Determination for the Contents of Neu5Ac, Neu5Gc and KDN in Organ Meats and Muscle of Pigs by LC-MS/MS

目的：建立液相色谱-三重四级杆质谱联用法（LC-MS/MS）测定猪内脏和肌肉组织中唾液酸N-乙酰神经氨酸（Neu5Ac）、N-羟乙酰神经氨酸（Neu5Gc）和脱氨神经氨酸（KDN）浓度的分析方法。方法：将内脏肉组织匀浆后,利用三氟乙酸（TFA）将结合态的唾液酸从糖脂或糖蛋白链上解离下来后进样分析。色谱条件：色谱柱为ZORBAX Eclipse Plus C18（5μm,4.6×250 mm,Waters）,流动相为0.1%醋酸铵-100%乙腈（9：1）,柱温：25℃,流速800μL·min~（-1）,进样量20μL。质谱条件：离子源为电喷雾化离子源（ESI）,扫描方式为多重反应监测（MRM）,监测离子对：Neu5Ac：307.7→87.0,Neu5Gc：323.3→116.0,KDN：266.7→87.0,13C3Neu5Ac内标品：310.9→90.0。结果：Neu5Ac、Neu5Gc和KDN分别在0.1~20.0、0.05~10.00和0.005~1.000μmol·L~（-1）的浓度范围内和对照品与内标峰面积比值线性关系良好,重复性平均RSD为1.2%,稳定性平均RSD为1.9%,日间和日内精密度试验RSD均小于6.7%,平均回收率为92.9~106.4%。结论：本方法简便、快速、灵敏度高,可广泛运用于组织和体液中唾液酸的测定。Objective： To establish an LC-MS/MS method for the detection of N-acetylneuraminic acid（Neu5Ac）,N-glycolylneuraminic acid（Neu5Gc） and 2-keto-3-deoxy-D-glycero-D-galactonononic acid（KDN） in organ meats and muscle of pigs.Methods： Tissues were acid hydrolyzed with trifluoroacetic acid（TFA） to get ganglioside-bound and glycoprotein-bound sialic acid,followed by injected and analyzed. The analysis was performed on an ZORBAX Eclipse Plus C18（5 μm, 4.6 ×250 mm, Waters）column, and the mobile phase consisted of 0.1 % ammonium~-100 % acetonitrile（9：1）, the flow rate was 800 μL/min, the temperature was maintained at 25 ℃, injection volume was set up 20 μL. Negative electrospray ionization（ESI） and multiple reaction monitoring（MRM） mode was used, MRM： Neu5Ac： 307.7→87.0, Neu5Gc： 323.3 →116.0, KDN： 266.7→87.0, 13C3Neu5Ac（IS）： 310.9→90.0.Results： The ratio of internal with standard sample peak area and content of Neu5 Ac, Neu5 Gc and KDN showed good linearity in the range of 0.1~20.0, 0.05~10.00, and 0.005~1.000 μmol/L, RSDs of the repeatability and stability were 1.2 % and 1.9 %, Intra-and inter-day RSDs were all lower than 6.7%, the recoveries of sialic acid were 92.9~106.4%. Conclusion： The established method is simple,accurate and rapid, higher sensitivity and can be applied to determination of Neu5 Gc, Neu5 Ac and KDN in tissues and fluid of animals and human.国家自然科学基金项目（31271038

Mechanism Study of the Promoting Action of Elongated Needle Acupuncture on Functional Repair in Rats with Acute Spinal Cord Injury

