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Objective To construct an eukaryotic expression vector pcDNA-UCA1a(CUDR)and to observe its expression in bladder cancer UM-UC-2 cells, and to provide experimental basis for study on the relationship between UCA1a(CUDR)gene and bladder cancer.Methods Human total length of UCA1a(CUDR)gene was obtained from the 5′-RACE-Ready cDNA of bladder cancer BLZ-2 1 1 cells by PCR and was inserted into pcDNA3.1 (+)vector.pcDNA-UCA1a(CUDR)was identified by digestion with EcoRⅠ and BamHⅠ.The bladder cancer UM-UC-2 cells were transfected stably with the constructed eukaryotic expression vector pcDNA-UCA1a(CUDR). The expressions of UCA1a(CUDR)gene in the UM-UC-2 cells transfected with pcDNA-UCA1a(CUDR)and the UM-UC-2 cells transfected with pcDNA3.1(+)(control vector)were detected by RT-PCR.Results The inserted fragment with 2 200 bp was successfully amplified, which was in accordance with the expected results. The eukaryotic expression vector pcDNA-UCA1a(CUDR)was constructed successfully after identified by double enzyme digestion and sequencing.The RT-PCR results showed that the expression of UCA1a(CUDR)gene in the cells transfected with pcDNA-UCA1 a (CUDR ) was significantly increased compared with the cells transfected with pcDNA3.1 (+). Conclusion The eukaryotic expression vector pcDNA-UCA1a (CUDR ) is successfully constructed.The UCA1a(CUDR)gene highly expresses in the UM-UC-2 cells transfected with the expression vector.
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[ ABSTRACT] AIM:To explore the role of superoxide dismutase ( SOD) and malondialdehyde ( MDA) in chronic pancreatitis ( CP) induced by dibutyltin dichloride ( DBTC) combined with ethanol, and the mechanisms for prevention and treatment of pancreatic fibrosis by Chaihushugansan.METHODS: The KM mice were randomly divided into control group, CP group ( DBTC combined with ethanol) and Chaihushugansan group ( CP+Chaihushugansan) .Except for control group, the mice in other groups were intravenously injected in tail with DBTC (8 mg/kg) and drank 10% ethanol.The mice in Chaihushugansan group were administered intragastrically with Chaihushugansan (6 g· kg-1 · d-1 ) at the follow-ing experimenal period.Before modeling and 1 week, 2 weeks, 4 weeks and 8 weeks after modeling, the mice were anes-thetized and sacrificed.The activity of amylase and the content of hyaluronic acid in the serum were measured.The mor-phology and the degree of fibrosis in the pancreas were observed by HE staining.The activity of SOD and the level of MDA in the pancreas homogenate were analyzed.The protein of pancreas was extracted to detect the expression of type I collagen by Western blotting.RESULTS:DBTC combined with ethanol induced CP with increased serum amylase and hyaluronic acid levels, while the serum amylase and hyaluronic acid levels in Chaihushugansan group were significantly lowered ( P<0.05).In 1 week, 2 weeks, 4 weeks and 8 weeks, the pancreas were obviously injured and appeared different degrees of fibrosis.The content of MDA and the expression of type I collagen in the increased significantly, but the SOD was de-creased.In Chaihushugansan group, the pathological damage and the degree of fibrosis of the pancreas were improved.The level of MDA and type I collagen expression in the pancreas were significantly reduced, but the SOD was increased.CON-CLUSION:The oxidative stress may take part in the development of CP.Inhibition of oxidative stress in the pancreas is one of the mechanisms that Chaihushugansan attenuates the development of CP.
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Objective:To evaluate the effect of shikonin on the expression of RANTES and chemotactic activity of monocyte in endometriosis.Methods:Established SCID endometriosis models and cultured U937 cells were treated by a series of concentration of shikonin.RANTES transcriptive expression was determined by Real-time PCR,and RANTES secretion was determined by ELISA.Furthermore,Chemotaxis assay in vitro was conducted to elucidate the effect of shikonin on chemotaxis of U937 cells by RANTES.Results:Shikonin improved the RANTES transcription of human endometrium transplanted to SCID mouse(P
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Objective:To investigate the expression of chemokine receptor in eupotic and ectopic endometrial tissues of women with endometriosis, and in endometrium of women without endometriosis.Methods:Normal endometrium, eutopic endometrium and endometriotic tissues were obtained from patients with endometriosis at laparoscopy. Total RNA was then extracted using the TRIzol reagent. The expression of chemokine receptors in these tissues were analyzed by way of semi-quantitative reverse transcriptase-polymerase chain reaction.Results:Compared to normal endometrium, the eutopic endometrium expressed significantly more CCR6, CCR8, CCR9 and CX3CR1(P