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1.
Talanta ; 265: 124865, 2023 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-37418960

RESUMO

The reliable and accurate detection of glyphosate is urgently demanded because it is related to food and environmental safety. In this contribution, a PDA-PEI/Cu2+ complex that possesses peroxidase-mimetic activity and stimulus-responsive fluorescence was fabricated by coordinating Cu2+ with polydopamine-polyethyleneimine copolymer dots (PDA-PEI CPDs). With the introduction of Cu2+, the fluorescence intensity of PDA-PEI CPDs dropped sharply owing to the electron transfer effect. As a peroxidase-mimicking nanozyme, the PDA-PEI/Cu2+ complex owns catalytic capacity to oxidize the colorless 3,3',5,5'-tetramethylbenzidine (TMB) into blue oxTMB, leading a further fluorescence quenching by internal filtering effect by oxTMB. Once the glyphosate participated, the fluorescence signal of PDA-PEI CPDs is recovered significantly because of the formation of more stable Glyp-Cu2+ complexes, meanwhile the peroxidase-mimicking activity of PDA-PEI/Cu2+ complex could be strongly hindered. According to this principle, a novel and extremely convenient 'turn off' colorimetric and 'turn on' fluorescence sensing platform can be established for dual-mode detection of glyphosate. The favorable sensitivity and selectivity and were verified in the analysis of glyphosate in the environment through the marriage of dual-signal sensing platform. The detection limit of the dual-mode glyphosate sensing platform was 103.82 ng/mL for colorimetric assay and 16.87 ng/mL for fluorescent assay, respectively. Satisfactory recoveries in the range of 96.40%-104.66% were obtained, indicating the potential of this method for application in complicated real sample. Thereby, this strategy broadens the applications of polydopamine nanomaterials and holds a promising application in determination of pesticide residues.


Assuntos
Colorimetria , Peroxidases , Peroxidase , Corantes Fluorescentes/química , Polietilenoimina/química , Glifosato
2.
World J Clin Cases ; 10(34): 12623-12630, 2022 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-36579105

RESUMO

BACKGROUND: Chronic intestinal pseudo-obstruction (CIPO) is a syndrome of intestinal motor dysfunction caused by intestinal nerve, muscle, and/or Cajal stromal cell lesions. CIPO is a serious category of gastrointestinal dynamic dysfunction, which can eventually lead to the death of patients with intestinal failure. Due to considerable phenotypic heterogeneity, the estimated incidence of CIPO is 1/476190 and 1/416666 in men and women, respectively. According to the etiology, CIPO can be divided into idiopathic and secondary, of which the latter is the most common, often secondary to tumor, virus infection, connective tissue disease, neurological diseases, and endocrine diseases. Idiopathic CIPO in the intestinal tract is divided into visceral myopathy, neuropathy, and stromal cell lesions according to the location. Surgery is usually not recommended for CIPO, because it often does not benefit patients with CIPO, and postoperative intestinal obstruction is likely to occur, which may even worsen the condition. CASE SUMMARY: Here, we describe the case of a 43-year-old male Han Chinese patient with a 15-year history of recurrent abdominal distention with no clear cause. The results of physical, biochemical, and other relevant examinations showed no clear abnormalities. Contrast-enhanced computed tomography (CT) indicated a large duodenum, clear expansion of the intestinal lumen, and CIPO. Whole exome sequencing (WES) of the patient and his mother confirmed the diagnosis of primary familial visceral myopathy type 2 chronic pseudoileus with a rare heterozygous gene mutation in MYH11. This is the second reported case of CIPO with a heterozygous MYH11 [NM_001040113.1: c.5819delC (p.Pro1940Hisfs*91)] mutation. CONCLUSION: This case report indicates that physicians can perform routine clinical examinations, CT, and WES to achieve a diagnosis and treatment of CIPO in early disease stages.

