FAM3A相互作用蛋白的检测

【目的】FAM3A（family with sequence similarity 3,memberA）为新近发现的细胞因子FAM3的家族成员之一,本研究检测FAM3A的相互作用蛋白。【方法】克隆FAM3A并连接到pGB载体,转化酵母Y190宿主菌,加入含主要信号传导细胞因子人cDNA文库,按标准程序进行酵母双杂交,以X-Gal显色评估活性。提取扩增阳性克隆,连接pACT2载体,测序、检索基因库确定阳性克隆基因。克隆阳性基因并与FAM3A质粒共转染酵母进一步确定相互作用。【结果】酵母双杂交检测到一个与FAM3A相互作用的阳性克隆,提取扩增后测序分析此相互作用蛋白为一睾丸特异性丝氨酸/苏氨酸激酶（TSSK4）,一种与精子的发育、成熟、及生育功能有关的蛋白质.共转染pGB-FAM3A质粒与pACT2-TSSK4质粒入Y190酵母证实上述相互作用。【结论】FAM3A可能通过与TSSK4的相互作用参与精子功能与生育能力的调节。     

应用酵母双杂交筛选与FAM172A相互作用蛋白的研究

目的 应用酵母双杂交技术（蛋白与蛋白的相互作用）,筛选人胎脑cDNA文库中与FAM172A蛋白相互作用的蛋白,为深入研究新发现的蛋白质FAM172A生物学功能及在疾病中的作用奠定基础.方法 构建pGB-FAM172A诱饵质粒,转化酵母菌株Y190.人胎脑cDNA转化诱饵酵母菌,于营养缺陷型培养基（SD/-Leu/-Trp/-His）上生长,从中筛选到35个单克隆进行β-半乳糖苷酶克隆转移滤纸实验,对蓝色克隆者进行质粒抽提,转入大肠杆菌DH/OB,进行抗性筛选,从中提取质粒,一对一与诱饵质粒pGB-FAM172A共转酵母细胞Y190,进行验证鉴定,提取质粒DNA进行测序并进行BLAST比对分析.结果 成功构建pGB-FAM172A质粒,经严格筛选,共有10个阳性克隆,分别进行测序、序列比对,成功筛选出6个与FAM172A存在相互作用的蛋白：RTCD1、MOCS2、A2M、KCNIP1、BTBD2和TOX2.结论 利用酵母双杂交系统筛选出6个可能与FAM172A相互作用的蛋白,为进一步研究FAM172A蛋白的生物学功能提供了线索.     

与杜氏盐藻驱动蛋白Ⅱ动力亚基C端相互作用的新蛋白的酵母双杂交筛选

目的:用酵母双杂交的方法筛选与杜氏盐藻驱动蛋白Ⅱ动力亚基(FLA8)C端相互作用的蛋白。方法:设计特异性引物(上下游分别引入EcoRⅠ和BamHⅠ酶切位点),扩增FLA8C端(433个氨基酸)cDNA,与穿梭载体pGBKT7连接以构建诱饵表达载体,然后转化酵母菌株Y187和AH109,Westernblot法检测诱饵蛋白在转化酵母菌株内的表达。通过自激活实验和毒性实验检测诱饵表达载体是否适合利用酵母双杂交的方法筛选相互作用蛋白。转化诱饵质粒的酵母菌株Y187与AH109(文库菌)杂交,待三叶形合子形成后用营养缺陷型培养基和α-半乳糖苷酶活性实验筛选阳性克隆,并对阳性克隆进行测序。结果:成功构建了pGBKT7-FLA8-C双杂交诱饵载体;经检测该载体的表达产物对Y187和AH109均无自激活和毒性作用,可用于酵母双杂交实验;杂交后筛选得到2个阳性克隆,测序结果显示为鞭毛结合蛋白107(FAP107)和含蛋白结合结构域的预测蛋白。结论:成功筛选得到了与杜氏盐藻驱动蛋白Ⅱ动力亚基C端相互作用的蛋白,分别为FAP107和预测蛋白。     

