RESUMO
Objective To eonstruct three eukaryotic expression vectors containing wilde-type hGHR gene and its mutants(hGHR-E42K and hGHR-H56R) related to congenital growth hormone insensitivity, then check their expression in CHO cells. Methods With the available PUC-hGHR vector, two mutate hGHR genes (hGHR-E42K and hGHR-H56R) were obtained through mutagenesis. Then three recombinants were cloned into eukaryotic expression vectors pcDNA3.1/zeo(+) with restriction enzymatic reactions.Then with Lipofectamine2000, we trans-fected expression vectors to CHO cells and screened the stably expressed CHO cells by Zeocin. RT-PCR and/or Western blotting were used to examine hGHR and STAT5-P. Results After sequencing, two mutations were introduced to hGHR, three eukaryotic expression vectors were identified. The transfected CHO cells expressed vd-hGHR or its mutants. Compared with hGH-wt, two mutate cells (E42K and H56R) had decreased phosphorylated STAT5 levels. Conclusion Three CHO cells which stably expresses wide type hGHR and its mutants were successfully established, E42K and H56R partly interfered the phosphorylation of STAT5.
RESUMO
Objective To compare the effects of upstream versus downstream tirofiban on tissure level perfu-sion and troponin Ⅰ release in high-risk non-ST-segment elevation acute coronary patients treated with percutaneous coronary intervention. Methods We randomized 138 non-ST-segment elevation acute coronary syndrome patients un-dergoing PCI to receive upstream(in the coronary care unit) and downstream(just prior to PCI) tirofiban. We com-pared the effects between the two drug regimens on tissue-level peffusion using Thrombolysis In Mycardial Infarction (TIMI) fram count,the TIMI myocardial perfusion grade(TMPG) and cardiac tropinin Ⅰ (cTn Ⅰ) release before and after PCI. Results There was no significant difference between two groups in TIMI 3 flow(P>0.05). High percent-age of TMPG 3 perfusion were observed in upstream tirofiban group than in downstream tirofiban group (P < 0.05). Post-procedual cTnI elevation was significantly less frequent among patients in the upstream group (P < 0.05). The cTnI level after PCI was significantly lower with upstream tirofiban compared with downstream tirofiban group (P<0.05). Conclusion Among high-risk non-ST-segment elevation ACS patients treated with an early invasive strategy, upstream tirofiban is associated with improved tissue-level perfusion and attenuated myocardial damage.
RESUMO
Objective To construct three eukaryotic expression vectors containing wilde-type hGHR gene and its mutants(hGHR-E42K and hGHR-H56R) related to congenital growth hormone insensitivity,then check their expression in CHO cells. Methods With the available PUC-hGHR vector,two mutate hGHR genes (hGHR-E42K and hGHR-H56R) were obtained through mutagenesis. Then three recombinants were cloned into eukaryotic expression vectors pcDNA3.1/zeo(+) with restriction enzymatic reactions. Then with Lipofectamine2000,we transfected expression vectors to CHO cells and screened the stably expressed CHO cells by Zeocin. RT-PCR and/or Western blotting were used to examine hGHR and STAT5-P.Results After sequencing,two mutations were introduced to hGHR,three eukaryotic expression vectors were identified. The transfected CHO cells expressed wt-hGHR or its mutants. Compared with hGH-wt,two mutate cells (E42K and H56R) had decreased phosphorylated STAT5levels. Conclusion Three CHO cells which stably expresses wide type hGHR and its mutants were successfully established,E42K and H56R partly interfered the phosphorylation of STAT5.