The method of multiplex PCR was set up to identify two or three transgenes in one reaction such as uidA and bar; uidA and IDx5 or uidA, bar and 1Dx5 genes. Three sets of primer pairs which was specific to each of these three genes respectively were designed and synthesized. Recombinant plasmids pAHC25 and p1Dx5 harboring uidA + bar and 1Dx5 gene separately were used as template DNA in the process of optimizing an multiplex PCR reaction. The optimal annealing temperature for uidA and bar MPCR is range from 57. 1℃-62. 3℃ , for uidA and 1Dx5 is range from 60℃ to 60. 6℃ , and for uidA、bar and 1Dx5 range from 57. 0℃-58. 4℃. The amount of template for MPCR is twice as much as that for simplex PCR, while the concentration of primers is the same with simplex one. Less than 50bp MPCR products can be separated clearly by 10% non-denaturalized polyacrylamid gel electrophoresis. Fourteen transgenic wheat lines were tested by multiplex and simplex PCR respectively, which shows the same results and hence presents that MPCR is the reliable, rapid and high-effective approach to detect foreign genes from transgenic plant.

Objective To explore the mechanism of the Jiedu Huayu Ⅱ Decoction in actue hepatic failure.MethodsThe acute hepatic failure rat models were induced with thioacetamide (TAA).84 SPF Wistar rats were divided into 7 groups randomly:blank group,model group,low dose group,middle dose group,high dose group,lactulose group,and the Angong Niuhuang Pill group.The drugs were administrated by gavage 3 d before model-making,5.5 d in total.The expression of Bcl-2,cytochrome C in mitochondrial and cytoplasm were measured with Western Blot.ResultsThe expression of Bcl-2 reduced in mitochondrial,the expression of cytochrome C increased in cytoplasm and reduce in mitochondrial in model group compare with that of blank group.Jiedu Huayu Ⅱ Decoction was able to enhance the expression of Bcl-2 and restrain cytochrome C release from mitochondrial to cytoplasm.Moreover,it shows dose-effect relation.ConclusionThe protection of Jiedu Huayu Ⅱ Decoction for hepatocyte in acute hepatic failure rats maybe relate to the increase of Bcl-2 in mitochondrial,blocking the mitochondrial permeability transition pore to arrest cytochrome C releasing.

Objective To investigate the effects of bone marrow mesenchymal stromal cells (BMSCs) transplantation after mannitol pretreatment on behavioral performance and synaptophysin expression in the CA3 region in hippocampus of vascular dementia (VD) rats.Methods The BMSCs of rats were isolated and purified by the whole bone marrow adherence method.The rats were subjected for permanent ligation of bilateral common carotid arteries at an interval of 3 days for each carotid artery.At the same time,Sham group was set in parallel.Four weeks after modeling,the VD rats were divided randomly into five groups:(1) VD control group; (2) culture media group; (3) mannitol group; (4) BMSCs group;(5) mannitol with BMSCs group.Morris water maze performance and synaptophysin expression in the CA3 region in hippocampus were observed at 4 weeks after transplantation.Results The morris water maze performance significantly improved in mannitol with BMSCs group when compared with BMSCs group,VD control group,culture media group,mannitol group.Moreover,the escape latency of fifth day decreased significantly ((9.3 ±2.9),(14.1 ±3.5),(23.5 ±4.4),(22.8 ±4.4),(23.2 ±2.8) s,F =43.900,P =0.000)),and the platform quadrant residence time increased significantly ((40.8 ± 6.3),(34.9 ±5.8),(26.4±4.8),(27.4 ±7.0),(28.5 ±6.2) s,F=13.000,P=0.000)).The synaptophysin expressions of the hippocampal CA3 region were significantly increased in the mannitol with BMSCs group (39 624 ± 7798) when compared with BMSCs group,VD control group,culture media group,mannitol group (27060 ±4668,18 294 ±6446,19 956 ±4244,18 946 ±4953,F =39.206,P =0.000).Conclusions Intravenous BMSCs transplantation after mannitol pretreatment improves the behavioral performance of VD rats and facilitates the synaptophysin expression of hippocampal CA3 region in VD rats than BMSCs transplantation alone.Mannitol pretreatment can amplify the therapeutic effect of intravenous BMSCs transplantation in VD rats.

