药用植物基因编辑种质创新应用与制度监管

药用植物资源是我国传统中医药学的重要组成部分,基因编辑技术能够在不引入外源基因的情况下进行基因组定点靶向突变,实现药用植物资源种质创新。CRISPR基因编辑技术是近年广泛应用的基因编辑技术,已在甜橙、铁皮石斛、罂粟、丹参、地黄、人参、灵芝、金针菇、竹黄、蛹虫草、茯苓等药用植物中研究和应用,构建了基因编辑体系,开展了活性成分关键基因与含量调控研究、表型性状关键基因与品质改良研究。中国、美国、日本、巴西、澳大利亚等国家陆续出台基因编辑种质创新相应的监管政策,促进了其产业化发展。建议进一步推进药用植物资源基因编辑种质创新基础研究、加强药用植物基因编辑后药物等效性评价、开展科普宣传和建立科学监管体系。     

药用植物丹参繁育技术的研究进展

丹参（ Salvia miltiorrhiza Bge．）是临床治疗心血管系统疾病的常用中药。由于生产中长期的只种不选引起品种退化,导致药材产量和品质均有较大程度下降。为改良丹参的药材种质,提高产量和药用成分含量,确保临床用药质量,有关丹参的品种选育与繁殖技术的研究报道不断推陈出新。从良种选育、田间管理措施与组织培养技术等方面,对近年来药用植物丹参的品种培育与繁殖现状展开综述,为实现丹参的规范化种植,提高中药材产量和药用成分含量提供技术依据,对进一步推动丹参植物资源开发的现代化进程提供借鉴。     

转基因苜蓿的研究进展

苜蓿(Medicago sativa)是一种优良的豆科牧草,应用基因工程技术对苜蓿进行开发和改造已成为国内外研究的热点.本文综述了病毒、细菌、寄生虫、抗逆基因、品质改良基因、抗除草剂基因、单克隆抗体和工业原料等转基因苜蓿的研究进展.     

丹参KSL基因多态性对其编码酶催化反应的影响

目的分析全国不同产区丹参KSL基因的变异类型,初步探究丹参KSL基因多态性对其编码酶催化效率的影响。方法设计特异性引物,同源克隆河南、山东、四川、湖北、山西、内蒙古、河北产区丹参KSL基因全长,比较变异类型,选取突变样品构建原核表达体系,进行体外诱导表达;通过气相色谱-质谱(GS-MS)技术检测该基因酶催化产物,进一步分析丹参KSL基因多态性对蛋白酶催化效率的影响。结果丹参KSL基因全长1 788 bp,共编码595个氨基酸,不同产区的丹参KSL基因存在基因多态性,并能够引起同义和非同义突变。丹参KSL不同基因型中催化效率明显不同,山西产区的催化效率最高,内蒙古产区的催化效率最低。其中,产自山西、湖北、山东、四川的丹参均包含1 047 bp处A型变异,其催化效率明显高于不包含A型变异的河南、河北、内蒙古3个产区的丹参的催化效率。结论不同产区丹参KSL基因存在多态性,由基因多态性引起的非同义突变进一步导致氨基酸序列的变化,最终使编码酶的催化效率产生差异。以丹参酮类成分合成途径中关键酶基因的多态性作为切入点,为丹参酮类成分积累机制的揭示及丹参优良种质的选育提供进一步的参考。     

白花丹参同源四倍体的诱导与鉴定

目的 对白花丹参种质资源进行遗传改良,拟创造出白花丹参同源四倍体新种质。方法 对未成熟种子,采用组织培养方法,用秋水仙碱进行诱变处理。结果 获得了17株同源四倍体,其中一株为四倍体非整倍体。经种植观察,四倍体白花丹参在株高、冠幅、叶片大小、叶片厚度、叶柄绒毛、花、花药、花粉粒大小等方面都明显大或高于原二倍体白花丹参;每株根条数和根粗与倍性成正相关,根部药材产量四倍体株系间虽有显著差异,但多数株系都高于二倍体对照,其中4～5号四倍体单株根部药材产量是二倍体白花丹参的 3.19 倍;丹参多酚酸、丹参酮等主要药物成分也都高于二倍体对照。结论 四倍体白花丹参新种质在丹参遗传改良和多倍体杂种优势利用方面具有重要应用价值。     

