HLA-A2肽四聚体的构建及其在乙、丙型肝炎中的初步应用

目的　分别构建乙型肝炎病毒 (HBV)和丙型肝炎病毒 (HCV)特异的HLA A2肽四聚体 ,为检测乙、丙型肝炎患者体内特异性的细胞毒T细胞 (CTL)提供直接、有效的方法 ,指导临床用药。方法　分别构建含有HLA A2 BSP(含有 15个可供识别生物素化的氨基酸位点 )和 β2微球蛋白(β 2m)基因的原核表达载体 ,并进行表达、复性、鉴定及纯化。再分别将HBV和HCV特异性短肽与HLA A2 BSP和 β 2m蛋白在体外进行耦合 ,该复合物经纯化浓缩后进行生物素化。生物素化的产物经纯化浓缩后形成单体 ,此单体再与藻红蛋白标记的链霉亲和素按一定比例耦合构建成四聚体 ,最后进行流式细胞仪检测。结果　获得了高效、稳定的pBV2 2 0 HLA A2 BSP和 pBV2 2 0 β 2m原核表达载体 ,表达量分别占菌体的 4 6 %和 2 6 %左右 ;纯度达 90 %以上。构建的四聚体可成功的检测急、慢性乙型和丙型肝炎患者中特异性的CTL ,急性HBV感染时CTL占 1 84 % ,慢性HBV感染时CTL占0 0 2 %～ 0 6 8% ,慢性HCV感染的CTL占 0 0 2～ 0 72 %。结论　高效、稳定的pBV2 2 0 HLA A2 BSP和 pBV2 2 0 β 2m原核表达载体大量的表达可用于构建各种肽特异性的四聚体复合物 ;在体外构建的MHC Ⅰ类分子四聚体 ,为特异性细胞毒T细胞的检测提供有效的工具。     

可溶性HLA-G1-肽复合物的体外折叠和鉴定

目的 合成可溶性HLA-G1-肽复合物。方法 利用原核高效表达的可溶性HLA-G1重链和轻链（β2m）,在人工合成的HLA-G1限制性九肽（KGPPAALTL）存在的条件下,通过稀释复性折叠成可溶性HLA-G1-肽复合物。利用特异性抗体（W6／32及兔抗人β2m抗体）进行免疫印迹及酶联免疫吸附试验,检测稀释复性的折叠产物。结果 折叠复合物含有35％可溶性HLA-G1-肽复合物、5％可溶性HLA-G1重链聚合体及60％复性的β2m3种成分。结论 通过原核表达、体外折叠能够获得大量可溶性HLA-G1-肽复合物。     

QLFATINSTL（93-102aa）是隐球菌抗原MP98CTL优势表位

目的鉴定隐球菌抗原MP98中的HLA—A＊0201限制性CD8^＋CTL表位。方法应用数据库SYFPEITHI预测隐球菌MP98中可能存在的HLA—A＊0201限制性CD8^＋CTL表位,经流式细胞术分析各抗原肽与HLA-A＊0201的亲合力,经时间分辨荧光法检测外周血单个核细胞（PBMC5）对各抗原肽产生的增殖反应,经细胞毒性实验研究各抗原肽诱导的特异性T细胞的细胞毒杀伤活性,逐步鉴定MP98的HLA—A＊0201限制性CD8^＋CTL表位。结果位于MP98氨基酸序列肽3（93—102aa）与HLA—A＊0201分子具有较高的亲合力,并都能刺激HLA—A＊0201阳性个体PBMC增殖,并诱导产生具有特异性杀伤活性的CTL。结论肽3QLFATINSTL（93—102aa）是抗原MP98上HLA—A＊0201限制性CD8^＋CTL的优势表位,可作为隐球菌疫苗设计的候选表位。     

