稳定表达外源野生型p53基因人胆囊癌细胞系的建立和鉴定

目的：建立稳定表达外源野生型p53（wtp53）基因的人胆囊癌细胞系。方法：在脂质体lipofectamine^TM 2000介导下，将含有wtp53的真核表达质粒pCMV-p53导入胆囊癌细胞系GBC—SD中。经G418筛选抗性克隆，扩大培养，建立转染克隆细胞系。用聚合酶链反应（PCR）证实外源野生型p53基因的整合，用反转录聚合酶链反应（RT—PCR）和蛋白印迹法分析目的基因及其蛋白表达。结果：野生型p53基因成功地导入胆囊癌细胞系GBC—SD，转染细胞中存在外源p53基因及蛋白的稳定表达。结论：GBC—SD胆囊癌细胞株能稳定表达外源野生型p53基因。     

野生型p53基因抑制胆囊癌细胞裸鼠体内生长的实验研究

目的：研究转染野生型p53（wtp53）基因对胆囊癌细胞裸鼠体内生长的影响。方法：在脂质体介导下将含有wtp53的真核表达质粒pCMV—p53，导入GBC—SD细胞中。用G418筛选，建立转染克隆细胞系。以PCR、RT—PCR和蛋白质印迹法证实外源p53基因的整合与表达。用裸鼠移植瘤试验检测体内致瘤性的影响。结果：野生型p53基因成功的导入胆囊癌细胞系，转染细胞中存在外源p53基因及蛋白的稳定表达。表达外源wtp53的GBC—SD—wtp53细胞，裸鼠体内致瘤性受到显著抑制。结论：野生型p53基因可有效抑制胆囊癌细胞在裸鼠体内的生长。     

膀胱癌P53基因突变与蛋白高表达的相关性研究

目的　探讨膀胱癌 p53基因突变及突变蛋白高表达情况和它们之间的相关性。 方法　采用PCR -SSCP方法检测 p53基因 5～ 8外显子突变 ,免疫组化研究 p53蛋白的表达情况。 结果　 75例膀胱移行细胞癌病例中 p53蛋白的阳性表达率为 4 4 .0 % ,PCR -SSCP检测 p53基因的点突变率为 4 5.3% ,且两者显著相关 (P <0 .0 1)。 11例出现在外显子 5～ 6,8例出现在外显子 7,15例出现在外显子 8。在有 p53基因突变的 34例膀胱移行细胞癌中 ,发现有 31例 p53蛋白阳性过表达。 结论　膀胱癌的发生与 p53基因突变和高表达关系密切。     

人类胚胎肌肉细胞体外自发恶性转化抑癌基因的改变

目的:研究人类胚胎肌肉源性自发恶性转化细胞系抑癌基因的改变.方法:运用免疫组化、PCR,PCR产物直接测序研究该类细胞系抑癌基因p16,p15,MTAP(甲硫氨酸磷酸化酶)和p53基因及蛋白的改变.结果:在染色体9021处存在纯合性片段缺失,包括p16,p15及MTAP;对p53基因突变率高发的外显子7,8及内含子7测序发现存在范围较大的突变,外显子突变率高达13%,皆为点突变,而内含子变异更甚,包括小片段缺失、插入、移位及点突变,其几与正常p53序列无明显相似性,在测序的6个细胞系中(含5个不同遗传背景)突变序列具有高度一致性.结论:多种抑癌基因的变异在正常胚胎肌肉细胞自发恶性转化进程中具有重要的作用.     

肝细胞癌p53基因的突变与表达

目的：对HCC患者的肝组织和外周血单核细胞（PBMC）进行突变p53基因、p53蛋白和HBX的检测并作比较,以助评估它们与HCC发生的关系。方法：从HCC患者肝组织和PBMC中提取DNA,用PCR/SSCP和PCR/RFLP法分析p53基因突变。HCC中HBx与p53蛋白表达用免疫组化法检测。结果：HCC标本35.8%（5/14)显示p53基因第7外显子突变发生在249密码子。同样的PBMC标本中亦测到p53基因第7外显子有突变。免疫组化分析HCC肝组织中p53蛋白检出率为28.6%,而HBx在大多数标本中均有表达。结论：在HCC,p53基因突变热点在第7外显子,p53基因突变率与p53蛋白检出率均约30%～40%,HBx较善遍地存在于所测的HCC标本中。这提示p53蛋白与HBx蛋白相互作用可能是HCC发病机制中的重要一步。     

