Objective To describe our experience and method of reconstruction after resection of tumors around inner canthus and nasion in the elderly. Methods The line between outer and inner canthus was considered during reconstruction.If the surface of wound after resection was above this line,glabellar flap was used to reconstruct the upper part and the residual defect was repaired with advancement of lower eyelid and rotation of flap.If the surface of wound was below this line,lower eyelid flap was used and the residual defect was reconstructed with glabellar flap. Results All flaps survived without any significant complications.9 cases among 15 patients were followed up from 1 to 24 months.Repaired tissues all matched well with surrounding tissue in color,texture,and outline.Eyelid and medial canthal were not disturbed and there was no tumor recurrence.Scars were smoothy,soft and hidden with satisfactory appearance and vision. Conclusions Single local flap or combined local skin flaps around the nasion and inner canthus is one of reconstruction methods to get satisfactory cosmetic effect or facial morphology.

Objective To investigate the inhibition of pathogenic viral gene expression in HPV6bE6-positive cell line by specific siRNA, which might have great potential for clinical use. Methods B16 cells were transfected with recombinant plasmid pcDNA3.1(+)-GFP/HPV6bE6, and the positive cell clones were selected by fluorescence protein observation and RT-PCR. Four specific siRNAs, none of which shares homology with exons of known human genes, were designed and synthesized to target HPV6bE6 mRNA. Quantitative real-time PCR was performed to measure the inhibition rates of target gene expression by comparing HPV6bE6 mRNA concentrations before siRNA transfection with those after transfection. The inhibition rates of target gene expression with different siRNA concentrations of 0.2 nmol/L, 1 nmol/L, 10 nmol/L, 50 nmol/L, 150 nmol/L and with different treatment time at 24 h, 48 h, 72 h and 96 h after transfection were measured respectively. Results More than 90% reduction of HPV6bE6 mRNA was observed following treatment with HPV6bE6-siRNA, and HPV-negative cells were apparently unaffected by HPV6bE6-siRNA. The decrease of HPV6bE6 mRNA was maximal at 24 h after siRNA treatment and sustained for at least 4 days. The minimal level of siRNA to efficiently silence the homogeneous target gene HPV6bE6 was 1 nmol/L. Conclusion HPV6bE6-siRNA can efficiently and specifically silence target genes and may be developed as a potential therapeutic approach for HPV infection.

OBJECTIVE: To evaluate the advantage of Gukangling Decoction (GKLD), a compound traditional Chinese herbal medicine, combined with technetium [(99)Tc] methylene diphosphonate injection ((99)Tc-MDP) in treating osteoporosis in rabbits. METHODS: A rabbit model of osteoporosis was established by intramuscular injection of dexamethasone (DX). Fifty-six rabbits were divided into 8 groups: Group A, B, C, D, E, F, G and H. Rabbits in groups A and B were intramuscularly injected normal saline as normal control, groups C and D were untreated groups, rabbits in group E were treated by (99)-MDP, rabbits in group F were treated by aminodiphosphate, rabbits in group G were treated by GKLD, and rabbits in group H were treated by (99)-MDP and GKLD. Rabbits in groups A and C were executed to demonstrate the establishment of the rabbit model of osteoporosis at the 8th week of experiment. Rabbits in the other six groups were executed after 16-week experiment (8-week treatment), and then bone structure and cell shape were observed by electron microscope, X-ray, CT and emission computed tomography (ECT). Bone density, biomechanical parameters, the levels of bone specific alkaline phosphatase (BALP) and bone Gla protein (BGP) were measured too. RESULTS: After 8-week of intramuscular injection of DX, the bone trabecula in group A were regular and showed normal configuration, while the bone trabecula in group C were sparse, ruptured and showed damaged form. The bone density and biomechanical parameters in group A were higher than those in group C, indicating that the rabbit model of osteoporosis was established successfully. At the 9th week of experiment, the results of cell pathology in group D showed that the bone trabeculas were sparse, ruptured, defected or had hollow section, but the bone trabeculas in group B were regular and dense. The bone trabeculas in groups H and E were restored, and were thicker than those in group D. The bone quality in groups H and E was better than group D significantly, the bone quality in group F was better than group G, and the bone quality in group G was better than group D slightly. CONCLUSION: GKLD combined with (99)-MDP had superiority in treating osteoporosis of rabbits as compared with the respective single therapy.

