RESUMO
Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease with an unclear pathogenesis. This study aimed to elucidate the function and potential mechanisms of TUG1 in IPF progression. Cell viability and migration were detected by CCK-8 and transwell assays. Autophagy, fibrosis, or EMT-related proteins were measured by Western blotting. Pro-inflammatory cytokine levels were assessed by ELISA kits. The subcellular localization of TUG1 was observed by FISH assay. RIP assay detected the interaction between TUG1 and CDC27. TUG1 and CDC27 was up-regulated in TGF-ß1-induced RLE-6TN cells. TUG1 depletion suppressed pulmonary fibrosis via attenuating inflammation, EMT, inducing autophagy and inactivating PI3K/Akt/mTOR pathway in vitro and in vivo. TUG1 knockdown prevented CDC27 expression. TUG1 silencing ameliorated pulmonary fibrosis by reducing CDC27 expression and inhibiting PI3K/Akt/mTOR pathway.
Assuntos
Fibrose Pulmonar , RNA Longo não Codificante , Subunidade Apc3 do Ciclossomo-Complexo Promotor de Anáfase/genética , Subunidade Apc3 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Metilação de DNA , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fibrose Pulmonar/genética , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , AnimaisRESUMO
OBJECTIVE: To evaluate the influence of epididymitis history on the results of microsurgical vasoepididymostomy and spontaneous pregnancy in patients with epididymal obstruction. METHODS: Totally 205 patients with epididymal obstruction underwent microsurgical two-suture longitudinal intussusception vasoepididymostomy from January 2014 to December 2016. After surgery, we evaluated the semen quality of the patients every 3 months till conception and compared the rates of patency and spontaneous pregnancy between the patients with and those without an epididymitis history. RESULTS: The patients ranged in age from 22 to 46 (mean 31) years, 37 (22.2%) with and 126 (77.8%) without an epididymitis history among the 163 patients for whom a 25.4-month follow-up (from 7 to 42 months) was completed. No statistically significant differences were observed postoperatively between the patients with and those without an epididymitis history in the patency rate (73.0% vs 81.7%, P = 0.243), sperm concentration (18 ï¼»1.3ï¼33.6ï¼½ vs 15.2 ï¼»0.8ï¼33.4ï¼½ ×106/ml, P = 0.710), percentage of progressively motile sperm (27.5 ï¼»0ï¼46.1ï¼½% vs 19.3 ï¼»0ï¼41ï¼½% (P = 0.592) or rate of spontaneous pregnancy (24.3% vs 38.9%, P = 0.104). CONCLUSION: Microsurgical vasoepididymostomy is an effective method for the treatment of epididymal obstruction, and epididymitis history does not affect the results of the strategy.
Assuntos
Epididimo , Epididimite , Gravidez , Feminino , Humanos , Masculino , Adulto , Epididimo/cirurgia , Epididimite/complicações , Epididimite/cirurgia , Análise do Sêmen , Resultado do Tratamento , Microcirurgia/métodos , Sêmen , Ducto Deferente/cirurgiaRESUMO
BACKGROUND: This systematic review will assess the effectiveness and safety neuromuscular electrical stimulation (NMES) for cancer pain (CP) in children with osteosarcoma. METHODS: This systematic review protocol will retrieve the following electronic databases from inception to June 1 in Cochrane Library, MEDLINE, EMBASE, Web of Science, Scopus, CNKI, and VIP database. Manual head-searching of reference lists and conference proceedings will be performed to further examine the articles of interest. No restrictions will be applied to language and publication status. We will utilize a 3-stage approach to scan titles, abstracts, and full-text studies against all eligibility criteria, and collect data from included trials. Study quality will be evaluated by the Cochrane Risk of Bias Tool. If possible, we will narratively summarize study results and carry out meta-analysis. RESULTS: This study will recapitulate the present high quality trials to appraise the effectiveness and safety of NMES for CP in children with osteosarcoma. CONCLUSION: The findings of this study will present evidence to determine whether NMES is effective and safe for CP in children with osteosarcoma.