脂肪组织源性肿瘤与抑癌基因p53关系的研究

从蛋白表达及基因水平探讨脂肪组织源性肿瘤与p53基因的关系。方法　LSAB免疫组化法 ,PCR SSCP及DNA序列分析方法。结果　p53蛋白只在脂肪肉瘤中表达 ,阳性率为 4 8 0 8% ( 2 5 52 )。不同类型脂肪肉瘤 ,分化良好者阳性率 30 0 0 %( 9 30 ) ,低于分化较低者 (P <0 0 0 5) .p53第 6、7、8外显子PCR SSCP分析 ,2例多形性脂肪肉瘤出现异常泳动带。DNA序列分析证实 1例第 8外显子第 2 68位编码区出现错义突变 (AACATC) ,另 1例第 6外显子第 2 2 1位编码区出现可疑杂合同义突变 (GAGGAA)。结论　p53蛋白与脂肪肉瘤的形成及分化和恶性程度有关。p53蛋白表达可作为判断脂肪肉瘤分化程度及恶性程度参考指标之一。脂肪肉瘤中p53基因第 6、8外显子分别存在点突变。     

miR-125b-5p调控HK2抑制胆囊癌细胞增殖和糖酵解

目的 研究miR-125b-5p通过HK2调控人胆囊癌细胞增殖和糖酵解的作用机制。方法 分别在人胆囊癌细胞GBC-SD中转染NC mimic、miR-125b-5p mimic、miR-125b-5p mimic+pcDNA-NC、miR-125b-5p mimic+pcDNA-HK2。实时荧光定量聚合酶链式反应（Real Time Quantitative Polymerase Chain Reaction,RT-qPCR）检测miR-125b-5p和HK2 mRNA的表达,Western blot检测HK2和LDHA、PDK1的蛋白表达。细胞计数试剂盒-8(cell countingKit-8,CCK-8)检测细胞增殖活力,糖酵解相关试剂盒检测乳酸生成、葡萄糖消耗以及ATP生成。双荧光素酶报告基因实验对miR-125b-5p和HK2的靶向关系进行验证。结果 miR-125b-5p在胆囊癌细胞系GBC-SD和NOZ(P <0.001)以及组织（P <0.01）中表达降低,且在GBC-SD细胞中低于NOZ细胞中。过表达miR-125b-5p可显著抑制GBC-SD细胞的增殖（...     

应用非同位素PCR-SSCP方法检测乳腺癌p53基因点突变

目的　探讨乳腺癌组织中p53基因点突变。方法应用非同位素PCR -SSCP技术检测 4 8例石蜡包埋乳癌组织中p53基因点突变。结果　 4 8例乳腺癌中 2 0例出现异常电泳 ,表明这些病例相应DNA片段中发生了点突变 ,其中 8例位于第 5- 6外显子 ,9例位于第 7外显子 ,3例位于第 8外显子。免疫组化检测突变p53蛋白的表达 2 5例为阳性 ( 52 % ) ,其中 5例SSCP未发现基因突变 ,突变可能发生在所检测的基因片点以外。结论　非同位素PCR -SSCP是一简便、快速、有效的检测基因点突变的方法 ,特别适用于大量标本基因点突变的筛选 ;p53基因点突变与突变型蛋白表达呈相关关系     

应用非同位素PCR—SSCP方法检测乳腺癌p53基因点突变

目的 探讨乳腺癌组织中p53基因点突变。方法 应用非同位素PCR－SSCP技术检测48例石蜡包埋乳癌组织中,p53基因点突变。结果 48例乳腺癌中20例出现异常电泳,表明这些病例相应DNA片段中发生了点突变,其中8例位于第5－6外显子,9例位于第7外显子,3例位于第8外显子。免疫组化检测突变p53蛋白的表达25例为阳性（52％）,其中5例SSCP未发现基因突变,突变可能发生在所检测的基因片点以外。结论 非同位素PCR－SSCP是一简便、快速、有效的检测基因点突点的方法,特别适用于大量标本基因点突变的筛选;p53基因点突变与突变型蛋白表达呈相关关系。     

利用RNA干扰技术沉默p53基因突变子175H的效果评价

目的：在p53基因缺失型细胞株H1299中转染p53基因突变子175H,构建H1299-175H细胞模型,并观察小发夹环RNA（shRNA）的基因沉默效果。方法：采用引物重叠PCR定点突变技术合成p53基因突变子175H,并利用基因重组技术构建出表达载体,以脂质体转染法建立细胞模型;合成针对p53-175H的shRNA并构建出反转录病毒表达载体Psuper-175HR,应用磷酸钙共沉淀法导入包装293T细胞,并收集假病毒颗粒感染H1299细胞。应用Western blotting法检测P53蛋白表达。结果：转染p53基因突变子175H的H1299-175H细胞模型P53蛋白表达阳性;H1299-175H细胞模型转染shRNA后,P53蛋白表达下调。结论：成功构建H1299-175H细胞模型;构建的Psuper-175HR基因沉默载体可以有效地抑制p53基因突变子175H的表达。     

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.     

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.     

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.