ABSTRACT
Objeetive To improve the method for the isolation and purification of rat hepatic stellate(HSC) cells and to provide a stable cell source for the research on liver-related diseases.Methods Rat liver was digested in situ by a two-step infusion assay under a strict control of the infusion temperature,flow rate and time with a combined utilization of Pronase E and Collagenase Ⅳ.And then,the HSC cells were separated by Percoll density gradient centrifugation.The cell growth curve and survival rate were measured by CCK-8 and trypan blue staining,respectively.The HSC cells were identified by flow cytometry and immunofluorescence cytochemistry.Results With the improved methods,there were (2.1 ± 0.2) × 107 HSC cells isolated from one rat and the survival rate was (96.2 ± 0.8) ％.The percentage of HSC cells with a spontaneous fluorescent characteristic from the isolated cells was 96.3％.The immunofluorescence cytochemistry was used to detect the expressions of the surface antigens α-SMA and Desmin in the isolated HSC cells.Conclusion By strict control of infusion temperature,flow rate and perfusion time as well as the combined application of Pronase E and Collagenase Ⅳ,there is an increased harvest of HSC cells with improved cell viability and purity,which is helpful for further research on HSC cells.
ABSTRACT
<p><b>OBJECTIVE</b>To study the effect of lovastatin on the expression of IkappaBalpha and cell-cycle regulating proteins in MCF-7 cells.</p><p><b>METHODS</b>MCF-7 cells were treated with 4, 8 and 16 micro mol/L lovastatin for 48 - 72 h. The distribution of cell cycles was assayed by flow cytometry (FCM). The protein expression of IkappaBalpha, CDK4, p16, pRb in cytoplasm and IkappaBalpha in the nucleus were detected by Western blot.</p><p><b>RESULTS</b>Lovastatin could arrest cellcycle in the G(0)/G(1) phase in a dose- and time-dependent manner, obviously lowering the expression of IkappaBalpha, CDK4 and pRb protein level in the cytoplasm and increasing IkappaBalpha in the nucleus, but not on p16 protein level.</p><p><b>CONCLUSION</b>Lovastatin can induce the arrest of cell cycle in G(0)/G(1) phase by affecting the expression of IkappaBalpha and cell-cycle regulating protein in MCF-7 cells.</p>
Subject(s)
Female , Humans , Antineoplastic Agents , Pharmacology , Breast Neoplasms , Metabolism , Pathology , Cell Cycle , Cell Cycle Proteins , Metabolism , Cell Line, Tumor , Cyclin-Dependent Kinase 4 , Cyclin-Dependent Kinase Inhibitor p16 , Metabolism , Cyclin-Dependent Kinases , Metabolism , I-kappa B Proteins , Metabolism , Lovastatin , Pharmacology , NF-KappaB Inhibitor alpha , NF-kappa B , Proto-Oncogene Proteins , Metabolism , Retinoblastoma Protein , MetabolismABSTRACT
Objective To investigate the expression of TGF-β and TGF-β receptor in human breast cancer cell Bcap-37 inhibited by soybean isoflavones. Methods mRNA and protein of TGF-β1、TGF-βRⅠ in Bcap-37 cells were examined with reverse transcription ploymerase chain reaction(RT-PCR) and immunohistochemistry after cells were treated with daidein or genistein for 1-4 d.The expression of TGF-β1 and TGF-β2 was determined with TGF-β resistance test. Results The TGF-β1, TGF-β2 and TGF-β recepor increased in Bcap-37 cells at a concentration of 3×10-5 mol/L of genistein. No changes was found when treated with daidzein. Conclusion Genistein may inhibit the proliferation of Bcap-37 cells and accompany with increasing expression of TGF-β1, TGF-β2 and TGF-β receptor.
ABSTRACT
Objective To investigate the expression of TGF-β and TGF-β receptor in human breast cancer cell Bcap-37 inhibited by soybean isoflavones. Methods mRNA and protein of TGF-β1、TGF-βRⅠ in Bcap-37 cells were examined with reverse transcription ploymerase chain reaction(RT-PCR) and immunohistochemistry after cells were treated with daidein or genistein for 1-4 d.The expression of TGF-β1 and TGF-β2 was determined with TGF-β resistance test. Results The TGF-β1, TGF-β2 and TGF-β recepor increased in Bcap-37 cells at a concentration of 3×10-5 mol/L of genistein. No changes was found when treated with daidzein. Conclusion Genistein may inhibit the proliferation of Bcap-37 cells and accompany with increasing expression of TGF-β1, TGF-β2 and TGF-β receptor.