Assuntos
Dor do Câncer/terapia , Terapia por Estimulação Elétrica/métodos , Osteossarcoma/patologia , Adolescente , Criança , Feminino , Humanos , Masculino , Osteossarcoma/epidemiologia , Segurança , Resultado do TratamentoRESUMO
The objective of this study was to explore the diagnostic value of the bronchoalveolar lavage fluid galactomannan (BALF GM) test for chronic respiratory disease with pulmonary aspergillosis and to establish the optimal cutoff value. Samples from a total of 309 chronic respiratory disease patients seen at the respiratory medicine department of Peking University Shenzhen Hospital from September 2016 to September 2019 were analyzed. According to the diagnostic criteria, we divided the patients into a case group (n = 79, comprising 25 proven cases and 54 probable cases) and a control group (n = 230). Bronchoalveolar lavage fluid was collected, and the BALF GM test results were analyzed. A nonparametric rank sum test showed that the mean rank of the case group was 255.30, which was higher than that of the control group (120.55). The Z-value was -11.567 (P = 0.000), indicating that the general distributions of BALF GM differed between the two groups. A BALF GM cutoff value of 0.88 showed the highest diagnostic efficacy for pulmonary aspergillosis. The sensitivity, specificity, positive predictive value, and negative predictive value were 77.2%, 93%, 79.2%, and 92.2%, respectively. As the cutoff value increased, the specificity and sensitivity of the BALF GM test increased and decreased, respectively. The BALF GM test can be used confirm the diagnosis of patients with pulmonary aspergillosis and chronic respiratory disease. The optimum BALF GM cutoff value is 0.88.
Assuntos
Líquido da Lavagem Broncoalveolar/química , Mananas/análise , Aspergilose Pulmonar/diagnóstico , Adulto , Idoso , Aspergillus , Biópsia , Broncoscopia , China , Ensaio de Imunoadsorção Enzimática , Feminino , Galactose/análogos & derivados , Humanos , Masculino , Pessoa de Meia-Idade , Aspergilose Pulmonar/microbiologia , Curva ROC , Sensibilidade e Especificidade , Adulto JovemRESUMO
OBJECTIVE: To evaluate the efficacy of microsurgical vasectomy reversal on patients after vasectomy. METHODS: In the study, 41 patients after vasectomy were admitted for microsurgical vasectomy reversal. The mean (range) age was 45 (29 to 66) years for the men. The mean duration of obstruction was 12 years. All the patients were seen by the surgeon for a complete history and physical examination. Semen analyses proved azoopermia, and their serum levels of sexual hormone were normal. They were all offered scrotal exploration. Microvasovasostomy was performed if the sperm was found in the material coming from the proximal vas lumen. The decision for microvasoepididymostomy was made during surgery, if the fluid was creamy, containing only debris or there was no fluid whatsoever when the vas was milked toward the cut end. Microvasovasostomy was performed in 40 patients, of whom 6 were with lateral microvasoepididymostomy and 1 with bilateral microvasoepididymostomy. The initial semen analysis was then done after usually 3 months postoperatively. Patency was arbitrarily defined as>10 000 sperm/mL in ejaculate in at least one semen analysis after surgery. Postoperative patency rate and postoperative impregnation rate were followed. RESULTS: The 41 patients were followed up for 3 to 72 months, and 5 cases were lost. In the 28 patients who had obstructions for less than 15 years, 3 were lost. Sperm was found by semen analysis in 21 patients. Their sperm density was 2×10(6) to 46×10(6) sperms/mL and motility was 0% to 60%. The semen analysis revealed azoospermia in 2 patients after 12 months, who were advised to undergo intracytoplasmic sperm injection. Natural conception occurred in 6 patients followed for more than 12 months. The overall patency rate was 84% (21/25). Among the patients with a follow-up of >6 months, the natural paternity rate was 29% (6/21). In the 13 patients who had obstructions for more than 15 years, 2 were lost. The patency and pregnancy rates were 64% (7/11) and 14% (1/7) respectively. Their sperm density was 0.02×10(6) to 30× 10(6) sperms/mL and motility was 0% to 43%. CONCLUSION: Microsurgical vasectomy reversal is an effective method to treat vasal obstruction due to vasectomy. The patency and pregnancy rates are related to the time of vasectomy. The patency and pregnancy rates are higher in patients with obstruction for less than 15 years than those for more than 15 years.
