RESUMO
Many studies have recognized that circular RNAs (circRNAs) can be promising targets for renal cell carcinoma (RCC) by acting as competing endogenous RNAs for miRNAs. This study intends to uncover the implication of a novel circRNA, circ_000926 in RCC, and how it affects tumorigenesis. Microarray-based circRNA/gene expression profiling of RCC was used to identify differentially expressed circRNAs/genes in RCC and normal tissues. miRNAs targeting the screened circRNAs/genes were predicted online, followed by analyzing circ_000926 expression in RCC. The crosstalk among circ_000926, miRNA-411 (miR-411), and CDH2 was then validated. The expression of circ_000926, miR-411, and cadherin 2 (CDH2) was up-regulated or down-regulated in RCC cells to unearth their effects on the biological behaviors of RCC cells. circ_000926 was highly expressed in RCC tissues and cell lines, whereas CDH2 was verified to be a target of miR-411. As a competing endogenous RNA, circ_000926 could directly bind to miR-411 to up-regulate CDH2. Down-regulation of circ_000926 resulted in inhibited growth, migration, and invasion abilities of RCC cells, as well as suppressed epithelial-mesenchymal transition and tumor growth. However, the inhibition of miR-411 or elevation of CDH2 reversed the antitumor effects induced by silencing circ_000926. Down-regulation of circ_000926 exerts an inhibitory effect on RCC progression through miR-411-dependent CDH2 inhibition, highlighting a potential target for RCC treatment.
Assuntos
Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Caderinas/metabolismo , Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , MicroRNAs/genética , RNA Circular/genética , Animais , Antígenos CD/genética , Apoptose , Biomarcadores Tumorais/genética , Caderinas/genética , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Adesão Celular , Movimento Celular , Proliferação de Células , Progressão da Doença , Transição Epitelial-Mesenquimal , Feminino , Seguimentos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
AIM: Adenovirus-mediated gene therapy is a novel therapeutic approach for the treatment of cancer, in which replication of the virus itself is the anticancer method. However, the success of this novel therapy is limited due to inefficient delivery of the virus to the target sites. In this study, we used dendritic cells (DCs) as carriers for conditionally replicating adenoviruses (CRAds) in targeting prostate carcinoma (PCa). METHODS: Four types of CRAds, including Ad-PC (without PCa-specific promoter and a recombinant human tumor necrosis factor, rmhTNF, sequence), Ad-PC-rmhTNF (without PCa-specific promoter), Ad-PPC-NCS (without an rmhTNF sequence) and Ad-PPC-rmhTNF, were constructed. The androgen-insensitive mouse PCa RM-1 cells were co-cultured with CRAd-loading DCs, and the viability of RM-1 cells was examined using MTT assay. The in vivo effects of CRAd-loading DCs on PCa were evaluated in RM-1 xenograft mouse model. RESULTS: Two PCa-specific CRAds (Ad-PPC-NCS, Ad-PPC-rmhTNF) exhibited more potent suppression on the viability of RM-1 cells in vitro than the PCa-non-specific CRAds (Ad-PC, Ad-PC-rmhTNF). In PCa-bearing mice, intravenous injection of the PCa-specific CRAd-loading DCs significantly inhibited the growth of xenografted tumors, extended the survival time, and induced T-cell activation. Additionally, the rmhTNF-containing CRAds exhibited greater tumor killing ability than CRAds without rmhTNF. CONCLUSION: DCs may be an effective vector for the delivery of CRAds in the treatment of PCa.
