Summary: To clone Uroplakin Ⅱ gene from Chinese transitional cell carcinoma (TCC) of bladder and construct its eukaryotic expression vector, the molecular cloning method was used to extract total RNA from a GⅢ/ T 3N 0M 0 tissue sample of the bladder TCC patients. The primers were designed by Primer 5.0 software. Full length cDNA of Uroplakin Ⅱ gene was amplified by reverse transcription polymerase chain reaction (RT-PCR), assayed by nucleic acid sequencing and then inserted between XbaⅠ and HindⅢ restrictive sites of eukaryotic expression vector pcDNA3.0. The recombinant was assayed by restricted enzyme digestion. Under the induction of Lipofectamine 2000, the recombinant was transfected into Uroplakin Ⅱ negative bladder cancer cell line EJ. Cellular expression levels of Uroplakin Ⅱ were detected by RT-PCR. The nucleic acid sequencing results indicated that Chinese Uroplakin Ⅱ cDNA (555 bp) was successfully cloned. The BLAST analysis demonstrated that the cloned sequence is 100 % homologous with sequences reported overseas. The GenBank accession number AY455312 was also registered. The results of restricted enzyme digestion indicated that eukaryotic vector pcDNA-UPⅡ for Uroplakin Ⅱ was successfully constructed. After being transferred with pcDNA-UPⅡ for 72 h, cellular Uroplakin Ⅱ mRNA levels were significantly improved (P<0.01). It is concluded that human Uroplakin Ⅱ gene was successfully cloned from Chinese TCC tissues, which provided a basis for further exploration of the roles of Uroplakin Ⅱ gene in TCC biological behaviors and potential strategies for targeted biological therapy of TCC.
Objective: To explore the growth inhibition effects and apoptosis inducing mechanisms of curcumin on human ovarian cancer cell line A2780. Methods: After treatment with 10-50 μmol/L curcumin for 6-24 h, the growth activity of A2780 cancer cells were studied by [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide （MTT） colorimetry. Cellular apoptosis was inspected by flow cytometery and acridine orange-ethidium bromide fluorescent staining methods. The fragmentation of cellular chromosome DNA was detected by DNA ladder, the ultrastructural change was observed under a transmission electron microscope, and the protein levels of nuclear factor-κappa B （NF-κB, P65） and cysteinyl aspartate specific protease-3 （Caspase-3） in ovarian cancer cells were measured by immunohistochemistry. Results: After treatment with various concentrations of curcumin, the growth inhibition rates of cancer cells reached 62.05%-89.24%, with sub-G1 peaks appearing on histogram. Part of the cancer cells showed characteristic morphological changes of apoptosis under fluorescence and electron microscopes, and the rate of apoptosis was 21.5%-33.5%. The protein expression of NF-κB was decreased, while that of Caspase-3 was increased in a time-dependent manner. Conclusion: Curcumin could significantly inhibit the growth of human ovarian cancer cells; inducing apoptosis through up-regulating Caspase-3 and down-regulating gene expression of NF-κB is probably one of its molecular mechanisms.
In order to study bladder intravesical instillation methods in pure line LEW rats and nude mice, female LEW rats and nude mice aged 2 to 4 weeks were sacrificed. Their urethra and bladder were observed under anatomical microscopy. A trochar was prepared according to the outline and an- gle of the urethra. Ink was poured into female rats and nude mice bladder though urethra. Filling and staining of bladder were observed and evaluated under anatomical microscopy. Status and urethral injury of rats and mice were observed. The results showed that urethra anatomic structure of rats and nude mice was different from that of human urethra. When bladder was filled with ink and became blue, liquid was not seen to leak out. The success rate of intubation was high (100%). Living activi- ties of animals weren’t influenced by intravesical instillation. It was concluded that bladder irrigation might be a kind of valid and utilizable method in pure line rat and nude mouse empirical study. The model may be a more effective tool for study of bladder tumor.
