Transfection and expression of Smad7 inhibits transforming growth factor-?_1 effects on renal tubular cells / 第三军医大学学报

Objective To explore the effect of Smad7 gene transfer on tubular cell cycle arrest, tubular apoptosis and fibronectin (FN) synthesis by transforming growth factor ? 1(TGF ? 1) induced. Methods Mouse Smad7 gene was transfected into renal tubular cells in primary cell culture by using Tfx 50 cationic liposome. Tubular cell proliferation was measured by MTT and cell cycle was observed by flow cytometry. The level of FN secretion in the supernatant was determined by ELISA. Results At 48 h after administration of 10 ng/ml TGF ? 1 to renal tubular cells, cell proliferation declined, G 0/G 1 arrested in cell cycle, and cell FN secretion increased significantly. These abnormalities were attenuated by liposome mediated Smad7 gene transfection. Conclusion Transfection and expression of Smad7 can markedly inhibit TGF ? 1 responses in renal tubular cells, which is helpful for further studies of Smad7 gene therapy in vivo .

An experimental study on the establishment of bladder cancer MDR cell line by gene transfection / 中华泌尿外科杂志

Objective To establish a bladder cancer MDR cell line by gene transfection and to lay a foundation for the research of occurrence and reversion mechanisms of MDR. Methods The invasive bladder cancer T24 cells were transfected with mdr1 cDNA by cationic liposome (DOTAP) introduced gene transfection.The adriamycin (ADM) resistant cells were screened as MDR cells,named TADM. The MDR phenotype of TADM was identified by MTT, immunohistochemistry,immunofluorescence,flow cytometry,PCR and RT PCR. Results The relative resistant index of TADM was 41.6,mdr1 cDNA being integrated into the genome of T24.As a result,the expression of P gp and mdr1 mRNA in TADM increased. Conclusions The integration of mdr1 cDNA into the T24 cells greatly increases the expression of P gp.TADM cells manifests excellent MDR phenotype.The establishment of MDR cell lines by gene tranfection has the benefits of less time consuming,stronger drug resistivity and more stable.

Reversal effects of carvedilol on multidrug resistance in bladder cell lines / 中国癌症杂志

Purpose:To study the reversal effects on multidrug resistance in multidrug-resistent bladder cell lines BIU-87/ADM. Methods:The degree of drug-resistence was detected by MTTmethod; the concentration of ADM in cells BIU-87 and BIU-87/ADM was detected after the cells were treated by carvedilol and ADM. Results:Pretreatment by carvedilol increased the concentration of ADM in BIU-87/ADM cells, ,and the concentration was much higher than that in those cells treated by ADM only. Conclusions:Carvedilol can enhance the cytotoxic effect of ADM,and can reverse the multidrug resistance in bladder cancer lines BIU-87/ADM.

Antigenic epitopes analyses and expression of P-glycoprotein / 中国癌症杂志

Purpose:To study antigenic epitopes and expression of P glycoprotein.Methods:The secondary structure and surface properties of P glycoprotein was studied by many methods , designed a pair of primers to amplify DNA with PCR. The product was inserted into a pGEM T vector, sequenced and cloned into pGEX 2T vector, established a high expression recombinant vector named pGEX Pgp,which was transferred into DH5? and expressed , identified and purified. Results:Antigenic epitopes in extracellular fragment of P glycoprotein were identified in amino acid residues 82 115(I),741 760(IV) and 800 816(V). SDS PAGE analysis indicated that the expressed protein was about 30?10 3 (30 kD).Conclusions:High level expression of the target gene fragment was established for preparation of its antibody and biospanning its specific peptide using random phage library , which became the basis for the targeting treatment of MDR. [

Implication of intracellular rhodamine-123 distribution model in bladder cancer resistant cells differing from sensitive cells / 中华泌尿外科杂志

Objective To observe the change of intracellular rhodamine 123 distribution model in bladder cancer sensitive cells(T24) and resistant cells(TADM),and to explore the cause of the change and its relation with multidrug resistance(MDR). Methods T24 and TAMD cells were cultured together with rhodamine 123 for a certain period.The intracellular distribution and intensity of fluorescence were detected by interactive laser cytometer(ILC). Results In T24 cells,rhodamine 123 was located mainly in the perinuclear membrane area,and in TADM cells,rhodamine 123 was concentrated mainly at the two poles of nucleus.After the extracellular rhodamine 123 was washed,in T24 cells ,the intracellular fluorescent intensity decayed slowly.It took more than 40 minutes for T24 cells to eliminate intracellular rhodamine 123 completely.By contrast,in TADM cells, the intracellular fluorescent intensity decayed rapidly.It took only 15 minutes for TADM cells to eliminate intracellular rhodamine 123 completely. Conclusions (1)Intracellular rhodamine 123 was rapidly pumped out,which is the main cause of MDR in TADM;(2)The intracellular rhodamine 123 distribution model in TADM differ from that in T24,which may be another cause of its MDR phenotype.