Assuntos
Adenoviridae/fisiologia , Células Dendríticas/virologia , Terapia Viral Oncolítica/métodos , Neoplasias da Próstata/terapia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Portadores de Fármacos/uso terapêutico , Terapia Genética/métodos , Humanos , Masculino , Camundongos , Regiões Promotoras Genéticas , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/uso terapêuticoRESUMO
Prostate cancer (PCa) patients with lymph node involvement (LNI) constitute a single-risk group with varied prognoses. Existing studies on this group have focused solely on those who underwent prostatectomy (RP), using statistical models to predict prognosis. This study aimed to develop an easily accessible individual survival prediction tool based on multiple machine learning (ML) algorithms to predict survival probability for PCa patients with LNI. A total of 3280 PCa patients with LNI were identified from the Surveillance, Epidemiology, and End Results (SEER) database, covering the years 2000-2019. The primary endpoint was overall survival (OS). Gradient Boosting Survival Analysis (GBSA), Random Survival Forest (RSF), and Extra Survival Trees (EST) were used to develop prognosis models, which were compared to Cox regression. Discrimination was evaluated using the time-dependent areas under the receiver operating characteristic curve (time-dependent AUC) and the concordance index (c-index). Calibration was assessed using the time-dependent Brier score (time-dependent BS) and the integrated Brier score (IBS). Moreover, the beeswarm summary plot in SHAP (SHapley Additive exPlanations) was used to display the contribution of variables to the results. The 3280 patients were randomly split into a training cohort (n = 2624) and a validation cohort (n = 656). Nine variables including age at diagnosis, race, marital status, clinical T stage, prostate-specific antigen (PSA) level at diagnosis, Gleason Score (GS), number of positive lymph nodes, radical prostatectomy (RP), and radiotherapy (RT) were used to develop models. The mean time-dependent AUC for GBSA, RSF, and EST was 0.782 (95% confidence interval [CI] 0.779-0.783), 0.779 (95% CI 0.776-0.780), and 0.781 (95% CI 0.778-0.782), respectively, which were higher than the Cox regression model of 0.770 (95% CI 0.769-0.773). Additionally, all models demonstrated almost similar calibration, with low IBS. A web-based prediction tool was developed using the best-performing GBSA, which is accessible at https://pengzihexjtu-pca-n1.streamlit.app/ . ML algorithms showed better performance compared with Cox regression and we developed a web-based tool, which may help to guide patient treatment and follow-up.
Assuntos
Excisão de Linfonodo , Neoplasias da Próstata , Masculino , Humanos , Prognóstico , Excisão de Linfonodo/métodos , Linfonodos/patologia , Neoplasias da Próstata/patologia , Antígeno Prostático EspecíficoRESUMO
LKB1 encodes a serine/threonine kinase generally inactivated in human lung cancers, which mediates cancer cell proliferation, migration and differentiation, but its biological function has not been completely elucidated. In this study, we demonstrated that LKB1 was associated with a substantial reduction of c-myc expression by using an inducible LKB1 expression system in the LKB1-null lung cell line A549. Nevertheless, the reduction of the c-Myc gene expression was not accompanied by corresponding reduction of mRNAs but protein, which can be abrogated by a proteosome inhibitor (MG132), suggesting that the reduction was associated with their increased degradation rather than transcriptional controls. Our results implied that the expression of c-Myc protein decreased by LKB1 in transfected cells may be a contributory factor in the process of cell proliferation. Overexpression of the LKB1 gene could inhibit the activation of ERK1/2 and STAT3 signaling pathways involved in the cell proliferation. Thus, LKB1-induced functional operation on c-Myc in promoting cell proliferation may occur in a novel mechanism, which may be regulated by ERK1/2 and/or STAT3 signal pathways in human lung carcinoma cells. Furthermore, our results give some insights into the understanding of how LKB1 inactivation contributes to lung carcinogenesis and emphasizes the central role played by LKB1 in lung cancer development.