白藜芦醇合酶基因的克隆及丹参的转化

目的为探索利用分子生物学技术培育新型药用植物的新途径,将外源的白藜芦醇合酶(RS)基因导入到丹参中表达,以改善或增加丹参的效用。方法根据已公布的核苷酸序列,利用引物悬挂延伸法,经过3次扩增,最终从葡萄基因组DNA中获得完整的RScDNA基因序列;以质粒pBin438为基础,构建含有RS目的基因的组成型植物表达载体。在根癌农杆菌介导下,利用叶盘法转化丹参,进而通过PCR、PCR-Southern杂交对不同的转基因植株进行筛选与检测。结果总共得到了45棵卡那霉素抗性植株,经PCR和PCR-Southern杂交检测,确定葡萄RS基因已整合到部分转基因丹参苗的基因组中。结论成功建立了白藜芦醇合酶基因转化丹参的体系,并获得了转基因植株。     

基于高分辨液质联用技术的不同产地鲜丹参药材品质分析

目的分析不同产地新鲜丹参中主要活性成分含量,合理评价鲜丹参药材品质。方法采用LTQ-Orbitrap XL高分辨质谱仪测定全国7个不同产地新鲜丹参中5个丹酚酸类和12个丹参酮类成分。在此基础上,进行主成分分析(PCA)和最小二乘法分析(PLS)。结果同一产地新鲜丹参主要活性成分的含量基本一致,不同产地新鲜丹参主要活性成分的含量差异显著;其中,湖北产区丹参的丹酚酸类成分含量最高,四川和河南产区丹参的丹参酮I和丹参酮IIA成分含量最高;PCA和PLS分析显示,大部分活性成分的含量分布具有明显的产地特异性。结论不同产地新鲜丹参质量差异显著并具有较显著的产地特异性,以新鲜丹参药材作为品质评价材料,是现今基于干药材为品质评价原料研究方法的有益补充,为更全面综合的评价丹参及其他中药材探索了新的研究途径。     

改良型TAT-VP3融合蛋白的基因合成、原核表达载体构建及表达的实验研究

目的 研究改良型TAT-VP3融合蛋白的基因合成、原核表达载体的构建及其原核表达.方法 首先通过基因拼接法合成VP3基因并委托生物技术公司合成改良型TAT基因.然后于原核表达载体pGEX-6p-1上构建改良型TAT-VP3融合蛋白的基因.最后将构建好的原核表达载体转导入基因工程菌DH-5α中并诱导其表达.结果 成功合成了VP3基因,构建了改良型TAT-VP3融合蛋白的原核表达载体,并经基因测序证明构建无误;成功的在基因工程菌DH-5α中诱导了改良型TAT-VP3融合蛋白的表达.结论 改良型TAT-VP3融合蛋白的基因合成、原核表达载体的构建及其原核表达是可行的,为进一步的蛋白制备及活性研究打下了基础.     

bZIP转录因子的生物学功能及其在中药材品质改良中的应用前景

植物来源中药材的品质与药用植物的生长及次生代谢密切相关。bZIP转录因子作为基因表达的关键调控因子,在植物生长发育、生物及非生物胁迫应答、次生代谢过程中发挥重要作用,对提高药用植物抗性、促进有效成分积累具有重要意义和价值。已证实通过调控bZIP转录因子的表达可有效提高青蒿、人参、丹参等中药材的品质。在阐明bZIP转录因子生物学功能的基础上,探讨其对药用植物品质(形态特征、抗逆反应、次生代谢)的影响,展望其在中药材品质改良中的应用前景,为深入探究bZIP转录因子在药用植物生长发育及有效成分积累中的作用提供参考,为优质中药材的培育和生产提供依据。     

