RESUMEN
OBJECTIVE: To establish the stably lower expression of vascular cell adhesion molecule-1 (VCAM-1) in MSC cell line (C3H10T1/2) by siRNA technology, and explore the effect of knockdown of VCAM-1 on the immunologic regulation capacity of murine MSC. METHODS: The mouse GV118-VCAM-1-RNAi retrovirus vector was constructed by gene recombination technology. The recombinant plasmid was identified by restriction analysis and sequencing, and then the recombinant plasmid GV118-VCAM-1-RNAi was transfected into 293 cells by Lipofectamine, and the supernatant was collected to transfect C3H10T1/2. Moreover, the VCAM-1 lower expression on MSC was evaluated by flow cytometry and fluorescent microscopy. The knockdown VCAM-1 MSC was sorted by flow cytometry. Furthermore, the inhibitory effect of the knockdown VCAM-1 MSC on lymphocyte proliferation was tested by lymphoblast transformation assay (LTT) and mixed lymphocyte reaction assay(MLR). RESULTS: The recombinant retroviral vector of knockdown VCAM-1 (GV118-VCAM-1-RNAi) was successfully constructed and transfected into mouse MSC cell line C3H10T1/2. The knockdown VCAM-1/MSC was obtained by flow cytometric sorting. The LTT and MLR assay showed that the immunosuppressive effect of MSC lower-expressing VCAM-1 dramatically decreased (P<0.05). CONCLUSION: Knockdown VCAM-1 in MSC can significantly down-regulate the inhibitory capability of MSC on the proliferation of T-cells. The data of this study laid an experimental foundation for studying effect of VCAM-1 transfecting into MSC on immune function.
Asunto(s)
Células Madre Mesenquimatosas , Animales , Línea Celular , Movimiento Celular , Proliferación Celular , Citometría de Flujo , Vectores Genéticos , Activación de Linfocitos , Ratones , Plásmidos , Interferencia de ARN , ARN Interferente Pequeño , Linfocitos T , Transfección , Molécula 1 de Adhesión Celular VascularRESUMEN
OBJECTIVE: To investigate the effect of vascular cell adhesion molecule-1 (VCAM-1) gene overexpression on adipogenic differentiation of mouse mesenchymal stem cells(MSC) and explore its molecular mechanism. METHODS: VCAM-1 overexpression MSC (MIGR1-VCAM-1/MSC) and the empty plasmid transfection MSC (MIGR1/MSC) were induced to adipogenic differentiation, oil-red-O staining and real-time PCR were used to detect the adipogenic differentiation ability and the mRNA expression level of key transcription factors C/EBP α and PPAR γ. The activation of P38, ERK and JNK pathways were analyzed by Western blot. Furthermore, the specific chemical inhibitors of MAPK pathway (SB203580, PD98059 and JNK inhibitor II) were added to the induced culture system and the alteration of the MSC adipogenic differentiation ability were evaluated. RESULTS: no matter in self or induced differentiation groups, the lipid droplets in MIGR1-VCAM-1/MSC became larger, the amount of adipocyte increased than that in MIGR1/MSC (P<0.01), the mRNA expression level of C/EBPα and PPARγ were upregulated, and JNK pathway were down-regulated while the P38 and ERK pathway were significantly up-regulated. The inhibition of JNK pathway of MIGR1-VCAM-1/MSC could lead to increased mRNA expression level of C/EBP α and PPAR γ, the amount of adipocytes increased (P<0.01), however, the inhibition of the P38 and ERK pathway of MIGR1-VCAM-1/MSC could lead to decreased mRNA expression level of C/EBP α and PPAR γ, and the lipid droplets and the number of adipocytes became smaller and less. CONCLUSION: The overexpression of VCAM-1 may promote MSC to differentiate into adipocytes through inhibiting JNK signaling pathway, activating P38 and ERK pathways.
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Diferenciación Celular , Células Madre Mesenquimatosas , Adipocitos , Animales , Proteína alfa Potenciadora de Unión a CCAAT , Regulación hacia Abajo , Sistema de Señalización de MAP Quinasas , Ratones , PPAR gamma , Reacción en Cadena en Tiempo Real de la Polimerasa , Transfección , Regulación hacia Arriba , Molécula 1 de Adhesión Celular VascularRESUMEN
The comparative effectiveness of aluminum hydroxide and aluminum chloride has been studied in the development of bacterial wilt infection on leaves of Nicotiana rustica cv. Gansu yellow flower. We have analyzed the changes of foliar H(2)O(2) content, as well as of non-enzymatic and enzymatic antioxidants under aluminum stress. Pretreatment with aluminum hydroxide before pathogen challenge reduced the development of Ralstonia solanacearum infection and decreased the extent of leaf injury. The pretreatment also reduced the Al uptake in comparison to pretreatment with aluminum chloride. H(2)O(2) generation was significantly enhanced by pretreatment with aluminum hydroxide. Increased NADPH oxidase and superoxide dismutase activities were correlated with limited infection. Aluminum hydroxide pretreatment shifted the leaf redox homeostasis of AsA/DHA and GSH/GSSG toward oxidation, yielding higher oxidant levels than aluminum chloride before bacterial inoculation. The results support the idea that aluminum hydroxide induced H(2)O(2) accumulation through non-enzymatic and enzymatic regulation, ultimately resulting in resistance to tobacco wilt disease.