BPHL与PML-C间相互作用的胞内、外验证

目的 通过酵母双杂交实验及免疫共沉淀技术验证人联苯样水解酶(BPHL)与缺失核定位信号的人急性早幼粒细胞白血病(PML)基因的coiled-coil的结构域(PML-C)蛋白之间的相互作用。 方法 将表达PML-C诱饵蛋白及BPHL靶蛋白的重组质粒pGBKT7-PML-C及pACT2-BPHL共转化AH109酵母菌,通过酵母双杂交实验验证两者在活细胞内的相互作用。构建能在人胚肾293细胞中表达带HA标签的PML-C融合蛋白的重组载体pCMV-HA-PML-C,经酶切鉴定正确后,和表达带myc标签的BPHL融合蛋白的重组真核表达载体pCMV-myc-BPHL,共转染人胚肾293细胞,利用免疫共沉淀技术验证BPHL与PML-C间的相互作用。 结果 pGBKT7-PML-C及pACT2-BPHL质粒共转化AH109酵母菌后,可见蓝色阳性克隆。构建的重组表达载体pCMV-HA-PML-C及pCMV-myc-BPHL经双酶切鉴定正确后共转染HEK293细胞,抗HA多克隆抗体沉淀HA-PML-C相互作用蛋白复合物后,用抗c-myc单克隆抗体进行Western blot检测,可以检测到myc-BPHL的表达蛋白。 结论 成功构建了pCMV-HA-PML-C及pCMV-myc-BPHL融合蛋白真核表达重组载体,利用酵母双杂交实验及免疫共沉淀技术证实BPHL与PML-C之间存在着相互作用。     

带核定位信号的维甲酸受体α 与Ubiquilin 1蛋白相互作用的验证

目的：验证带有核定位信号的维甲酸受体α（nuclear localization signal-retinoic acid receptor α, NLS-RARα）与Ubiquilin 1（UBQLN1）蛋白之间的相互作用。方法：将表达NLS-RARα诱饵蛋白和UBQLN1靶蛋白的两种重组表达质粒pGBKT7-NLS-RARα及pACT2-UBQLN1共转化AH109酵母菌,通过酵母双杂交技术验证两者在活细胞内的相互作用;构建真核细胞表达载体pCMV-HA-NLS-RARα 和pCMV-Myc-UBQLN1,经酶切鉴定后,共转染HEK293细胞,利用抗HA多克隆抗体沉淀,抗Myc单克隆抗体检测验证他们之间的相互作用。结果：pGBKT7-NLS-RARα及pACT2-UBQLN1质粒共转化AH109酵母菌后,可见蓝色阳性克隆;构建的重组表达载体pCMV-HA-NLS-RARα和pCMV-Myc-UBQLN1经酶切鉴定成功,转染HEK293细胞后,用免疫共沉淀技术检测到Myc-UBQLN1蛋白的表达。结论：酵母双杂交和免疫共沉淀技术可验证NLS-RARα与UBQLN1之间存在相互作用。     

Sedlin突变体S124A对其与PAM14相互作用的影响

目的 构建Sedlin的点突变体,研究其与PAM14在酵母中的相互作用,寻找其相互作用的位点.方法 用蛋白质在线序列分析软件CPHmodels预测Sedlin蛋白的磷酸化位点,可能的磷酸化位点主要有S2、S30、S119、S124、Y115,针对其中一个磷酸化位点(S124)设计定点突变引物;以pAS-Sedlin为模板,以两条含有突变位点的互补序列为引物进行PCR扩增,将得到的PCR产物用Dpn Ⅰ酶切除去模板,消化产物转化大肠杆菌感受态DH5α,对得到的阳性克隆进行测序;将构建的定点突变质粒pAS-sedIins与pACT2-PAM14共转化至酵母Y190中,并对生长至合适大小的克隆进行B-半乳糖苷酶活性分析.结果 构建了pAS-Sedlin的定点突变质粒pAS-SedlinS,其与pACT2-PAM14共转克隆的β-半乳糖苷酶活性反应呈阳性,并且Sedlin S124A蛋白与PAM14蛋白的相互作用比野生型Sedlin蛋白与PAM14蛋白相互作用强.结论 成功构建了Sedlin S124A,Sedlin蛋白的S124并不是其与PAM14在酵母中相互作用的特异性结合位点.     

COX-2诱饵载体的构建及其在酵母双杂交系统中自激活作用的检测

目的构建环氧化酶-2(cyclooxygenase-2,COX-2)的诱饵载体,为应用酵母双杂交系统(yeast two-hybrid system)筛选与COX-2相互作用的蛋白建立实验基础。方法 PCR扩增COX-2基因编码区全长,引入酶切位点EcoRⅠ、BamHⅠ,将COX-2基因克隆至载体pGBKT7中,将构建好的诱饵载体pGBKT7-COX-2转化到酵母细胞Y190中,检测pGBKT7-COX-2在Mathmaker GAL4Two-Hybrid System3中有无自激活作用。结果 成功扩增了COX-2基因编码区全长,并克隆入pGBKT7中,测序结果正确,并成功转化到酵母细胞Y190中,检测无自激活作用。结论 成功构建了COX-2的酵母诱饵载体,为进一步运用酵母双杂交技术筛选与之相互作用的蛋白奠定了基础。     

