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AIM: To detect the effect of conjunction matrigel with mammary fad pat(MFP)implantation on the tumorigenesis, proliferation, apoptosis and metastasis of Her2 positive and negative breast cancer model. METHODS: The Her2 positive BT 474 and Her2 negative MDA-MB 231 breast cancer cells were injected into MFP of nude mice with or without matrigel to establish breast cancer model. The tumor volume was measured every 3 d. Followed up for 30 d after implantation, the nude mice were killed and the tumors and associated organs were dissected for pathological sectioning and staining with hematoxylin and eosin. The time of tumor formation and the tumorigenesis were determined after implantation. The tumor volume and metastasis rate were calculated and compared with each other. The proliferation and apoptosis of Her2 positive and negative tumors were also determined. RESULTS: Matrigel and MFP implantation technology shortened the time of tumorigenesis significantly(P<0.01). The tumorigenesis rate of BT 474 and MDA-MB 231 breast cancer cells did not show any different(P>0.05). The metastasis rate of MDA-MB 231 breast cancer cells were improved from 25.0% to 37.5%(P<0.05). CONCLUSION: Matrigel and MFP implantation can be combined to shorten the time of tumor formation by two kinds of breast cancer cells, and improve the metastasis of Her2 negative MDA-MB 231 cells. Using matrigel does not show any effect of proliferation and apoptosis on Her2 positive and negative breast cancer cells.
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AIM:To study the blocking effect of shRNA on the expression of PSMA gene in LNCaP cell line by using shRNA eukaryotic expression vector.METHODS:Three pairs of DNA templates coding shRNA,synthesized against PSMA and cloned into the vector pSilencer 2.1-U6-neo,which was named pSilencer 2.1-U6-neo-shRNA,were identified by restriction endonuclease digestion analysis and DNA sequencing.LNCaP cells were then transfected with these three pSilencer 2.1-U6-neo-shRNAs and the negative control pSilencer 2.1-U6-neo-NC.After G418 selection,the cells were selected and the interfering effect was detected by RT-PCR and Western blotting.The biological behaviours of the transfected LNCaP cells were also tested.RESULTS:Restriction endonuclease digestion analysis and DNA sequencing results all showed that the 3 target segments were cloned into pSilencer 2.1-U6-neo vector respectively.After transfected into LNCaP cells,the inhibitory ratio of PSMA mRNA was 33.15%,9.26% and 41.97% respectively,and that of PSMA protein was 26.26%,6.47%,40.69% respectively.The p-shRNA3 was chosen to test the cell growth and its invasive power in vitro.The results showed that after interfering,the invasiveness of LNCaP cells were enhanced.CONCLUSION:The vector-based shRNA on PSMA gene effectively knocks down the PSMA gene expression.The successful construction of PSMA shRNA makes it possible for further study of the interaction between PSMA and prostate cancer.
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AIM: To find out the gene structure and diversity of protate specific membrane antigen(PSMA) alternative spliced variants, and probe into the pathogenesis of prostate cancer.METHODS: 5'-RACE and 3'-RACE methods were used to amplify the 5' and 3'end of alternative spliced variant and then those viariants were sequenced for analyzing the gene stucture and diversity of PSMA alternative spliced variants of prostate cancer tissues.RESULTS: Four new alternative spliced variants of PSMA were discovered from prostate cancer tissues.Compared with reported PSMA alternative spliced variants,different insertions and deletions existed in different sites of those new variants.CONCLUSION: The discovery of the new variants confirms the diversity of PSMA spliced variants and provides the clues for seeking the target of diagnosis and therapy of prostate cancer.
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AIM: To discuss the relationship between prostate specific membrane antigen(PSMA) and prostate cancer and to seek a target for diagnosis and therapy of prostate cancer.METHODS: A pair of primers was designed according to the published PSMA mRNA sequence.Total RNA was extracted from prostate cancer tissues and was reversely transcribed into cDNA,which was used as a template for PCR to amplify the PSMA gene.The recombinant was sequenced and the result was analyzed by BLAST.The PSMA5 gene specific primers were designed to identify its expression in different cells and prostate tissues.RESULTS: A new alternatively spliced variant of PSMA named PSMA5 was discovered when sequencing the recombinant.PSMA5 showed well pathological tissue-specificity,and its expression rate in prostate cancer,benign prostatic hyperplasia of prostate,and normal prostate tissue were 92.6%,78.8% and 10.0%,respectively.It expressed specifically in Pca cell line LNCaP,not in cell lines of PC3,bladder carcinoma,renal carcinoma,or hepatoma.CONCLUSION: A new alternative spliced variant of PSMA named PSMA5 was discovered,which was well correlated with prostate cancer and benign prostatic hyperplasia.This finding may give a new clue to the evolution of prostate cancer and may provide a target for the diagnosis and therapy of prostate cancer.
