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Objective To evaluate the growth inhibition of human gastric carcinoma cell lines SGC 7901 in vitro and the expression of bcl-2, bcl 2l12 and bax with docosahexaenoic acid (DHA) and 5-fluorouracil (5-FU). Methods The effect of DHA and 5-FU was measured by trypan blue, and the interaction between two agents was judged by combination index (CI). Cells were observed by inverted microscope. Flow cytometry was used for analysis of apoptosis by PI staining and Annexin-V/PI. RT-PCR was used to analyze the levels of bcl-2, bcl 2l12 and bax mRNA. Results DHA significantly inhibited the growth of SGC 7901 cells in a dose- and time-dependent way ( P < 0. 05 ), the IC50 of 24 h and 48 h was 67. 81 μg/ml and 45.76 μg/ml, and a strong synergism was found in the combination of DHA and 5-FU (CI < 1 ,P <0. 01 ). Treated by DHA and 5-FU for 48 h, cells became sparse under inverted microscope. DHA or 5-FU was able to induce apoptosis and the effect became even more significant by the combination of DHA and 5-FU. Cells were holted in phase of G01/G1 and S. RT-PCR showed that DHA or 5-FU down-regulated the expression of bcl-2 and bcl 2l12 mRNA, while bax mRNA expression was not downregnlated. Conclusions DHA could inhibit the growth of gastric carcinoma cells, DHA and 5-FU had synergetic effect in the inhibition of the cells growth and blockage of the cell cycles possibly by down-regulating the expression of bcl-2 and bcl 2l12.
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Objective To study the human telomerase reverse transcriptase (hTERT) and spleen tyrosine kinase (SYK) activity expression and molecular margin in the tumor center and peripheral tissues of patients with colorectal cancer, and to explore the security of tumor surgical margins. Methods The expressions of hTERT and SYK were examined by SABC and Supervisan immunohistochemistry in 20 specimens of colorectal cancer and tissues 1 cm, 2 cm, 3 cm, 4 cm and 5 cm, away from proximal and distal margin of the tumor respectively. Results The positive expression rate of hTERT in colorectal cancer was 80.00 %, the rate in tissues 3 cm, 4 cm and 5 cm away from the tumor margin was 0. The positive expression rate of hTERT in tissues from the cancer center to 3 cm away from the tumor margin was gradually diminishing (P<0.01). The positive expression rate of SYK in colorectal cancer was 10.00 %, the positive expression rate of SYK in tissues from the cancer center to 3 cm away from the tumor margin was gradually increased (P<0.05). Conclusion There is molecular margin around colorectal cancer tissues. Considering hTERT and SYK expressions, 3 cm can be defined as the safe molecular margin for the radical operation of colorectal cancer.
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AIM: To obtain nerve growth factor subunit ?(NGF ?) gene from Chinese fetal hippocampus tissue, prove its sequence, clone the mature peptide sequence, and make it express in E.coli .METHODS: Total RNA was extracted, amplified by RT-PCR method. Its 650 bp DNA sequence was inserted into PCR Ⅱ vector. PCR/NGF ? vector was used as the template to amplfy the C-terminate mature peptide sequence, then subcloned it into PG5 vector. The recombinant was transferred into E.coli BL21. BL21 was cultured and induced by IPTG. The activity of the expressed product was measured after purified and refolded.RESULTS: A complete cDNA sequence was determined as 1 047 nucleotides. The cloned 636 bp encoding 212 amino acids was proved homological to Genbank by sequence analysis. The expressed mature peptide showed an clear band of the prospected 14 kD by SDS-PAGE electrophoresis, and was testified by Western blot. The expression level was about 10% of the total cell lysate. CONCLUSION: The chinese NGF ? gene was homological to the foreigners'. The recombinant NGF ? was efficiently expressed in E.Coli and the recombinant protein has high immunological activities.
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AIM:To detect the treatment of K562 leukemia cells with bortezomib altering the expression of genes fas,bcl-2,bcl2l12,bim,bax,caspase-9 and caspase-3.METHODS:MTT assay was used to detect the inhibition of proliferation.Apoptosis was detected by Annexin-V staining and mitochondrial transmembrane potential(??m).RT-PCR was used to analyze the mRNA expressions of fas,bcl-2,bcl2l12,bim,bax,caspase-3 and caspase-9.RESULTS:Bortezomib caused a time-and dose-dependent inhibition of cell proliferation and IC50 of 24 h and 48 h were 161.41 nmol/L and 96.33 nmol/L,respectively.At the concentration of 104 nmol/L,bortezomib induced apoptosis in a time-dependent manner,including increasing annexin-V positivity and decreasing the ??m.RT-PCR showed that bortezomib up-regulated the mRNA expression of fas,bcl2l12,caspase-9 and caspase-3,but mRNA expressions of bcl-2,bim and bax did not changed obviously.CONCLUSION:Bortezomib inhibits the proliferation of K562 and induces apoptosis,in which fas,bcl2l12,caspase-9 or caspase-3 gene is one of the main genes taking part in.
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AIM: To establish a fluorogenic quantitative polymerase chain reaction (FQ-PCR) method for the routine examination of c-erbB-2 gene expression in breast cancer. METHODS: The c-erbB-2 standard gene was obtained by in vitro amplification of cloned c-erbB-2 fragment in plasmid PGEM-T easy vector. FQ-PCR product was detected by using a 7700 ABI PRISM sequence detector system and c-erbB-2 standard curve was obtained to quantity c-erbB-2 in unknown samples. RESULTS: “S” kinetics curve of FQ-PCR amplification was generated by relating the fluorescence signal intensity (△Rn) to the cycle number. The standard curve of c-erbB-2 was constructed by the linear relationship between the cycle threshold (ct) and the log of starting copy number. The high correlation (0.999) revealed the reliability of FQ-PCR. CONCLUSION: The FQ-PCR is a rapid, sensitive, reliable method for quantity of c-erbB-2 gene expression.