Objective To investigate the effects of substance P on anoxia/reoxygenation (A/R) injury to cardiomyocytes of neonatal rats.Methods Cardiomyocytes of neonatal rats were isolated from Sprague-Dawley rats,aged 1-3 days,and were cultured in 6-well plates for 72 h.The cardiomyocytes were then assigned into 4 groups (n =3 each) using a random number table:control group (group C),A/R group,substance P group (group SP) and substance P + D-SP group (group SP + D-SP).The cells were cultured routinely for 6 h in group C and the cells were exposed to 99.9 ％ N2 in an incubator at 37 ℃ for 3 h followed by reoxygenation for 2 h in the other groups.The cells were incubated with substance P with the final concentration of 10-7 mol/L for 1 h before anoxia in group SP.The cells were incubated for 1 h with substance P with the final concentration of 10-7 mol/L and D-SP (a specific antagonist of neurokinin-1 receptor) with the final concentration of 10-6 mol/L before anoxia in group SP + D-SP.The apoptosis rate and lactate dehydrogenase (LDH) activity were detected at the end of reoxygenation using TUNEL assay and LDH assay kit,respectively.Results Compared with C group,the apoptosis rate and LDH activity were significantly increased in A/R,SP and SP+ D-SP groups.Compared with A/R group,the apoptosis rate and LDH activity were significantly decreased in SP and SP + D-SP groups.Compared with SP group,the apoptosis rate and LDH activity were significantly increased in SP + D-SP group.Conclusion Substance P can attenuate A/R injury to cardiomyocytes of neonatal rats,and activation of neurokinin 1 receptors is involved in the mechanism.

Objective To evaluate the effect of calcitonin gene-related peptide (CGRP) on anoxiareoxygenation (A/R)-induced injury to neonatal rat cardiomyocytes incubated in high glucose medium.Methods Cardiomyocytes were obtained from 1-3-day old Sprague-Dawley rats and cultured in the culture medium containing 15％ bovine calf serum and then seeded onto 6-well plates at a density of 10 × 105/ml (3 ml/well).The cells were randomly divided into 5 groups (n =9 each):normal glucose medium control group (NG group),high glucose medium group (HG group),high glucose medium + A/R group (HG+ A/R group),high glucose medium + A/R + CGRP group (HG + A/R + CGRP group),and high glucose medium + A/R + CGRP+ CGRP8-37 group (HG + A/R + CGRP + CGRP8-37 group).The cells were incubated in normal glucose (5.5 mmol/L) medium for 72 h in NG group.In HG group,the cells were incubated in high glucose (25.0 mmol/L) medium for 72 h.In HG + A/ R group,the cells were incubated in high glucose medium for 72 h and then exposed to 3 h of anoxia followed by 2 h of reoxygenation.In group HG + A/R + CGRP,the cells were incubated in high glucose medium for 72 h,CGRP (final concentration 10-8 mol/L) was then added to the culture media and the cells were incubated for 1 h and then underwent A/R.In HG + A/R + CGRP + CGRP8-37 group,the cells were incubated in high glucose medium for 72 h,CGRP (final concentration 10 8 mol/L) was then added to the culture media and the cells were incubated for 1 h and then underwent A/R.In HG + A/R + CGRP + CGRP8-37 group,the cells were incubated in high glucose medium for 72 h,CGRP8-37 (final concentration 10-8 mol/L) and CGRP8-37 (CGRP receptor antagonist,final concentration 10-7 mol/L) was then added to the culture media and the cells were incubated for 1 h and then underwent A/R.Apoptosis in cardiomyocytes was detected using TUNEL and apoptosis index (AI) was calculated.The lactate dehydrogenase (LDH) activity in the culture medium was analyzed.Results AI and LDH activity were significantly higher in HG group than in NG group,and in HG + A/R group than in HG group.Compared with HG + A/R group,AI and LDH activity were significantly decreased in HG + A/R + CGRP group,while no significant changes were found in HG + A/R + CGRP + CGRP8-37 group.Compared with HG + A/R + CGRP group,AI and LDH activity were significantly increased in HG + A/R + CGRP + CGRP8-37 group.Conclusion CGRP attenuates A/R-induced injury to neonatal rat cardiomyocytes incubated in high glucose medium via combing with CGRP receptor.