3.
Hua Xi Kou Qiang Yi Xue Za Zhi ; 39(3): 313-319, 2021 Jun 01.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-34041881

RESUMO

OBJECTIVES: The proliferation, migration capacity, and expression of activation-related proteins of NHGFs+Cal27-exo were determined by coculturing Cal27 exosome (Cal27-exo) with normal human gingival fibroblasts (NHGFs) to explore the effects of Cal27-exo on the activation and biological behavior of NHGFs. METHODS: Cal27-exo was extracted using supercentrifugation, and exosomes were identified using Western blot, transmission electron microscopy (TEM), and particle size detection. Cal27-exo was cocultured with NHGFs to detect the uptake of Cal27-exo by NHGFs, and the proliferation and migration capacity of NHGFs+Cal27-exo were detected using CCK8 and wound healing tests, respectively. The expression levels of NHGF activation-related proteins, i.e., matrix metalloproteinase-9 (MMP-9), fibroblast-activating protein (FAP), alpha smooth muscle actin (αSMA), and transforming growth factor-ß (TGF-ß), were detected using real-time quantitative polymerase chain reaction (qRT-PCR). RESULTS: Cal27-exo was extracted u-sing supercentrifugation, and Western blot showed the positive expression levels of Alix and CD63. TEM showed that Cal27-exo had a circular double-layer vesicle. The particle size was between 30 and 150 nm. Cal27-exo labeled with PKH67 entered NHGFs after the coculture method. The wound healing test showed that the migration capacity of NHGFs+Cal27-exo was stronger after the scratch compared with that of NHGFs. CCK8 results showed that the proliferation activity of NHGFs+Cal27-exo was enhanced. qRT-PCR results showed that the MMP-9 levels of NHGFs+Cal27-exo were upregulated, whereas the TGF-ß and αSMA mRNA levels of NHGFs+Cal27-exo were downregulated (P<0.05). CONCLUSIONS: The proliferation and migration ability of NHGFs+Cal27-exo are enhanced, and the mRNA expression of related proteins is changed. Cal27-exo can activate NHGFs, which suggests that Cal27-exo has potential significance in tumor invasion and metastasis.


Assuntos
Exossomos , Proliferação de Células , Fibroblastos , Gengiva , Humanos , Metaloproteinase 9 da Matriz
4.
Dent Mater J ; 39(2): 200-205, 2020 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-31694998

RESUMO

This study was to prepare and screen a novel root canal sealing agent modified by polyhexamethylene biguanide (PHMB) that was in accordance with the ISO 6876:2001 standard and to study its physical and antimicrobial properties. The modified sealers were produced by mixing a certain amount of zinc oxide with eugenol containing different concentrations of PHMB (0.05, 0.1, 0.2, 0.4, 0.6 and 0.8%) at a ratio of 1:1 (w/v). The setting time, flow, film thickness, solubility and dimensional change after solidifying were assessed to screen out the modified sealing agents that the physical properties met the mentioned standards. The modified direct contact test (DCT) was used to evaluate the antimicrobial activity against Enterococcus faecalis. The results suggested that when the concentrations of PHMB were 0.05, 0.1 and 0.2%, the modified root canal sealers showed the best performance in physical and antimicrobial properties.


Assuntos
Anti-Infecciosos , Materiais Restauradores do Canal Radicular , Óxido de Zinco , Biguanidas , Eugenol , Cimento de Óxido de Zinco e Eugenol
5.
Hua Xi Kou Qiang Yi Xue Za Zhi ; 37(1): 25-30, 2019 Feb 01.
Artigo em Chinês | MEDLINE | ID: mdl-30854814

RESUMO

OBJECTIVE: This study aims to establish an effective and stable periodontal ligament cell line stably expressing human telomerase reverse transcriptase (hTERT) gene by using the adenovirus method. METHODS: Polymerase chain reaction (PCR) was used to amplify the full length of hTERT gene to construct recombinant adenovirus plasmid pAd-pshuttle-cmv-hTERT. Packaged adenovirus particles were used for infection of human periodontal ligament cells. The expression levels of hTERT and osteogenic genes, such as alkaline phosphatase, Runt-related transcription factor 2, bone sialoprotein, osteocalcin, osteopontin, and collagen Ⅰ mRNA, were detected by quantitative real-time PCR (qRT-PCR). The ability of osteogenic differentiation was observed by alizarin red staining, and the cell proliferation was determined by CCK-8. RESULTS: Adenovirus particles containing the hTERT gene were successfully constructed and infected with periodontal ligament cells. The infected cells were similar to normal periodontal ligament cells. The qRT-PCR results showed that hTERT and osteogenesis-associated genes were highly expressed in the periodontal ligament cell lines constructed by adenoviruses. Alizarin red staining showed that the periodontal ligament cell line had strong osteogenic differentiation capability. CCK-8 showed that the periodontal ligament cell line had strong proliferation capability. CONCLUSIONS: The human periodontal ligament cell line with high efficiency and stable expression of hTERT was established by the adenovirus method, thereby providing an ideal cell line for studying the mechanism of periodontal regeneration.