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AIM: To amplify from leader peptide region an d obtain human monoclonal anti-D variable region gene with high specificity and affinity, and analyze the nucleotide and deduced amino acid sequences.ME THODS: The total RNA was extracted from an Epstein-Barr-virus-transforme d cell line secreting monoclonal anti- (rhesus D) antibody. The leader region pri mers containing a ribosome recognition site were designed. By using PCR method, the cDNA of human anti-(rhesus D) antibody (IgM ?) variable region gene was amp lified. Cloning and subsequent sequence analysis of the variable region gene was performed. The deduced amino acid sequence was also compared and analyzed with previ ously published sequences.RESULTS: A band of approximate 440 and 410 base pairs were amplified using heavy chain primers and light chain primer s, respectively. Sequence analysis indicated that the deduced amino acid sequenc e w as in agreement with the characterization of the amino acid present in the human Ig variable region. CONCLUSION: The cloning and sequencing of a human anti- (Rhesus D) antibody variable region cDNA will make benefits for pro duction of recombinant anti-(Rhesus D) antibody and prevention of Rh haemolytic disease in newborns.
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AIM: To investigate the effect of norcantharidin(NCTD)on proliferation and invasion of human breast cancer cell line SKBR3 in vitro and its anticancer mechanisms.METHODS: MTT assay was used to determine SKBR3 cell proliferation. Light and FACScan were used to detect apoptosis and cell cycle. The invasiveness of SKBR3 was evaluated by the adhesion test,Matrigel experiment and the crossing-river test.RESULTS: NCTD had inhibitive effects on growth of SKBR3 cells in a dose and time-dependent manner, with the IC50 value of 12.5 mg/L at 24 h.The cells treated with 10 mg/L NCTD for 24 h and 48 h showed typical apoptotic morphology and hypodiploid peak before G1 phase. The cell cycle was arrested at G2/M phase. The apoptosis percentage was up to 3.44% and 6.17%, and the G2/M percentage was up to 35.82% and 38.70%. NCTD also could inhibit obviously the adhesion, movement and invasive capability simulating human basement membrane of SKBR3. Its effect was also in a dose-dependent manner. In the NCTD-treated group, crossing-river time was prolonged significantly and passing-membrane cells markedly decreased. CONCLUSION: NCTD in vitro inhibits not only the proliferation and growth of human breast cancer cells but also invasion and metastasis of the cells at relatively low concentration. NCTD shows prominent anti-tumor effects.
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AIM: To obtain eukaryotic expression vector of Chinese prostate-specific membrane antigen. METHODS: Chinese prostate-specific membrane antigen (PSMA) cDNA was amplified by RT-PCR from prostate cancer tissues, then cloned into eukaryotic expression vector pcDNA3 0 and sequenced. RESULTS: Seven bases in Chinese PSMA cDNA sequence were found different from those reported by Israeli, which lead to two different amino acids. CONCLUSION: We have obtained the PSMA cDNA, and the recombinant eukaryotic expression vector was successfully constructed. The study lays foundation for DCs vaccine modified by PSMA gene for the treatment of prostate neoplasms.
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AIM: To clone and express a human monoclonal anti-D Fab fragment in E. coli and make benefits for the expression of the whole immunoglobulin molecules of anti-D. METHODS: The gene of anti-D Fab fragment was cloned into the phagemid vector pComb3. After analyzing by PCR and restriction site analysis, the recombinant was expressed in E. coli and the expressed protein was analyzed by SDS-PAGE and ELISA. RESULTS: The result of SDS-PAGE confirmed that E.coli expressed a 48 kD protein. The ELISA result demonstrated that the cell culture supernatant reacted with Rh+ group O human erythrocytes, but was not recognized by Rh-group O human erythrocytes. CONCLUSION: Expressed Fab fragment has the antigenic specificity for human erythrocytes.