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Objective:To purify H5N1 influenza virus concentrate prepared by MDCK cells with a new mixed-mode chromatography medium Capto Core700 and the traditional medium Sepharose 4FF, and to compare the separation and purification efficacy of the two media.Methods:Capto Core700 and Sepharose 4FF were used to purify inactivated H5N1 influenza virus concentrate. The morphology of virus particles in different samples was then observed under a transmission electron microscope. Single radial immunodiffusion (SRID), Folin-Phenol (Lowry) method, double-antibody sandwich ELISA and qPCR were used to detect hemagglutinin, total protein, host cell protein (HCP) and host cell DNA (HCD) before and after purification. The recovery rate of virus antigen and the removal rate of impurities were calculated. The immunogenicity of the viruses purified with different media was analyzed using animal experiments. Difference in the purification efficacy of the two chromatography media was analyzed by t-test. Results:H5N1 influenza viruses purified by Capto Core700 or Sepharose 4FF showed the typical influenza virus morphology under transmission electron microscope. There was no significant difference in the recovery rate of hemagglutinin between the two chromatography media ( P>0.05), but compared with Sepharose 4FF, Capto Core700 had a higher removal rate of impurities (total protein, HCP, HCD) and the difference was statistically significant ( P<0.05). Animal experiments showed that the viruses purified by the two chromatography media had good immunogenicity. Conclusions:Compared with Sepharose 4FF chromatography medium, Capto Core700 could more effectively remove process-related impurities such as HCP, HCD and total protein without affecting the recovery rate of viral antigen. This study provided reference for the development of purification technology in the production of H5N1 influenza virus vaccine in MDCK cells.
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Objective:To investigate the clinical effect of lobulated chimeric perforator flap pedicled with descending branch of lateral circumflex femoral artery (d-LCFA) in repair of multiple composite tissue defects of the foot and ankle.Methods:From February 2017 to March 2021, a total of 6 patients with foot and ankle multiple site deficiency injuries were treated in the Department of Hand Surgery, Shenzhen Hospital of Integrated Traditional Chinese and Western Medicine. The area of the soft tissue defect was 14.0 cm×9.0 cm~28.0 cm×9.0 cm. The size of the flap were 15.0 cm×10.0 cm-29.0 cm×10.0 cm. In all the cases, the wounds were complicated with bone defects and different degrees of infection. After primary debridement, VSD was used for continuous lavage and drainage. In the second stage, the defect was repaired with a lobulated chimeric perforator flap of d-LCFA. The colour, texture and sensory recovery of flap were observed in the scheduled postoperative follow-up.Results:All flaps survived smoothly without vascular crisis. After 10-12 months of follow-up, no recurrence of infection was found. The flaps had no pigmentation, soft in texture, with non-bloated appearance, good wear resistance, and recovered part of sensation. According to the British Medical Research Council (BMRC) hand sensory function evaluation standard, the sensation recovered to S 1-S 2. Conclusion:The lobulated chimeric perforator flap pedicled with d-LCFA only requires microanastomosis with 1 vascular pedicle. It simultaneously covers wounds in different depths at multiple sites and is also anti-infective. It is an ideal flap for repairing multi-site composite tissue defects of foot and ankle.
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Objective:To investigate the clinical effect of microdissected peroneal artery perforator flap in repair of soft tissue defect of dorsal side of the fingers.Methods:From August 2015 to July 2020, 19 patients with soft tissue defects on dorsal fingers were treated with microdissected peroneal artery perforator flap. The area of wound defect was 3.8 cm×1.5 cm-5.8 cm×3.0 cm, with exposure of phalanges and tendons. The size of flaps was 4.0 cm×1.8 cm-6.0 cm×3.3 cm. According to the size of soft tissue defects on the dorsal side of the fingers, the flaps were designed with the perforating branch of peroneal artery in the centre. The length and width of a flap were 0.2-0.3 cm bigger and wider than the area of defect. The perforator vessels with a length of 2.0-3.0 cm were arvested in the superficial layer of deep fascia. Most of the adipose tissues of the flap were removed under microscope, and the small arteries between adipose tissues were protected. The flaps were used to cover the defects of fingers. The perforator artery of the flap was anastomosed with the proper palmar digital artery of the recipient site, the accompanying vein of the perforator artery was anastomosed with the dorsal digital vein of the recipient site, and the cutaneous nerve in the flap was anastomosed with the dorsal digital nerve. The donor sites were directly pulled together and sutured intermittently. Outpatient and WeChat follow-up were conducted after operation, including wound healing, flap survival, flap sensation, donor site recovery, and flexion and extension functions of the fingers. Functional recovery was evaluated according to the Evaluation Standard of Upper Limb Partial Functional of Hand Surgery of Chinese Medical Association.Results:All wounds healed in Ⅰ stage, and all 19 flaps survived. The follow-up ranged from 9 to 25 months, with an average of 11.5 months. The appearance of the flaps was satisfactory and the texture was good. Sensation recoveried to S 4 in 4 paitients, S 3 in 9 patients and S 2 in 6 patients, and with only a linear scar was left in the donor sites. The hand function recovery was evaluated according to the Trial Criteria of Upper Limb Function Evaluation of the Hand Surgery Society of the Chinese Medical Association, with 18 cases were excellent and 1 was good. Conclusion:The microdissected peroneal artery perforator flap is an ideal surgical method to repair the soft tissue defect of dorsal side of the fingers, which has good shape and simple operation, avoids the secondary thinning and plastic surgery and offers good therapeutic effects.