Assuntos
Ligamento Periodontal , Telomerase , Adenoviridae , Fosfatase Alcalina , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Humanos , Osteogênese
6.
Oncol Lett ; 8(5): 2096-2102, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25295097

RESUMO

Pancreatic cancer (PC) is the fourth leading cause of cancer-related mortality in the United States. There is no effective serum biomarker for the early diagnosis of PC at present. Although serum UL16-binding protein 2 (ULBP2) and macrophage inhibitory cytokine-1 (MIC-1) levels are reported to be elevated in PC patients, the diagnostic and prognostic value of ULBP2 and MIC-1 alone or in combination remains unknown. The aim of the present case-control study was to compare the diagnostic value of ULBP2, MIC-1 and carbohydrate antigen 19-9 (CA19-9) in 359 serum samples, consisting of 152 cases of PC, 20 cases of pre-pancreatic cancer, 91 cases of chronic pancreatitis (CP) and 96 normal controls (NC). All patients were followed up for a median of 2 years. It was found that the serum levels of ULBP2, MIC-1 and CA19-9 were significantly higher in the PC patients compared with those in the NC group. In distinguishing PC from the CP, the highest sensitivity and specificity were ULBP2 (0.878) and CA19-9 (0.816), respectively. The area under the receiver operating characteristic curve of ULBP2 was 0.923, which was the highest of the three biomarkers. MIC-1 was the optimal choice for the diagnosis of early-stage PC (area under the curve, 0.831). Overall, MIC-1 in combination with ULBP2 improved the diagnostic accuracy in differentiating PC from CP and NC. In addition, a higher level of MIC-1 was correlated with a poorer prognosis, as calculated by the Kaplan-Meier test (P=0.039). Patients with serum MIC-1 levels of ≥1,932 ng/ml had a median survival time of 15.62±2.44 months (mean ± standard deviation) vs. 18.66±2.43 months in patients with a lower level of MIC-1. Overall, combined detection of serum MIC-1 and ULBP2 improved the diagnostic accuracy in differentiating PC from CP and NC, and serum MIC-1 level alone was a predictor of survival in the patients with PC.

7.
World J Gastroenterol ; 20(32): 11241-8, 2014 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-25170208

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer. Substantial progress has been made in the understanding of the biology of pancreatic cancer, and advances in patient management have been significant. However, most patients (nearly 80%) who present with locally advanced or metastatic disease have an extremely poor prognosis. Survival is better for those with malignant disease localized to the pancreas, because surgical resection at present offers the only chance of cure. Therefore, the early detection of pancreatic cancer may benefit patients with PDAC. However, its low rate of incidence and the limitations of current screening strategies make early detection difficult. Recent advances in the understanding of the pathogenesis of PDAC suggest that it is possible to detect PDAC in early stages and even identify precursor lesions. The presence of new-onset diabetes mellitus in the early phase of pancreatic cancer may provide clues for its early diagnosis. Advances in the identification of novel circulating biomarkers including serological signatures, autoantibodies, epigenetic markers, circulating tumor cells and microRNAs suggest that they can be used as potential tools for the screening of precursors and early stage PDAC in the future. However, proper screening strategies based on effective screening methodologies need to be tested for clinical application.