Assuntos
Vasectomia , Vasovasostomia , Adulto , Idoso , Azoospermia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Gravidez , Taxa de Gravidez , Contagem de Espermatozoides , Injeções de Esperma Intracitoplásmicas , Espermatozoides , Fatores de TempoRESUMO
We retrospectively evaluated the clinical outcome of penile prosthesis implantation (PPI) in Chinese patients with severe erectile dysfunction (SED). From July 2000 to December 2011, 224 patients (mean age: 35.9±11.8 years, range: 20-75 years) with SED underwent PPI by experienced surgeon according to standard PPI procedure at our centre. A malleable prosthesis (AMS 650) was implanted in 45 cases (20.1%), and a three-piece inflatable prosthesis (AMS 700 CXM or AMS 700 CXR) was implanted in 179 cases (79.9%). Surgical outcomes, including postoperative complications, clinical efficacy and couple satisfaction, were evaluated over than 6 months postoperatively using medical record abstraction, IIEF-5, quality of life (QoL) scores, and the patient/partner sexual satisfaction score proposed by Bhojwani et al. Of the 224 patients eligible for the study, 201 subjects (89.7%) completed follow-up. All of patients could perform sexual intercourse post PPI with the mean postoperative IIEF-5 and QoL scores were 20.02±2.32 and 5.28±0.76, respectively, which were significantly improved compared with the preoperative scores (6.29±1.5 and 2.13±0.84, P<0.01). Of the 201 men, mechanical malfunction occurred in four cases (2.0%) and three cases were re-implanted new device, and two cases (1.0%) developed a mild curvature of the penis. Scrotal erosion with infection occurred in one case with diabetes mellitus (0.5%) and required complete removal of the implanted AMS 700 CXM. Satisfactory sexual intercourse at least twice per month was reported by 178 men (88.6%), and overall satisfaction with the PPI surgery was reported by 89.0% of men and 82.5% of partners. Patient satisfaction in the three-piece inflatable prosthesis group was higher than in the malleable prosthesis group (P<0.05). Satisfaction, however, between the types of prostheses, did not differ in the partner survey. PPI is a safe and effective treatment option for Chinese patients with SED and experienced surgeon perform PPI according to standard PPI procedure could reduce the postoperative complications of PPI and could improve patient satisfaction ratio and QoL.
