核基质MINT C端片段(2226-2959)相互作用分子的筛选与鉴定

目的:为了研究MINT的功能及其转录抑制的机制,筛选与MINT相互作用的蛋白.方法:以MINT第2226-2959氨基酸(F5为诱饵,用酵母双杂交法筛选小鼠胚胎9 d的cDNA文库.用诱饵质粒和文库先后转化酵母宿主后,对4.5×106个酵母克隆进行了营养筛选和α-半乳糖苷酶筛选,获得51个阳性克隆.从中提取质粒进行酶切鉴定,获得10个非重复克隆,对此10个克隆进行序列分析.结果:筛选到的3个基因分别为MINT,α-晶状体蛋白结合蛋白1(A1phaA-CRYBP1)和核受体辅抑制因子1(N-CoR1).在酵母中分析了N-CoR1与MINT的作用区段,N-CoR1与MINT的2226-2959和2960-3576两个片段均有相互作用.结论:①MINT在细胞内可能形成二聚或多聚体;②除了RBP-J和MSX2外,MINT还可能调节AlphaA-CRYBP1的活性;③MINT可能通过N-CoR1募集组蛋白去乙酰化酶(HDAC)进行转录抑制.     

MINT蛋白(1～365氨基酸)与Vav1相互作用及其位点的验证

目的：明确MINT(Msx2-interacting nuclear target protein)N-末端与Vav1之间的相互作用，进一步探讨MINT的转录抑制调控功能及其机制．方法：以克隆有MINT—F1片段(1—365氨基酸)的pGBKT7-F1质粒作为诱饵，分别与pACT2-V23#， pGADT7-V620，pACT2-V8900．pGADT7-SH2和pGADT7-SH3几个克隆有Vav1不同结构域的质粒，用酵母双杂交方法分析它们之间的相互作用．结果：pACT2-V23#与pGBKT7-F1之间、pACT2-V8900与pGBKT7-F1之间有比较明确的相互作用，而pACT2-V23#与pGBKT7-F2-F6、pGBKT7-F1与pGADT7-V620、pGADT7-SH2和pGADT7-SH3之间均无相互作用．结论：①MINTN-端的F1段与Vav1发生相互作用；②Vav1与MINT的F1段发生相互作用的区域位于其SH2-SH3-C端结构域，而且独立的Vav1 SH2或SH3-C端结构域均不能发挥与MINT的F1段产生相互作用的功能。     

核基质MINT N端片段(365～1084aa)相互作用蛋白的筛选及鉴定

目的:确定核蛋白MINT(Msx2-interacting nuclear target Protein)的功能及作用机制. 方法:利用酵母双杂交的方法,以MINT N端第365～1084位氨基酸(MINT-F2)为诱饵用酵母双杂交法筛选小鼠胚胎9日的cDNA文库. 用诱饵质粒和文库先后转化酵母宿主后,对1.0×108个酵母克隆进行营养筛选和β-半乳糖苷酶筛选,获得128个阳性克隆. 并提取质粒进行酶切鉴定,将获得的4个非重复克隆并进行序列分析. 结果:筛选出4个与MINT-F2相互作用的蛋白,它们分别是:67 ku层黏连蛋白受体,,2个16 S核糖体RNA,精氨酰-tRNA合成酶. 结论:成功筛选出与MINT-F2相互作用的蛋白,为进一步研究MINT的功能及作用机制打下基础.     

PER1与RACK1蛋白作用位点分析

目的 筛选人下丘脑视交叉上核(SCN)区域内与PERIOD1(PER1)相互作用的新蛋白,研究RACK1与PER1的作用特点,明确其结合的关键结构域.方法 采用酵母双杂交方法,筛选得到人SCN区域内与PER1-PAS结构域相互作用的新蛋白,并构建5种表达不同长度RACK1片段的酵母文库质粒与PER1诱饵质粒共转染酵母AH109进行杂交,通过营养缺陷筛选、报告基因检测获得阳性克隆,并采用体外转录、免疫共沉淀实验证实阳性克隆蛋白间的相互作用.结果 酵母双杂交筛选得到人SCN区域内表达RACK1蛋白的克隆,重组RACK1表达质粒与PER-PAS诱饵质粒进行酵母双杂交筛选后得到三个阳性克隆:RACK1(WD1-7)、RACK1(WD4-7)和RACK1 (WD5-7),β-半乳糖苷酶测试阳性证实报告基因表达,免疫共沉淀结果显示阳性克隆与PER1蛋白间存在相互作用.结论 RACK1与PER1存在直接相互作用,RACK1含有7个WD40结构域,本研究发现与PER1结合的最小区域位于Ⅴ、Ⅵ、Ⅶ三个WD40结构域,提示其碳端序列为其结合的关键部位.     

应用酵母双杂交系统筛选LKB1相互作用蛋白

目的 应用酵母双杂交技术研究与抑癌基因LKB1发生相互作用的蛋白.方法 应用酵母双杂交系统,以人 LKB1为诱饵,筛选人类全基因组开放阅读框酵母双杂交文库,寻找能与之相互作用的蛋白,并且通过免疫共沉淀和 GST-Pull down实验证实其之间的相互作用.结果 经过酵母双杂交共获得17个克隆,经过验证分析,最终确定1个无重复阳性克隆.结论 获得的1个基因编码的蛋白可能揭示LKB1新的作用机制.     