结核杆菌抗原Ag85C的HLA-A*0201限制性CD8+CTL表位的预测及鉴定

目的：预测及鉴定结核杆菌（Mycobacterium tuberculosis，Mtb）抗原Ag85C的HLA-A＊0201限制性CD8^＋CTL表位，为基于表位的结核疫苗研究提供依据。方法：应用SYFPEITHI数据库预测结核杆菌抗原Ag85C序列中可能存在的HLA-A＊0201限制性T细胞表位，利用T2细胞株分析各预测的抗原肽与HL-A＊0201分子的亲合力，选取高亲合力肽诱导特异性CTL细胞，检测各高亲合力肽刺激其特异性CTL细胞分泌的IFN-γ水平、在体外的CTL增殖反应以及细胞杀伤毒性，逐步鉴定出Ag85C的HLA-A＊0201限制性CD8^＋CTL表位。结果：SYFPEITHI数据库从Ag85C序列中预测到14条能够结合HLA—A＊0201分子的抗原肽，其中3个抗原肽（170～178aa、317～325aa和144～153aa）显示与T2细胞上HLA-A＊0201分子有高结合力，而抗原肽FLTREMPAWL（144～153aa）能够诱导大多数HLA—A＊0201阳性结核患者及PPD（＋）健康志愿者产生特异性CTL细胞，并分泌大量的IFN-γ，能够诱导CTL体外发生增殖，能够产生特异性杀伤活性。结论：成功鉴定出抗原肽FLTREMPAWL（144～153aa）结核杆菌抗原Ag85C的HLA-A＊0201限制性CD8^＋CTL表位，可作为结核疫苗设计的候选表位，为进一步研发新型有效的抗结核疫苗奠定了基础。     

可溶性HLA-G-肽复合物的体外制备与纯化

目的 探讨可溶性HLA-G-肽复合物的体外制备与纯化.方法 原核高效表达的HLA-G重链及轻链(β2m)在抗原肽(人工合成九肽NH2-KGPPAALTL-COOH)的存在下,通过稀释复性折叠成HLA-G-肽复合物,采用凝胶过滤层析对折叠复合物进行纯化.利用特异性抗体(mAb W6/32和兔抗人β2m抗体)进行ELISA和Western blot对纯化产物进行鉴定.结果 折叠复合物中,主要含有HLA-G重链聚合体、HLA-G-肽复合物及β2m 3种成分,纯化后主要为HLA-G-肽复合物.结论 成功地获得纯度较高的可溶性HLA-G-肽复合物单体,为进一步研究HLA-G的生物学功能奠定了基础.     

乙型肝炎病毒c抗原特异性T细胞系的建立及CTL克隆的筛选

目的：建立乙型肝炎病毒（HBV）抗原特异性T细胞系，并筛选得到抗原肽特异性细胞毒性T淋巴细胞（CTL）克隆。方法通过流式细胞染色方法鉴定出1例急性乙型肝炎患者的人类白细胞分化抗原（HLA）为HLA‐A2型，分离患者外周血单个核淋巴细胞（PBMCs）；用重组人HBVc抗原（HBcAg）刺激PBMCs建立抗原特异性T细胞系；用HBcAg抗原肽刺激和有限稀释方法，筛选出HBcAg抗原肽特异性CTL克隆；以固相酶联免疫斑点技术（ELISPOT）和乳酸脱氢酶（LDH）细胞毒试验对HBcAg特异性T细胞系和CTL克隆进行功能鉴定。结果建系后HBcAg特异性T细胞明显得到富集，有限稀释后获得15株CTL克隆，其中14株表现出明显的HBcAg抗体肽特异性，包括抗原肽Core18‐27特异性CTL克隆8株，抗原肽Core107‐115特异性CTL克隆4株，另外2株CTL克隆对2条HBcAg抗原肽有交叉反应性。结论成功建立HBcAg抗原特异性T细胞系和CTL克隆，为后续试验奠定基础。     