人FAM172A基因慢病毒载体的构建及其在巨噬细胞中的表达

目的 构建人FAM172A基因慢病毒载体,并包装慢病毒FAM172A感染人巨噬细胞予以鉴定.方法 以PDC315-FAM172A为模板扩增FAM172A基因全长序列后,与慢病毒载体pLenti6.3/V5-GFP经酶切、连接转化大肠杆菌,挑取阳性克隆,行菌液PCR、酶切及测序鉴定.将鉴定正确的FAM172A基因慢病毒载体和病毒包装质粒共转染FT293细胞包装慢病毒FAM172A,测定病毒效价.Western blot和QPCR验证慢病毒FAM172A感染巨噬细胞效果.结果 菌液PCR、酶切及测序结果显示FAM172A基因成功插入到慢病毒载体中.病毒包装后荧光显微镜观察到FT293细胞中有大量绿色荧光,病毒效价为1.2×108TU/ml.Western blot和QPCR结果显示,慢病毒FAM172A侵染靶细胞后可明显增加目的基因FAM172A的表达.结论 成功构建慢病毒FAM172A,为后续FAM172A基因功能研究奠定了基础.     

分化抑制因子1相互作用蛋白AGGF1的筛选

目的 利用酵母双杂交方法,筛选成人肺组织文库中与分化抑制因子1(Id1)相互作用的蛋白.方法 PCR方法扩增Id1的编码序列并定向克隆入诱饵质粒pHybLex/Zeo,构建重组诱饵质粒pHybLex/Zeo-Id1,酶切鉴定后转化人酵母株EGY48/pSH18-34,检测重组诱饵质粒有无非特异激活报告基因Leu2、LacZ作用;扩增并提取成人肺组织文库质粒,将文库质粒及重组诱饵质粒转化入酵母细胞,依次筛选Leu2 ,Leu2 LacZ 克隆;设置阴性及阳性对照,重复筛选Leu2 LacZ 克隆并排除假阳性克隆,获取真阳性文库质粒,进行测序和同源性比对.结果 酶切证实,重组诱饵质粒构建成功.重组诱饵质粒转化入酵母细胞后.经检测无非特异激活报告基因作用.扩增并筛选成人肺组织文库.获得198个Leu2 及19个Leu2 LacZ 克隆.经排除假阳性克隆,最终获得1个Leu2 LacZ 真阳性克隆,经测序和同源性比较证实该克隆与蛋白AGGF1高度同源(556/559,99.5%),Westem blotting证实其在酵母细胞中有表达.结论 利用酵母双杂交方法,通过筛选成人肺组织文库,发现Id1与AGGF1存在相互作用.     

酵母双杂交诱饵蛋白RGS4融合表达质粒的构建及鉴定

目的研究糖皮质激素受体各个结构域与G蛋白信号转导负调节因子4（RGS4）之间是否存在相互作用,构建酵母双杂交系统诱饵蛋白融合质粒。方法采用RT-PCR方法扩增RGS4片断全长,酶切回收,将其连接至酵母双杂交系统诱饵蛋白质粒载体pGBKT7,构建重组质粒pGBKT7-RGS4,并经酶切和测序等方法鉴定后,使用Y187酵母菌检测重组质粒的自激活现象及毒性。然后,提取酵母总蛋白,采用Western blot方法检测重组质粒在Y187内的表达情况,以鉴定其作为诱饵蛋白的可行性。结果RT-PCR扩增的RGS4基因片断电泳后,大小正确,重组质粒,pGBKT7-RGS4测序结果与GenBank中RGS4基因序列比对完全正确。重组质粒pGBKT7-RGS4双酶切鉴定、体外转录翻译实验,Western blot等方法均证实重组质粒中的RGS4基因能够正确合成RGS4蛋白。转化酵母后排除重组质粒的自激活作用。结论构建重组质粒pGBKT7-RGS4,能够在Y187内正确表达,可作为酵母双杂交系统诱饵蛋白使用。     

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.     

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.     

Expression of OPGmRNA and ODFmRNA in the patients of hip fracture due to osteoporosis

Objective：To investigate the gene expression of osteoprotegerin（OPG） and osteoclast differentiation factor（ODF） in the bone tissue of patients with hip fracture due to osteoporosis. Methods：OPGmRNA and ODFmRNA in the bone tissue in 50 cases of osteoporosis sufferers（over 50 years old） with hip fracture（Observer Group） and 30 cases of hip facture sufferers with no osteoporosis（Control group） were analyzed with the Semi-Quantitative RT-PCR method. Results：The mRNA expressed of ODF, OPG were both high in the patients with hip fracture. In the control group, the expression of OPG mRNA was observed, while the expression of ODF mRNA was very slight. Conclusion：Aged patients contained all signals including OPG, ODF that are essential for inducing osteoclastogenesis and promoting bone resorption.     