目的 观察芒针对急性脊髓损伤炎症反应和神经细胞凋亡的影响,研究PI3K/Akt和MAPK/ERK信号转导途径是否参与芒针的神经保护作用,探讨芒针促脊髓损伤修复的具体作用机制.方法 将150只成年雄性SD大鼠随机分为A组、B组、C组、D组和E组,每组30只.采用改良Allen＇s法制作大鼠脊髓中度损伤模型,A组为假手术组,不予以损伤脊髓及芒针治疗;B组造模后不予以芒针治疗;C组造模后采用芒针治疗;D组造模前0.5 h鞘内注射LY294002,造模后采用芒针治疗;E组造模前0.5 h鞘内注射PD98059,造模后采用芒针治疗.分别采用BBB评分检测大鼠的自发活动;ELISA检测炎性因子TNF-α 、IL-6、IL-1β、NF-κB的含量;TUNEL检测细胞凋亡的程度;免疫组化检测Bcl-2和Bax阳性细胞水平;Western印迹分析p-Akt和p-ERK在脊髓组织的表达,RT-PCR分析Cyt-C和Caspase-3在体内的表达.相对的下行Akt和ERK信号通路通过LY294002和PD98059特异性抑制剂处理分析在体内的抑制作用.结果 炎症反应和PI3K/Akt和MAPK/ERK信号通路抑制的神经元凋亡参与了脊髓损伤大鼠模型的损害,芒针介导的神经保护作用与Bax蛋白阳性神经元数目的减少及Bcl-2蛋白阳性神经元数量的增加有关.芒针治疗能改善大鼠的运动功能,PI3K/Akt和MAPK/ERK信号通路中p-Akt和p-ERK的激活,通过下调Bax蛋白和Bcl-2表达上调,抑制了线粒体凋亡途径关键因子Cyt-C的表达.TUNEL法检测并抑制激活神经元凋亡的Caspase-3的级联.PI3K/Akt和MAPK/ERK信号通路特异性抑制剂LY294002和PD98059的应用抑制了p-Akt和p-ERK的表达.结论 芒针促脊髓损伤修复的神经保护作用可能与炎症反应和PI3K/Akt和MAPK/ERK信号通路的激活、通过下调Bax蛋白和Bcl-2表达上调以及抑制线粒体途径诱导的凋亡有关.Objective To observe the effect of elongated needle acupuncture on inflammatory reactions and apoptosis in acute spinal cord injury, investigate if PI3K/Akt and MAPK/ERK signal transduction pathways are involved in the neuroprotective effect of elongated needle acupuncture and explore the promoting action of elongated needle acupuncture on spinal cord injury repair.Method One hundred and fifty adult male SD rats were randomized into groups A, B, C, D and E, 30 each. A model of moderate spinal cord injury was made by modified Allen＇s method. Group A received a sham operation without spinal cord injury and no elongated needle acupuncture. Group B did not receive elongated needle acupuncture after model making. Group C received elongated needle acupuncture after model making. Group D received an intrathecal injection of LY294002 at 0.5 hour before model making and elongated needle acupuncture after model making. Group E received an intrathecal injection of PD98059 at 0.5 hour before model making and elongated needle acupuncture after model making. Rat spontaneous activity was examined using the BBB rating. Inflammatory cytokines TNF-α, IL-6, IL-1β and NF-κB contents were measured by ELISA. The degree of apoptosis was determined by TUNEL. Bcl-2- and Bax-positive cell levels were measured by an immunohistochemical method. Spinal p-Akt and p-ERK expressions were determined by Western blot. In vivo expressions of Cyt-C and Caspase-3 were determined by RT-PCR. The in vivo inhibitory effect on downstream Akt and ERK signaling pathways was investigated using specific inhibitors LY294002 and PD98059.Result Inflammatory reactions and neuronal apoptosis due to the inhibition of PI3K/Akt and MAPK/ERK signal pathways were involved in the damage in a rat model of spinal cord injury. The neuroprotective effect of elongated needle acupuncture was related to a decrease in the number of Bax protein-positive neurons and an increase in the number of Bcl-2 protein-positive neurons. Elongated needle acupuncture treatment improved r浙江省中医药科研计划项目（2008CB067

Vegetation carbon stocks and net primary productivity of the mangrove forests in Shenzhen,China