Assuntos
Neoplasias Pulmonares/prevenção & controle , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fator de Transcrição STAT3/fisiologia , Quinases Proteína-Quinases Ativadas por AMP , Linhagem Celular Tumoral , Proliferação de Células , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Humanos , Neoplasias Pulmonares/genética , Sistema de Sinalização das MAP Quinases , Proteínas Serina-Treonina Quinases/genéticaRESUMO
BACKGROUND: Circulating tumor cells (CTCs) and relevant autophagy Beclin-1 genes expression are critical biomarkers for tumorigenesis and tumor progress. Here we investigated the relationship of dynamic changes of CTCs and Beclin-1 expression of CTCs with renal cell carcinoma (RCC) prognosis. MATERIALS AND METHODS: A total of 69 patients with RCC were enrolled and divided into two groups based on the postoperative status of distant metastasis, including metastasis-free group (n = 58) and metastatic group (n = 11). Demographic characteristics of each patient were recorded in detail. All 69 enrolled patients had received multiple CTC tests and peripheral blood samples were obtained at three different time points (1 day before operation, 6 months and 12 months after operation). Peripheral blood samples were drawn before each time point and CTCs were separated by using Can Patrol CTC enrichment technique. CTCs were divided into epithelial, mesenchymal and mixed phenotype based on different surface biomarkers. RNA in situ hybridization assay was used to detect the expression of Beclin1 gene. RESULTS: The percentages of epithelial, mesenchymal and mixed CTCs were 11.64%, 28.04% and 60.32%, respectively. There were no significant differences of initial CTCs counts between metastasis-free group (8.43 ± 5.15) and metastatic group (7.71 ± 3.82) (P > 0.05). As for metastatic group, the number of mixed CTCs at 12 months postoperatively was significantly higher than that of mixed CTCs preoperatively and 6 months postoperatively (P < 0.05). In the metastatic group, the number of Beclin1 positive CTCs was significantly higher than that of Beclin1 negative CTCs preoperatively (P < 0.05), moreover, there were several significantly changes of Beclin1 positive CTCs with different types and at different time points. CONCLUSION: The recurrence or metastasis of RCC was uncorrelated with initial CTCs counts, but probably related with the variation trend of CTCs, especially mesenchymal CTCs and Beclin1 positive CTCs.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma Papilar/secundário , Carcinoma de Células Renais/patologia , DNA de Neoplasias/genética , Neoplasias Renais/patologia , Recidiva Local de Neoplasia/patologia , Células Neoplásicas Circulantes/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Papilar/genética , Carcinoma Papilar/cirurgia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/cirurgia , DNA de Neoplasias/análise , Transição Epitelial-Mesenquimal , Feminino , Seguimentos , Humanos , Neoplasias Renais/genética , Neoplasias Renais/cirurgia , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/cirurgia , Células Neoplásicas Circulantes/metabolismo , Nefrectomia , Fenótipo , Prognóstico , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
Armadillo repeat-containing protein 8 (ARMc8) is a key factor in regulating cell migration, proliferation, tissue maintenance, and tumorigenesis. However, its role in bladder cancer remains unknown. Thus, in this study we sought to investigate the effect of ARMc8 on the epithelial-to-mesenchymal transition (EMT) progress in bladder cancer cells induced by transforming growth factor-ß1 (TGF-ß1). Our results found that ARMc8 was highly expressed in bladder cancer cell lines. ARMc8 silencing inhibited the TGF-ß1-induced migration and invasion and suppressed the EMT progress in bladder cancer cells. Furthermore, ARMc8 silencing inhibited the TGF-ß1-induced expression of ß-catenin, cyclin D1, and c-myc in bladder cancer cells. In conclusion, the present study demonstrates a novel function for ARMc8, which acts as a mediator for TGF-ß1-induced cell migration/invasion through modulation of the Wnt/ß-catenin signaling pathway in bladder cancer cells. This study suggests that ARMc8 may be a potential therapeutic target for the development of therapies for bladder cancer.