基于自噬基因Beclin-1与LC3探讨丹参素对H/R大鼠心肌细胞ATP5G1表达的影响

目的探讨丹参素对缺氧/复氧(H/R)大鼠心肌细胞的保护作用,并揭示其作用机制。方法将培养的心肌细胞分为3组,正常组(N组),模型组(M组),丹参素组(D组)。正常组以完全培养基37℃、5%CO2条件下持续培养7 h;模型组、丹参素组更换无糖培养基,转移至缺氧培养箱中培养(5%CO2+95%N2),进行缺氧刺激4 h后,丹参素组更换含有浓度为10μM丹参素的完全培养基;模型组更换不含有丹参素的完全培养基,然后将模型组、丹参素组细胞恢复正常培养3 h(37℃、5%CO2)。采用CCK-8法检测大鼠心肌细胞活力;采用RTPCR法检测Bcl-2、Bax、LC3、Beclin-1、ATP5G1的基因表达。结果与正常组比较,模型组大鼠心肌细胞活力下降,抑制凋亡基因Bcl-2的mRNA表达下调,促进凋亡基因Bax上调,自噬基因LC3、Beclin-1表达上调,ATP5G1基因表达上调,差异具有统计学意义(P 0. 01);与模型组相比较,丹参素组大鼠心肌细胞活力升高,抑制凋亡基因Bcl-2的mRNA表达上调,促进凋亡基因Bax下调,自噬基因LC3、Beclin-1表达下调,ATP5G1基因表达下调,差异具有统计学意义(P 0. 05,P 0. 01)。结论丹参素具有提升H/R大鼠心肌细胞活力、抑制凋亡的作用,这可能与丹参素调节自噬、下调ATP5G1的基因表达有关。     

Value of D-Dimers in patients with acute aortic dissection

Objective: To evaluatel the value of D-dimers in patients with acute aortic dissection (AAD). Methods: This study consisted of 16 patients with AAD and 27 non-AAD patients. Serum D-dimets were measured by Sta-Liatest D-DI immunoturbidimetric assay. Results: D-dimer level was higher (P ＜ 0.001) in patients with AAD(7.91 ± 5.52 μg/ml) than that in non- AAD group(1.57±1.24 μg/ml). D-dimer was positive (＞0.4 μg/ml) in all patients with AAD and in 10 control group patients (37%). Among patients with acute AAD, D-dimers tended to be higher in Stanford A than in Stanford B (8.67 ± 4.31 μg/ml vs. 3.24±1.27 μg/ml, P ＜0.01). D-dimer values tended to be higher in more extended disease(3.84 ± 1.65 μg/ml, 8.57 ± 3.58 μg/ml and 11.87 ± 5.69 μg/ml in thoracic aorta, thoracic and abdominal aorta, thoracic and abdominal aorta and iliacal arteries, respectively, P ＜ 0.05 for both 8.57 ± 3.58 and 11.87 ± 5.69 vs. 3.84 ± 1.65 ). Including the control group into the analysis, we found a sensitivity of 100%, a negative predictive value of 100%, and a specificity of 66% and a positive predictive value of 64% for D-dimer in diagnosis of AAD in our patients with suspected AAD. Conclusion: D-dimer was elevated in patients with AAD. A negative D-dimer test result could be useful in excluding AAD.     

Research of combined liver-kidney transplantation model in rats

Objective： To set up a simple and reliable rat model of combined liver-kidney transplantation. Methods： SD rats served as both donors and recipients. 4℃ sodium lactate Ringer＇s was infused from portal veins to donated livers,and from abdominal aorta to donated kidneys, respectively. Anastomosis of the portal vein and the inferior vena cava （IVC） inferior to the right kidney between the graft and the recipient was performed by a double cuff method, then the superior hepatic vena cava with suture. A patch of donated renal artery was anastomosed to the recipient abdominal aorta. The urethra and bile duct were reconstructed with a simple inside bracket. Results： Among 65 cases of combined liver-kidney transplantation, the success rate in the late 40 cases was 77.5%. The function of the grafted liver and kidney remained normal. Conclusion： This rat model of combined liver-kidney transplantation can be established in common laboratory conditions with high success rate and meet the needs of renal transplantation experiment.     