红树林是滨海湿地"蓝碳"的主要类型之一.准确和定位评估不同植物群落的固碳能力,对于红树林保育管理和恢复造林具有指导作用.本研究对深圳福田红树林4种代表性群落(白骨壤群落、秋茄群落、海桑群落、无瓣海桑群落)的各个植被碳库组分(乔木植物生物量碳库、林下灌丛碳库、呼吸根碳库、枯立木碳库、枯倒木碳库和枯枝落叶层碳库等)进行调查,计算各群落的植被碳储量,并通过生长增量-凋落物产量法计算得到各群落的净初级生产力.结果表明:白骨壤群落、秋茄群落、海桑群落和无瓣海桑群落的植被碳储量分别为28.7、127.6、100.1、73.6 t C·hm~(-2),各群落的净初级生产力分别为8.75、7.67、9.60、11.87 t C·hm~(-2)·a~(-1).位于深圳市中心的福田红树林,每年固定大气CO2高达4000 t.本研究结果将为红树林"蓝碳"碳汇功能的评估提供理论指导,并为我国红树林碳汇林建设提供依据.Mangroves are the most important coastal blue carbon sinks. The accurate estimation on the carbon sequestration capacity of plant communities would guide the mangrove conservation,afforestation and management. This study investigated the vegetation carbon stocks of dominant mangrove communities,which were Avicennia marina,Kandelia obovata,Sonneratia caseolaris,and Sonneratia apetala in Futian Nature Mangrove Reserve in Shenzhen,Guangdong Province of China.Vegetation carbon stock consisted of living trees( aboveground and belowground biomass),understory,pneumatophore,standing dead trees,fallen dead trees and litter in these communities. The net primary productivity( NPP) was calculated from the litterfall and incremental growth in the same year of each community. Our results showed that the vegetation carbon stocks for A. marina,K. obovata,S. caseolaris,and S. apetala communities were 28. 7,127. 6,100. 1,and 73. 6t C·hm~(-2),and the NPP were 8.75,7.67,9.60,and 11.8 t C·hm~(-2)·a~(-1),respectively. Therefore,acting as urban forests,Futian mangroves in Shenzhen assimilated about 4000 t CO2·a-1.These results provided guidance for mangrove blue carbon assessment,and theoretical basis for the construction of coastal carbon sequestration forests in China.国家自然科学基金项目(31270581);; 福建省自然科学基金项目(2013J01145);; 深圳城市管理局科研项目(201406)资助~

Effects of electro-acupuncture on neuronal apoptosis and associative function in rats with spinal cord injury

