal lesions were examined under the slit lamp microscope every day,and cornea was pictured by laser scanning confocal microscope on the 3rd,7th,14th,21st and 28th day individually after CXL.All rabbits were sacrificed and corneal tissues were obtained 4 weeks after treatment,and the collagen fiber diameter and fibrocytes were observed under the scanning electron microscope.Results Fungal corneal ulcer models were successfully established by corneal scratching and decellularized ostrich cornea covering.The gray ulcer lesions and hypbae like bean pod were seen by slit lamp microscope and laser scanning confocal microscope 3 days after modeling.Corneal ulcer deepened and expanded 1 week later,and there were a large number of spore and hyphae criss-crossing as short rod in shallow stroma.Inflammatory cells were observed in corneal endothelial cells and ocular anterior chamber.In the CXL treatment group,the range of corneal epithelial deficiency was less than that in the nontreatment group on the 3rd,7th,14th,and 21st (all at P＜ 0.05).The diameters of collagen fibers were (24.6± 1.8) nm,(24.9 ± 1.9) nm and (43.0 ± 7.4) nm in the normal control group,non-treatment group and CXL treatment group,showing a significant difference among the 3 groups (F =27.05,P =0.00),and the collagen diameters were thicker in the CXL treatment group than those in the normal control group and non-treatment group (t =5.40,-5.30,both at P＜0.05),and fibrocytes were seen among the collagen fibers.No significant difference was found in the collagen diameters between the non-treatment group and normal control group,and the fibrocytes were less in the non-treatment group.Conclusions CXL therapy can treat fungal corneal ulcer by enhancing collagen,promoting fibrocytes proliferation,suppressing fungus and inflammatory response and accelerating tissue repair.

Objective:To investigate the effect of verbascoside (VB) on epithelial-mesenchymal transformation and its potential molecular mechanism in glioblastoma cells.Methods:(1) Glioblastoma cell lines T98 and U251 were routinely cultured in vitro. Cells were divided into 4 groups and treated with 0 μmol/L (controls), 20 μmol/L, 40 μmol/L, and 80 μmol/L VB for 24 h, respectively; CCK-8 was used to detect the cell viability; Transwell assay was used to evaluate the migration and invasion abilities; reverse transcription (RT)-PCR and Western blotting were conducted to examine the mRNA and protein expressions of transforming growth factor (TGF)-β, vimentin and Snail, respectively. (2) T98 and U251 cells were divided into 4 groups: control group (cells being transfected with empty plasmid), VB group (cells being transfected with empty plasmid and treated by 40 μmol/L VB), TGF-β group (cells being transfected with TGF-β overexpression plasmid), TGF-β+VB group (cells being transfected with TGF-β overexpression plasmid and treated by 40 μmol/L VB); CCK-8 was used to detect the cell viability; Transwell assay was used to evaluate the migration and invasion abilities; RT-PCR and Western blotting were conducted to examine the mRNA and protein expressions of TGF-β, vimentin and Snail, respectively. (3) U251 cells were implanted subcutaneously into the nude mice; the mice were divided into VB group and control group ( n=8). The mice in the VB group were intraperitoneally injected with 100 mg/kg/d VB, and mice in the control group were injected with the same volume of PBS. Tumor size and mice body mass were recorded. At the end of the experiment, mice were sacrificed; tumors were removed and weighed. The protein expressions of TGF-β, vimentin and Snail in tumor tissues were detected by Western blotting. Results:(1) As compared with that in the 0 μmol/L VB group, the cell survival rate in the 20 μmol/L and 40 μmol/L VB group showed no significant difference ( P>0.05), while that in the 80 μmol/L VB group showed significant difference ( P<0.05). As compared with that in the 0 μmol/L VB group, the cell number migrating and invading into the lower chamber in the 20 and 40 μmol/L VB groups was significantly smaller as concentration increased, and the mRNA and protein expressions of TGF-β, Snail and vimentin were also downregulated successively, with signficant differences ( P<0.05). (2) As compared with the control group, the cell number that migrating and invading into the lower chamber, and protein expressions of TGF-β, vimentin, and Snail in the VB group were significantly decreased ( P<0.05); while as compared with the control group, the cell number that migrating and invading into the lower chamber, and protein expressions of TGF-β, vimentin, and Snail in the TGF-β group were significantly increased ( P<0.05); as compared with those in the VB group, the cell number that migrating and invading into the lower chamber, and protein expressions of TGF-β, vimentin, and Snail in the TGF-β and TGF-β+VB groups were significantly increased ( P<0.05). (3) As compared with the control group, the tumor volume and tumor weight in the VB group were statistically decreased, and the TGF-β, vimentin and Snail protein expressions in the VB group were significantly downregulated ( P<0.05); however, there was no significant difference in mice body weight between the two groups ( P>0.05). Conclusion:VB inhibits epithelial-mesenchymal transformation in glioblastoma by suppressing the TGF-β expression.