A strain of Mucor named M2,which could produce protease,was isolated from a traditional fermented soybean product.Culture conditions of proteases produced by M2 were studied therefore.The results showed that nitrogen source and carbon source preferred for protease production were soybean protein isolate and glucose,while inorganic salts preferred were KH2PO4,CaCl2 and MgCl2.The suitable culture conditions for protease production were as follows:culture temperature was 28℃,inoculation volume was 2%,liquid level was 100 mL in 300 mL triangle bottle at pH 5,rotating speed was 150 r/min and culture time was 48 h.The obtained protease activity in culture was about 4.35 U/mL.The protease produced by Mucor was analyzed with SDS-PAGE.The protease had a molecular weight of 36.4 kD.

Objective To establish characteristic spectrum of ginsenosides in Panax quinquefolium from Liuba. Methods Using C_(18) solid phase-extraction cartridges, main ginsenosides from 40% ethanol extracts of P. quinquefolium were purified. Then the samples were analyzed by HPLC-EMS. Results(From the) total ion spectrum of P. quinquefolium, 3 stronger peaks were selected. Based on them, characteristic corresponding spectrum of ginsenosides in P. quinquefolium from Liuba was established. Conclusion This method has reliable reproducibility and precision. Its simple pretreatment, easily operation, and rapidly analytic procedure show that this method is suitable for identifying P. quinquefolium.

AIM:To investigate the effects of 17-AAG on apoptosis and cell cycle of HCT-15 cells and to clar-ify the related mechanisms .METHODS: MTT method was employed to evaluate the inhibitory effects of 17-AAG with Aifferent time and different doses on the proliferation of HCT-15 cells.The cells were stained with Annexin V-FITC/propid-iumiodide and measured by flow cytometry .The expression of STAT3, cyclin D1, Cyt C, caspase 9 and caspase 3 at mR-NA and protein levels was determined by RT-PCR and Western blotting .RESULTS:Treatment with 17-AAG at concentra-tion of 1.25~20 mg/L for 24 h and 48 h significantly inhibited the activity of HCT-15 cells at both time-and concentra-tion-dependent manners .Treatment with 17-AAG at concentrations of 0.425, 0.85 and 1.7 mg/L for 48 h significantly in-duced apoptosis and cell cycle arrest of HCT-15 cells.The exposure of 17-AAG at concentrations of 0.425, 0.85 and 1.7 mg/L for 48 h to the HCT-15 cells significantly down-regulated the expression of STAT 3 and cyclin D1 at mRNA and pro-tein levels, but up-regulated Cyt C, caspase 9 and caspase 3 mRNA and protein in a concentration-dependent manner . CONCLUSION:17-AAG inhibits the cell activity , induces apoptosis and G 1 arrest by down-regulating the expression of cyclin D1, and promoting the mitochondria apoptosis through STAT 3 pathway.

A high performance liquid chromatography-electrospray ionization mass spectrometry- charged aerosol detection ( HPLC-MS-CAD) method was established for the simultaneous quantitative analysis of four Lignans in Magnoliae Flos extract. The components were separated on a YMC-Pack ODS-A column (250 mm× 4. 6 mm, 5 μm) by gradient elution with methanol and water as the mobile phase at aflow rate of 1. 0 mL/min. Then the elution solution was routed into MS equipment at a flow rate of 0. 3 mL/min and CAD detector at a flow rate of 0. 7 mL/min by a split ratio of 3:7 for the further detection. The column temperature was 25 ℃ and the detection wavelength was 278 nm. A method was developed for the quantitative analysis of muti-components by single maker ( QAMS) to determine pinoresinol dimethylether, magnoli, 1irioresinol B dimethylethe and epi-magnoli A . Magnoli was selected as internal standard and the relative correction factors ( RCF) of the four Lignans were calculated. The contents of the four Lignans in Magnoliae Flos extract were determined by both external standard method and QAMS. The QAMS method was evaluated by comparison of its assay result and that of external standard method. Under the selected chromatographic condition, the limits of detection of pinoresinol dimethylether, magnoli, lirioresinol B dimethylethe and epi-magnoli A were 0. 34, 0. 55, 0. 50 and 0. 58 mg/L, respectively, while the linear range were within 6. 8-270 mg/L, 11-546 mg/L, 2. 0-101 mg/L and 2. 3-116 mg/L. The recoveries ( n=9 ) were 98. 2%-99. 5%, and the correlation coefficient were 0 . 9995-0 . 9998 . No significant differences were found between the quantitative results of external standard method and QAMS method. The developed method is accurate, feasible, and can be used for quality evaluation of Magnoliae Flos .