目的 :探讨电针对大鼠急性脊髓损伤后膀胱功能改善的影响及作用机制。方法 :取健康成年雄性SPf级Sd大鼠60只,体重220~250 g,适应性饲养1周后,将大鼠按照随机数字表法随机分为假手术组、模型组、电针组和电针对照组,各15只。假手术组不予任何刺激,模型组、电针组和电针对照组大鼠采用改良AllEnS法制作脊髓损伤中度损伤Sd大鼠模型,模型组不予治疗,电针组给予秩边与水道穴电针治疗,电针对照组给予秩边与水道穴旁开0.5寸电针治疗,频率2/100 Hz,电流1 M A,刺激15 MIn,电针左右隔次交替,每日1次,共7次;分别于术后1、7 d观察大鼠残余尿量、排尿量的变化;术后7 d处死大鼠取伤段脊髓观察各组大鼠凋亡情况,检测bCl-2、bAX、bAd含量的变化。结果:造模后3组大鼠均出现不同程度的膀胱功能障碍。术后7 d,电针组、电针对照组残余尿量较术后1 d明显降低(P<0.001),且电针组与电针对照组比较差异有统计学意义(P<0.01);电针组、电针对照组较模型组在术后7 d排尿量增加,且电针组与电针对照组比较差异有统计学意义(P<0.001);TunEl发现电针可以抑制脊髓神经细胞的凋亡,电针组、电针对照组与模型组相比在术后7 d脊髓神经细胞凋亡率显著增加(P<0.01,P<0.05),且电针组与电针对照组比较差异有统计学意义(P<0.05);与模型组相比,电针组、电针对照组bAX、bAd的阳性表达率降低(P<0.01,P<0.05),而bCl-2的阳性表达率升高(P<0.01);且电针组与电针对照组比较差异有统计学意义(P<0.05)。结论 :电针能明显促进急性脊髓损伤的修复,其机制可能为通过增加bCl-2、抑制bAX、bAd的表达,从而抑制脊髓神经元细胞的凋亡发生作用的。Objective:To explore the effect of electro acupuncture to improve the bladder function after acute spinal cord injury in rats and its possible mechanism.Methods:Sixty healthy adult male SD rats of SPF grade,with body weight of 220 to250 g,one week after feeding adaptation,were randomly divided into sham operation group,model group,electro acupuncture group,electro acupuncture control group with 15 rats in each group.Sham operation group underwent no stimulation,and the moderate damage model of spinal cord injury were made in other three groups according to modified Allens method.The model group were not treated,electro acupuncture group were treated with electro acupuncture on Zhibianxue and Shuidaoxue,and electro acupuncture control group were treated with electro acupuncture on 0.5 inch next to Zhibianxue and Shuidaoxue.The frequency of 2 / 100 Hz,current of 1 m A,stimulation time of 15 min,once a day,left and right alternately stimulate every time,for a total of 7 times.The changes of residual urine volume and urine output in rats at the 1st and the 7th days after operation were observed.And 7 d later,the rats were sacrificed and the injured spinal cord were taken out to observe the apoptosis,and to detect the changes of Bcl-2,Bax,Bad content.Results:After modeling,the rats of three groups showed different bladder dysfunction.In electro acupuncture group and electro acupuncture control group,the residual urine volume of the 7th day after operation was significant lower than the 1st day after operation(P<0.001),and there was statistically significant difference on the 7th day after operation between two groups(P<0.001).Compared with model group,the urine output of electro acupuncture group and electro acupuncture control group was significantly increased on the 7th day after operation,and there was significant difference between electro acupuncture group and electro acupuncture control group(P<0.001).Electro acupuncture can inhibit apoptosis of spinal cord neurons by TUNEL detection.Postoperative at 7 d,the rate of nerve cell apoptosis in electro acupuncture group and electro acupuncture control group was significant increased than model group(P<0.01,P<0.05),and there was significant difference between electro acupuncture group and electro acupuncture control group(P<0.005).Compared with model group,the positive expression rate of Bax,Bad decreased(P<0.01,P<0.05),and Bcl-2 increased(P<0.01)in electro acupuncture group and electro acupuncture control group,there was significant difference between electro acupuncture group and electro acupuncture control group(P<0.01).Conclusion:Electro acupuncture can obviously promote the repair of acute spinal cord injury,its mechanism may be through increasing Bcl-2,inhibiting the expression of Bax,Bad,which inhibits the apoptosis of spinal cord neurons.浙江省中医药科研计划项目(编号:2008CB067)~

国内首例宫颈癌自体卵巢组织冻存移植成功妊娠

目的探讨卵巢组织冻存及自体移植手术保护子宫颈癌患者生育力与卵巢内分泌功能的疗效。方法对1例26岁子宫颈癌ⅡA1期患者在癌症手术过程中进行卵巢组织取材、冻存，待其癌症完全缓解后进行冻存卵巢组织自体移植手术，随访监测患者的绝经相关症状、激素水平及卵泡发育情况。结果卵巢组织移植第6个月卵泡刺激素降低至6.60 U/L，雌二醇由＜10.00 ng/L升高至89.00 ng/L。移植第10个月，B超监测到移植卵巢组织有卵泡发育且能完成生理排卵功能。移植第15个月，卵泡刺激素维持在7.24 U/L，雌二醇水平升至368.00 ng/L。移植2年余，患者通过辅助生殖技术成功诞下了一名健康的婴儿。结论冻存卵巢组织移植后卵巢恢复内分泌功能及排卵功能，并成功妊娠，证明卵巢组织在临床上移植成功

