Acupuncture Therapy ; Animals ; Female ; Humans ; Polycystic Ovary Syndrome ; therapy

Aim To ascertain whether defibrase has the protective effect against reperfusion injury after cerebral ischemia.Methods 70 renovascular hypertensive rats(RHR) were randomly divided into defibrase group, control group and sham-operated group.Reversible middle cerebral artery occlusion(MCAO) models were produced by the modified. Longa's method,and reperfusion was begun 2 hours after occlusion.Rats in the defibrase group were given defibrase 10 U?kg-1 body weight via femonal intraveneous injection, and in the control group with the same amount of saline. The brain pieces were processed by TTC and HE staining and the infarct size,brain microvessels damage and secondary bleeding were compared between the two groups. Results The volume of infarction in the defibrase group was obviously smaller than in the control group, the damage of brain microvessels was less severe, and the bleeding lesions under optical microscope were less than in the control group. Conclusion Defibrase has protective effect against reperfusion injury post cerebral ischemia.

OBJECTIVE To observe the effect of natrin from Naja naja atra(Chinese cobra)on intracellular free calcium overload,and to discuss the protective effect and the possible mechanism of natrin on myocardium calcium(Ca2+)and potassium(K+)ion channels in the primary cardiomyocytes of SD neonatal rats. METHODS The primary cardiomyocytes of SD neonatal rats were used,which were respectively pretreated with natrin 5,25 and 125 mg · L-1 for 24 h before injury was induced by H2O2 0.3 mmol · L- 1. The dynamic variation of intracellular calcium was monitored by laser confocal microscopy using Fluo-3 as Ca2+fluorescence probe. Additionally,the cardio myocytes of neonatal rats were pretreated for 24 h using different concentrations of natrin 5,25,125 mg · L-1 and verapamil 5 nmol · L-1,followed by exposure to H2O2 0.3 mmol · L-1 for 15 min. Then,the mRNA expressions of calcium channels subunits Cav1.2,Calm,RyR2 and potassium channel Kir6.2 were analyzed by FQ-PCR method. RESULTS Laser confocal microscopy revealed that H2O2 obviously caused calcium overload in cardiomyocytes, giving rise to 49.37% fluorescence increase in intracellular calcium compared with the control group(P<0.01). However,natrin 5,25 and 125 mg·L-1 resulted in 27.52%, 12.71% and 5.15% fluorescence increase in intracellular calcium,respectively,compared with the control group(P<0.01). Moreover, the PCR results showed that the mRNA expressions of Cav1.2, Calm and RyR2 in the myocardial cells treated with H2O2 were increased 2.78,2.26,and 5.34 times as compared with the control group,while Kir6.2 displayed a 1.79-fold expression level(P<0.01). By contrast, the combination of natrin and verapamil significantly decreased the mRNA expression of Cav1.2,Calm and RyR2,compared to the H2O2-treated group(P<0.01). Meanwhile,the expression of Kir6.2 was considerably higher than that of the H2O2-treated group(P<0.05). CONCLUSION Natrin can reduce the intracellular calcium overload of cardiomyocytes induced by H2O2 and shows a protective effect against oxidative damage for cardiomyocytes. The possible mechanism is that natrin can decrease the mRNA expression of Cav1.2,Calm,RyR2 and increase the expression of Kir6.2 of the H2O2-induced cardiomyocytes.

Objective To investigate the curriculum construction and evaluation of teaching effectiveness of simulation and comprehensive experiment for clinical nursing. Methods Based on the investigation of hospital, taking working progress and working task of nurses as orientation, we constructed the curriculum of simulation and comprehensive experiment for clinical nursing, and unfold in junior class (before clinical practice) for student nurses. The teaching effect was evaluated. Results Unfolding simulation and comprehensive experiment could effectively enhance the nursing students' ability to transform theoretical knowledge into clinical nursing practice, and was favorable to change the role of nurses and improve the satisfaction degree of clinical practice.Conclusions Unfolding simulation and comprehensive experiment for clinical nursing for student nurses before clinical practice is practicable and essential.

AIM:To study the mechanism of the unique export of one of human basic fibroblast growth factor (hbFGF) forms lacking the N-terminal nuclear localization signal (NLS),we high expressed and purified this hbFGF form in E.coli strain BL21(DE3).METHODS:The cDNA fragment of the hbFGF amplified by polymerase chain reaction (PCR) was cloned into the expression vector pET3c and expressed in BL21(DE3) by IPTG induction.The expressed hbFGF was purified by ionic exchange and heparin affinity chromatography from the supernatant of bacteria lysate.The mitogenic activity was measured by MTT.RESULTS:The expression level of hbFGF in E.coli was about 20% of the total cellular protein.The appreciable mitogenic activity of the purified hbFGF was comparable to that of hbFGF standard.CONCLUSION:The BL21(DE3)/ pET3c expression system could be used to high express hbFGF lacking NLS.The purified recombinant hbFGF was prepared and sufficient for further study.

