Objective To obtain recombinant human granulysin using prokaryotic expression system. Methods Total RNA was extracted from cultured PBMC. Granulysin gene segments were obtained with granulysin-specific primers by RT-PCR and then inserted into pET32a(+) plasmid. After identification by DNA sequence, pET-GN-LY9K and pET-GNLY15K were transferred to E. Coli Rosetta (DE3). The fusion protein was identified by SDS-PAGE and Western blot. The bioactivity of granulysin fusion protein was measured by MTT assay. Results The prokaryotic expression vectors pET-GNLY9K and pET-GNLY15K were successfully constructed. The corresponding protein was highly expressed in E. Coli. Recombinant protein was specifically bound by anti-granulysin antibody. GNLY9K fusion protein significantly inhibited the growth of A549 cells in a dose-dependent manner, while GN-LY15K had little effect on the growth of A549. Conclusion Granulysins with different mw were successfully expressed using prokaryotic expression system, which might be helpful for the further study of granulysin.

Objective To obtain recombinant human granulysin using prokaryotic expression system.MethodsTotal RNA was extracted from cultured PBMC. Granulysin gene segments were obtained with granulysin-specific primers by RT-PCR and then inserted into pET32a(+) plasmid. After identification by DNA sequence,pET-GNLY9K and pET-GNLY15K were transferred to E. coli Rosetta (DE3). The fusion protein was identified by SDS-PAGE and Western blot.The bioactivity of granulysin fusion protein was measured by MTT assay.Results The prokaryotic expression vectors pET-GNLY9K and pET-GNLY15K were successfully constructed.The corresponding protein was highly expressed in E.coli. Recombinant protein was specifically bound by anti-granulysin antibody. GNLY9K fusion protein significantly inhibited the growth of A549 cells in a dose-dependent manner,while GNLY15K had little effect on the growth of A549.Conclusion Granulysins with different mw were successfully expressed using prokaryotic expression system,which might be helpful for the further study of granulysin.

Spleen cells from Balb/c mice immunized with HBsAg were fused with HGPRT deficiencyhybridoma cells that secret anti-horseradish peroxidase (HRP) monoclonal antibodies (McAb),and two bispecific McAb secreting triomas were obtainedit is reacted with HRP.

Objective: This present study is to establish a rat model of intravenous heroin self-administration and observe the effect of electro-acupuncture on the heroin-seeking behavior of the heroin self-administrating rat. Methods: Six Male Wistar rats were surgically implanted with an intra-jugular intravenous catheters under sodium pentobarbital anesthesia,following a minimum recovery of 7 days, the rats were trained by nose-poke response with one daily 4-hour session for 14 consecutive days to self administer heroin (50 μg /kg / per injection), the session ended after 25 infusions were earned or 4 hours had passed, whichever came first, the reinforcement schedule was fixed ratio 1 (FR1) that the rat received a heroin injection for each nose poke response. And then the rats were given electro-acupuncture treatment daily 30min prior to each session and then were trained with one daily 4-h session for 7 consecutive days. Results: Stable intravenous heroin self-administration behavior of rats was established after 7-8 days' drug training, electro-acupuncture given to the stable heroin self-administrating rat 30 min prior to each session could reduce the drug self-administration behavior, namely the total heroin intake in each session was reduced, especially the 1st hour heroin intake, the initiation of first nose-poke response in each session was relayed, and the haste heroin-seeking behavior also became slower. Conclusions: The above results suggested that the electro-acupuncture had better detoxification effect.

Objective To investigate the analgesic effect of oxysophoridine(OSR)and the influence of verapamil(Ver)on the antinociception of OSR when two drugs were co-administrated in mice.Methods The number of writhing within 15 min after ip different doses of OSR was observed in painful mouse mo- dels caused by acetic acid.The hot plate method was used to assess nociceptive sensitivity of CaCl_2 and Ver before ip OSR.Nitric oxide(NO)in serum was measured by spectrophotometry.Results The number of writhing was decreased and the latency of licking the hind paws was prolonged in a dose-dependent manner after ip OSR.The antinociception of OSR could be antagonized by CaCl_2 and enhanced by Ver.No inter- ference was detected in serum volume of NO.Conclusion These results suggest that OSR can antagonize the acute pain caused by acetic acid and hot plate in a dose-dependent manner in mice.Calcium channel blocker could enhance the effect of OSR.

Objective： To elucidate the role of bone marrow stromal cells in cooperation with exogenous cytokines in hematopoiesis. Methods： Fetal bone marrow stromal cells (FBMSC) was combined with cytokines including SCF,IL-3,IL-6,GM-CSF in a 5-day liquid culture system of adult bone marrow mononuclear cells, then we cultured bone marrow derived CD34+-enriched cells with FBMSC+SCF+IL-3+IL-6+G-CSF+EPO for 2 weeks. Results：FBMSC were in good cooperation with above mentioned exogenous cytokines. When CD34+-enriched cells from adult bone marrow were cultured with combinations of FBMSC, SCF, IL-3, IL-6, G-CSF and EPO, total nucleated cells, CFU-GM, BFU-E and CD34+ cells were increased by 119.6±30.9, 54.6±17.4, 25.2±4.4, 11.1±4.2 folds, respectively. Conclusion：FBMSC in cooperation with exogenous cytokines support the in vitro expansion of human hematopoietic progenitor cells efficiently.