THE CLINICAL SIGNIFICANCE OF TELOMERASE ACTIVITY IN LIVER CANCER AND PRE CANCEROUS LESION

目的　探讨端粒酶检测用于早期诊断原发性肝癌的可能性。方法　采用TRAP法分别检测 2 2例原发性肝癌及癌旁组织标本、12例肝硬变组织、6例腺瘤样增生组织和 10例正常肝组织中的端粒酶活性。结果　 2 2例原发性肝癌中有 2 0例端粒酶表达阳性 ,阳性率为 90 .9% ;2 2例癌旁组织中有 14例阳性 ,阳性率为 6 3 .6 % ;12例肝硬变组织中有 10例阳性 ,阳性率为 83 .3 % ;6例腺瘤样增生组织中 5例阳性 ,阳性率为 83 .3 % ;而 10例正常肝组织的端粒酶检测均为阴性。结论　端粒酶活性表达可能发生在肝组织癌变过程中 ,并在其中扮演重要角色 ,因此端粒酶有可能成为肝癌早期诊断以及疗效和预后判断的一种标志物Objective To investigate the variety of telomerase activity in the course of liver cancer development, and the possibility of using telomerase as a marker of HCC. [WT5”HZ]Methods [WT5”BZ]Human liver specimens, comprising 22 HCC and adjacent peritumoral tissues, 12 liver cirrhosistissues, 6 nodulat regenerative hyperplasia (NRH) tissues and 10 normal liver tissues, were examined for telomerase activity by TRAP assay based on PCR. [WT5”HZ]Results [WT5”BZ]Twenty of 22 HCC and 14 of 22 adjacent tissue specimens were positive for telomerase activity with a positive rate of 90.9% and 63.6% respectively. Ten of 12 liver cirrhosis tissues were positive with a positive rate of 83.3%. 5 of 6 NRH were positive with a positive rate of 83.3%. Telomerase activity was negative in 10 normal liver tissues. [WT5”HZ]Conclusion [WT5”BZ]Telomerase may occur in the progress of hepatocarcinogenesis. Telomerase can be used as a tumor marker of HCC

生物质催化热解的TG-FTIR研究

借助综合热分析仪和傅立叶红外联用技术（TG-FTIR），考察了HUSY、REY和HZSM-53种分子筛以及重油催化裂化催化剂MLC和馏分油催化裂解催化剂CIP对生物质催化热解的活性和选择性。研究结果表明，分子筛对生物质热解产物的脱氧活性顺序为：REY≈HUSY〉HZSM-5，生成高辛烷值烃类的选择性顺序为：REY〉HUSYHZSM-5；MLC催化剂和CIP催化剂都表现出较高的选择活性，前者的脱氧活性略高于后者；择形分子筛HZSM-5的引入对调整催化剂酸强度、提高催化转化选择性和抑制焦炭生成产生一定作用

基于3D-药效团的P糖蛋白和细胞色素P4503A4的协同作用机理研究

P糖蛋白(P-glycoprotein,Pgp)和细胞色素P4503A4(CYP3A4)是决定药物ADME性质的两个重要蛋白,目前还无法通过实验方法,从分子水平清晰阐明这两个蛋白采取怎样的互补作用机理来降低外来药物的生物利用度.通过3D-药效团模建方法,提取Pgp和CYP3A4的共同底物的特征阐明这两个蛋白可能的协同作用模式.所得的药效团有助于理解药物分子同这两个蛋白的作用模式,同时该模型可以指导新药设计和改造,从而提高药物的生物利用度