Objective To investigate the cellular immune response to the recombinant DNA vaccine CRT120/HPV6bE7 in mice. Methods The recombinant encoding HPV6bE7, linked with CRT120, was constructed in pcDNA3.1 eukaryotic vector with the report gene GFP. The pcDNA3.1-GFP -HPV6bE7 was transfected into B16 cells by a lipofectamine kit. The C57BL/6 mice were vaccinated with recombinant DNA plamids. The T-cell phenotype in the peripheral blood lymphocytes of the immunized mice was measured by flow cytometry. The CTL activity of the lymphocytes from the spleens and lymph nodes, and the levels of IL-2 and IFN-? were analyzed. Results The constructed recombinant plasmid CRT120/HPV6bE7 was analyzed. Positive transfected cell clones were established and could stably express the target gene HPV6bE7. Compared with HPV6bE7, CRT120/HPV6bE7 plasmid enhanced to a greater extent CD8+ T-lymphocyte differentiation, the number of TCR?? T-lymphocytes and the levels of IL-2 and IFN-? (all P

Objective To investigate the effects of crude extracted proteins from lesions of condyloma acuminata on immunophenotypes of dendritic cells.Methods Plastic-adherent mononuclear cells(MNCs)were isolated from umbilical cord blood or peripheral blood and cultured in media containing cytokines(GM-CSF,IL-4and LPS).The morphology and phenotypes of these cells were analyzed by flow cytometry and microscopy in12day's culture.Cells on the fourth day were incubated with crude extracts from lesions of condyloma acuminata,foreskin proteins,and PBS,respectively,followed by phenotypic analysis after9-12days' culture.Results Expression of antigens CD1a,CD80,CD86,MHC-I,MHC-II,CD14,CD54was detected after12days' cul-ture.It was shown that MNCs could be induced to differentiate to mature dendritic cells in our culture system.After incubation with crude extracts from lesions of condyloma acuminata for another9-12days,expression of CD86and HLA-DR was increased on dendritic cells.Conclusions Compared to foreskin and PBS pulsed dendritic cells,expression of CD86and HLA-DR is upregulated on dentritic cells after pulsing with condyloma acuminata lesion proteins.The data suggest that crude extracts from lesions of condyloma acuminata might enhance antigen-pre-senting capacity of dendritic cells and strengthen activation of T lymphocytes.

Objective To screen and identify the predicted epitopes of synthesized HLA-A*0201restricted CTL derived from HPVll E7 antigen.Methods Five HPVll E7 CTL epitope peptides and terramers consisting of HLA-A*0201 were selected by way of computer and synthesized by Sanquin company,including HPVllE7 7-15(TLKDIVLDL),15-23(LQPPDPVGL),47-55(PLTQHYQIL),81-89(DLLLGTLNI)and 82-90(LLLGTLNIV).These peptides binding to human peripheral blood-derived DCs were tested for their ability to activate T cells isolated from peripheral blood lymphocytes of HLA-A*0201 healthy individuals.the number of specific tetramer+CD8+T cells by flow cytometry,the level of the section of IFN-γ by ELISA,and the ability of the CTL to kill the target cells were observed.Results The immature DCs could be fully activated by all the five HPV11 E7 peptides.Peptide-loaded mature DCs were able to stimulate the epitope-specific T cells responses in vitro.An increased frequency(P<0.05)of T ceils specific for the E7 7-15 epitope compared to other epitopes of HPV11E7.The epitope-specific CTL of E7 7-15 induced by the activated DCs specifically killed HPV11E7 expressing 293 cell line,and in a ratio of 50:1,the specific cytolytic activity was the strongest than the others(P<0.05).Conclusion DCs loaded with HPV11 E7 7-15(TLKDIVLDL)peptide can induce highly effective and specific ectogenic processed epitopespecific CTL responses in vitro.This peptide may be the candidate for development of CTL based vaccine in the treatment of HPV infeetions.