Assuntos
Carcinoma Ductal Pancreático/diagnóstico , Detecção Precoce de Câncer/métodos , Neoplasias Pancreáticas/diagnóstico , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Carcinoma Ductal Pancreático/sangue , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/mortalidade , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/cirurgia , Diabetes Mellitus/epidemiologia , Testes Genéticos , Humanos , Estadiamento de Neoplasias , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/cirurgia , Valor Preditivo dos Testes , Fatores de Risco
8.
Ann Surg Oncol ; 20(12): 3809-16, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23943021

RESUMO

PURPOSE: To demonstrate the effect of diabetes mellitus (DM) (stratified by long-term/new-onset presurgical diabetes, resolved/unresolved postsurgical diabetes) on prognosis for pancreatic ductal cell adenocarcinoma (PDAC) after radical resection. METHODS: One hundred ninety-nine patients who underwent radical resection for PDAC between 2007 and 2011 at Ruijin Hospital (Shanghai, China) were retrospectively analyzed. Clinical and pathologic characteristics, surgical and adjuvant chemotherapy related outcomes, disease-free survival (DFS), and postoperative survival were compared among patients with long-term (≥2 years)/new-onset (<2 years) presurgical diabetes and resolved/unresolved postsurgical diabetes. Univariate and multivariable analysis was performed to determine factors associated with DFS and overall survival (OS). RESULTS: Of 199 patients, 90 (44.7%) had DM, 64 of which were new onset and 26 of which were long-standing. Resolution of DM after radical pancreatic resection was observed in 65% (42 of 64) in the new-onset group, but in none of the long-standing group. Resolved new-onset DM patients had larger, well-differentiated tumors compared to patients with unresolved new-onset DM. Patients with long-standing DM had shorter postoperative DFS and OS than nondiabetic/new-onset DM, whereas postoperative resolved new-onset DM is associated with longer DFS and OS than unresolved DM. Morbidity was higher and postoperative hospital stay was longer in patients with new-onset DM compared with patients with long-standing DM and patients without DM. There was no difference in the adjuvant chemotherapy toxicity rate among patients with long-standing or new-onset DM and those without DM. CONCLUSIONS: Different status of DM has different effects on outcome after resection for PDAC. Long-standing DM is related to progression of disease, whereas postsurgical resolved new-onset DM is a favorable prognostic factor.


Assuntos
Carcinoma Ductal Pancreático/mortalidade , Diabetes Mellitus/mortalidade , Neoplasias Pancreáticas/mortalidade , Idade de Início , Carcinoma Ductal Pancreático/complicações , Carcinoma Ductal Pancreático/cirurgia , Diabetes Mellitus/etiologia , Diabetes Mellitus/cirurgia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Neoplasias Pancreáticas/complicações , Neoplasias Pancreáticas/cirurgia , Prognóstico , Estudos Retrospectivos , Fatores de Risco , Taxa de Sobrevida , Neoplasias Pancreáticas
9.
Oncol Lett ; 5(1): 255-260, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23255931

RESUMO

Resistance to 5-fluorouracil (5-FU) in patients with gastric cancer is a serious therapeutic problem and major efforts are underway to understand the underlying mechanisms. We have previously identified RhoGDI2 as a contributor to 5-FU resistance in colon cancer cells using 2D electrophoresis and mass spectrometry and the current study aimed to further investigate this role. The expression of RhoGDI2 in seven gastric cancer cell lines was positively correlated with resistance to 5-FU. Lower 5-FU sensitivity of isolated tumor cells from patients with gastric cancer was also associated with higher RhoGDI2 expression. Ectopic expression of RhoGDI2 in gastric cancer cells increased the resistance to 5-FU and reverted low dose 5-FU-induced G2/M phase arrest without affecting the population of sub-G1 cells. Overall, these findings suggest that RhoGDI2 is associated with 5-FU resistance and is a potential therapeutic target for enhancing chemotherapy efficacy in gastric cancer.

