小鼠共刺激分子B7-1的分子克隆、测序与真核表达载体的构建

目的：克隆小鼠共刺激分子B7－1基因，测序并构建其真核表达载体。方法：从小鼠脾组织中提取总RNA，用RT－PCR方法将B7－1的DNA扩增出，克隆到真核表达载体pcDNA3中，测定其cDNA序列。结果：测序结果表明我们克隆的B7－1基因序列与GenBank中序列有1个碱基不同，所编码的氨基酸发生突变。结论：已成功构建小鼠B7－1的真核表达载体，为进一步应用B7－1进行肿瘤基因治疗打下基础。     

人酪氨酸酶相关蛋白-2的cDNA克隆及表达

目的 克隆酪氨酸酶相关蛋白－2（TRP－2）编码基因，表达TRP－2蛋白。方法 从培养的人黑素细胞中提取总RNA，反转录成cDNA，通过PCR方法扩增TRP－2编码基因，克隆至pUC19载体并测序，再亚克隆至谷胱甘肽巯基转移酶（GST）融全表达载体pGEX-4T-1，转化大肠杆菌，IPTG诱导表达TRP－2／GST融合蛋白。结果 从培养的人黑素细胞中扩增出编码TRP－2的cDNA，序列测定证实与文献报道的序列一致；成功构建了GST融合表达载体pGEX-4T-1/TRP-2,表达了TRP－2／GST融合蛋白。结论 成功克隆了人TRP－2基因，构建了GST融合表达载体并表达了TRP－2／GST融合蛋白。     

人CD137分子基因克隆及其在COS-7细胞中表达的研究

目的：克隆人CD17分子的编码序列，将其重组人真核表达载体，并表达于COS－7细胞，方法：从活化的T细胞cDNA文库中以PCR方法克隆编码人CD137的编码序列，对其序列进行测定。将测定正确的CD137编码序列克隆入真核表达载体PCI－neo，构建重组表达载体。采用质体法转染COS－7细胞，用流式细胞仪检测CD137分子的表达。结果：PCR方法扩增出一800bp左右的基因片段，插入PCI－neo构建重组表达载体后，经序列测定证实扩增的片段为人CD137编码序列，将重组子转染COS－7细胞，流式细胞仪检测显示有27．11％的细胞表达人CD137分子。结论：成功地克隆并在COS－7细胞中表达了CD137分子。     

大鼠共刺激分子B7 cDNA的克隆和骨肉瘤细胞转染

目的：为了研究共测激分子B7增强针对大鼠骨肉瘤的特异性细胞免疫反应，首先需获得大鼠B7分子的cDNA克隆，方法：应用反转录PCR的方法，从大鼠脾细胞mRNA中扩增特异性片段，结果：经测序表明，所获得的cDNA克隆与已经发表的B7－1和B7－2的c-DNA序列完全一致，并将其表达载体转染UMR－106细胞系，结论：成功获得大鼠B7共刺激分子的cDNA克隆，其转染的UMR－106大鼠骨肉瘤细胞可用于免疫诱导研究。     

恶性疟细胞毒性T淋巴细胞单表位疫苗抗原递呈细胞模型的建立

目的 构建恶性疟细胞毒性T淋巴细胞（cytotoxic T lymphocyte,CTL）单表位疫苗及建立抗原递呈细胞模型。方法 采用中国人群常见的HLAⅠ类分子A11限制的恶性疟（CTL）抗原表位(VTCGNGIQVR),合成其DNA序列并克隆于真核表达载体,构建CTL单表位DNA疫苗。将上述克隆在HLA－A11表型细胞株中进行表达,流式细胞仪检测细胞表面HLAⅠ类分子表达水平。结果 上述CTL单表位DNA疫苗编码的短肽在细胞内的表达明显促进HLA－A11分子在细胞表面的表达,用流式细胞仪测定平均荧光强度可量化表达水平（P＜0.05）。结论 成功构建CTL单表位短肽表达载体,模拟体内环境建立了抗原递呈细胞模型,提示CTL表位在该细胞模型内被内源性加工和递呈,以该表位为基础的疫苗可以为HLA－A11遗传背景的人群提供免疫防护。     

重组人补体C3片段抑制小鼠内毒素休克

目的 利用补体灭活片段ｉＣ３ｂ上含精氨酸－甘氨酸－天冬酰胺（ＲＧＤ）序列的重组蛋白，探索其抗炎治疗的应用价值。方法 利用基因重组技术构建表达载体，在大肠杆菌中表达了含ＲＧＤ序列的重组蛋白（简称Ｃ３３），并进行体内外活性分析。结果 电泳分析Ｃ３３分子量约１５０００，纯化后纯度可达９５％以上，氨基酸组成分析与理论计算值相符。低剂量醋酸苯汞刺激Ｕ９３７细胞可粘附于包被的Ｃ３３，抗ＣＤ１１ｂ的单克隆抗体可     