Assuntos
Disfunção Erétil/cirurgia , Implante Peniano , Pênis/cirurgia , Adulto , Idoso , China , Humanos , Masculino , Pessoa de Meia-Idade , Satisfação do Paciente , Implante Peniano/métodos , Prótese de Pênis , Implantação de Prótese , Qualidade de Vida , Estudos Retrospectivos , Parceiros Sexuais , Resultado do TratamentoRESUMO
OBJECTIVE: To investigate the injected autologous adipose-derived stem cells (ADSCs) in improving stress urinary incontinence in a rodent model of parturition-related stress incontinence and the possible mechanism. METHODS: The 40 rats were developed stress urinary incontinence models by postpartum balloon dilation of the vagina for 4 hours followed by bilateral ovariectomy. ADSCs were isolated from the peri-ovarian fat and labeled with thymidine analog 5-ethynyl-2-deoxyuridine (EdU). Twenty stress urinary incontinence rats received peri-urethral injection of phosphate-buffered saline as the negative controls and the other 20 stress urinary incontinence rats received peri-urethral injection of EdU-labeled ADSCc. Twenty control rats underwent sham ovariectomy without balloon dilation and served as positive controls. Four weeks later, voiding function was assessed by cystometry. Urethral histologic examination (Masson trichrome stain, picrosirius red stain, Hart elastin stain, Gordon and Sweet stain, and immunohistochemical stain) and Western blot were performed on urethral tissues. RESULTS: Both leak point pressure and bladder capacity were significantly increased in ADSC-treated rats, compared to the balloon-injured ovariectomized rats. Histologic examination revealed normalized appearance of the fibromuscular structure of the urethra as well as increased peri-urethral blood vessel density in ADSC-treated rats. On Western blot, vascular endothelial growth factor and P-extracellular signal-regulated kinases (ERKs)1/2 protein was expressed at a higher rate in tissues from ADSC-treated rats compared to phosphate-buffered saline-treated rats. CONCLUSION: Peri-urethral injection of ADSCs is associated with more normal urinary function and urethral structure in rats with parturition-related incontinence. The activation of vascular endothelial growth factor and ERK1/2 may be responsible for the paracrine effects from ADSCs.
Assuntos
Tecido Adiposo/transplante , Sistema de Sinalização das MAP Quinases , Transplante de Células-Tronco , Uretra/fisiopatologia , Incontinência Urinária por Estresse/fisiopatologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Modelos Animais de Doenças , Feminino , Músculo Liso/patologia , Músculo Estriado/patologia , Neovascularização Fisiológica , Ovariectomia , Parto , Ratos , Ratos Sprague-Dawley , Uretra/irrigação sanguínea , Uretra/metabolismo , Uretra/patologia , Bexiga Urinária/fisiologia , Incontinência Urinária por Estresse/metabolismo , Incontinência Urinária por Estresse/terapia , Micção/fisiologia , UrodinâmicaRESUMO
OBJECTIVE: To evaluate the changes in the number and maturation of lung tissue dendritic cells (DCs) and to assess the chronic inflammation in a cigarette smoke-induced COPD model in rats. METHODS: Thirty male F344 rats were randomly divided into 3 groups (n = 10): a control group, a smoke-exposure group and a smoking cessation group. Rat lung pathomorphological changes were observed by hematoxylin-eosin (HE) stain. Lung tissue CD(11c)(+) DCs, CD(86)(+) DCs and CD(8)(+) T cell numbers were observed by immunohistochemistry method. Flow cytometry was used for detection of CD(11c)(+)/CD(86)(+) DCs and CD(11c)(+)/MHCII(+) DCs proportions. RESULTS: The airway inflammatory pathological score and the mean linear intercept (MLI) obtained from he smoke-exposure group and the smoking cessation group (390 ± 33, 324 ± 28) and [(68 ± 11) µm, (58 ± 9) µm] were higher than those in the control group (56 ± 13) and (36 ± 6) µm (F = 459.85 and 34.03, all P < 0.05). In the smoke-exposure group and the smoking cessation group, the positive rate of CD(11c)(+) DCs [(1.47 ± 0.12)%, (1.30 ± 0.17)%], and the positive rate of CD(86)(+) DCs [(1.26 ± 0.18)%, (1.02 ± 0.08)%] were higher than those in the control group [(0.96 ± 0.08)%, (0.65 ± 0.03)%] (F = 6.55 and 30.26, all P < 0.05), but there was no significant difference between the smoke-exposure group and the smoking cessation group (t = 1.10 and 1.47, all P>0.05). In the smoke-exposure group and the smoking cessation group, CD(8)(+) T positive rate [(2.72 ± 0.15)%, (2.35 ± 0.23)%] was higher than that in the control group [(1.39 ± 0.11)%] (F = 16.07, P < 0.05). CD(11c)(+)/CD(86)(+) DCs and CD(11c)(+)/MHCII(+)DCs percentages [(5.5 ± 0.4)%, (4.8 ± 0.4)%], [(4.2 ± 0.4)%, (3.3 ± 0.3)%] decreased in the smoke-exposure group and the smoking cessation group as compared to the control group [(8.0 ± 0.5)%, (6.1 ± 0.5)%] (F = 14.34 and 12.82, all P < 0.05). There was no significant difference between all the above index from the smoke-exposure group and the smoking cessation group (t = 1.10 and 2.11, all P > 0.05). CONCLUSIONS: Cigarette smoke exposure induced increased DCs transmigrated and influenced the maturation of DCs in COPD rats. Even after smoking cessation, non-specific chronic inflammation was still present, suggesting that DCs number and maturation abnormality may be involved in the chronic inflammation of COPD.