Assuntos
Proteínas do Domínio Armadillo/genética , Transição Epitelial-Mesenquimal/genética , Inativação Gênica , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Expressão Gênica , Humanos , RNA Interferente Pequeno/genética , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
OBJECTIVE: To investigate the role of simultaneous blockade of CD40/CD40L and B7/CD28 pathways in the immune tolerance via co-expression of sCD40LIg and CTLA4Ig mediated by replication-defective adenovirus. METHODS: Ad-sCD40LIg-IRES(2)-CTLA4Ig, replication-defective adenovirus co-expressing sCD40LIg and CTLA4Ig, was constructed and identified. The co-expression of sCD40LIg and CTLA4Ig was evaluated with confocal laser scanning microscope and Western blotting. Skin transplantations of C57BL/6 to BALB/c mice were performed. PBS, Ad-Shuttle-CMV and Ad-sCD40LIg-IRES(2)-CTLA4Ig were administered. Skin graft survival was monitored and the mRNA expression of both genes was evaluated in the skin allografts. RESULTS: Ad-sCD40LIg-IRES(2)-CTLA4Ig was constructed successfully and identified. The co-expression of sCD40LIg and CTLA4Ig was identified with confocal laser scanning microscopy and Western blotting. Compared to the skin graft mean survival time (MST) of non-treated group ((5.75+/-0.71) d) or Ad-Shuttle-CMV-treated group ((5.50+/-0.53) d), the skin graft MST was dramatically prolonged in the Ad-sCD40LIg-IRES(2)-CTLA4Ig-treated group ((16.38+/-1.19) d, P<0.001). The mRNA expression of both genes was detected. CONCLUSION: Ad-sCD40LIg-IRES(2)-CTLA4Ig, a replication-defective adenovirus carrying genes encoding sCD40LIg and CTLA4Ig, was constructed. Simultaneous blockade of CD40/CD40L and B7/CD28 costimulatory pathway mediated by replication-defective adenovirus significantly prolonged skin allograft survival in mice.
Assuntos
Sobrevivência de Enxerto/imunologia , Imunoconjugados/imunologia , Proteínas Recombinantes de Fusão/imunologia , Transplante de Pele/métodos , Abatacepte , Adenoviridae/genética , Animais , Efeito Citopatogênico Viral , Imunoconjugados/genética , Imunoconjugados/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante de Pele/imunologia , TransfecçãoRESUMO
To investigate all the predictors of operative duration, hospital stay and stone-free rate post-minimally invasive percutaneous nephrolithotomy (MPCNL) and to establish a logistic regression formula to predict the probability of stone-free post-MPCNL. From August 2009 to August 2012, 396 patients were enrolled in the present study. The patients' characteristics, history, laboratory examination and imaging information were used as independent variables, and operative duration, hospital stay, residuals (≥4 mm) as outcomes. Univariate and multiple regression analysis were performed to determine independent variables. According to the result of multiple logistic regression analysis, a logistic regression formula was established to predict the stone-free probability post-MPCNL. The predictors of operative duration were type of stones and stone burden. Factors affecting hospital stay included leucocytes, stone burden, type of stones and hemoglobin. Residuals were significantly related with stone burden and type of stones. With the above results, we developed a logistic regression formula, which can effectively predict the probability of stone-free with 91.7% of sensitivity, 70.4% of specificity and 85.9% of overall accuracy.
Assuntos
Técnicas de Apoio para a Decisão , Cálculos Renais/cirurgia , Nefrostomia Percutânea , Adulto , Algoritmos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise de RegressãoRESUMO
PATIENT: Male, 24 FINAL DIAGNOSIS: Urethral stricture Symptoms: - MEDICATION: - Clinical Procedure: - Specialty: Urology. OBJECTIVE: Unusual or unexpected effect of treatment. BACKGROUND: The most dependable management of anterior urethral stricture is the complete excision of the area of fibrosis, with a primary reanastomosis of the normal ends of the anterior urethra. CASE REPORT: A 24-year-old man had urethral stricture in the penoscrotal junction caused by catheterization approximately 3 years ago. After the resection of the urethral stricture segment and the end-to-end anastomosis were performed, in addition to stricture, urethrocutaneous fistula formation as another complication in the penoscrotal junction was confirmed. The direct vision internal urethrotomy did not improve all the above symptoms. The retrograde urethrogram and voiding cysto-urethrogram showed complete obliteration in the penile urethra, urethrocutaneous fistula, and proximal urethral bifurcation singularity. Intraoperatively, we found that the distal urethral end had been anastomosed to the proximal false passage in the initial surgery and the proximal urethra was located in the dorsal side of the false passage. Then, tubularized preputial flap urethroplasty was performed. The patient was followed up for 10 months. His peak urinary flow was 18.3 milliliter per second. CONCLUSIONS: We would remind urologists that urethral end intraoperatively anastomosed to the false passage is a rare, serious, avoidable, and elementary medical error. Urethroplasty is one of the curative choices for treatment of this unexpected condition.
