RESUMO
OBJECTIVE: To determine the sperm mtDNA content, mtDNA4977bp deletion and ROS in the seminal plasma of normal and leukocytospermia men, and to investigate the correlation of the changes of sperm mtDNA with the increase of leukocytes and reactive oxgygen species (ROS) in the seminal plasma. METHODS: Seventy-eight semen samples from leukocytospermia patients and 31 from healthy donors were divided into 3 layers, supernatant fluid, 30% sperm and 80% sperm, by Percoll gradient centrifugation, their sperm mtDNA content and mtDNA4977bp deletion quantitatively analyzed by real-time PCR, and the level of ROS determined by flow cytometry. RESULTS: The ROS in the seminal plasma and the sperm mtDNA contents of the three layers were all significantly higher in the leukocytospermia group than in the healthy control (P < 0.01). In the supernatant fluid and 80% layers, mtDNA4977bp deletion showed no obvious difference between the control and the leukocytospermia group, but was significantly higher in the 30% layer of the latter (P < 0.01). The ROS level was found positively correlated with the mtDNA content in the 30% (r = 0.347, P < 0.01) and the 80% layer (r = 0.456, P < 0.01), but not in the supernatant layer. CONCLUSION: The increase of leukocytes and ROS may be one of the causes of the enhanced sperm mtDNA content, but has no significant impact on the mtDNA4977bp deletion.
Assuntos
DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Infertilidade Masculina/fisiopatologia , Espermatozoides/metabolismo , Adulto , Citometria de Fluxo , Humanos , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Leucócitos/química , Leucócitos/metabolismo , Leucocitose/genética , Leucocitose/metabolismo , Leucocitose/fisiopatologia , Masculino , Reação em Cadeia da Polimerase , Espécies Reativas de Oxigênio/metabolismo , Deleção de Sequência , Contagem de Espermatozoides , Espermatozoides/citologiaRESUMO
OBJECTIVE: To develop a new denaturing high performance liquid chromatograph (DHPLC)-based method to screen patients with EXT gene mutation and to study the gene mutation in three families with multiple exostoses. METHODS: All the exons of EXT gene, including the intro-exon boundaries, were amplified by PCR. Linkage analysis and DHPLC screening were carried out to identify the mutations. DNA sequencing was used to confirm the mutations. RESULTS: Two known splice site mutations, IVS2+1 G to A and IVS7+1 G to T, and two SNPs have been detected in EXT2 or EXT1 gene. CONCLUSION: The transversions of IVS2+1 G to A and IVS7+1 G to T in EXT2 gene are suggested to be the disease-causing mutations and the DHPLC is a high throughout, sensitive, simple, quick, economical method to screen gene mutation in hereditary multiple exostosis.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Exostose Múltipla Hereditária/genética , Mutação , N-Acetilglucosaminiltransferases/genética , Adulto , Análise Mutacional de DNA , Eletroforese em Gel de Poliacrilamida , Éxons/genética , Feminino , Humanos , MasculinoRESUMO
To screen differentially expressed genes involved in osteogenic differentiation of human bone marrow stromal cells (BMSCs) at defined stages, subtractive cDNA library was established by means of suppression subtractive hybridization. The BMSCs cultured for 12 and 21 d were used as driver and tester, respectively. A subtract library was successfully constructed and five positive clones were selected from the library. Sequencing analysis and homology comparison showed that the five clones differentially expressed in BMSCs cultured for 21 d were at least 90% homologous with the known genes in human GenBank. It was interestingly found that the osteogenic BMSCs cultured for 21 d differentially expressed decorin and Bax inhibitor 1. RT-PCR was performed to confirm the differentially expressed genes. The results showed that the expression of Bax inhibitor 1 was significantly higher in the cells of 21-day than that of 12-day, while the expression of decorin was only detected in the cells of 21-day.