Objective To establish the method of contact heat evoked potential(CHEP)and to explore the value of this evoked potential in pain testing of patients with cerebral infarction.Methods A total of 100 healthy volunteers and 30 patients were examined.The healthy volunteers were divided into 3 groups according to the length of their arms:(Group A:56.0～65.0 cm ;Group B :65.5～74.0 cm ;Group C :74.5～83.0 cm).A recently de- veloped heat-foil technique with a rapid temperature rising rate at 70℃/s was used to elicit pain and contact heat e- voked potentials.Contact heat was delivered via one circular thermode(diameter 27 mm,area 573 mm~2)and set at two intensity levels(49.5℃and 54.5℃)to three body sites:the thenar eminence,the dorsum of hand and proximal volar forearm.The subjects were asked to rate the pain with numerical rating scale after each stimulus and CHEP was recorded from Cz and Pz.The association between stimulus intensities and pain rating was explored,the main compo- nents of the evuked potential were watched.CHEP,sensory conduction velocity(SCV)and somatosensory evoked potentials(SEP)were performed in patients with hemi-anesthesia caused by cerebral infarction.Results The pain intensity ratings were 3.2?0.3 and 4.4?0.5 at thenar eminence,5.0?0.7 and 6.3?0.8 at the dorsum of hand and 5.3?0.6 and 7.2?0.5 at the proximal volar forearm when the temperature of 49.5℃and 54.5℃was applied, respectively;Three components,Cz/N550,Cz/P750 and Pz/P1000,were identified in the evoked potentials.Cz/ N550 and Cz/P750 appeared when the dorsum of hand and proximal volar forearm were stimulated.In contrast,Pz/ P1000 could be identified when nociceptors of thenar eminence and proximal volar fbrearm were excited.In the pa- tients with cerebral infarction,CHEP disappeared or became abnormal on one side,while SCV and SEP were normal on that side.Conclusion It was suggested that CHEP could be elicited reliably in the controls.CHEP is helpful in the assessment of analgesia in patients with cerebral infarction.

Objective To evoke cerebral potentials by stimulating nociceptive fibers with contact heat evoked potentials stimulator (CHEPS)and estimate the nerve conduction velocities of peripheral nerve fibers mediating these responses.Methods Subjects were set in supine position.A heat-foil technology with a rapid rising speed at 70 ℃/s was used to elicit pain and contact heat evoked potentials(CHEP).Contact heat was delivered via one circular thermode (diameter 27 mm,area 573 mm~2).Thermal stimuli were sent at two intensity levels (49.5 ℃ and 54.5 ℃) to three body sites:thenar eminence,the dorsum of hand and proximal volar forarm.Contact heat evoked potentials were recorded from Cz and Pz.A systemic effect between stimulus intensities and pain rating were observed,the main components of this evoked potential were observed.Nerve conduction velocity was calculated from latency difference of CHEP and center to center distance of distal and proximal stimulus arrays.Results The pain intensity rating was 3.2?0.3 and 4.4?0.5 when thenar eminence was stimulated at the temperature of 49.5 ℃ and 54.5 ℃ respectively;the rating was 6.3?0.8 and 7.2?0.5 when the dorsum of hand and proximal volar forarm were stimulated at the temperature of 54.5 ℃ respectively.Three components,Cz/N550,Cz/P750 and Pz/P1000,were found in the evoked potentials.Nerve conduction velocities of the fibers were (12.9?7.5) and (1.7?0.4) m/s respectively,which were corresponding to those of A8 fiber and C fiber.Conclusions CHEPs can be elicited reliably and stably.Velocities of peripheral nerve fibers demonstrate that A8 fiber and C fiber mediate the response.