This study was aimed to compare the antioxidant activity of puerarin and 3 other flavonoid compounds, and to investigate their structure-activity relationship. The intragastric administration of 4 kinds of typical flavonoids compounds (soybean element, puerarin, quercetin and rutin) were given to mice, respectively. The model mice of acute hepatic injury were established with intraperitoneal injection of 0.1% carbon tetrachloride (CCl4) after 7 days. After 18 h fasting, liver tissues were removed. The histomorphology was observed after paraffin sectioning and hematoxylin-eosin staining. The activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT) and alkaline phosphatase (ALP) in serum were detected with automatic biochemical analyzer. The content of malondialdehyde (MDA), the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in liver tissues were detected with homogenization. The pathological results of liver tissues showed that hepatic damages were decreased in all 4 medicine treatment groups compared with the model group, but there were no significant differences among these treatment groups. The results of blood serum bio-chemical analysis showed that compared with the model group, puerarin and quercetin could decrease the activities of ALT, AST and GGT in serum significantly (P < 0.05, orP < 0.01). There were no content changes of ALP. In the soybean element group, only the activities of ALT and AST decreased obviously (P < 0.05, orP < 0.01). There was no obvious change in the serum of mice in the rutin treatment group. The homogenate detection results of liver tissues showed that compared with the model group, quercetin and rutin significantly lowered MDA (P < 0.05), increased SOD and GSH-Px (P < 0.05, orP < 0.01); while soybean element and puerarin only improved GSH-Px levels (P < 0.05, orP < 0.01). It was concluded that the antioxidant capacity of quercetin was better than that of soybean element, puerarin and rutin, which may be related to its structure. Compared with 3 other chemical compounds, quercetin had more polyhydroxies and its polyhydroxies were not glycosylated, which suggested that the structure of quercetin may be closely related to its antioxidation activity.

Objective To construct four expression plasmids, pcDNA3.1-GFP/HPV6bE6, pcDNA3.1-GFP/HPV6bE7, pcDNA3.1-GFP/HPV11E6, pcDNA3.1-GFP/HPV11E7 and their transfected murine cell lines. Methods The Four recombinant expression plasmids comprising HPV6bE6,HPV6bE7,HPV11E6 and HPV11E7 linked with GFP, respectively, were constructed and transfected to B16 cells by lipofectamine kit. Positive clones were selected by G418 and observed by fluorescent microscopy and identified by RT-PCR. Results The four constructed recombinant plasmids were authenticated by restriction enzyme digestion and DNA sequencing. Under the fluorescent microscope, the green fluorescence could be observed in cytoplasm and nucleus of four transfected B16 cell lines. The RNA extracted from positively transfected clones resistant to G418 were analyzed by RT-PCR, which demonstrated the presence of four expected fragments. Conclusions The transfected murine cell lines B16 can express HPV6bE6,HPV6bE7,HPV11E6 and HPV11E7 gene. These transfected cell lines can be further transplanted to mice in order to investigate the biological properties and immunological mechanisms of these genes in vivo.