BACKGROUND: Our previous studies have demonstrated that cryopreserved bone marrow stromal cells (BMSCs) still maintain high survival rate, cell proliferation and osteogenic differentiation potentials after thawing. However, this result needs confirmed in vivo environment. OBJECTIVE: To explore the effects of cryopreserved BMSCs and collagenic membrane BME-10X complex on type Ⅰ collagen synthesis in vivo. METHODS: Beagle dog BMSCs were cultured in vitro and cryopreserved for 12 months, which were thawed and prepared complexes with collagenic membrane. The complexes were cultured with mineralization induction medium or normal medium for 5 days, followed by implanting into nude mice. The specimens were harvested and analyzed by gross observation, histopathological and immunohistochemistry at 4 weeks after implantation. The collagenic membrane cultured with mineralization induction medium served as controls. RESULTS AND CONCLUSION: In the control group, the boundary of collagenic membrane was distinctly, without cell growth around boundary or intra collagenic membrane, additionally, there was little type Ⅰ collagen. In the non-induction group, cells grew into collagenic membrane, trabes-like collagen formed, and type Ⅰ collagen distribution increased at 4 weeks. In the induction group, scaffold degraded, more cells grew, and plenty of collagen formed osteoid-like tissues. The distribution of typeⅠcollagen was obviously increased than that of other groups. The findings demonstrated that cryopreserved BMSCs possess strong osteogenic differentiation potentials after proliferation and induction combined with collagenic membranes in vitro.

Objective To investigate the sustained release of BSA from chitosan-OREC/BSA films coated mats in vitro.Methods The negatively charged cellulose acetate (CA) fibrous mats were modified with multilayers of the positively charged chitosan or chitosan-OREC intercalated composites and the negatively charged bovine serum albumin (BSA) via electrostatic layer-by-layer (LBL) self-assembly technique.The adsorption and rinsing steps were repeated until the desired number of deposition bilayers was obtained.The in vitro BSA encapsulation and release experiments demonstrated that OREC could affect the degree of protein loading capacity and release ficiency of the LBL films coating.Results In the pH-gradient release assay,only a small amount of BSA was released from the mats in 1 h.As the time increased,the release rate of BSA of all the samples gradually went up to the maximum data within 8 h.For the samples with identical number of bilayers and record time,obvious increasing of the release amount could be seen in pH 7.4,in comparison with pH 1.2.Besides,doubling bilayers film-coated mats generally.Meanwhile,it was slightly distinguishable between 5 and 5.5 as well as 10 and 10.5 bilayers (t=0.651～ 1.324,P＞0.05).Interestingly,it could be seen that protein release of the chitosan-OREC/BSA films coated mats remarkably increased compared with that of chitosan/BSA films coated mats(t=2.264～ 2.305,P＜0.05).Conclusion The release of protein in the initial time could be controlled by adjusting the number of deposition bilayers,the outmost layer and the composition of coating bilayers.

The introduction of stapling instruments and improved understanding of pathology has resulted in a greater proportion of low rectal cancer patients undergoing sphincter-preserving resection.A variety of alternative techniques have been proposed to avoid a permanent stoma,including abdominal pull through,abdominal trans-sphincteric resection and intersphincteric resection.However,these damages always inflicted on the anal sphincters with poor functional results.More recently,the anterior perineal plane for ultra-low anterior resection of the rectum (APPEAR) technique was developed which approaches the anurectum via an anterior transperineal approach and exploits an anatomic space within the pelvic floor musculature termed “rectal no-man's laud”.The ability to access this segment of distal rectum by the perineal approach may determine whether a sphincter-saving resection can be performed for a proportion of patients who would otherwise require a permanent stoma.We performed laparoscopic APPEAR for a 46-year old woman with low rectal cancer with satisfactory results.

To make a portable apparatus for the patient self-doing joint voluntarily exercise that is keeping with the training demand of human body potential and muscle strong. Different kinds of spring belts are used for joint exercise apparatus according to the needs of joint exercise. Though active joint extension and flexion by using the method of Chinese physical and breathing exercises, the exercise was done by drawing the spring belt. The extremity multifunctional training box is easy for carrying and sutble for each mussel be trained. Because of the wide range of back spring power, it is easy to reach the best training mount and to do the continually training under the biggest training mount. It is beneficial to the joint active function improvement. From June 2006 to February 2008, this kind of apparatus was used in 30 patients with footdrop at the Beijing Tongren Hospital, Capital Medical University. The design of the extremity multifunctional training box is reasonable, and it is easy for carrying and operating. It is also helpful for the patient to do muscle exercise themselves, and the joint function can be persisted and improved by continual exercise using this apparatus.