Through the comparative analysis of undergraduate specialty settings of medical colleges and universities in minority areas in the different periods,this paper reveals the characteristics of undergraduate specialty settings.It is necessary to follow the demand for social and economic development,and adhere to the medical and national characteristics,promoting the durable development of undergraduate specialty settings of medical colleges and universities in minority areas.

Protective effect and mechanism of electroacupuncture (EA) on acute reperfusion ventricular arrhthmia was investigated. Ventricular arrhythmia was induced by occlusion of the proximal left anterior descend (LAD) branch of coronary artery for 5 min and followed with 15 min reperfusion. EA on acupoint "Neiguan", "Jianshi" was performed at 30 min before ligation and continued another 5 min during ischemia. Isoprenaline (20, 30 and 50 microg/kg) or atropine (1 mg/ kg) was intravenously injected at 5 min before ischemia. The results showed that EA significantly decreased the incidence of ischemia/reperfusion (I/R) induced ventricular tachycardia (VT), ventricular fibrillation (VF) and mortality as compared to I/R group. Atropine partially suppressed the EA's effect of antiarrhythmia; Isoprenaline increased the incidence and severity of reperfusion arrhythmia, which was inhibited by EA, but this inhibition of EA was blocked with increasing dose of isoprenaline. The results indicated that EA treatment could prevent the occurrence of reperfusion ventricular arrhythmia in rats with myocardial ischemia, and its mechanism might be related to the regulation of EA on the beta-adrenoceptors and M-cholinergic receptor activation in myocardium.

OBJECTIVETo observe the clinical efficacy of acupuncture at Hegu (LI 4) on central facial nerve paralysis after ischemic stroke, and explore dose-effect relationship among different stimulation intensities of acupuncture at Hegu (LI 4) as well as its optimal treatment plan.

METHODSAccording to different acupuncture stimulation intensities which were based on treatment time and needle insertion direction, fifty patients were randomly divided into a Hegu 1 group, a Hegu 2 group, a Hegu 3 group, a Hegu 4 group and a control group, ten cases in each one. Different stimulation intensities of acupuncture at Hegu (LI 4) combined with facial paralysis acupoints, including Yingxiang (LI 20), Dicang (ST 4), Jiache (ST 6) and Quanliao (SI 18), were applied in Hegu 1 to 4 groups; meanwhile acupuncture at stroke acupoints, including Neiguan (PC 6), Shuigou (GV 26) and Sanyinjiao (SP 6), and medication treatment were adopted. Except acupuncture at Hegu (LI 4), the treatment of the control group was identical as Hegu groups. The treatment duration lasted for 14 days. The House-Brackmann facial never grading systems (H-B), Toronto facial grading system (TFGS), degrees of facial never paralysis (DFNP), facial disability index (FDI) and clinical efficacy were compared among groups.

RESULTS(1) Compared before the treatment, H-B, TFGS, DFNP and physical function score in FDI were all improved significantly in the Hegu 1 to 4 groups (all P < 0.05), but social function score in FDI was not obviously improved (all P > 0.05); all the scores in the control group were not evidently changed (all P > 0.05). (2) Compared with the control group, differences of H-B before and after treatment in the Hegu 1 to 4 groups, differences of TFGS in the Hegu 2 group and differences of DFNP in the Hegu 1 and Hegu 2 group were significantly improved (all P < 0.05). The differences of any scale among Hegu 1 to 4 groups were not significant (all P > 0.05), in which the most evident change was found in Hegu 2 group. (3) The total effective rate was 90.0% (9/10), 100.0% (10/10), 90.0% (9/10) and 80.0% (8/10) in Hegu 1 to 4 groups, which were significantly higher than 60.0% (6/10) in the control group (all P < 0.05).