人白细胞介素—18的基因克隆及原核表达

目的 为进一步研究人白细胞介素（ｈＩＬ－１８）的生物学特性打下基础，进行ｈＩＬ－１８的基因克隆及原核表达。方法 采用反转录ＰＣＲ（ＲＴ－ＰＣＲ）法从正常人外周血白细胞中克隆ｈＩＬ－１８基因，并重组于ＰＶＢ２２０表达载体上，经酶切初步鉴定后进行ＤＮＡ序列分析，继而观察重组ｈＩＬ－１８基因在大肠杆菌中不同诱导时间表达量的差异。结果 所克隆的基因与预期结果一致，并在４２℃诱导５ｈ时表达量最高。结论 成功     

可溶型白细胞相关抗原B27分子基因的克隆与表达

目的 克隆和表达可溶型白细胞相关抗原（ＨＬＡ）－Ｂ２７分子的基因。方法 通过三轮聚合酶链反应（ＰＣＲ）方法选择性删除编码跨膜单位的第５外显子而克隆得到可溶型ＨＬＡ－Ｂ２７基因,并以电转法将其转入人Ｂ淋巴细胞系,使其高效表达。结果 人Ｔ、Ｂ及单核细胞系及小鼠Ｌ细胞系在正常培养条件下均存在选择性剪切现象,而产生可溶型ＨＬＡ－Ｉ类分子。经ＰＣＲ方法克隆得到的可溶型ＨＬ－Ｂ２７基因,经酶切图谱鉴定及序列分析证实确为删除了第５外显子的ＨＬＡ－Ｂ２７分子的基因,将此克隆产物转入不含ＨＬＡ－Ｂ２７基因的细胞系Ｃ１Ｒ中,在其培养上清中检测到高水平的可溶型ＨＬＡ－Ｂ２７分子,其产量受培养温度、培养条件等因素影响。结论 ＨＬＡ－Ｂ２７分子前ｍＲＮＡ发生选择性剪切而导致跨膜单位丢失可产生溶型ＨＬＡ－Ｂ２７分子,该细胞系为将来研究环境     

人血管内皮生长因子基因cDNA的克隆及其在COS-7细胞中的表达

目的：克隆和在COS－7细胞中表达人血管内皮生长因子165（VEGF165）。方法：利用RT－PCR方法，从新鲜卵巢癌组织中扩增到人VEGF165的全长cDNA，并测定其核酸序列；利用基因重组技术构建VEGF165的真核表达载体pcDNA3.1-VEGF165；应用脂质体介导的基因转移术将这一表达载体导入COS－7细胞后，免疫组化（SP法）检测瞬时表达的产物。结果：经酶切鉴定和基因测序证实，克隆的基因片段为人VEGF65cDNA，重组质粒pcDNA3．1－VEGF165转染COS－7细胞后，免疫组化检测有VEGF表达，结论：成功克隆人VEGF165基因，并在COS－7细胞获得表达。     

人白细胞介素18（hIL—18）的cDNA基因克隆及序列测定

目的：克隆人白细胞介素18（IL－18）全长cDNA基因,进一步探讨人白细胞介素18的功效。方法：采用RT－PCR的方法从人肝组织中扩增出人白细胞介素18（IL－18）基因,并克隆到真核表达的绿色荧光蛋白报告载体pEGFP-NI中,结果：经PCR及DNA序列测定得以证实其序列与文献报导一致,结论：该实验成功地克隆了IL－18cDNA,并将其重组构建了含有IL－18cDNA的真核表达载体pEGFP-N1-IL18。     

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.     

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…     

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.     

Acupuncture Combined with Spinal Tui Na for Treatment of Primary Dysmenorrhea in 30 Cases

Objective: To observe the therapeutic effects in acupunture treatment of primary dysmenorrhea combined with spinal Tui Na, and study its mechanism. Methods: Thirty cases of the treatment group were treated by acupuncture combined with spinal Tui Na, and thirty cases in the control group were treated by routine acupuncture. Results: The total effective rate was 93.3% in the treatment group, and 73.3% in the control group, with a significant difference between the two groups (P<0.05). Conclusions: Acupuncture combined with spinal Tui Na has good prospects for treatment of primary dysmenorrhea.     

Luo Lingjie's Experience in Treating Chronic Nephropathy

In treating chronic nephropathy,Luo Lingjie,a chief physician,pays attention to regulating the balance between yin and yang,treating infection if present,and removing pathogenic factors.He prescribes gentle drugs and uses carefully strongly warming-tonifying ones,emphasizes the importance of persuading the patient to persist in treatment with medication and nurse one's health for recuperation,and is good at combined use of TCM and western medicine therapy and brings the merits of various therapies into full play,with obvious theraoeutic effects.     

Dr.Zhang Ren's Experience in the Acupuncture Treatment of Different Diseases with the Same Therapeutic Principle

Dr.Zhang Ren,the chief physician,is the chairman of Shanghai Acupuncture and Moxibustion Association.Having been engaged in medicine for about 40 years,he is experienced in treating various intractable diseases.In his long years of clinical practice,he advocates taking the TCM differentiation as the basis to seek for the acupuncture method for treatment of modern intractable diseases.The author of this essay had the fortune to follow Dr.Zhang in study.The following is a summary of Dr.Zhang's experience in the acupuncture treatment for different intractable diseases with the same therapeutic principle.     

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.