BLOOD PRESSURE CHANGE WITH AGE IN SALT-SENSITIVE TEENAGERS

Objective To observe blood pressure change with age in salt-sensitive teenagers whose salt sensitivity were determined by repeated testing.Methods Salt sensitivity was determined through intravenous infusion of normal saline combined with volume-depletion by oral diuretic furosemide in 55 teenagers. After five years, salt sensitivity was re-examined and subject blood pressure was followed up. Blood pressure changes in salt-sensitive teenagers were compared to that of non-salt sensitive teenagers over five years.Results After 5 years, the repetition rate of salt sensitivity determined by intravenous saline loading is 92.7%. In teenagers with salt sensitivity on the baseline, both the systolic blood pressure increments and increment rates were much higher than non-salt sensitive teenagers (12.7±12.1 mmHg vs. 2.8±5.2 mmHg, P＜ 0.01; 12.2%± 12.0% vs. 2.5% ±4.4%, P＜ 0.001,respectively). There was a similar trend for diastolic blood pressure (8.4 ± 6.4 mmHg vs. 3.7 ± 6.4 mmHg, P = 0.052; 13.2% ±10.6 % vs. 6.8%± 10.1%, P = 0.053, respectively).Conclusions Salt sensitivity determined by intravenous saline loading showed good reproducibility. Blood pressure increments with age were much higher in salt-sensitive teenagers than non-salt sensitive teenagers, especially in terms of systolic blood pressure.     

APPLICATION OF LORNOXICAM TO PATIENT-CONTROLLED ANALGESIA IN PATIENTS UNDERGOING ABDOMINAL SURGERIES

FOR anesthesiologis s ,treatingpostoperativepainhas alwaysbeen a problem.Althoughopioidshave been provedtobe effective,theirsideeffectscouldnotbeignored.With thedevelopmentofscienceand pharmacology,many drugs with aspectsof satisfactoryanalgesicefficacyand couldbe welltoleratedby patientshave been developed.And lornoxicamisone of them, which isa non-steroidalanti-inflammatorydrug (NSAID ), with analgesic, anti-infl-ammatory,andantipyreticproperties.Itseliminationhalf-time(3 to 5 hours) isle…     

安心颗粒对急诊经皮冠状动脉介入术后生活质量影响的研究

目的：评价使用安心颗粒对急诊经皮冠状动脉介入术（PPCI）术后生活质量的影响.方法：将160例接受PPCI的急性ST段抬高型心肌梗死患者随机分为安心颗粒组（术前顿服安心颗粒8.8g,术后安心颗粒4.4 g/次,每日2次）和对照组（仅接受基础药物治疗）.所有患者均服用阿司匹林、氯吡格雷和阿托伐他汀.分别在入院时、出院前1d、出院后180 d时,应用心肌梗死多维度量表（MIDAS）、中文版SF-36评价量表对患者生活质量评分.并观察术后30 d以内的出血并发症、血小板减少症发生情况.结果：入院时和出院前1d,两组患者的心肌梗死MIDAS、SF-36量表评分比较无差异（P＞0.05）;出院后180 d时,与对照组比较,安心颗粒组MIDAS、SF-36评分明显减低（P＜0.05）;组内与入院时比较,两组出院前1d、出院后180 d时,MIDAS、SF-36评分均降低（P＜0.05）.两组患者在随访期间均无大量出血、少量出血、重度和极重度血小板减少症发生,安心颗粒组有4例、对照组有7例发生不明显出血（P＞0.05）.两组发生轻度血小板减少症的患者数比较无差异（P＞0.05）.结论：PPCI使用安心颗粒,能改善急性ST段抬高型心肌梗死患者的生活质量,且不增加出血风险.     

The comparative studies of the influences of Urapidil and Nicardipine on sino-atrial node function, atrio-ventricular node function and hemodynamics