The clinicopathological and immuohistochemical analysis of solid-pseudopapillary tumor of the pancreas： report of 9 cases

Objective：To investigate the clinical features, pathological characteristics and immunophenotype of solid-pseudopapillary tumor of the pancreas（SPTP）. Methods：Nine surgically treated cases of SPTP were retrospectively reviewed. Hematoxylin and Eosin（HE） staining and immunohistochemical staining were used to analyze all cases, and the general clinical data was collected. Results：Six patients were asymptomatic except for a palpable mass. Two patients complained of vague-epigastric pain. One patient appeared jaundice. The tumor was encapsulated and solid tissues alternately with cystic tissues. Histologically, the histological structure of solid portion was pseudopapillary with a fibrovascular core. Tumor cells were uniform and medium-sized which were arranged in sheets ets or nests or pseudopapillary patterns. Immunohistochemical studies demonstrated that SPTP proved positive in vimentin（9/9 cases）, AAT（9/9 cases）, NSE（9/9 cases）, ACT（7/9 cases）, CK20（2/9 cases）, CgA（1/9 cases）, S-100（3/gcases）, PR（4/gcases）, Syn（3/9 cases） and CD56（5/9cases）, negative in CEA and ER. Conclusion：SPTP is a tumor predominantly occurring in young women frequently without special symptoms. This tumor has various characteristical histological patterns with different immunophenotype.     

Electro-Acupuncture Combined with the Trigger Point Needle-Embedding for Treatment of Primary Trigeminal Neuralgia in 31 Cases

In recent years, the author of this essay has applied electro-acupuncture combined with the trigger point needle-embedding for treatment of primary trigeminal neuralgia in 31 cases, yielding satis- factory results as reported in the following.     

Investigation of Matrix Metalloproteinase -1, 2 and tissue inhibitor of matrix metalloproteinases-1 in Endometriosis

Objective： To explore the role of matrix metalloproteinase-1,2 （MMP-1, MMP-2） and tissue inhibitor of matrix metalloproteinases-1 （TIMP-1） in endometriosis. Methods： The eutopic and ectopic endometria from 40 subjects suffering from endometriosis and regular.endometria from 40 subjects （excluding endometriosis） were collected and examined by in situ hybridization technology and western blot assay. Results： Both expressions of MMP-1 and -2 were stronger in ectopic endometrium and eutopic endometrium than in normal endometrium. On the contrary, the expression of TIMP-1 in ectopic endometrium and eutopic endometrium was lower. The differences were significant （P 〈 0.01 ）. Moreover, there was no relationship among the expressions of MMP-1, 2 and TIMP-1 in ectopic endometrium. Conclusion： The expressions of MMP-1, 2 and TIMP-1 lose balance and lack of periodic changes in ectopic endometrium , which explains the biological invasive behavior of endometriosis. It was suggested-that regulating the balance between the MMPs and TIMP-1 should be an ideal therapeutic target to endometriosis.     

Shi Da-zhuo (史大卓) —An outstanding cardiologist of integrative medicine

Prof. SHI Da-zhuo, Ph.D., male, was born on March 20, 1960. Prof. SHI entered the Ph.D. program in 1990 at the China Academy of Chinese Medical Sciences under the supervision of Prof. CHEN Ke-ji, majoring in the treatment of cardiovascular diseases. After receiving his Ph.D. degree in 1993, Prof. SHI started working at the Cardiovascular Center in Xiyuan Hospital affiliated to China Academy of Chinese Medical sciences.     

Persist in and Carry Forward the Integrative Medical Research——Speech at the 6th National General Congress of Chinese Association of Integrative Medicine by Academician CHEN Zhu,Minister of Ministry of Health

The 6th National General Congress of Chinese Association of Integrative Medicine （CALM） was convened at 19-20, April 2008 in Beijing. Academician CHEN Zhu, the minister of Ministry of Health indicated at the congress that the integration of Chinese and Western medicine is very well in keeping with the situation of our country and the general rule of development in medical science; and as a good integration of Chinese medicine and Western medicine, it is mutually beneficial and advantageous to both of them. Seeing the creativity shown in integrative medical investigation in theoretic and methodological sides, we should and must persist in and develop it.