Assuntos
Células Dendríticas/patologia , Inflamação , Doença Pulmonar Obstrutiva Crônica/patologia , Fumar/efeitos adversos , Animais , Contagem de Células , Células Dendríticas/citologia , Modelos Animais de Doenças , Pulmão/citologia , Masculino , Ratos , Ratos Endogâmicos F344 , Abandono do Hábito de FumarRESUMO
OBJECTIVE: To evaluate the intraoperative factors affecting the patency of microsurgical vasoepididymostomy. METHODS: From January 2009 to July 2010, 73 men with obstructive azoospermia had undergone longitudinal intussusception vasoepididymostomy (LIVE). The mean age (range) was 31 years (23 to 48) for the patients and their mean infertility was 50.9 months (2 to 180). All the patients were included in three groups by the clinical variables in surgery: anastomosis number, anastomosis sites, epididymal fluid analysis. Bilateral and lateral anastomosis was in 50 and 23 cases, respectively. The anastomosis on caput, corpus and caudal was in 19, 46, and 8 cases respectively. Motile sperm in the epididymal fuild continuing flowage could be found in 55 patients. By contrast, immotile sperm could be found in 18 patients. Semen analysis was done at the end of 3 months, and patency was arbitrarily defined as >10 000 sperm/mL in ejaculate in at least one semen analysis after surgery. RESULTS: Of all the patients, 53 were followed up. Sperm was present in the ejaculate in 38 patients postoperatively. The overall patency rate was 71.7% (38/53). Patency rates of bilateral surgery and of lateral surgery achieved 80.7% and 27.6%; of caput anastomosis 38.5%, of corpus anastomosis 78.8%, of caudal anastomosis 100%, of motile sperm with epididymal fluid continuing flowage 83.7% and of immotile sperm 40%, respectively. In the study, 17 patients (33.3%, 17/53) reported spontaneous pregnancy. CONCLUSION: Except for microsurgical technology, bilateral anastomosis, corpus or caudal anastomosis, and motile sperm with epididymal fluid continuing flowage may improve patency rate postoperatively.
Assuntos
Azoospermia/cirurgia , Epididimo/cirurgia , Microcirurgia/métodos , Ducto Deferente/cirurgia , Vasovasostomia/métodos , Adulto , Anastomose Cirúrgica/métodos , Azoospermia/diagnóstico , Epididimo/patologia , Humanos , Masculino , Resultado do Tratamento , Ducto Deferente/patologia , Adulto JovemRESUMO
This study sought to investigate the clinical efficacy and safety of combined oral therapy with sildenafil and doxazosin GITS compared to sildenafil monotherapy in treating Chinese patients with erectile dysfunction (ED) and lower urinary tract symptoms secondary to benign prostatic hyperplasia (BPH/LUTS). The trial was conducted in hospitals in Beijing, Shanghai, Changsha, Wuhan and Guangzhou, five major cities in China. A total of 250 patients diagnosed with ED and BPH/LUTS aged 50-75 years, and who had International Index of Erection Function-5 (IIEF-5) scores ≤21 and International Prostate Symptom Score (IPSS) ≥10 points, were enrolled and randomly divided into Group A (168 cases; doxazosin GITS 4 mg once daily plus sildenafil 25-100 mg on demand) and Group B (82 cases; sildenafil 25-100 mg on demand). Efficacies were evaluated by IIEF-5 and IPSS scores and a quality of life (QoL) questionnaire, and adverse effects were evaluated during the treatment period. There were no statistically significant differences in mean age, and IIEF-5, IPSS and QoL scores pre-treatment between the two groups. After treatment, IIEF-5, IPSS and QoL scores were significantly improved in Group A, while only IIEF-5 scores were significantly improved in Group B compared with pre-treatment. There were no significant differences in side effects between the two groups. The results indicated that combined therapy with sildenafil and doxazosin GITS for the treatment of ED and BPH/LUTS is safe and effective compared to sildenafil monotherapy.
