RESUMO
INTRODUCTION: Tubularized urethroplasty is commonly performed in clinical practice using genital skin flaps, bladder mucosa, and buccal mucosa. However, the long-term effects are not satisfying, and donor site morbidities remain a problem. Besides, those grafts are unavailable with malignant conditions of the urinary tract, a history of lichen sclerosis, or oral disease. OBJECTIVE: An autologous granulation tissue tube of any required length and diameter can be produced by implanting foreign objects subcutaneously (Summary Fig.). The current study aimed to investigate to what extent of length this fully autologous tissue could be used for tubularized urethroplasty, satisfying urethral patency and tissue regeneration, in male rabbits. STUDY DESIGN: Twenty-seven New Zealand male rabbits were randomly divided into three groups. Silastic tubes were implanted subcutaneously in Group 1 and Group 2. By 2 weeks the granulation tissue encapsulating the tubes was harvested. In Group 1, pendulous urethral segments of 1 cm were excised, and urethroplasty was performed with the granulation tissue tube in an end-to-end fashion. In Group 2, a pendulous urethral segment of 1.5 cm was replaced with the tissue tube. In Group 3, a pendulous urethral defect of 1 cm was repaired by re-anastomosis as control. Serial urethrograms were performed at 1, 2 and 6 months postoperatively. Meanwhile, the neo-urethra were harvested and analyzed grossly and histologically. RESULTS: The urethrograms showed that all animals in Group 1 maintained a wide urethral caliber. In contrast, animals in Group 2 and Group 3 developed progressive strictures. Histologically, an intact urothelium with one to two cell layers lined the graft by 1 month, which was surrounded by increasing organized smooth muscle in Group 1. By 6 months, the grafts were completely integrated into native urethra. Nevertheless, extensive fibrosis occurred in Group 2 and Group 3. DISCUSSION: The tissue successfully maintained patency and guided urethral regeneration across a distance of 1 cm. As an epithelium-free graft, the tissue showed better results than acellular matrix for tubularized urethroplasty compared with previous studies. Nevertheless, several limitations existed: (1) the urethral defect was created in healthy urethra, which could not fully simulate the clinical situation; (2) as a small animal model, rabbit was less informative for clinical problems; (3) the tissue was inadequate for long segmental urethral replacement. Further study is needed before the procedure is used clinically. CONCLUSION: An autologous granulation tissue tube grown subcutaneously could be successfully used to repair urethral defects of 1 cm in male rabbits.
Assuntos
Tecido de Granulação/transplante , Engenharia Tecidual , Uretra/cirurgia , Procedimentos Cirúrgicos Urológicos/métodos , Animais , Biópsia por Agulha , Modelos Animais de Doenças , Rejeição de Enxerto , Sobrevivência de Enxerto , Imuno-Histoquímica , Masculino , Coelhos , Distribuição Aleatória , Recuperação de Função Fisiológica , Medição de Risco , Coleta de Tecidos e Órgãos , Transplante Autólogo/métodos , Resultado do Tratamento , Uretra/anormalidadesRESUMO
The NS1 gene of the H5N1 subtype avian influenza virus was amplified by RT-PCR, and the am-plified product was cloned into the eukaryotic expression vector pCMV-Myc, then it was transfected into A549 cells. After 48 h, the expression of NS1 was detected by Western blot. Fluorescence and electron microscopy and flow cytometry showed that the NS1 gene of H5N1 avian influenza virus could induce apop-tosis in human pulmonary carcinoma cell line A549.
Assuntos
Apoptose , Virus da Influenza A Subtipo H5N1/genética , Proteínas não Estruturais Virais/genética , Anexina A5/análise , Linhagem Celular Tumoral , Clonagem Molecular , Humanos , Virus da Influenza A Subtipo H5N1/patogenicidade , Neoplasias Pulmonares/patologia , Proteínas não Estruturais Virais/fisiologiaRESUMO
Human transferrin receptor (TfR) was isolated from homogenates of placental tissues by affinity chromatography on transferrin-Sepharose, and then used to screen human scFv against it from a fully-synthesized phage scFv library. After verifying the specificity, gene fragment of one of the selected scFv was inserted into the plasmid pET22b(+) and transformed into E. coli BL21(DE3) . Expression of scFv in transformant was induced with 0.5mmol/L IPTG. ELISA assay on HeLa cells showed that scFv protein could recognize and bind to TfR on the surface of HeLa cells. The scFv was purified by one-step affinity chromatography with Ni+ -NTA agarose, and injected into Kunming mouse via tail veins. This scFv was detected in brain tissues 1h later by capillary depletion method, which indicates that scFv protein can permeate through the blood brain barrier by mediation of the TfR receptor. Our works lay the foundation for the treatment of tumors and central nervous system diseases.