Objective:To explore the effects ofbufalin (BUF) combined with doxorubicin (DOX) on the proliferation and apoptosis in human lung cancer cell line A549 in vitro.Methods:Methyl thiazolyl tetrazolium (MTT) assay was used to measure the inhibitory effects of BUF,DOX and their combination on the growth ofA549 cells.Hoechst 33342 staining was used to observe the changes of nucleus.Flow cytometry was used to investigate the apoptosis and cell cycle distribution of A549 cells.Western blot was used to examine the expression of apoptotic protein.Results:BUF and DOX showed inhibitory effect on the A549 cells in a dose and time-dependent manner.Compared with BUF or DOX alone,combination of BUF (1,20,100 nmol/L) with DOX (1.0 μg/mL) could significantly increase the growth inhibition rate ofA549 cells at 24,36,72 h,respectively (all P＜0.05).BUF and DOX alone could induce apoptosis,and their combination could significantly increase the apoptosis ratio.In addition,BUF combined with DOX could block the cell stage of A549 cells,keep the cell stage stay in S stage and up-regulate the expression of caspase-3.Conclusion:BUF combined with DOX can significantly inhibit the proliferation ofA549 cells,which might be related to the induction of apoptosis,cell cycle S phase arrest and caspase-3 up-regulation.

Objective To construct an overexpression lentiviral vector of CD44 and obtain an androgen-dependent prostate cancer LNCaP cell line with CD44 overexpression.Methods CD44 gene sequence was obtained by PCR amplification.The synthesized oligonucleotides were cloned in the lentiviral vector LV5 after digested with NotI and NsiI enzyme.Recombinant plasmids were verified by enzyme digestion analysis and sequencing,and were transfected into 293T package cells.Supernatant was collected and concentrated to obtain lentivirus solution at a high titer.The titer of virus was identified by multiple proportion dilution methods.Then the recombinant viruses were transfected into LNCaP cells.Results Fluorescence intensity in infected LNCaP cells was increased,detected by a fluorescence microscope.And QRT PCR and Western blot results showed that the expression of CD44 was significantly increased(P＜0.05).Conclusions The overexpression lentiviral vector of CD44 is successfully constructed,and the LNCaP cell line with CD44 overexpression is obtained.

To investigate the clinical manifestations, molecular genetics and gonadal pathology characteristics of patients with disorders of sex development (DSD), and to summarize the clinical experience of identifying rare diseases from common symptoms.