RESUMO
OBJECTIVE: The aim of the present work was to study the prevention of liver cancer angiogenesis via miR-126. For this purpose, experimentations were conducted. MATERIALS AND METHODS: The precursor sequence of miR-126 was amplified in the DNA of human liver cancer cell lines. We, therefore, constructed the overexpression and interference vectors of miR-126 in vitro; which were respectively transferred to liver cancer cells in the logarithmic phase and inoculated under both sides of the back skin of Balb/c-nu nude mice aged 4-6 weeks with 10 mu l (1 x 105) cell suspension. The experiment consisted of non-vector control group, miR-126 overexpression group, and miR-126 inhibition group. Eight weeks later, the mice were sacrified; the tumor volumes and serum ALT, AFP, VEGF levels were compared. VEGF expression, as well as the microvascular density of the liver tissues, was detected via immunohistochemistry. RESULTS: Tumors volumes, serum ALT, AFP and VEGF levels and positive rates of VEGF were low in the miR-126 overexpression group and high in the miR-126 inhibition group, the difference being statistically significant (p < 0.05). CONCLUSIONS: At the end of this study, we conclude that miR-126 inhibits liver cancer angiogenesis.
Assuntos
Neoplasias Hepáticas/patologia , MicroRNAs/metabolismo , Alanina Transaminase/sangue , Animais , Antagomirs/metabolismo , Células Hep G2 , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/irrigação sanguínea , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Nus , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Neovascularização Patológica/prevenção & controle , Transplante Heterólogo , Carga Tumoral , Fator A de Crescimento do Endotélio Vascular/sangue , alfa-Fetoproteínas/análiseRESUMO
OBJECTIVE: To analyze the sequence difference of the SSU rDNA variable regions of Leishmania isolates from hilly foci and plain foci of China. METHODS: Specific SSU rDNA fragments from nuclear DNA of five Leishmania species and isolates were amplified by PCR. The amplified DNA fragments were cloned into pGEMR-T Easy vector. The specific fragments were sequenced by the automated DNA sequencer. RESULTS: Sequence analysis showed that the amplified DNA fragments of five Leishmania species and isolates were all 392 bp in length, point mutations were located in the two unique sequence (UQ-I and UQ-II); L. d. SC10 and L. d. GS7 had two same point mutations in UQ-II, only L. d. GS7 had one in UQ-I; no insertion/deletion. CONCLUSION: Sequence difference of the SSU rDNA variable region existed between Leishmania isolates from hilly foci and plain foci; The sequences of the SSU rDNA variable regions of L. d. SD2 isolate and L. infantum were identical.
Assuntos
DNA de Protozoário/química , DNA Ribossômico/química , Leishmania donovani/genética , Leishmaniose Visceral/parasitologia , Animais , Humanos , Mutação Puntual , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
OBJECTIVE: By sequencing of SSU rRNA gene cloning from Xinjiang cutaneous leishmaniasis pathogen (XJCLP) to provide evidence for identification of the pathogen. METHODS: By PCR assay with primers R222 and R333, the specific fragment had been produced from SSU rRNA gene of XJCLP, L. infantum, L. tropica and cloned into pGEM-T Easy vector. The clones were sequenced by the Sanger dideoxy-mediated chain termination method, analysis of SSU rRNA gene sequences from XJCLP, L. tropica, L. infantum with DNASIS. RESULTS: Sequence analysis showed that the specific fragment of SSU rRNA gene from XJCLP, L. infantum, L. tropica, were all 394 bp in length. There were 391 bases identical and three point mutations between the sequences of XJCLP and L. tropica, the similarity being 99.2%; 390 bases identical and three point mutations and one insertion/deletion between the sequences of XJCLP and L. infantum, the similarity being 99.0%. One insertion/deletion between the sequences of L. tropica and L. infantum, the similarity being 99.7%. The primary and secondary structures of SSU rRNA gene from XJCLP differed from those of L. infantum and L. tropica. A retrieval from GenBank confirmed that these 394 bp sequence are new gene sequences. CONCLUSION: The primary and secondary structures of SSU rRNA gene from XJCLP, L. infantum, L. tropica were different. 394 bp sequence from SSU rRNA gene of XJCLP is a new gene sequence.
Assuntos
Leishmania/genética , Leishmaniose Cutânea/parasitologia , RNA de Protozoário/química , RNA Ribossômico/química , Animais , Sequência de Bases , China , Clonagem Molecular , Humanos , Leishmania/classificação , Dados de Sequência Molecular , Mutação PuntualRESUMO
In this report, we described the extraction of L. donovani genomic DNA and construction of L. donovani genomic library in order to isolate individual gene and its products that can be the candidate to develop serum diagnostic and protective products for this disease. We prepared L. donovani promastigote genomic DNA using the protocols of extracting genomic DNA of cultivated cells by centrifugation (15,000 g, 60 min) to get rid of kinetoplast DNA. Genomic DNA was then partially digested with restriction enzymes Hae III, the digests were size-separated by gradient centrifugation, then ligated to EcoRI linkers, inserted to lambda gt11 arms, and packaged with pack gene in vitro. This procedure resulted in 2.28 x 10(6) phages and the insert proportion was 87%.
