ABSTRACT
The establishment of in vitro model will provide optimal conditions for the study of human papillomavirus (HPV)-associated cervical cancer. In this study, E6 and E7 gens of HPV31 were cloned and expressed in E. coli. The recombinant proteins were purified and used as antigens to immunize mice for the production of polyclonal antibody. Mammalian expression plasmid pBudCE4. 1-HPV31-E6/E7 was also constructed and transfected into C33A cells. The transfected cells were then selected by Zeocin. The expressions of the E6 and E7 mRNAs and proteins were detected by RT-PCR and Western blot respectively. A stable cervical cancer cell line was established as an in vitro model for the study of human papillomavirus type 31(HPV31) associated cervical cancer.
Subject(s)
Animals , Female , Humans , Mice , Cell Line , Virology , Human papillomavirus 31 , Genetics , Metabolism , Oncogene Proteins, Viral , Genetics , Metabolism , Papillomavirus E7 Proteins , Genetics , Metabolism , Papillomavirus Infections , Virology , Recombinant Proteins , Genetics , Metabolism , TransfectionABSTRACT
This study was aimed to detect the expression of leukemia stem/progenitor cell (LSPC) related genes (ABCB1, BMI-1, HOXB4) in the patients with acute leukemia, and to explore its clinical significance in acute leukemia. Bone marrow samples were collected from de novo acute leukemia patients (41 cases), patients with complete remission (CR, 16 cases) and the patients with non-malignant hematologic diseases (10 cases) respectively. And the expressions of ABCB1, BMI-1, HOXB4 genes were detected by comparative real-time quantitative PCR (RQ-PCR) with SYBR Green assay. The results showed that the expressions of ABCB1, BMI-1, HOXB4 were not detected in the patients with non-malignant hematologic diseases, but were higher (relative expressive level: 4.26 ± 2.26, 3.72 ± 1.91, 3.74 ± 2.38) in de novo acute leukemia patients and lower (relative expressive level: 2.14 ± 1.47, 2.07 ± 0.99, 1.47 ± 0.89) in the acute leukemia patients with CR (p < 0.05). The expressions of LSPC related genes were lower (relative expressive level: 1.77 ± 1.29, 2.09 ± 1.26, 1.78 ± 1.49) in the patients acquired CR/partial remission (PR) than those in the patients not acquired CR/PR (relative expressive level: 7.23 ± 1.78, 3.96 ± 0.92, 4.48 ± 2.57) (p < 0.01). Univariate analysis revealed that there were more cases with the expression of LSPC immunophenotype (CD34(+)CD38(-)CD96(+) and CD34(+)CD38(-)CD123(+)) and more hyperleukocytosis cases in patients with any higher expression of LSPC related gene (p < 0.05). Analysis of multiple parameters discovered larger significance (p < 0.01). It is concluded that there is a good relationship between LSPC related genes (ABCB1, BMI-1, HOXB4) and LSPC immunophenotype. The expression of LSPC-related genes is higher in de novo acute leukemia patients, and lower in patients acquired CR/PR. The patients with higher expressed LSPC-related genes display worse response to chemotherapy, lower CR/PR rate and higher leukocytosis, the analysis of multiple parameters may be a good method for assessing the therapeutic efficacy/prognosis of acute leukemia.
Subject(s)
Adolescent , Adult , Aged , Female , Humans , Male , Middle Aged , Young Adult , ATP Binding Cassette Transporter, Subfamily B , ATP Binding Cassette Transporter, Subfamily B, Member 1 , Genetics , Metabolism , Gene Expression , Homeodomain Proteins , Genetics , Metabolism , Immunophenotyping , Leukemia, Myeloid, Acute , Genetics , Metabolism , Neoplastic Stem Cells , Metabolism , Polycomb Repressive Complex 1 , Genetics , Metabolism , Transcription Factors , Genetics , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To investigate the in vitro effect of iron overload on the generation of reactive oxygen species (ROS) and of bone marrow (BM) cell function.</p><p><b>METHODS</b>BM mononuclear cells (BMMNCs) were cultured with ferric citrate (FAC) at different concentrations and for different time to create iron overload and confirmed by the detection of cellular labile iron pool (LIP). The changes of ROS, apoptosis, hematopoietic colony formation (CFU-E, BFU-E, CFU-GM and CFU-mix) and the percentage of the CD34 + cells percentage were analyzed. The differences of these index were tested after the iron overload treated with deferasirox (DFO) or antioxidants (N-acetyl-L-cysteine, NAC).</p><p><b>RESULTS</b>1) When BMMNCs were cultured with FAC, the LIP was found to increase in a time and concentration dependent manner. The intracellular LIP reached maximum at 400 micromol/L of FAC for 24 hours. 2) The ROS of total cells, leukocytes and erythrocytes increased to 1.77, 1.75 and 2.12 fold respectively compared with that of normal control when cells were cultured at 400 micromol/L of FAC for 24 hours . DFO and NAC could reduce the ROS efficiently (P<0.05). 3) The apoptotic rates of the FAC treated cells [(24.80 +/- 2.99)%] increased significantly compared with that of normal control [(8.90 +/- 0.96)%]. The capacity of hematopoietic colony formation in FAC treated cells decreased markedly compared with that of normal control (P<0.05). The percentage of CD34+ cells of FAC treated cells [(0.39 +/- 0.07)%] also decreased significantly compared with that of normal control [(0.91 +/- 0.12)%]. And these changes could be recovered by addition of NAC or DFO.</p><p><b>CONCLUSION</b>Iron overload can affect the hematopoiesis by inducing the generation of ROS and this damage could be corrected by removing the excess iron and ROS of the BM cells. These findings might improve the treatment of dyshematopoiesis in patients with iron overload.</p>
Subject(s)
Humans , Bone Marrow Cells , Physiology , Cells, Cultured , Culture Media , Chemistry , Erythrocytes , Ferric Compounds , Pharmacology , Hematopoiesis , Iron Overload , Reactive Oxygen Species , MetabolismABSTRACT
PBD-1 is an antibacterial peptide that plays an important role in defence system of porcine. To produce PBD-1 with bioactivity in Pichia pastoris, according to published amino acid sequence of porcine beta-defensin 1(PBD-1) and the partiality codon of yeast, the PBD-1 gene was synthesized by PCR and cloned into pPIC9K to construct the recombinant expression vector pPIC9K-PBD-1, the obtained recombinant plasmid was linearized by Sal I, and then transformed into SMD1168 by electroporation. Under the control of the promoter AOX1, an approximately 4.5 kD PBD-1 peptide was expressed. Antibacterial activity assay shows that the PBD-1 has the antibacterial activity on Staphylococcus aureus. This is the first secreted expression of porcine beta-defensin 1 gene in Pichia pastoris.
Subject(s)
Animals , Anti-Bacterial Agents , Pharmacology , Cloning, Molecular , Gene Expression , Genetic Vectors , Genetics , Pichia , Genetics , Protein Engineering , Staphylococcus aureus , Swine , Genetics , beta-Defensins , Genetics , PharmacologyABSTRACT
A fragment containing amino acid residues 561～578 of HSV-2 glycoprotein G(gG2) was obtained by PCR assembling technique,and doubly cloned into vector pET-KDO.The recombinant plasmid was transformed to BL21(DE3)plysS.Fusion protein,of molecular weight about 39kDa was highly expressed by induction of IPTG.Western blot result showed the fusion protein had good antigenicity.After putification and digestion,the purity reached 95%.The digested purified protein was analysed by ELISA and showed good sensitivity and specificity.The recombinant protein should be useful for type-specific serodiagnosis of HSV-2.