10.
Front Biosci (Landmark Ed) ; 17(7): 2541-9, 2012 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-22652796

RESUMO

The emerging roles of bone morphogenetic proteins (BMPs) in the initiation and progression of multiple cancers have drawn great attention in cancer research. We hypothesized that BMP2 promotes cancer metastasis by modulating MMP-2 secretion and activity through intracellular ROS regulation and ERK activation in human pancreatic cancer. Our data show that stimulation of PANC-1 cells with BMP2 induced MMP-2 secretion and activation, associated with decreased E-cadherin expression, resulting in epithelial-to-mesenchymal transformation (EMT) and cell invasion. Blockade of ROS by the ROS scavenger, 2-MPG, abolished cell invasion, inhibited the EMT process and decreased MMP-2 expression, suggesting ROS accumulation caused an increase in MMP-2 expression in BMP2-stimulated PANC-1 cell invasion. Furthermore, treatment of PANC-1 cells with 2-MPG or ERK inhibitor PD98059 reduced the phosphorylation of ERK, resulting in attenuation of BMP2-induced cell invasion and MMP-2 activation. Taken together, these results suggest that BMP2 induces the cell invasion of PANC-1 cells by enhancing MMP-2 secretion and acting through ROS accumulation and ERK activation.


Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteína Morfogenética Óssea 2/farmacologia , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/fisiologia , Humanos , Sistema de Sinalização das MAP Quinases , Invasividade Neoplásica/patologia , Invasividade Neoplásica/fisiopatologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais
11.
Front Biosci (Landmark Ed) ; 17(7): 2559-65, 2012 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-22652798

RESUMO

Vascularization is crucial for tumor growth and metastasis. Angiogenesis and vasculogenesis are widely accepted processes of tumor vascularization, particularly for endothelium-dependent vessels. In both these processes, the tumor vascular endothelial cells are derived from the host cells, including cells in normal tissues around the tumor or endothelial progenitor cells. In addition, the mosaic vessels occur as a transitional pattern between endothelium-dependent vessels and vasculogenic mimicry (VM), wherein both host endothelium and tumor cells participate in tumor vascularization. VM provides a special passage not involving endothelial cells and is conspicuously different from angiogenesis and vasculogenesis. The biological features of the tumor cells that form VM remain unknown. Tumor stem cells may participate in VM. In this review, we discuss the patterns involved in the origin of vascularization in tumors.


Assuntos
Neoplasias/irrigação sanguínea , Neovascularização Patológica/etiologia , Animais , Diferenciação Celular , Células Endoteliais/patologia , Humanos , Modelos Biológicos , Neoplasias/patologia , Células-Tronco Neoplásicas/patologia
12.
Hua Xi Kou Qiang Yi Xue Za Zhi ; 29(3): 289-93, 2011 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-21776858

RESUMO

OBJECTIVE: To study the effects of Saccharomyces albicans (S. albicans) on the cell cycle distribution and proliferation of human umbilical vein endothelial cell ECV304 cells in vitro. METHODS: The line of ECV304 cultured in vitro were divided into four groups which were treated by S. albicans supernatant, S. albicans inactivated bacilli, supernatant and inactivated bacilli mixture, normal culture medium. The proliferous effect of ECV304 induced by supernatant, inactivated bacilli, supernatant and inactivated bacilli mixture using the methods of MTT, cell count, microscope and flow cytometry were conducted. RESULTS: In the condition of different times and different culture concentrations, ECV304 cells incubated with 4-fold diluted S. albicans supernatant for 48 h increased the proliferation rate. The S and G2/M population of ECV304 cells increased after incubated with S. albicans supernatant for 40 h, which showed significant increasing cell proliferation index (PI) (P < 0.05). The PI of the cells treated by inactivated bacilli showed no significant change (P > 0.05). CONCLUSION: S. albicans could induce ECV304 cell proliferation which depends on the release of metabolic products of S. albicans.