酵母双杂交系统筛选Mps1相互作用蛋白

目的：对前期筛出的与Mps1可能有相互作用的两种蛋白用酵母双杂交回转，验证与Mps1有相互作用的蛋白，为染色体分离机制研究提供线索，并为蛋门质相互作用网络提供资料。方法：应用酵母双杂交技术，将前期以pDBLeu—Mps1为诱饵质粒筛选成人肝脏cDNA文库，得到的Leu＋LacZ＋阳性克隆进一步回转酵母进行验证，即将文库质粒与诱饵质粒一对一重新转入酵母体内对相互作用进行验证。并对验证后的阳性克隆的外源片段进行测序及同源性分析。结果：用同源性分析，从人胚胎cDNA文库筛选到的与Mps1有相互作用的两个蛋白在酵母双杂交回转实验中仅有一个为阳性，另一个相互作用为阴性。结论：应用酵母双杂交系统从人胚胎cDNA文库中筛选并初步验证了与Mps1有相互作用的蛋白，为Mps1蛋白的功能研究奠定了基础．     

人巨细胞病毒UL135蛋白结合蛋白的酵母双杂交筛选

目的 利用酵母双杂交系统从人胎脑cDNA文库中筛选与人巨细胞病毒(HCMV)UL135编码蛋白相互作用的蛋白.方法 将成功构建的酵母诱饵表达载体pGBKT7-UL135转化到酵母菌AH109中,再将人胎脑文库DNA转化到含有pGBKT7-UL135的酵母细胞中,筛选与HCMV UL135编码蛋白相互作用的细胞蛋白,并对阳性克隆进行测序和生物信息学分析.结果 最终确认60个克隆与HCMV UL135编码蛋白相互作用,其中2个克隆与Thy-1高度同源.结论 成功应用酵母双杂交系统筛选出与HCMV UL135编码蛋白相互作用的蛋白,其中Thy-1在HCMV感染致病过程中可能起重要作用.     

应用酵母双杂交方法筛选LRP16相互作用蛋白

目的:应用酵母双杂交技术筛选人乳腺癌细胞MCF-7中可与LRP16相互作用的蛋白。方法:将诱饵质粒pGBKT7-LRP16、MCF-7双链cDNA文库片段及线性化质粒pGADT7-Rec共转化AH109酵母菌,营养缺陷型培养基(SD/-Leu/-Trp/-His/-Ade)筛选阳性菌落。提取阳性克隆质粒进行PCR,将PCR产物测序,并将酵母质粒转化感受态大肠杆菌Top10获得与诱饵质粒有相互作用的单个文库质粒,测序后回复验证其在酵母中的相互作用。结果:获得8个与LRP16相互作用的候选蛋白,选取4个在酵母中进行回复验证,其中有3个可生长出阳性克隆。结论:应用酵母双杂交技术筛选出一族功能基本明确的可与LRP16相互作用的候选蛋白,为研究LRP16的病理生理功能和分子机制奠定了基础。     

脆性X智力低下蛋白与NDK／Nm23—H2的相互作用

目的筛选与脆性X智力低下蛋白(FMRP)相互作用的蛋白,为FMRP生理功能的阐释提供新线索.方法以5月龄人胚海马mRNA为模板制备cDNA,重组于DNA激活结构域载体pGAD10,构建酵母双杂交文库,从中筛选与FMRP相互作用的蛋白.应用酵母双杂交体系对FMRP的相互作用区域进行定位.结果 (1)所构建的5月龄人胚海马cDNA酵母双杂交文库含1×106克隆,插入片段平均长度约1.5 kb,有效克隆90%;(2)在该文库中筛选到一个与FMRP相互作用的阳性克隆.序列分析表明该克隆为人的核苷酸二磷酸激酶亚基B(NDK/Nm23-H2)cDNA;(3)NDK/Nm23-H2与缺少外显子12、14-17的FMRP异构体,以及含FMRP外显子1-12的克隆相互作用,而与FMRP外显子1-6、外显子2-7不能直接结合.结论人Nm23-H2在酵母细胞内能与FMRP结合,其相互作用区域定位于FMRP的前1-11个外显子.Nm23-H2具反式调控因子特性,FMRP与Nm23-H2的相互作用提示FMRP参与基因表达调控.这一发现有助于进一步研究FMRP在细胞核内的生物学功能.     

酵母双杂交技术筛选与HCMV-UL146编码产物相互作用蛋白

目的 应用酵母双杂交技术,在人胎脑文库中筛选与人类巨细胞病毒(human cytomegalovirus,HCMV) UL146基因编码蛋白相互作用蛋白,以探讨HCMV-UL146的生物功能.方法 应用酵母双杂交系统,构建pGBKT7-UL146诱饵质粒,转化酵母AH109,与含人胎脑cDNA文库质粒的酵母相互作用,于涂有X-α-gal营养缺陷型培养基上筛选生长.挑选蓝色克隆,提取此酵母克隆的质粒转化大肠杆菌,提取质粒DNA进行测序分析.结果 pGBKT7-UL146构建成功.经严格筛选,共有200余个阳性克隆,分离测序80个克隆,成功筛选出30个与HCMV-UL146特异性相互作用的蛋白.结论 HCMV-UL146与多种蛋白有相互作用,提示其生物学功能复杂.此结果为进一步研究HCMV-UL146基因功能提供依据.     

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…     

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     

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.     

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.