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Objective To clone and express Dengue virus nonstructural protein 4A (NS4A) gene and express in eukaryotic cells.Then,to isolate and purify and isolate cellular proteins interacted with NS4A.Methods With specific primers,NS4A gene fragment tagged with FLAG and HA (FLAG-NS4A-HA) was amplified by PCR and cloned into an expression vector,pSG5 vector.Recombinant plasmid was transfected into A549 cells by LipofectAMINETM2000.Transient expression of FLAG-NS4A-HA was detected by Western blot.The NS4A interacting proteins were isolated and purified by tandem affinity purification (TAP) system using HA and FLAG antibodies,and then assayed by silver stained SDS-PAGE.Results Dengue virus NS4A gene tagged with FLAG and HA was successfully constructed into pSG5 vector and expressed in A.549 cells.Silver stained SDS-PAGE showed that the expressed NS4A and two potential interacting proteins that interact with NS4A were isolated after TAP purification and SDS-PAGE.Conclusion Cellular proteins that potentially interacted with Dengue virus NS4A were successfully purified and isolated,which provided a basis for further research.
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Aim The pharmacodynamics and mechanism of Kadsura heteroclita on the Alzheimers disease (AD) model mice were studied. Methods The Kunming mice were treated with D-galactose and AlCl 3(90 d)to make the AD animal model. The therapy groups were treated with HS 2 (po, 40 d) respectively since d51. The Morris water-maze test, the expressions of PS1, BACE, as well as the pathological observation of the hippocampus and cerebral cortex were carried out to evaluate the effect of HS 2 on the AD model mice. Resluts By oral administration with HS 2, the capacity of learning and memory of the AD model mice was improved. HS 2 can downregulate the expression of PS1, BACE and decrease the amount of senile plaque in their brains. Conclusion HS 2 can meliorate AD model mice's learning and memory deficiency as well as decrease the mRNA content of PS1, BACE and senile plaque in their brains.
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Aim This study was designed to investigate the inhibition of matrine on U251 glioma cell line and its mechanism. Methods MTT was used to measure the levels of the proliferation of U251 cultured with matrine in different concentrations.The effects of matrine on cell cycle of U251 were observed by FCM. The expression of proto oncogenes c myc was measured by RT PCR. Results The proliferation of U251 was obviously inhibited by matrine in a dose dependent manner. The inhibitory rate was (53 7?6 0)%,when cultured with matrine at 0 10 g?L -1 . The outcome of FCM showed that the proportion of G 0/G 1 phase cells were decreased. The proportion of S phase cells were reduced obviously,when cultured with matrine at 0 10 g?L -1 in 3 days.The outcome of RT PCR showed that the expression of proto onco gene C myc was notably decreased, when the dose of matrine was increased. Conclusion Matrine can inhibit the proliferation of U251 and inhibit the expression of proto onco gene C myc.
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AIM: This study was designed to investigate the inhibition of tanshinone ⅡA on C6 glioma cell line and its mechanism. METHODS: MTT was used to measure the levels of the proliferation of C6 cultured with tanshinone ⅡA at different concentrations. The effects of tanshinone ⅡA on cell cycle of C6 were observed by FCM. The change of DNA was observed by Sepharose electrophoresis. The expression of proto-oncogenes c-myc was measured by RT-PCR. RESULTS: The proliferation of C6 was obviously inhibited by tanshinone ⅡA in a dose-dependent manner. The outcome of FCM showed that the apoptotic cell rate was 7.7%, when cultured with tanshinone ⅡA at 1.0 mg/L for 3 days. The apoptotic cell rate was 21.6%, when cultured with tanshinone ⅡA at 2.0 mg/L in 3 days. CONCLUSION: Tanshinone ⅡA inhibits the proliferation of C6 cells, induces apoptosis and inhibits the expression of proto-oncogene c-myc.
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AIM: To investigate the inhibitory effect of TanshinoneⅡA on U251 glioma cell line and its mechanism. METHODS: MTT was used to measure the levels of the proliferation in U251 cultured with TanshinoneⅡA at different concentrations. The effects of TanshinoneⅡA on cell cycle of U251 were observed by FCM. The expression of proto-oncogene c-myc was measured by RT-PCR. RESULTS: The proliferation of U251 was obviously inhibited by TanshinoneⅡA in a dose dependent manner. The inhibitory rate came to the peak at (54 2?0 9)%, when cultured with TanshinoneⅡA at 0 10 g/L. The outcome of FCM showed that the proportion of G 0/G 1 phase cells was increased and the proportion of S phase cells was reduced obviously, when cultured with TanshinoneⅡA at 0 10 g/L for 3 days. The RT-PCR experiment showed that the expression of proto-oncogene c-myc was notably decreased, when the dose of TanshinoneⅡA was increased. CONCLUSION: TanshinoneⅡA inhibited the proliferation of U251 and the expression of proto-oncogene c-myc.