Assuntos
Doxazossina/administração & dosagem , Disfunção Erétil/tratamento farmacológico , Piperazinas/administração & dosagem , Hiperplasia Prostática/tratamento farmacológico , Sulfonas/administração & dosagem , Transtornos Urinários/tratamento farmacológico , Idoso , Povo Asiático , China , Humanos , Masculino , Pessoa de Meia-Idade , Hiperplasia Prostática/complicações , Purinas/administração & dosagem , Qualidade de Vida , Citrato de Sildenafila , Resultado do Tratamento , Transtornos Urinários/etiologiaRESUMO
In this study, we investigated the safety and efficacy of a poly acid-co-glycolide biodegradable scaffold (Maxpol-T) coated by autologous fibroblasts (AF) for penile girth enlargement in small penis syndrome (SPS). Eighty patients with SPS were enrolled in a clinical study at 2 medical centers; 69 patients completed the study protocol. Scrotal skin was harvested under local anesthesia, and AFs were cultured and seeded on a Maxpol-T scaffold; the cografted scaffold was implanted under the Buck's fascia of penile shaft via a circumcising incision. Patients were followed up at 1, 3, and 6 months to evaluate penile girth changes. Patient satisfaction was assessed via Visual Analogue Scale and scored on the International Index of Erectile Function-5 (IIEF-5). Mean preoperative penile girth in the flaccid and erect state was 8.18 ± 0.83 cm and 10.26 ± 1.22 cm, respectively. At the 6-month postoperative follow-up, mean penile girth in the flaccid and erect state was increased to 12.19 ± 1.27 cm and 13.18 ± 1.31 cm, respectively (P < .001 for change in both flaccid and erect state). Sixty-five patients (94.2%) reported satisfaction with the procedure. Among them, 4 cases (5.8%) were dissatisfied, 7 cases (10.1%) were satisfied, 26 cases (37.7%) were very satisfied, and 32 cases (46.4%) were extremely satisfied. All men maintained IIEF-5 scores of more than 22. Complications included prolonged subcutaneous edema in 3 patients (4.3%) and pinpoint erosion at the suture area in 3 patients (4.3%). Implantation of autologous fibroblasts seeded on a Maxpol-T collagen scaffold holds promise as a safe and novel technique for penile girth enhancement in patients with SPS.