Objective:To investigate the influences of urapidil and nicardipine on rabbit sinus function,atrio-ventricular node function and hemodynamics.Methods:Thirty-two Angora's rabbits were selected and randomly divided into four groups.U1 group:urapidil 0.25 mg/kg;U2 group:urapidil 0.5 mg/kg;N1 group:nicardipine 10 μg/kg;N2 group:nicardipine 20 μg/kg.All these medicine were administrated within 30 seconds.Measurements were taken before and after the administration of urapidil or nicardipine for the following data:mean blood pressure(MAP),heart rate(HR),sino-atrial conduction time(SACT),maximal sinoatrial recovery time(SNRTmax)corrected sinus node recovery time(CSNRT),index of sinus node recovery time(SNRTI),Wenckebach A-V conduction frequency (WB),and P-R interval.Results:Significant MAP and HR changes were identified in all of the four groups before and after administration of both urapidil and nicardipine.No significant changes could be found in the rest of the parameters.Intergroup analysis showed that SACT and CSNRT of N1 and N2 groups were shorter than those of the U2 group(P＜0.01);the MAP decreased(P＜0.01)and the HR increased drastically(P＜0.01).Conclusions:Neither urapidil(0.25 mg/kg,0.5 mg/kg)nor nicardipine(10μg/kg,20μg/kg)has any significant influence on rabbit sinus function or rabbit atrio-ventricular node function.Nicardipine could be a better choice than urapidil for parafunctional sinus node patients.     

Dynamic Changes in Serum Estradiol and Progesterone levels in Patients of Premenstrual Syndrome with Adverse Flow of Liver-qi

Objective:To probe into the influence of changes of ovarian hormones on the pathogenesis of the specific sub-type premenstrual syndrome(PMS)and reveal partial microcosmic mechanisms of adverse flow of liver-qi.Methods:Estradiol(E2)and progesterone(P)levels in serum were determined at different phases of menstrual cycle by radioimmunoassay.Results:In the group of PMS with adverse flow of liver-qi.the secretive peak value Of E2 and P at the follicular phase significantly decreased,and the secretive peak value at the luteal phase did not come into being.Conclusions:Low E2 and P secretive peak at the follicular phase and absence of secretive peak at the luteal phase is one of the microcosmic mechanisms of PMS with adverse flow of liver-qi.One of the pathophysiologic mechanisms of specific sub-type PMS is probably the continuous low level of E2and P.     

Real-time Three-dimensional Echocardiography in Assessment of Left Ventricular and Right Ventricular Volumes

Real-time three-dimensional echocardiography (RT3DE)is a new ultrasound technique that enables dynamic threedimensional visualization and quantification of the heart in real time. Investigation of feasibility and methodology of RT3DE in determining left ventricular (LV) and right ventricular (RV) volumes, RT3DE was performed in 35 normal adults using Philips SONOS 7500 system with a 2-4 MHz matrix array transducer. The 60°×60° "pyramid" volume database was obtained and analyzed on a TomTec echo workstation. Both LV and RV volumes were calculated with four 3DE methods (i.e. apical 2, 4, 8, and 16-plane) through manually tracing ventricular endocardial borders in end diastole and end systole. Stroke volumes were then calculated. LV volume was also measured by 2DE Simpson's rule using GE VIVID 7 ultrasound machine.     

SCREENING FOR A 21-CHROMOSOME ABNORMALITY IN PREIMPLANTED EMBRYOS OF ELDERLY WOMEN

Increasing maternal age is the only etiological factor unequivocally linked to Down's syndrome in humans. The occurrence rate of newborns with Down's syndrome is about 1/220 in women over 35 years old. However, the occurrence rate in embryos fertilized in vitro, of the elder woman is unclear. Using FISH we screened the number of chromosome 21 in preimplanted embryos of 5 elderly women (average age, 38.4 years) to study the feasibility and necessity of screening trisomy 21 in embryos in patients over 35 years old at the in vitro fertilization (IVF) center.     

Scientific principles and rigorous processes should be followed in developing clinical guidelines for therapeutic interventions of integrative medicine

A clinical guideline for the therapeutic interventions of integrative medicine may be defined as a written document which states a series of recommendations on therapeutic interventions of integrative medicine for a special disease or condition. The guideline may provide assistance to medical professionals in making clinical decisions aimed at improving the clinical outcome of patients and reducing the costs of medical care（~＇4~. Recommendations issued by a guideline should be based on the best available evidence in both Western and Chinese medicine. For fulfilling this purpose, the development of clinical guidelines for therapeutic interventions in the field of integrative medicine should follow scientific principles and undergo a rigorous processes.