CONCLUSIONAcupuncture at Hegu (LI 4) has affirmative clinical efficacy on central facial nerve paralysis after ischemic stroke, in which oblique insertion along the opposite direction of meridian for 5 s of twirling manipulation has the best clinical effect.

Acupuncture Points ; Acupuncture Therapy ; Adult ; Aged ; Facial Paralysis ; etiology ; therapy ; Female ; Humans ; Male ; Middle Aged ; Stroke ; complications

OBJECTIVETo study the effect of ephedrine on neural plasticity after hypoxic-ischemic brain damage (HIBD) in neonatal rats.

METHODSixty SD rats, aged 7 days, were made as HIBD model, which were randomly divided into following 4 groups, an ephedrine group, a D-amphetamine (D-AMPH) group, a cytodine triphosphate-2Na (CTP) group and a ganglioside (GMI) group. Changes in the expression of growth-associated protein-43 (GAP-43) and synaptophysin (SYP) in the hippocampal area CA3 were detected with immunohistochemical method. Four weeks after the operation, learning and memory test in Morris water maze was performed for 5 days.

RESULT(1) The GAP-43 and SYP expression levels in hippocampal area CA3 in the ephedrine group were higher than those in the spontaneous recovery group (P < 0.05), with no significantly different from those the CTP group and the D-AMPH group. (2) The average escape latency in the ephedrine group, the D-AMPH group and the CTP group were significantly shorter than that in the spontaneous recovery group (P < 0.05), and the frequency passing the original platform in the 3 treatment groups were significantly more than those in the spontaneous recovery group (P < 0.01). The escape latency was longer and the frequency passing the original platform was less in the ephedrine group than that in the GM1 group, with no significant differences as compared with the CTP group and the D-AMPH group.

CONCLUSIONEphedrine can enhance spatial orientation and learning and memory abilities of HIBD rats in later life. This protective effect is associated with decrease of neuronal loss after HIBD, and promotion of the expression of GAP43 and SYP. Ephedrine can exert the same protection against HIBD as D-AMPH and CTP do, but the amelioration of spatial orientation and learning and memory abilities by ephedrine in later life in rats after HIBD is slightly weaker than that by GM1, which is possibly related with the dose of ephedrine.

Animals ; Animals, Newborn ; Ephedra sinica ; chemistry ; Ephedrine ; isolation & purification ; pharmacology ; GAP-43 Protein ; metabolism ; Hippocampus ; drug effects ; metabolism ; Hypoxia-Ischemia, Brain ; metabolism ; physiopathology ; Maze Learning ; drug effects ; Neuronal Plasticity ; drug effects ; Neuroprotective Agents ; pharmacology ; Plants, Medicinal ; chemistry ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Synaptophysin ; metabolism

BACKGROUND:Our previous studies have found that polygonatum sibiricum polysaccharide (PSP) promotes osteogenic differentiation of bone marrow mesenchymal stem cel s (BMSCs) by Wnt/β-catenin signaling pathway, but the molecular mechanism is unclear.OBJECTIVE:To investigate the effect of PSP promoting the osteogenic differentiation via Wnt signaling pathways in BMSCs after LRP5 silencing. METHODS:LRP5 interference vectors were constructed and then transfected into C57BL/6 mouse BMSCs cultured in vitro. The transfection efficiency of cel s was calculated under fluorescence inverted microscope and the expression of LRP5 protein was detected by western blot assay. The osteogenic potential of BMSCs after LRP5-siRNA transfection was analyzed by alkaline phosphatase staining, alizarin red staining and western blot assay. Effect of PSP on the osteogenic differentiation of LIRP5-silenced mouse BMSCs was detected by real-time PCR and dual luciferase assay. RESULTS AND CONCLUSION:Compared with the control group, the mineralization ability, the mRNA expressions of Runx2 and Osterix, and the protein expression of LRP5 were significantly decreased in the LRP5-siRNA group (P<0.05). PSP could promote LRP5-siRNA transfected mouse BMSCs differentiating into osteoblasts and significantly upregulated the expressions ofβ-catenin and Osterixin, and also induced the high expression of luciferase reporter gene (TOPFlash) containing wild type TCF binding sites (P<0.05). To conclude, LRP5 plays an important role in the process of mouse BMSCs differentiating into osteoblasts. PSP can promote the osteogenic differentiation of mouse BMSCs by activating the Wnt/β-catenin signaling pathway independent on LRP5.