To establish a LC-MS/MS method for quantification of chlorogenic acid, caffeic acid, 3,4-DCQA, ferulic acid and cinnamic acid in rats plasma and study its pharmacokinetics after administration of Mailuoning injection at a single dose to rats. Plasma samples were acidified with hydrochloric acid and extracted with ethyl acetate. The analytes were determined by LC-MS-MS using a ZOBAX SB C18 column with a mobile phase of methanol-water (containing 2 mmol x L(-1) ammonium acetic) (60:40)at a flow rate of 0.5 mL x min(-1) and detected using ESI with negative ionization mode. Ions monitored in the multiple reaction monitoring (MRM) mode were m/z 353.1/191.0 [M-H]- for chlorogenic acid, m/z 178.9/134.9 [M-H]- for caffeic acid, m/z 515.2/353.0 [M-H]-for 3,4-DCQA, m/z 193.0/133.9 [M-H]-for ferulic acid, m/z 146.9/102.9 [M-H]- for cinnamic acid and m/z 246.0/125.8 [M-H]- for tinidazole (IS). After administration of Mailuoning injection at a single dose to eight Sprague-Dawley rats, the concentrations of chlorogenic acid, caffeic acid, 3,4-DCQA, ferulic acid and cinnamic acid in plasma were determined by LC-MS/MS method. The main pharmacokinetics parameters of measured data were caluculated by using DASver 1.0 software. The linear concentration ranges of the calibration curves for chlorogenic acid, caffeic acid, 3,4-DCQA and cinnamic acid were 2.006-1,027 microg x L(-1) (r = 0.999 6), 1.953-1,000 microg x L(-1) (r = 0.999 7), 28.51-1.459 x 10(4) microg x L(-1) (r = 0.998 9), 1.836-940.0, g x L(-1) (r = 0.997 7) and 4.780-2,447 microg x L(-1) (r = 0.998 6) respectively. The inner and inter-days relative standard deviations were both less than 5.0%, indicating legitimate precise and accuracy to the requirement of biological sample analysis. For chlorogenic acid, the pharmacokinetic parameter t1/2, AUC0-t, and CL were (49.78 +/- 12.81) min, (123.55 +/- 14.82) mg x min x L(-1) and (0.004 3 +/- 0.000 5) L x min(-1), respectively. For caffeic acid, the pharmacokinetic parameter t1/2, AUC0-t, and CL were (36.65 +/- 10.59) min, (91.67 +/- 11.77) mg x min L(-1) and (0.005 7 +/- 0.000 7) L x min(-1), respectively. For 3,4-DCQA, the pharmacokinetic parameter t1/2, AUC0-t, and CL were (50.08 +/- 13.78) min, (278.34 +/- 31.82) mg x min x L-1 and (0.001 6 +/- 0.000 2) L x min(-1), respectively. For ferulic acid, the pharmacokinetic parameter t1/2, AUC0-t, and CL were (51.39 +/- 15.52) min, (34.72 +/- 4.67) mg x min x L(-1) and (0.000 4 +/- 0.0001) L x min(-1), respectively. For cinnamic acid, the pharmacokinetic parameter t1/2, AUCo-t, and CL were (74.42 +/- 18.32) min, (34.63 +/- 4.82) mg x min x L(-1) and (0.007 7 +/- 0.001 1) L x min-', respectively. The assay method is proved to be sensitive, accurate and convenient. It can be applied to the pharmacokinetic study of chlorogenic acid, caffeic acid, 3,4-DCQA, ferulic acid and cinnamic acid.
Animals ; Chromatography, Liquid ; methods ; Drugs, Chinese Herbal ; administration & dosage ; pharmacokinetics ; Female ; Hydroxybenzoates ; blood ; pharmacokinetics ; Male ; Rats ; Rats, Sprague-Dawley ; Tandem Mass Spectrometry ; methods

Objective: To compare the effects of pericardium patch aortoplasty and pulmonary patch aortoplasty for treating the infants with aorticcoarctation (AC) combining hypoplastic aortic arch in order to provide a better surgical choice in clinical practice.
Methods: A total of 57 patients with AC combining hypoplastic aortic arch treated in our hospital from 2009-01 to 2014-12 were retrospectively studied. The patients were divided into 2 groups: Pericardium patch aortoplasty group,n=26 and Pulmonary patch aortoplasty group,n=31. The changes of the pressure gradient at post-operation and follow-up period were compared.
Results: There were 2/57 (3.5%) patients died, 1 in Pericardium patch aortoplasty group by pulmonary hypertension crisis, the other 1 in Pulmonary patch aortoplasty group by respiratory distress syndrome. No renal failure or neurological complication occurred in neither groups. The cardiopulmonary bypass time, aortic clamping time, ventilator time and ICU stay time were similar between 2 groups,P>0.05. Selective cerebral perfusion time in Pericardium patch aortoplasty group was shorter than Pulmonary patch aortoplasty group (30.5 ± 8.6) s vs (35.6 ± 10.3) s,P<0.05. By ultrasound estimation, the post-operative AC pressure gradients were decreased than they were before, as in Pericardium patch aortoplasty group (9.5 ± 7.5) mmHg vs (39.9 ± 15.5) mmHg and in Pulmonary patch aortoplasty group (11.8 ± 11.3) mmHgvs (39.2 ± 14.5) mmHg, bothP<0.05; while post-operative pressure gradients were similar between 2 groups,P>0.05. Follow-up study was conducted in 51 patients for (17.6 ± 16.6) months, Pericardium patch aortoplasty group had 6 patients with re-stenosis, 3 of them would receive balloon angioplasty and 3 would be continuously followed-up; Pulmonary patch aortoplasty group had 6 patients with re-stenosis, 2 of them ifnished balloon angioplasty and their pressure gradients were obviously decreased, 4 would be continuously followed-up. Kaplan-Meier curves presented that Pulmonary patch aortoplasty group was superior to Pericardium patch aortoplasty group in re-stenosis occurrence during follow-up period.
Conclusion: Both pericardium patch aortoplasty and pulmonary patch aortoplasty were effective for treating the patients with AC combining hypoplastic aortic arch, the early post-operative efifcacy was similar, while the mid-term follow-up result was better in pulmonary patch aortoplasty.