Expression of TopoⅡ gene in human bladder cancer multidrug-resistant cells / 中国癌症杂志

Purpose:To study the expression of TopoⅡ gene in multidrug resistant cells of human bladder cancer. Methods:The degree of drug resistance was detected by MTT method ;the expression of TopoⅡ gene in cell lines BIU 87/ADM and BIU 87 was detected with reverse transcriptase polymerase chain reaction. Results:The cell lines BIU 87/ADM were 56.4 times more resistant to ADM than the cell lines BIU 87;the expression of TopoⅡ gene was poorly positive in BIU 87/ADM but strongly positive in BIU 87 cells. Conclusions: The decreased expression of TopoⅡ gene in BIU 87/ADM cells might contribute to the development of multidrug resistance of human bladder cancer.

PAI-1,TIMP-1 gene expression in renal tubulointerstitial fibrosis of ureteral obstruction and the interfering effects of HGF treatment / 中国病理生理杂志

AIM: The aim of this study was to determine the relationship between PAI-1,TIMP-1 gene expression and renal tubulointerstitial fibrosis(TIF) of ureteral obstruction ,and the interfering effects of hepatocyte growth factor (HGF) treatment. METHODS: Sixty rats were divided into normal control, sham operation,unilateral ureteral obstruction(UUO),and rhHGF treated groups (received 0 5 mg?kg -1 ?d -1 HGF for twenty-one days), and were sacrificed at postoperative day 3, 7, 14, 21. The levels of PAI-1, TIMP-1 mRNA were measured by RT-PCR. MMP2 and MMP9 activities were detected by substrate zymography, and renal fibrosis was assessed by measuring tissue hydroxyproline. RESULTS: Compared to controls, expression levels of PAI-1,TIMP-1 mRNA were significantly increased in UUO rats, and this was accompanied by decreased activities of MMP2 and MMP9 and increase in tissue hydroxyproline content. HGF treatment significantly decreased expressions of PAI-1, TIMP-1 mRNA, increased MMP2 and MMP9 activities,and decreased tissue hydroxyproline content in the obstructive kidney. CONCLUSIONS: These results indicate that the increases in PAI-1, TIMP-1 mRNA expression may be the major cause of sustained decreased matrix degradation during the development of tubulointerstitial fibrosis after unilateral ureteral obstruction. rhHGF efficiently ameliorates renal tubulointerstitial injury by the reduction of PAI-1,TIMP-1 mRNA expression, and increasing MMP2, MMP9 activities. [

Localization of Smad 2, 3, 6, 7 and their changes of expression in experimental interstitial fibrosis model in rats / 中华肾脏病杂志

Objective To investigate the localization of Smad protein 2, 3, 6, 7 and their changes of expression in experimental interstitial fibrosis model in rats. Methods Thirty-six rats were divided into normal control, sham operation, and unilateral ureteral obstruction(UUO) groups, and were sacrificed at postoperative day 3, 7, 14, 21. The level of TGF-?1 mRNA was examined by RT-PCR. The sites and levels of expression of Smad protein 2, 3, 6, 7 were examined by immunohistochemistry staining and Western blot . Renal fibrosis was assessed by measuring tissue hydroxyproline. Results Compared to sham operation group, TGF-?1 mRNA was significantly increased in UUO rats, and this trend was positively correlated to increased hydroxyproline content. Immunohistochemistry staining studies indicated that Smad protein 2, 3 mainly expressed in renal tubular cells, rarely in glomeruli, and Smad protein 6, 7 were presented in both the glomeruli and proximal renal tubular cells. Expression of the Smad protein 2, 3 were significantly increased from day 3 to 21 after UUO, while the Smad protein 6, 7 were significantly reduced in the obstructive kidney. Conclusions TGF-?1/Smad signaling is involved in the progression of renal tubulointerstitial fibrosis. The absence of up-regulation of these anti-Smads proteins may be the major cause of the interstitial fibrosis in this model.

Expression of fused P-glycoprotein in E. coli DH5? / 第三军医大学学报

Objective To study the expression of the extracellular fragment of P glycoprotein in the bacteria. Methods According to the P glycoprotein antigen analyses and mdr1 gene structure, a couple of primers were designed to amplify the extracellular DNA fragment of the protein with PCR. The product was inserted into pGEM T vector followed by sequencing. The sequencing verified vectors were cloned into pGEX 2T to get a highly expression recombinant vector pGEX Pgp. The recombinant was transferred into E. coli DH5?, and its expression, identification and purification were studied. Results The highest level of recombinant expression was achieved at 4 h after IPTG induction. SDS PAGE analysis indicated that the expressed protein was about 30?10 3. Conclusion The target gene fragment expression at high level is established for the preparation of its antibody and biospanning of its specific peptide by using random phage library, founding a basis for the targeting treatment of multidrug resistance.