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AIM: To study the suppressive effect of glycogen synthase kinase-3β( GSK-3β) knockdown by RNA interference on the formation of keloid .METHODS:Human keloid fibroblasts ( KFB) in vitro were transfected with 3 pairs of specific GSK-3βsmall interfering RNA (siRNA).The best siRNA to inhibit the GSK-3βexpression in human KFB was screen by RT-PCR and Western blot .The expression of GSK-3βand related proteins at mRNA and protein levels in the KFB was determined by RT-PCR and Western blot .RESULTS: The GSK-3βsiRNA1434 remarkably inhibited the expression of GSK-3βat mRNA and proteins levels in the human KFB .After transfection with GSK-3βsiRNA, the protein levels of β-catenin, p-GSK-3β, Wnt2 and cyclin D1 were all decreased.KFB growth became slow.With the extension of time, the inhibition of cell growth increased , and the cell doubling time was significantly delayed .CONCLUSION:siRNA targeting GSK-3βefficiently knocks down the expression of GSK-3βin the human KFB, and inhibits the activation of Wnt signaling pathway , thus inhibiting the growth of keloid .GSK-3βmay be a potential therapeutic target for keloid .
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Objective To explore promoter methylation of HIC1 gene and the expression of HIC1/SIRT1 related to the occurrence, development, and metastasis of papillary thyroid carcinoma. Methods Using Bisulfite sequencing PCR to analyze the promoter methylation of HIC1 gene. Using quantitative real-time PCR and Western blot to analyze expression differences of HIC1 and SIRT1 genes in tissues of papillary thyroid carcinoma(40 cases) and in adjacent normal thyroid(40 cases), of which datas were analyzed by statistics. Results The degree of HIC1 gene promoter methylation was significantly higher than that in adjacent normal tissues(P<0. 01). The degree of HIC1 gene promoter methylation in papillary thyroid carcinoma was related to lymph node metastasis, age, and the tumor-node-metastasis stages(P<0. 01). Compared with the expression of HIC1 mRNA and protein in adjacent normal thyroid tissue, that in papillary thyroid carcinoma was significantly lower(P<0. 01), while the expression of SIRT1 mRNA and protein in papillary thyroid carcinoma was significantly higher(P<0. 01). The lower expression of HIC1 mRNA and protein in the tumor tissues was related to the stage of lymph node metastasis, age, and the tumor-node-metastasis stages(P<0. 05). There was a strong negative correlation between the degree of HIC1 gene promoter methylation and expression of HIC1 in papillary thyroid carcinoma(P<0. 05). The expression of HIC1 mRNA and protein between that of SIRT1 also showed a strong negative correlation(P<0. 01). Conclusion Promoter hypermethylation of HIC1 and aberrant expression of HIC1/SIRT1 in papillary thyroid carcinoma may play a significant role in the oncogenesis and progress of papillary thyroid carcinoma. HIC1 is expected to become a new marker for prevention and treatment of papillary thyroid carcinoma.
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Objective To study the expression of WWOX and C-JUN in keloid and to approach their role and mechanism in the pathogenesis of keloid.Methods Immunohistochemical SP methods were used with computer pathological image analysis.Western blot and reverse transcription polymerase chain reaction (RT-RCR) were performed to detect the expression of WWOX and C-JUN in keloid and normal skin with statistical analysis.Results In keloid,the expression of WWOX protein was located in the cytoplasm of fibroblasts,and the expression of WWOX protein and its mRNA decreased,with significantly statistical difference (P＜0.05) compared to normal skin in the control group; the expression of C-JUN protein was located in the cell nucleus and cytoplasm of fibroblasts,with increased expression of C-JUN protein and its mRNA,with significantly statistical difference (P＜0.05) in comparison to normal skin in the control group.The expression of both was negative correlation (r=-0.626,P＜0.01).Conclusions Both WWOX with low expression and C-JUN with high expression are keloid-related genes,having significantly negative correlation between them,which may be one of the mechanisms for the keloid formation.It indicates that the WWOX protein may be an inhibitory factor to the expression of C-JUN protein,and the genes may play a major role in the pathogenesis of keloiod through fibroblasts.