RESUMO
In the present study, we compared the expression of miRNAs and angiogenesis-related genes in the renal tumors and adjacent normal renal tissues of patients with clear cell renal cell carcinoma (ccRCC). The first part of the present study was a preliminary analysis of 4 patients with stage T1a/b ccRCC that measured the levels of angiogenesis and expression of angiogenesis-related genes and miRNAs in the tumors and adjacent normal renal tissues. The second part of this study was an analysis of 30 patients with stage T1, T2 or T3 ccRCC that employed qPCR to characterize expression of angiogenesis-related miRNAs in the tumors and adjacent normal tissues. The first part of this study indicated that all 4 patients had increased levels of CD34 in tumors, indicating elevated angiogenesis. However, quantitative analysis of microvessel density and expression of miRNAs indicated highly variable results among these patients. The data of all patients in the present study indicated that more patients with stage T1 ccRCC had higher expression of miR-126 and miR-378 in their normal tissues, whereas more patients with stage T2/3 ccRCC had higher expression of these miRNAs in their tumor tissues. The tumors of patients with ccRCC had lower expression of miR-126 and miR-378 during the early stages of disease (T1), but higher expression of these miRNAs during the later stages of disease (T2/T3).
Assuntos
Carcinoma de Células Renais/genética , Neoplasias Renais/genética , MicroRNAs/genética , Neovascularização Patológica/genética , Idoso , Antígenos CD34/metabolismo , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Rim/irrigação sanguínea , Rim/patologia , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica/metabolismoRESUMO
LKB1 encodes a serine/threonine kinase generally inactivated in many human cancers, which mediates cancer cell proliferation, migration and differentiation. Recent studies indicated that LKB1 exhibits potent anti-metastatic activity. However, the underlying molecular mechanisms of this activity remain unclear. In this study, we reintroduced LKB1 into A549 lung cancer cells that lack the LKB1 gene to investigate how LKB1 affects tumor invasiveness and metastasis. We demonstrated that overexpression of the LKB1 protein in lung cancer cells resulted in significant inhibition of invasion. Furthermore, transfected lung cancer cells with LKB1 suppressed tissue factor (TF) and vascular endothelial growth factor (VEGF) expression at both the mRNA and protein levels. Here, we provided evidence showing that downregulation of TF and VEGF by LKB1 is correlated well with the inhibition of cell invasion. Overexpression of the LKB1 protein in human lung cancer is significantly associated with a decrease in activity and expression of the transcription factor SP1. Constitutive activation of the transcription factor Sp1 plays a critical role in TF and VEGF overexpression. We conclude that suppression of lung cancer cell invasion by LKB1 through downregulation of TF and VEGF may partly depend on its inhibitory effect on the transcription factor Sp1. Collectively, our data provide a novel molecular mechanism for the antitumor activity of LKB1 and may help further improve its effectiveness in controlling lung cancer growth and invasion.
Assuntos
Movimento Celular , Neoplasias Pulmonares/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Transcrição Sp1/metabolismo , Tromboplastina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Adesão Celular , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/metabolismo , Proteínas Serina-Treonina Quinases/genética , Fator de Transcrição Sp1/genética , Tromboplastina/genética , Transfecção , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Ceftriaxone is known to cause biliary pseudolithiasis and, rarely, nephrolithiasis mainly in children. However, we reported the development of bilateral distal ureteral ceftriaxone-associated lithiasis in 7 adults, which suggests that the risk of ureterolithiasis impaction should be considered when treating patients with ceftriaxone, even in adults. To avoid strengthening greater renal damage, ureteroscopic insertion of double J stents may be an alternative management for patients with ureteral ceftriaxone-associated lithiasis.