OBJECTIVESTo investigate the changes in intrahepatic portal systemic shunt flow (IHSF) and their relationship with microspheres induced acute portal hypertension.

METHODSFollowing acute intrahepatic presinusoidal obstruction by intraportal injection of 15 microm diameter microspheres in male Wistar rats, functional hepatic blood flow (FHBF) and IHSF were determined by hepatic sorbitol uptake methods. The percentage of large shunts of diameter > 15 microm were estimated by intraportal injection of 51Cr labeled 15 mum diameter microspheres.

RESULTSIn normal control rats, hepatic sorbitol uptake was 97.9%+/-0.5% and IHSF was negligible, with FHBF equaling total hepatic blood flow [(2.52 +/- 0.23) ml/min x 100 g body weight-1]. Microsphere injection decreased sorbitol uptake to 12.8% +/- 4.3% and further to 4.1% +/- 0.7% when followed by hepatic arterial ligation. In the latter two groups, IHSF (1.46 +/- 0.15 and 1.16 +/- 0.19 ml/min x 100 g body weight-1, respectively) was not significantly different from portal venous flow [(1.36 +/- 0.20) and (1.20 +/- 0.20) ml/min x 100 g body weight-1, respectively; t = 2.013 and t = 2.116]. Portal venous flow remained at 70% of basal values and portal venous pressure only increased by 50% from baseline. 51Cr labeled microsphere shunt fraction through large shunts (> 15 microm) was less than 1.0%.

CONCLUSIONIntrahepatic portasystemic shunts in the normal rat liver predominantly have diameters less than 15 microm and, when activated by intraportal injection of microspheres, they divert up to 70% of portal venous blood flow away from hepatic sinusoids and thereby they reduce acute increases in portal venous pressure.

Animals ; Hepatic Artery ; physiopathology ; Hepatic Veins ; physiopathology ; Hypertension, Portal ; chemically induced ; physiopathology ; Microspheres ; Portal Vein ; physiopathology ; Random Allocation ; Rats ; Rats, Wistar

OBJECTIVE This study aimed to optimize polysaccharides extraction from Urena lobata L.and investigate its antioxidant activity.METHODS The mathematical model was established by re-sponse surface method (RSM) based on the results of single factor experiments, using polysaccha-rides extraction rate as response value,and using the ratio of water to material,cellulase concentra-tion,extraction temperature and time as experimental factors,which was used to screen optimum poly-saccharide extraction conditions from Urena lobata L.. Antioxidant activity of polysaccharides was stud-ied by DPPH and ·OH free radical elimination method. RESULTS The optimum conditions obtained by RSM were as follows:the cellulase level was 10.8 g·L-1,extraction time duration was 72 min,the ra-tio of water to feedstock was 7 mL·g-1,extraction temperature was 43℃,the pH value was 5.0.Under the optimal conditions, there was a difference of less than 5％ between predicted extraction rate 13.37％ and experimental extraction rate 13.32％. The polysaccharide yield was most significantly af-fected by cellulase concentration,followed by extraction time,water to material ratio and extraction tem-perature.IC50of DPPH and·OH were 1.082 g·L-1and 3.202 mg·L-1,respectively.Antioxidant activity of sample polysaccharides was weaker than those of vitamin C. CONCLUSION The polysaccharide extraction process from Urena lobata L. by cellulase enzymolysis approach was obtained, which was convenient and feasible,and extracted polysaccharides had good free radical scavenging activity.