丙型肝炎病毒HLA-A＊1101和A＊2402限制性CD8＋T细胞表位的研究

目的：鉴定出丙型肝炎病毒（HCV）特异性HLA-A＊1101限制性和A＊2402限制性CD8＋T细胞表位.方法：采用T细胞表位预测软件SYFPEITHI预测HCV特异性CD8＋T细胞表位,合成HLA-A＊1101限制性、A＊2402限制性候选表位;建立永生化的HLA-A＊1101阳性、A＊2402阳性B细胞株,采用竞争性肽结合实验检测候选表位与HLA-A＊1101或A＊2402分子的结合力;候选表位体外分别刺激HLA-A＊1101阳性或A＊2402阳性HCV感染者外周血单个核细胞（PBMCs）后,采用酶联免疫斑点（ELISPOT）和细胞内细胞因子染色（ICS）实验分别检测肽特异性分泌γ-干扰素（IFN-γ）的细胞的水平和（肝） 异性IFN-γ＋CD8＋T细胞的水平.结果：5条HLA-A＊1101限制性候选表位中,NS3_609（ITLTHPITK）和NS2_165（VVFSDMETK）与HLA-A＊1101分子具有高结合力.3条HLA-A＊2402限制性候选表位中,NS3_373 （KCDELASKL）和NS5b_382 （YYLTRDPTI）与HLA-A＊2402具有高结合力;在HLA-A＊1101阳性HCV感染者PBMCs中存在NS3 609和NS2_165特异性分泌IFN-γ的CD8＋T细胞,而在HLA-A＊2402阳性HCV感染者PBMCs中存在NS3_373和NS5b 382特异性分泌IFN-γ的CD8＋T细胞.结论：本研究证实NS3_609（ITLTHPITK）和NS2_165（WFSDMETK）为全新的HCV特异性HLA-A＊1101限制性CD8＋T细胞表位,NS3_373 （KCDELASKL）和NS5b_382 （YYLTRDPTI）为全新的HCV特异性HLA-A＊2402限制性CD8＋T细胞表位.     

人可溶性单链β2m-HLA-A2-BSP融合蛋白表达载体的构建及其表达

目的 构建并表达了人可溶性单链β2m-HLA-A2-BSP（scHLAA-A2）融合蛋白表达载体．为进一步制备HLA-A2四聚体及其功能研究奠定了基础。方法 应用重叠延伸PCR（overlap-PCR）对编码HI，A-A2的cDNA序列进行一个碱基的同义突变．并将β2m和HLA-A2胞外段亚单位两段编码序列的cDNA通过一疏水性多肽接头（Gly．Ser）。的DNA序列进行前后拼接．并利用引物所带的BSP编码序列．构建单链β2m-HLA-A2-BSP融合基因．将测序正确的β2m-HLA-A2-BSP融合基因插入原核表达载体pET-22b中．转化BL21（DE3）细菌．SDS-PAGE检测其表达．融合蛋白于体外抗原肽存在条件下稀释复性后由Western blot鉴定其空间构象。结果 经DNA序列分析表明：同义突变与预期结果一致．β2m和HLA-A2-BSP、linker的连接顺序、方向及序列完全正确．表达载体转化BL21（DE3）细菌后可表达β2m-HLA-A2-BSP融合蛋白．SDS-PAGE显示该融合蛋白分子量为45kD．与理论值一致．将融合蛋白与特异性抗原肽在体外进行稀释复性后经Western blot鉴定表明该复合物能与HLA-1类分子特异性结合的单克隆抗体W6／32结合。结论 人可溶性单链β2m-HLA-A2-BSP融合蛋白的构建及其表达获得成功．经体外复性可获得HLA-1类分子天然构象。     

抗原肽HPV18 E77-15诱导产生特异性细胞毒T细胞的实验研究

目的探讨HLA—A2限制性表位HPV18E77—15的免疫原性。方法对抗原肽HPV18E7-15进行T2结合分析;通过体外细胞培养技术抗原肽诱导特异性CTL扩增;采用RPE标记的表位特异性五聚体和FITC标记的CD8＋抗体通过流式细胞仪检测抗原肽所诱导产生的特异性CTLs。结果抗原肽HPVl8E77—15可将T2细胞表面HLA—A＊0201分子的表达提高2．6倍;在流式细胞仪检测中,抗原肽HPV18E77—15刺激诱导产生CD8＋五聚体＋双阳性的抗原肽特异性CTLs为1．87％。结论抗原肽HPV18E77—15能够诱导HLA—A2阳性正常人外周血淋巴细胞产生特异性CTLs,表明HPV18E77-15具有一定的免疫原性。     