Assuntos
Células Endoteliais da Veia Umbilical Humana , Saccharomyces , Ciclo Celular , Divisão Celular , Proliferação de Células , Humanos , Veias Umbilicais
13.
J Dig Dis ; 12(2): 131-7, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21401899

RESUMO

OBJECTIVE: DJ-1 is an oncoprotein secreted by cancer cells. Therefore, it might be a diagnostic or prognostic biomarker for pancreatic cancer (PC). METHODS: The study involved 47 patients with PC, 43 with chronic pancreatitis, and 40 healthy subjects. We assayed the serum level of DJ-1 and the conventional tumor marker carbohydrate antigen 19-9 (CA 19-9) to define the diagnostic and prognostic value of DJ-1 for PC. RESULTS: Serum DJ-1 level was elevated in patients with PC compared with those with chronic pancreatitis and healthy individuals. The area under the curve (AUC) of serum DJ-1 was higher than CA 19-9 (DJ-1 vs. CA19-9, 0.8735 ± 0.0356 vs. 0.6647 ± 0.0572 ng/mL), and an 87.5% sensitivity was reached with a combination of serum DJ-1 and CA19-9. No association of serum DJ-1 level with tumor node metastasis (TNM) classification or tumor resectability was found. However, after resection, the median serum DJ-1 level was decreased from 2.00 to 0.78 ng/mL. In addition, higher serum DJ-1 was correlated with shorter overall survival as analyzed by both Kaplan-Meier test (P = 0.018) and COX regression analysis (P = 0.013). The median overall survival time of PC patients with serum DJ-1 level greater than or equal to 2.06 ng/mL was 7.00 ± 1.11 months, whereas that of patients with lower DJ-1 levels was 13.0 ± 2.5 months. CONCLUSIONS: These findings indicate the potential clinical significance for serum DJ-1 level to be used for the diagnosis and prognosis prediction of patients with PC.


Assuntos
Biomarcadores Tumorais/sangue , Peptídeos e Proteínas de Sinalização Intracelular/sangue , Proteínas Oncogênicas/sangue , Neoplasias Pancreáticas/diagnóstico , Pancreatite Crônica/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/sangue , Prognóstico , Proteína Desglicase DJ-1 , Adulto Jovem
14.
Hua Xi Kou Qiang Yi Xue Za Zhi ; 27(3): 350-2, 2009 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-19637495

RESUMO

Hypohidrotic ectodermal dysplasia (HED) is a hereditary disorder characterized by abnormal development of tissues derived from ectoderm. A case of hypohidrotic ectodermal dysplasia was reported, and the molecular biological progress in this area were reviewed.


Assuntos
Displasia Ectodérmica Anidrótica Tipo 1 , Humanos
15.
Hua Xi Kou Qiang Yi Xue Za Zhi ; 27(6): 610-3, 2009 Dec.
Artigo em Chinês | MEDLINE | ID: mdl-20077893

RESUMO

OBJECTIVE: The purpose of this study was to clone and analyze mutation in the eda-A1 gene for hypohidrotic ectodermal dysplasia (HED), and to construct a new recombined eukaryotic expression vector (mutant M, wild W) as a basis for further study on the genetic function. METHODS: After total mRNA was extracted from peripheral blood lymphocytes from the HED affect patient and control, eda-A1 gene was amplified by reverse transcription polymerase chain reaction (RT-PCR) with a pair of specific primers containing the constriction enzyme sites of BamH I and Hind III. When the vector pcDNA3.1(-) and eda-A1 (M/W) were digested by BamH I and Hind III respectively, eda-A1 (M/W) fragment was then ligated to vector pcDNA3.1 (-) and the new vector was named as pcDNA3.1 (-)-eda-A1-M/W. RESULTS: eda-A1 gene was successfully cloned and a novel missence mutation was identified, which changes the codon 306 from glutamine to proline. PCR, restrictive endonuclease analysis and DNA sequencing were then performed to identify the recombinant eukaryotic expression vector pcDNA3.1 (-)-eda-A1-M/W, and the results were surely confirmed. CONCLUSION: Our result indicates that the novel missense mutation in eda is associated with the isolated tooth agenesis and provide preliminary explanation for the abnormal clinical phenotype at a molecular structural level. And also, the recombinant eukaryotic expression vector pcDNA3.1 (-)-eda-A1-M/W was successfully constructed, which will be thereafter taken use of further study on eda gene in odontogenesis.


Assuntos
Displasia Ectodérmica Anidrótica Tipo 1 , Vetores Genéticos , Humanos , Mutação , Odontogênese , RNA Mensageiro , Análise de Sequência de DNA
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