Assuntos
Antibacterianos/efeitos adversos , Ceftriaxona/efeitos adversos , Ureterolitíase/induzido quimicamente , Dor Abdominal/etiologia , Adulto , Anuria/etiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Radiografia , Infecções Respiratórias/tratamento farmacológico , Ultrassonografia , Ureter/diagnóstico por imagem , Ureterolitíase/complicações , Ureterolitíase/terapia , Ureteroscopia , Adulto JovemRESUMO
Retroperitoneal pseudocysts are rare entities. Egg shell-like calcified retroperitoneal pseudocyst is rarer. We report a 75-year-old woman with an egg shell-like calcified retroperitoneal pseudocyst. Subsequently, the calcified pseudocyst was dissected and excised completely through laparoscopy via a retroperitoneal approach.
Assuntos
Calcinose/diagnóstico por imagem , Cistos/diagnóstico por imagem , Espaço Retroperitoneal/diagnóstico por imagem , Idoso , Calcinose/patologia , Calcinose/cirurgia , Cistos/patologia , Cistos/cirurgia , Feminino , Humanos , Tomografia Computadorizada por Raios XRESUMO
AIM: To isolate and culture primary human umbilical vein endothelial cells (HUVECs) and then to coat rat islets in vitro. METHODS: Primary HUVECs were isolated and cultured, then they were identified by detecting human VIII factor associated antigen with immunohistochemical method. Weible-palade bodies were observed by transmission electron microscope and the phagocytosis for DiI-Ac-LDL was detected by fluorescent microscopy. Rat islets were isolated and coated with HUVECs coexpressing sCD40L-Ig and CTLA4-Ig in a heparinized culture dish. The coated islets were observed via morphology and high glucose challenge test. RESULTS: The human VIII factor associated antigen, Weible-palade bodies and the phagocytosis for DiI-Ac-LDL were found in HUVECs. The islets coated by HUVECs coexpressing sCD40L-Ig and CTLA4-Ig were identified by green fluorescence proteins. Compared with that of the untreated islets (4.09), the release index of the coated islets was 3.94. There was no difference in insulin release between coated islets and untreated islets under glucose challenge. CONCLUSION: HUVECs have been isolated, cultured and identified successfully. The rat islets were coated with HUVECs in vitro function well.
Assuntos
Antígenos CD/imunologia , Ligante de CD40/imunologia , Células Endoteliais/metabolismo , Células Endoteliais/transplante , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Ilhotas Pancreáticas/citologia , Veias Umbilicais/citologia , Animais , Antígeno CTLA-4 , Células Cultivadas , Células Endoteliais/citologia , Fator VIII/genética , Fator VIII/metabolismo , Humanos , Imunoglobulina G/isolamento & purificação , Imuno-Histoquímica , Transplante das Ilhotas Pancreáticas , Masculino , Microscopia de Fluorescência , Modelos Animais , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: To construct a replication-incompetent recombinant adenovirus mediating short hairpin RNA (shRNA)-induced tissue factor gene silencing in the islet. METHODS: Four pairs of complementary oligonucleotides were designed and synthesized to create double-stranded oligonucleotides (ds oligo). The ds oligos were cloned into Pentr/U6 vector to construct the shuttle plasmid pENTR/U6-shRNA, which was transduced into human islets via liposome after sequence verification. The plasmid with the best silencing effect was identified by real-time RT-PCR, followed by homologous recombination with the adenovirus backbone plasmid. The functional clone was transfected into 293A cells to amplify the adenovirus, whose silencing effect against TF expression was tested using real-time RT-PCR and Western blotting. RESULTS: The pENTR/U6-shRNA shuttle plasmid was constructed and verified by sequencing. The recombinant adenovirus-mediated shRNA against TF was constructed, and real-time RT-PCR and Western blotting demonstrated that the strongest silencing effect of the adenovirus against TF occurred on the 4th day following islet transfection. CONCLUSION: Replication-incompetent recombinant adenovirus-mediated shRNA against TF has been successfully constructed, which has good silencing effect against TF expression in human islet in vitro.
