ABSTRACT
Objective To study the antiendotoxin activity of P1 and P2 based on the lipopolysacchride binding protein.Method P1 and P2 were designed and obtained.In vitro test,peripheral blood mononuclear cell(PBMC)were extracted from volun-teer 100ml venous blood,the experiment group was arranged as:control group,positive control group,LPS group,LPS+P1 (2 mg/L,5 mg/L,12.5 mg/L)group and LPS+P2(2 mg/L,5 mg/L,12.5 mg/L)group,TNF-α and IL-6 of supernatant liquor in every group were detectd by ELISA.In vivo,40 kunming rice were randomly divided into four groups with ten rice each group:control group,model group,LPS+P1 and LPS+P2 group,Pathologic changes of lung and liver tissues were ob-served by hematoxylin and eosin(HE)staining.Results The serum level of IL-6 and TNF-αin 12.5 mg/L P1 and 2 mg/L, 5 mg/L,12.5 mg/L P2 treatment group were lower than that in model group,the difference was statistically significant(all P<0.01).Serum level of TNF-αor IL-6 in 12.5 mg/L P2 treatment group were similar to that in PMB treatment group, and there was no statistically significant difference(P>0.05).Histologymorphology finding showed that central veins of liver and hepatic sinusoid congestion,hepatic cellular edema existed,occasionally,acidophilic change and spotty necrosis were found,pulmonary interstitial edema,focal hemorrhage,alveolar space stenosis existed.As regards 10 mg/kg P1 treatment group mice,hepatic cellular edema and pulmonary interstitial edema ameliorated.About 10 mg/kg P2 treatment group mice, veins of liver and hepatic sinusoid congestion obviously ameliorated,mild pulmonary interstitial edema exsited.Conclusion The results indicated P1 and P2 had antiendotoxin effect,in vivo and vitro,for 12.5 mg/L P2,its inhibition effect for TNF-αor IL-6 release was positive.
ABSTRACT
This study was aimed to compare the differential expressions of calcineurin (PP2B, PP3) in the mouse Pre-B cell lines (S9) and the tumor cell lines (S4C2) derived from pre-B lymphocytes, and to clarify its possible mechanism involving in the leukemia cell apoptosis. The quantitative real-time PCR was used to detect the differential expressions of H2AX-associated phosphakinase ATM, ATR, DNA-PKs, JNK1, P38 and the γ-H2AX-related phosphatase PP1, PP2A, calcineurin, PP4, PP6, PP5 between S9 and S4C2 cell lines. CCK-8 assay and flow cytometry were used to detect the effect of imatinib (IM) and cyclosporine A (CsA) on cytotoxicity and apoptosis of 2 cell lines. The Western blot was used to detect the effects of 2 drugs on apoptosis of S9 and S4C2 cell lines. The results showed that the expression level of calcineurin gene in the leukemia cell S4C2 was about 3.5 times of that in S9 cells, while the expression of other genes in these 2 kinds of cells was not significantly different. The apoptosis and toxicity of IM and CsA on S4C2 cells was significantly stronger than that on S9 cells. The expression level of calcineurin in S4C2 cells was higher than that in S9 cells.When CsA inhibited the calcineurin activity, the expression of DNA damage marker γ-H2AX in S9 cells was significantly lower than that in S4C2 cells,while the expression level of γ-H2AX between the two cell lines was no significantly different after treatment with imatinib, the expression level of γ-H2AX in S9 cells was lower than that in S4C2 cells when the two drugs were combined. It is concluded that the calcineurin plays a role of anti-apoptosis in B leukemic cells, cyclosporine A can promote the leukemia cell apoptosis.
Subject(s)
Animals , Mice , Apoptosis , Calcineurin , Metabolism , Cell Line, Tumor , Cyclosporine , DNA Damage , Flow Cytometry , Leukemia , Metabolism , Precursor Cells, B-Lymphoid , Metabolism , Real-Time Polymerase Chain ReactionABSTRACT
MUC1 mucin is a high molecular weight, type I transmembrane glycoprotein. High and aberrant expression of MUC1 is observed in various types of tumors, which make it an ideal target for tumor biotherapy as well as a biomarker for tumor diagnosis and prognosis. MUC1/Y is an isoform of MUC1 generated by alternative splicing. Specific expression of MUC1/Y in breast cancer as well as its involvement in tumor cell signal transduction have been reported. In order to purify peptides containing MUC1/Y-specific epitope in E. coli and prepare MUC1/Y-specific antibody, DNA fragment encoding the MUC1/Y-specific peptide was amplified by PCR using MUC1/Y full length cDNA as the template and cloned into fusion expression vector pGEX-2T, resulting pGEX-Y30. DNA sequencing was performed to confirm the correct amplification and orientation of the target sequence. Competent E. coli DH5alpha was transformed with pGEX-Y30 and the expression was induced for 4-5 hours in 0.2 mmol/L IPTG at 30 degrees C and 37 degrees C. Expressed proteins were released from the cells by ultrasonication or B-PER II reagent treatments. The fusion protein GST-Y30 were purified by affinity and anion exchange columns and identified by SDS-PAGE and Western-blotting. Polyclonal antibody was prepared by immunizing rabbits with the GST-Y30 protein for 4 times with intervals of 3 weeks and purified by GST column. Western blotting, ELISA and immunohistochemistry analysis were carried out using the purified antibody to confirm its MUC1/Y-binding capacity and specificity. The expressed fusion protein GST-Y30 is about 31 kD in size and represented about 20% of total cellular proteins. The majority of the GST-Y30 protein existed as soluble form when the induction was carried out at both 30 degrees C and 37 degrees C. After the two-step purification, the purity of GST-Y30 was about 94%. The titer of polyserum generated by GST-Y30 immunization was 1:320,000 by ELISA. The antiserum showed MUC1/Y specificity and can recognize MUC1/Y on MCF7 cell. The MUC1/Y-specific polyclonal antibody can be used for studying the role of MUC1/Y in carcinogenesis.
Subject(s)
Animals , Humans , Rabbits , Antibodies , Metabolism , Antibody Specificity , Cell Line, Tumor , Metabolism , Epitopes , Chemistry , Genetics , Metabolism , Escherichia coli , Genetics , Metabolism , Immunohistochemistry , Models, Genetic , Mucin-1 , Chemistry , Genetics , Metabolism , Peptides , Chemistry , Genetics , MetabolismABSTRACT
Objectives:To study the expression of GST-pi and MDR1 genes in operative specimens of ovarian cancer,and to analyze the possible clinical role of GST-pi and MDR1. Methods:Eighteen frozen specimens of ovarian carcinoma and ten specimens of normal ovarian tissues from patients were examined for the expression of GST-pi and MDR1 genes by means of RT-PCR, and quantitative analysis was performed using β-actin as internal contrast.Results: Positive expression rate of GST-pi and MDR1 in ovarian carcinoma were 61.1% and 33.3%,respectively,and in contrast, 20% and 10% in normal ovarian tissues respectively. The level of GST-pi gene expression in ovarian carcinoma was obviously higher than that in normal ovarian tissue (P<0.05)and MDR1 gene also had high level expression in ovarian carcinoma, but had no statistical significantance. Four patients with ovarian carcinoma had GST-pi and MDR1 coexpression. Expression levels of GST-pi mRNA were lower than that of protein. Conclusions: (1) GST-pi and MDR1 had higher level expression in ovarian carcinoma than in normal ovarian tissues. (2) GST-pi and MDR1 may have same regulating factors but different mechanisms of action. (3)Processing after transcription and/or regulation of translation level may exist in GST-pi expression.