Assuntos
Fibroblastos/transplante , Pênis/cirurgia , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos , Procedimentos Cirúrgicos Urológicos Masculinos/instrumentação , Procedimentos Cirúrgicos Urológicos Masculinos/métodos , Adulto , Humanos , Ácido Láctico , Masculino , Pessoa de Meia-Idade , Satisfação do Paciente , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Síndrome , Alicerces Teciduais , Adulto JovemRESUMO
OBJECTIVE: To study the curative effect of operation to flatfoot deformity with concurrent spinal disease in children. METHODS: A retrospective survey was performed in 8 flatfoot patients with concurrent spinal disease from January 2004 to January 2008. Every case received special operative treatment. After casting, achilles tendon prolongation, reefing or tendon transfer, rehabilitation therapy was performed sequentially. Radiographs and clinical outcomes were evaluated during the follow-up. RESULTS: Six patients were followed up. There were 2 feet in 1 female and 7 feet in 5 males. The complications included tethered cord syndrome (n = 1), T12-S1 vertebral canal arachnoid cyst (n = 1), cerebral palsy and scoliosis (n = 1) and scoliosis and spinal bifida occult (n = 3). The average age was 8.4 (4 - 14) years old and the average follow-up time 22 (14 - 64) months. All cases were satisfied with the appearance and function according to Maryland foot score. Five were excellent, 2 good and 2 fair postoperatively versus 2 fair and 7 failed preoperatively. The total excellent and good rate was 77.8%. In the early stage after tendon transfer in 4 patients, the lateral view of height arch, talus-first metatarsal angle, calcaneus-navicular-first metatarsal angle and talonavicular coverage angle improved. Yet it lost the improved angle and reverted to the pre-operative state. No degenerative change was detected in tarsus joints during the long follow-up. CONCLUSION: The special and sequential surgical procedure may be an effective regimen in the treatment of flatfoot deformity with concurrent spinal disease in children.
Assuntos
Pé Chato/complicações , Pé Chato/cirurgia , Doenças da Coluna Vertebral/complicações , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Estudos RetrospectivosRESUMO
To study the function of mitochondrial PTEN in mediation of cellular apoptosis, the adenoviral recombinant of Mito-PTEN, which contains CoxVII (subunit VII of Cytochrome C Oxidase) gene in N-terminus, were generated. Using CoxV II-PTEN-EYFPN1 as a template, Cox VII-PTEN was cloned into the shuttle vector pAdTrack-CMV with the restriction endonuclease sites Xho I and Xba I . The shuttle plasmid was linearize with Pme I and co-transformed with adenoviral backbone vector pAdeasy-1 into E. coli BJ5183. Following selection and identification, the positive recombinant plasmids were transformed into E. coli Dalpha for propagation. To package the adenoviruses, recombinant plasmid candidate was linearize using Pac I and transfected into HEK-293A cells with Lipofectamine 2000. Through freeze-thaw-vortex cycles, recombinant viral particles were collected and harvested, and utilized to infect 293A cells for further amplification. The method of TCID50 was employed to determine virus titers. With green fluorescent protein (GFP) as marker, the efficiency of transfection and infection was monitored by fluorescence microscopy, and the apoptosis of A431 cells after infection of Mito-PTEN-Ad was analyzed by flow cytometry. Adenoviral recombinant of Mito-PTEN was packaged successfully with the TCID50 as 10 pfu/mL and the expressed protein was detected by western blot. In addition, it has been demonstrated that Mito-PTEN promoted apoptosis of A431 cells. Take together, the successful generation of adenoviral recombinant of Mito-PTEN, which could induce apoptosis in A431 cells, sets up a basis for further functional studies of mitochondrial PTEN and provides us a potential tool for cancer treatment in future.
Assuntos
Apoptose , Mitocôndrias/fisiologia , PTEN Fosfo-Hidrolase/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Adenoviridae/genética , Linhagem Celular Tumoral , Complexo IV da Cadeia de Transporte de Elétrons/genética , Humanos , Plasmídeos , Reação em Cadeia da PolimeraseRESUMO
OBJECTIVE: To study the expression and localization of ATP50 by construction of ATP50-pEYFP-N1 in primary cultured mouse Leydig cells. METHODS: Primary cultured mouse Leydig cells were confirmed by 3B-HSD staining. ATP50 was cloned into pEYFP-N1 between Bam HI and Eco RI sites. Cell-transfection and living-cell fluorescence imaging microscopy were employed to investigate the sub-cellular localization of YFP-ATP50 in TM3 mouse Leydig cells. RESULTS: ATP50 green fluorescent protein was well co-localized with red fluorescence mitochondrion marker-Mitotracker in TM3 mouse Leydig cells. CONCLUSION: ATP50 was expressed in primary cultured mouse Leydig cells. The fluorescent expression vector of ATP50 was constructed successfully and YFP-ATP50 was located in mitochondria in TM3 mouse Leydig cells, which provided a useful clue for further research on the steroidogenesis dysfunction in aging males.