Assuntos
Adenoviridae/genética , DNA Recombinante/genética , Engenharia Genética/métodos , Ilhotas Pancreáticas/metabolismo , Interferência de RNA , Tromboplastina/deficiência , Tromboplastina/genética , Adenoviridae/fisiologia , Sequência de Bases , Linhagem Celular , Expressão Gênica , Humanos , Sequências Repetidas Invertidas , Plasmídeos/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Carga Viral , Replicação ViralRESUMO
OBJECTIVE: To establish a stable method for obtaining large quantity of highly purified immature dendritic cells (imDCs) in vitro, and identify the morphology, function and surface markers of the cells. METHODS: CD117(+) hemopoietic stem cells (HSCs) were isolated and purified from the bone marrow of healthy C57 mice by magnetic affinity cell sorting. After cell expansion by treatment with stem cell factor (SCF) and interleukin-3 (IL-3), the HSCs were induced for directional differentiation into imDCs by treatment with GM-CSF, IL-4 and IL-10. The imDCs obtained were identified by morphological and functional observation under inverted microscope, scanning electron microscope and transmission electron microscope, followed by detection of the expressions of the surface markers using flow cytometry. RESULTS: After 3, 5 and 7 days of culture in the presence of SCF+IL-3, the cells were expanded by 10.34-/+1.43, 22.65-/+2.71 and 54.39-/+3.08 folds, respectively. The HSCs were successfully induced to differentiate into imDCs with phagocytotic activity. The dendrites of the imDCs were short small, and appearing spinous. The expressions of surface markers were detected from the cells showing the phenotype of CD11c(+), I-A/I-E(low), CD40(-), CD80(-), CD86(-). CONCLUSION: The method described allows steadily acquisition of large quanty of highly purified imDCs and of their effective identification in vitro.
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Separação Celular/métodos , Células Dendríticas/citologia , Células-Tronco Hematopoéticas/citologia , Animais , Células Cultivadas , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Proteínas Proto-Oncogênicas c-kitRESUMO
AIM: To construct adenovirus expressing secretory human CD40L-Ig (sCD40 ligand Ig fusion protein). METHODS: The genes encoding the extracellular domain of human CD40L and IgG Fc were amplified with PCR and inserted into shuttle vector pAdTrack-CMV which then was transformed into E. coli pAdEasy-1-BJ5183 to produce recombinant Ad plasmid. The recombinant plasmid was digested with Pac I and transfected into 293 cells to generate recombinant adenovirus. The recombinant adenovirus was then used in mixed lymphocyte reaction (MLR) to test its function. RESULTS: The recombinant adenovirus sCD40L-Ig was produced. It could inhibit the lymphocyte proliferation in MLR. CONCLUSION: The sCD40L-Ig adenovirus is prepared successfully and its inhibition of MLR is confirmed.
Assuntos
Adenoviridae/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Animais , Western Blotting , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Teste de Cultura Mista de Linfócitos , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/genéticaRESUMO
AIM: To set up a method of isolation and culture of Sertoli cells from adult testis and to investigate their immune privilege mechanism. METHODS: Adult testis Sertoli cells were prepared successfully by digestion with trypsin, collagenase, hyaluronidase and DNase, and then the expression of Fas-L and TGF-beta1 was examined by SABC staining. The Sertoli cells were cocultured with adult splenocytes to detect the inhibitory effect of Sertoli cells on splenocyte proliferation by MTT colorimetry. RESULTS: In cultured cells, Sertoli cells accounted for more than 80% of total cells. The activity of Sertoli cells reached 90%. Sertoli cells expressed Fas-L and TGF-beta1 and could inhibit the proliferation of cocultured splenocytes in vitro. CONCLUSION: A method of isolation and culture of Sertoli cells from adult testis has been established. The immune privilege mechanism of Sertoli cells may be related to the expression of Fas-L and TGF-beta1.