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Compuestos de Aluminio/metabolismo , Hidróxido de Aluminio/metabolismo , Cloruros/metabolismo , Nicotiana/metabolismo , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/prevención & control , Hojas de la Planta/metabolismo , Cloruro de Aluminio , Compuestos de Aluminio/farmacología , Hidróxido de Aluminio/farmacología , Cloruros/farmacología , Regulación de la Expresión Génica de las Plantas , Peróxido de Hidrógeno/análisis , Peróxido de Hidrógeno/metabolismo , NADPH Oxidasas/análisis , NADPH Oxidasas/metabolismo , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/microbiología , Ralstonia solanacearum/crecimiento & desarrollo , Superóxido Dismutasa/análisis , Superóxido Dismutasa/metabolismo , Nicotiana/efectos de los fármacos , Nicotiana/microbiologíaRESUMEN
We used conventional methods to investigate the mechanism by which Acidithiobacillus ferrooxidans colonizes a solid surface by assessing pili-mediated sliding, twitching motility, and adherence. A. ferrooxidans slided to form circular oxidized zones around each colony. This suggested that slide motility occurs through pili or flagella, though A. ferrooxidans strains ATCC 19859 and ATCC 23270 lack flagella. The results of reverse transcription-PCR demonstrated that the putative major pili gene of A. ferrooxidans strains ATCC 19859, ATCC 23270, and BY3 genes were transcribed. Culture of A. ferrooxidans between silicone gel and glass led to the production of type IV pili and the formation of rough twitching motility zones. When the bacteria were grown on lean ore cubes, pyrite was colonized readily by A. ferrooxidans and there is a correlation between pilus expression and strong attachment. However, non-pili bacteria attached minimally to the mineral surface. The results show a correlation between these functions and pilus expression.
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Acidithiobacillus/fisiología , Adhesión Bacteriana/fisiología , Fimbrias Bacterianas/metabolismo , Flagelos/metabolismo , Acidithiobacillus/ultraestructura , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/ultraestructura , Flagelos/ultraestructura , Regulación Bacteriana de la Expresión GénicaRESUMEN
The genetic variability among 32 Chinese Acidithiobacillus spp. environmental isolates and four reference strains representing three recognized species of the genus Acidithiobacillus was characterized by using a combination of molecular methods, namely restriction fragment length polymorphisms of PCR-amplified 16S rRNA genes and 16S-23S rRNA gene intergenic spacers, repetitive element PCR, arbitrarily primed PCR and 16S rRNA gene sequence analyses. 16S rRNA gene sequences revealed that all Acidithiobacillus spp. strains could be assigned to seven groups, three of which encompassed the Acidithiobacillus ferrooxidans strains from various parts of the world. A comparative analysis of the phylogenetic Group 1 and 2 was undertaken. Restriction fragment length polymorphism results allowed us to separate the 35 Acidithiobacillus strains into 15 different genotypes. An integrated phenotypic and genotypic analysis indicated that the distribution of A. ferrooxidans strains among the physiological groups were in agreement with their distribution among the genomic groups, and that no clear correlation was found between the genetic polymorphism of the Acidithiobacillus spp. strains and either the geographic location or type of habitats from which the strains were isolated. In addition, five unidentified sulfur-oxidizing isolates may represent one or two novel species of the genus Acidithiobacillus. The results showed that the Chinese Acidithiobacillus spp. isolates exhibited a high degree of genomic and phenotypic heterogeneity.
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Acidithiobacillus/clasificación , Acidithiobacillus/genética , Microbiología Ambiental , Variación Genética , Acidithiobacillus/aislamiento & purificación , Análisis del Polimorfismo de Longitud de Fragmentos Amplificados , China , Análisis por Conglomerados , Dermatoglifia del ADN , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , ADN Espaciador Ribosómico/química , ADN Espaciador Ribosómico/genética , Genotipo , Geografía , Secuencias Repetitivas Esparcidas , Datos de Secuencia Molecular , Oxidación-Reducción , Filogenia , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Longitud del Fragmento de Restricción , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Azufre/metabolismoRESUMEN
The complete sequences of 32 intergenic spacer regions (ISR) from Acidithiobacillus strains, including 29 field strains isolated from coal, copper, molybdenum mine wastes or sediment of different geoclimatic regions in China, reference strain ATCC19859 and the type strains of the two species were determined. These data, together with other sequences available in the GenBank database, were used to carry out the first detailed assessment of the inter- and intraspecific genomic variability of the ISR sequences and to infer phylogenetic relationships within the genus. The total length of the 16S-23S rRNA intergenic spacer regions of the Acidithiobacillus thiooxidans and Acidithiobacillus ferrooxidans strains ranged from 451 to 490 bp, and from 434 to 456 bp, respectively. The degree of intrageneric ISR sequence similarity was higher than the degree of intergeneric similarity, and the overall similarity values of the ISRs varied from 60.49% to 84.71% between representatives of different species of the genus Acidithiobacillus. Sequences from the spacer of the A. thiooxidans and A. ferrooxidans strains ranged from 86.71% to 99.56% and 92.36% to 100% similarity, respectively. All Acidithiobacillus strains were separated into three phylogenetic major clusters and seven phylogenetic groups. ISR may be a potential target for the development of in situ hybridization probe aimed at accurately detecting acidithiobacilli in the various acidic environments.