AIM:To synthesize the human platelet factor-4(hPF4) gene with a convenient and effective approach, and high express the hPF4 gene in E. coli BL21 (DE3). METHODS: According to the primary structure of hPF4, the nucleotide sequence was synthesized using touch-down PCR method. The resultant gene fragment containing EcoR Ⅰ and Xho Ⅰ overhangs at 5' and 3' ends was cloned into the expression vector pGEX-4T-1 to construct the recombinant plasmid pGEX-4T-1-hPF4,which was then transformed into the E. coli strain BL21 (DE3). RESULTS: hPF4 gene was successfully synthesized by touchdown PCR method. A fusion protein composed of glutathione S-transferase (GST) and the recombinant hPF4 was expressed in BL21(DE3) by IPTG induction. The expression level of the fusion protein in E. coli was about 30% of the total cellular protein. CONCLUSION: Touch-down PCR may provide a convenient and effective approach to obtain other target genes. The expressed fusion protein forms the inclusion bodies, providing sufficient material for further purification and biological activities process.

In order to decrease the potential side-effects of human basic fibroblast growth factor (hbFGF) caused by its broadspectrum mitogenic activity, a single residue of hbFGF, the residue serine 108, was replaced with neutral alanine residue to construct a mutant of hbFGF (mhbFGF) with reduced mitogenic activity. The mutant was overexpressed in Escherichia coli BL21(DE3) by IPTG induction. The expression level of mhbFGF was about 30% of the total cellular protein. The expressed mhbFGF was purified by ionic exchange and heparin affinity chromatography from the supernatant of bacteria lysate. Measured by MTT method, the effect of mhbFGF on Balb/c 3T3 cell proliferation was much lower than that of wild-type hbFGF. The purified recombinant mhbFGF was prepared and sufficient for the following pharmacological and safety studies.

This paper is to report the study of the pharmacokinetics of a fusion protein TAT-haFGF(14-154) for human acidic fibroblast growth factor and transcriptional activator protein in rat plasma, and the investigation of their penetration across blood-brain barrier in mice and rats, in order to provide a basis for clinical development and treatment of Alzheimer's disease. Enzyme-linked immunosorbent assay (ELISA) was used to determine concentration of TAT-haFGF(14-154) in rat plasma and in mouse brain homogenate; and immunohistochemistry was used to analyze the distribution in brain. The concentration-time curve fitted two-compartment open model which was linear kinetics elimination after a single intravenous injection of TAT-haFGF(14-154) in rat at the dose of 300 microg x kg(-1). The half life time was 0.049 +/- 0.03 h for distribution phase and 0.55 +/- 0.05 h for elimination phase, and the weight was 1/C2. The result showed that TAT-haFGF(14-154) could be detected in the brain by ELISA and immunohistochemistry, the elimination of TAT-haFGF(14-154) in rat was swift, and TAT-haFGF(14-154) could penetrate across the blood-brain barrier, distribute in pallium and hippocampus and locate in the nucleus.

AIM:The present study was designed to establish H2O2-induced NRK52E cell apoptotic model and to investigate the effects and the mechanism of nmhaFGF on the NRK52E cell apoptosis induced by H2O2. METHODS: In vitro experiment, a apoptotic model of normal rat kidney epithelium (NRK52E) was made by MTT method, Hoechst33342 dyeing and flow cytosorting (FCR). NRK52E cells were cultured with different concentrations of nmhaFGF and haFGF for 24 h before H2O2 was added. The apoptotic rate was detected with FCR method. RESULTS: H2O2 at concentration of 0.4 mmol/L, the optimal condition to establish the apoptotic model, was used to treat NRK52E cells for 18h. Different doses of nmhaFGF (0.01, 0.03, 0.10, 0.30, 1.00 mg/L) reduced the apoptotic rates with the dose rising. However, the decreasing tends of apoptotic rates with dose increasing for haFGF was not so obvious. CONCLUSION: nmhaFGF protects the NRK52E cells against apoptosis. The mechanism might be connected with its non-mitogenic property.

AIM: To observe the effects of the human acidic fibroblast growth factor mutant (mhaFGF), lacking 27 amino acids at N-terminal, on the proliferation and signal transduction of the hepatocarcinoma cells. METHODS: The hepatocarcinoma cells were treated with human acidic fibroblast growth factor (haFGF) and mhaFGF, respectively. The expression levels of the signal proteins, Grb2 and Erk1/2, in the hepatocarcinoma cells were detected by semi-quantitative Western-blotting after treated for 15 min. The mitogenic activity of both haFGF and mhaFGF was detected by MTT method and the cell cycle was analysed by flow cytometer (FCM) after treated for 48 h. RESULTS: The mitogenic activity and the ratio of G 1 and S phase cells in mhaFGF-treated cells were markedly lower than that of the haFGF, and close to that of the control group. The expression level of both Grb2 and Erk1/2 in the mhaFGF-treated cells were lower than those in the haFGF- treated cells. CONCLUSION: The decrease in the mitogenic activity of mhaFGF is probably associated to its down-regulating the expression of the signal molecular, MAPK-ERK1/2 and Grb2.