树突状细胞吞噬PLA-AFP 218-226微球后诱导细胞毒性T淋巴细胞反应

目的研究树突状细胞（DC）吞噬包被有甲胎蛋白HLA—A2限制性表位肽（AFP218-226,LLNQHACAV）的聚乳酸微球（PLA—AFP218-226）后诱导的特异性细胞毒性T淋巴细胞（CTL）对肝癌细胞株HepG2和负载有AFP218-226的T2细胞株的毒性作用。方法用GM—CSF和IL-4诱导HLA—A2^＋的健康志愿者外周血单核细胞,LPS诱导成熟,使其分化为高纯度DC,在诱导过程中加入PLA—AFP218-226微球,用吞噬了PLA—AFP218-226微球的DC诱导自身T淋巴细胞,使其成为肝细胞癌（HCC）特异性CTL。用流氏细胞仪检测DC膜分子标志,T2细胞与AFP218-226结合实验检测HLA—A2分子与AFP218-226之间的亲合力,MTT法检测特异性CTL杀伤HepG2和T2细胞株的能力。结果AFP218-226与HLA—A2分子具有较高的亲合力,吞噬微球后的成熟DC高表达CD83、CD86、CD40等膜分子,其诱导的特异性CTL对HepG2和负载有AFP218-226的T2细胞株具有强的细胞毒作用。结论PLA—AFP218-226被DC细胞吞噬后,能够诱导HCC特异性CTL的产生,可能成为一种新型的抗肿瘤表位肽疫苗,在肝癌的防治中得到应用。     

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…     

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.     

Development on Adjustable Calibration Marker for Shock Wave Focus

Shock wave lithotripsy （SWL） is a treatment of choice for upper urinary stones. However, this procedure is inappropriate for obese patients because the focus is often unable to reach the target owing to the limited focal distance in shock wave source. Although treating such patients in a blast path may increase the application length of shock wave source, it＇s difficult to find this path on the lithotripter monitor. For this reason, we invented an adjustable calibration marker in order to set an effective focus in the shock wave hath.     

Study on the TCM Pathogenesis of Angiopathic Complications of Type II Diabetes

Excess production of reactive oxygen species(ROS)of mitochondrion mediated by hyperglycemia is the common pathogenesis of angiopathic complications of diabetes.TCM holds that the damp from the dysfunction of spleen.kidney and liver is the causative factor of complications of diabetes.This is similar to the mechanism of Ros resulting in angiopathic complications of diabetes.When the angiopathic complications of type II diabetes mellitus(T2DM)are difierentiated as caused by turbid damp in TCM can be explained as ROS.Since the obstruction of pathogenic damp in channels and collaterals is said to be the main pathogenesis,the treating principle should be dissolving the damp to remove the obstruction.     

Leptin, Ghrelin and TNF-α before and after Hypo-caloric Traditional Chinese Diet and Auricular Acupuncture

INTRODUCTION Obesity is a complex emergent problem, which can be possibly solved not only by the diet but also by the life style and promotion of a constant physical exercise. 1, 2 No doubt careful attentions must be given to the nutritional condition of obese people, the dietary habits, the somatic build (i.e. distribution of fat mass) and the organic functions linked to formation of the fat mass. All the parameters should be constantly monitored before, during and after a diet treatment. 3, 4, 5     

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.     

Relationship between Dysglycemia and Carotid Atherosclerosis in Tibetan Population

People with dysglycemia are at high risk for atherosclerotic diseases. This study aims at investigating the atherosclerotic vascular damage in dysglycemia and its metabolic origin in Tibetan population.     

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.