Assuntos
Adenosina Trifosfatases/biossíntese , Proteínas de Transporte/biossíntese , Células Intersticiais do Testículo/metabolismo , Proteínas de Membrana/biossíntese , Adenosina Trifosfatases/genética , Animais , Proteínas de Transporte/genética , Células Cultivadas , Clonagem Molecular , Vetores Genéticos , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Masculino , Proteínas de Membrana/genética , Camundongos , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras , Proteínas Recombinantes de Fusão/biossíntese , TransfecçãoRESUMO
OBJECTIVE: To explore the ability of insulin-like growth factor-I (IGF-I) and hyaluracan acid in prompting chondrogenesis of engineering cartilage tissue. METHODS: Human articular chondrocytes were isolated and cultured in DMEM plus 10% fetal bovine serum. They were divided into three groups: hyaluracan acid + chondrocytes + IGF-I group (IGF-I group), hyaluracan acid + chondrocytes group (cell group), hyaluracan acid group (control group). The ability of chondrogenesis was investigated by HE and toluidine blue staining, human collagen II immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Both cell group and IGF-I group could develop into cartilage tissue in the sixth week while control group could not. The number of cartilage lacuna in IGF-I group were more than that in cell group. Human collagen II immunohistochemistry showed that there were stronger positive cell in IGF-I group than in cell group, collagen II mRNA expression was more higher and collagen I mRNA expression was lower in IGF-I group than in cell group. CONCLUSION: Insulin growth factor-I can prompt chondrogenesis of engineering cartilage tissue and ameliorate the quality of engineering cartilage tissue in vitro.
Assuntos
Cartilagem Articular/citologia , Condrócitos/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Engenharia Tecidual , Cartilagem Articular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Condrócitos/fisiologia , Colágeno Tipo II/metabolismo , Humanos , Imuno-Histoquímica , Polímeros/metabolismoRESUMO
OBJECTIVE: To investigate the expression and role of adrenomedullin (ADM) and adrenomedullin receptor (ADMR) in patients with chronic obstructive pulmonary disease (COPD). METHODS: The first study included 9 COPD patients (group I) and 7 control patients (group II) who had underwent pulmonary lobe resection. Small pulmonary artery remodeling was observed with morphometric analysis. The expressions of ADM and ADMR mRNA in lung tissue were examined by hybridization in situ. The second study recruited 22 patients with COPD. Cardiac catheterization was performed to monitor the changes of hemodynamic parameters and the subjects were divided into two groups according to the mean pulmonary artery pressure (mPAP): patients without pulmonary hypertension (PH, group A, n = 10) and patients with PH (group B, n = 12). The levels of pulmonary arterial plasma ADM were measured by radioimmunoassay. Arterial blood gas was also measured. RESULTS: (1) In patients underwent pulmonary lobe resection: (1) Morphometry studies showed that the ratio of vascular wall thickness to external diameter (MT%) and ratio of vascular wall area to total area (MA%) increased in group I (P < 0.01); (2) Hybridization in situ showed that ADM/ADMR mRNA expressions were significantly higher in pulmonary artery walls of group I as compared with group II (P < 0.01). (2) In COPD patients without pulmonary lobe resection, the plasma ADM level of group B was significantly higher than that of group A (P < 0.05), and the plasma ADM level had a significantly positive correlation with mPAP and pulmonary vascular resistance (PVR), but a negative correlation with PaO(2). CONCLUSION: ADM may play an important protective role as a local autocrine/paracrine factor in the development of COPD and PH.