Objective:To explore the protective mechanism of tea polyphenols (TP) on mouse oral cancer.Methods:A total of 50 mice were divided into control group, model group, TP group, Selisistat group, TP+ Selisistat group, with 10 mice in each group. The control group was gavaged with physiological saline, while the model group, TP group, Selisistat group, and TP+ Selisistat group were gavaged with 300 mg/L 4-NQO to establish a mouse oral cancer model. Physiological saline, 200 mg/kg TP, 0.01 mg/kg Selisistat, and 200 mg/kg TP+ 0.01 mg/kg Selisistat were gavaged respectively. The weight changes of each group of mice were compared; HE staining was used to observe the morphology of mouse oral tumor tissue; Enzyme linked immunosorbent assay was used to detect the levels of malondialdehyde (MDA) and superoxide dismutase (SOD) in serum; Immunoblotting and immunohistochemistry were used to detect the expression of silencing information regulatory factor (Sirt1) and nuclear factor E2 related factor 2 (Nrf2) proteins in mouse oral tissues.Results:Compared with the control group, the model group mice had a decrease in body weight [(23.19±1.36)g], a decrease in serum SOD level [(91.64±8.75)U/ml], an increase in MDA level [(5.18±0.46)nmol/ml], a decrease in Sirt1 (0.38±0.05) and Nrf2 (0.36±0.05) protein expression in oral tissue, and an increase in Nrf2 acetylation level (0.84±0.11) (all P<0.05). Compared with the model group, the TP group mice had an increase in body weight [(25.28±1.25)g], elevated serum SOD levels [(121.24±10.68)U/ml], decreased MDA levels [(3.89±0.42)nmol/ml], increased expression of Sirt1 (0.61±0.09) and Nrf2 (0.58±0.06) proteins in oral tissue, and decreased Nrf2 protein acetylation levels (0.39±0.05); The Selisistat group mice showed a decrease in body weight [(21.41±1.07)g], a decrease in serum SOD levels [(72.16±7.43)U/ml], an increase in MDA levels [(5.87±0.41)nmol/ml], a decrease in Sirt1 (0.23±0.04) and Nrf2 protein (0.24±0.03) expression in oral tissue, and an increase in Nrf2 acetylation levels (1.12±0.14) ( P<0.05). The body weight [(23.32±1.27)g], serum SOD levels [(92.58±8.13)U/ml], and oral Sirt1 (0.41±0.06) and Nrf2 (0.38±0.05) protein expression in the TP+ Selisistat group mice were higher than those in the Selisistat group, while MDA [(5.11±0.38)nmol/ml] and Nrf2 acetylation levels (0.82±0.09) were lower than those in the Selisistat group (all P<0.05). Conclusions:Tea polyphenols can alleviate oral tissue damage and alleviate oxidative stress in mice with oral cancer, and their mechanism may be related to the upregulation of the Sirt1/Nrf2 pathway.

Background: The newly released Rome criteria in 2016 has a stricter and more precise definition of functional gastrointestinal disorders (FGIDs) when compared with Rome III criteria. The adjustment and improvement of diagnostic criteria by Rome criteria may affect the clinical diagnosis of FGIDs. Aims: To investigate the differences and the similarities between Rome III and Rome criteria in the diagnosis of FGIDs in college students. Methods: The FGIDs database of college students in Zhejiang Province established by our previous research team were further evaluated and analyzed by Rome criteria, and the incidence, psychological symptom score, overlapping of disease of FGIDs were calculated, and compared with Rome III criteria. Results: Of the 1 870 cases in database, 1 025 (54.81%) met Rome criteria of FGIDs; while 1 111 (59.41%) met Rome III criteria, the difference in detection rate was statistically significant (P <0.01). In Rome group, incidences of belching disorders (2.14% vs. 5.83%, P<0.01), irritable bowel syndrome (IBS) (2.78% vs. 6.90%, P<0.01), functional abdominal bloating/distension (1.28% vs. 4.12%, P<0.01) were significantly lower than those in Rome III group, while incidence of functional diarrhea was significantly higher (3.85% vs. 0.70%, P<0.01). Patients met Rome criteria showed a higher score of obsession⁃compulsion, depression and anxiety (P<0.05). Rome criteria caused 33 (25.58%) original IBS patients included in functional diarrhea, and 6 (4.65%) original IBS patients included in function constipation. The diagnosis of functional bowel disease overlapping with other FGIDs (belching disorders, functional dyspepsia) according to Rome III and Rome criteria were statistically different (P<0.01, P<0.05). Conclusions: Rome criteria has a stricter and more accurate definition of FGIDs, reflecting a more accurate psychological and clinical features, and identification of patients who really need treatment, resulting in a more efficient and feasible application in clinical practice and scientific research.