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Objective To investigate the expression and clinical significance of zinc finger gene 217 (ZNF217) in human papillary thyroid carcinoma.Methods The expressions of ZNF217 mRNA and protein were detected by quantitative realtime PCR and Western blot in papillary thyroid carcinoma tissues (n =20) and adjacent normal tissues (n =20),and the data were analyzed.Results The expressions of ZNF217 mRNA and protein in papillary thyroid carcinoma were significantly higher than those in adjacent normal tissues (96.72 ± 44.19 vs 4.86 ±3.55,0.994 ± 0.172 vs 0.195 ± 0.061,both P＜0.01),being higher in the papillary thyroid carcinoma with capsule invasion compared with that without capsule invasion (P＜0.01).The expressions of ZNF217 mRNA and protein in papillary thyroid carcinoma were not related to gender,age,tumor size,TNM stage or lymph node metastasis (all P＞0.05).Conclusions The overexpression of ZNF217 may be associated with the oncogenesis and progress of papillary thyroid carcinoma and capsule invasion,and thus is expected to become a new target for prevention and treatment of papillary thyroid carcinoma.
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Silent information regulator 1 (SIRT1) is a kind of histone deacetylase which dependents on nicotinamide adenine dinucleotide.It is involved in the occurrence and development of tumors.Data show that SIRT1 is highly expressed in many tumors,such as gastric cancer,breast cancer,prostate cancer and so on.On the other hand,the expression of SIRT1 is lower in many other types of tumors,including glioblastomas,bladder cancer,ovarian cancer and so on.Therefore,the role of SIRT1 in tumor formation process is still uncertain and controversial.
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Objective To study the expression of ZNF217 and EF1α gene in the pathological scars and to investigate role and probable mechanism in the pathogenesis of abnormal scar.Methods Quantitative real-time PCR and Western blot were performed to detect the expression and distribution of mRNA and protein of ZNF217 and EF1α in hypertrophic scar (10 cases),keloid (10 cases),normal scar (10 cases),and normal skin (10 cases),and statistics was used to analyze the data.Results The expression of ZNF217 mRNA and protein in the normal skin,normal scar,hypertrophic scar and keloid were 1.46±0.397,1.45±0.265,4.49±0.999,5.47±0.808; 0.276±0.0211,0.299±0.0150,0.743t0.0509 and 0.747±0.0377,respectively.The expression of EF1α mRNA and prorein in the normal skin,normal scar,hypertrophic scar,and keloid were 1.47±0.469,1.47±0.218,5.10±1.68,5.74±1.92; 0.505±0.0371,0.518±0.0153,0.780±0.0369 and 0.792±0.0290,respectively.The positive rate of mRNA and protein of ZNF217 and EF1α was not statistically different between the hypertrophic scar and keloid (P＞0.05),while they were all remarkably significant in comparison between normal scar and abnormal scar (P＜0.01).In pathological scar mRNA and protein of ZNF217 and EF1α showed a strong positive correlation.Conclusions The expression of ZNF217 and EF1α is increased in pathological scar.Therefore,ZNF217 and EF1α overexpression may play an important role in the proliferation of fibroblasts and in the pathogenesis of pathological scar.
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Objective To evaluate the role of miR-143, miR-145 in the development of gastric gastrointestinal stromal tumor. Methods The expression levels of miR-143 and miR-145 in 21 cases of gastric gastrointestinal stromal tumor and the matched non-tumor adjacent tissue specimens were examined by stem-loop real-time RT-PCR, and its correlation with clinicopathologic features of gastric gastrointestinal stromal tumor were analyzed. Results Expression level of miR-145 were significantly higher in tumor than adjacent normal tissues (P＜0.01 ) and that with mitotic count ≥ 5/50HPF cases was significantly lower than that with mitotic count ＜5/50HPF cases (P=0.02). miR-145 expression in huge tumor (＞10 cm)was significantly lower than that in the large tumor (5～10 cm) and small tumor (2～5 cm) (P=0.048).By Fletcher risk stratification system, miR-145 expression in high-risk cases was significantly lower than that in the intermediate-risk and low-risk cases (P=0.048). While the expression of miR-145 in low-risk group was significantly different compared to that in intermediate-risk group and high-risk group (P=0.01).There was no difference between the expressions of miR-143 in tumor and that in normal tissue(P=0.06).Conclusion In gastric gastrointestinal stromal tumor, MiR-145 expression is significantly higher in tumor than adjacent normal tissues. miR-145 is closely associated with tumor size. mitotic counts and Fletcher risk stratification system.
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Objective To study the expression of eukaryotic translation initiation factor 4E(eIF4E)in the pathological scars and its probable role in the pathogenesis of pathological scars.Methods Immunohistochemiscal technique was performed to detect the expression and distribution of eIF4E protein in hypertrophic scars(14 cases),keloids(25 cases),mature scars(20 cases)and normal skins(20 cases).Reverse transcription polymerase chain reaction(RT-PCR)was used to detect the eIF4E mRNA level in hypertrophic scars(7 cases),keloids(8 cases),mature scars(8 cases)and normal skins(8 cases).Results Thepositive rate of eIF4E protein expression was remarkably significant difference between normal scars and pathological scars(P＜0.05).The level of eIF4E mRNA in pathological scars 1.73±0.31was higher than that in control group 0.99±0.28.There was significant difference between two groups (P＜O.05).Conclusions The expression of eIF4E is increased in pathological scar.eIF4 E expression is closely associated with the development of pathological scar.Therefore,eIF4E overexpression may play an important role in the proliferation of fibroblasts and in the pathogenesis of pathological scar.