ABSTRACT
AIM: To construct eukaryotic expression vector of human CD34 and transfect it to 3T3 cells so as to establish stably transfected 3T3 cell line. METHODS: The RNA was extracted from KG1a. CD34 gene was amplified by RT-PCR. With the double-enzyme digestion, CD34 gene was cloned into pCI-neo eukaryotic expression vector, yielding pCI-CD34. The pCI-CD34 was transfected into 3T3 cell by electroporator. Stably transfected 3T3 cell line was established, and the CD34 expression in the transfected cells was detected by RT-PCR and FACS. RESULTS: The eukaryotic expression vector pCI-CD34 was constructed, and stably transfected 3T3 cell line was established. CONCLUSION: Construction of eukaryotic expression vector of CD34 and the establishment of stably transfected 3T3 cell line are helpful to preparation of anti-CD34 mAbs and further functional study of CD34.
Subject(s)
Antigens, CD34/biosynthesis , Antigens, CD34/genetics , Genetic Vectors/chemical synthesis , Genetic Vectors/genetics , 3T3 Cells , Animals , Antigens, CD34/chemistry , Cell Line , Cloning, Molecular/methods , Flow Cytometry/methods , Genetic Vectors/chemistry , Humans , Mice , Reverse Transcriptase Polymerase Chain Reaction/methods , TransfectionABSTRACT
A series of N-((2-methyl-4(3H)-quinazolinon-6-yl)methyl)dithiocarbamates 5a-w were synthesized and evaluated for their cytotoxic activity against five human cancer cell lines. We found that compound 5k inhibited proliferation of A549, MCF-7, HeLa, HT29 and HCT-116 cells with IC(50) values of 5.44, 7.15, 12.16, 10.35 and 11.44 microM, respectively. Compound 5i was the most potent with an IC(50) value of 3.65 microM against proliferation of MCF-7 cells, while 5n was the most potent with an IC(50) value of 5.09 microM against proliferation of A549 cells. Cell cycle analysis showed that both 5i and 5k arrested A549 cells at S and G2/M phases, suggesting that these compounds act through mechanisms different from 5-fluorouracil, which arrests cells at S phase only.
Subject(s)
Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Thiocarbamates/chemical synthesis , Thiocarbamates/pharmacology , Antineoplastic Agents/chemistry , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Thiocarbamates/chemistryABSTRACT
AIM: To prepare and characterize the specific monoclonal antibodies (mAbs) that can recognize the native conformation of HER2 so as to guide the clinical application of Herceptin. METHODS: Hybridomas were generated by the fusion with the NS1 myeloma cell and spleen cell, which were from mice immunized by HER2 recombinant protein. After cell fusion, ELISA, Yeast cell base ELISA and immunohistochemistry were used to screening clones respectively. RESULTS: Eight clones that could react with SK-Br-3 and yeast displaying HER2 were selected from 168 clones which were positive in initial screening by ELISA. CONCLUSION: Successfully prepared 8 mAbs that can recognize the native conformation of HER2, which should provide useful reagent for personalized therapy against breast cancer with Herceptin.
