人结肠癌细胞Dickkopf-1的表达水平及意义

目的检测六株结肠癌细胞中Dickkopf-1（DKK1）的表达并探讨其意义。方法在6株结肠癌细胞（SW480、SW620、HT29、
LS174T、LOVO、HCT116）分别应用qPCR方法检测DKK1的mRNA表达水平。用Western blot方法和细胞免疫组化方法检测
DKK1的蛋白表达情况。结果荧光定量分析显示,DKK1在HT29、HCT116中的表达高于其他四株细胞（P<0.05）;Western blot
显示,DKK1在HCT116、HT29中的表达同样高于其他四株细胞;细胞免疫组化染色显示,DKK1在六株结肠癌细胞株中呈阳性
表达。结论DKK1在6株人结肠癌细胞系中的表达具有差异性。
     

丁酸钠诱导结肠癌细胞分化过程中NDRG2的表达及意义

目的：研究丁酸钠诱导结肠癌细胞分化过程中抑癌候选基因NDRG2的表达特点及意义．方法：采用丁酸钠诱导结肠癌HT29,SW480,SW620细胞分化,通过碱性磷酸酶（AKP）测定和透射电镜评价结肠癌细胞的分化程度,应用Real time RT—PCR和Western Blot法测定不同分化点（0,1,2,3,4,5,6d）NDRG2mRNA和蛋白的表达特点．结果：经丁酸钠作用24h-6d后,结肠癌细胞中NDRG2基因mRNA和蛋白水平均显著高于对照组水平．结论：丁酸钠诱导结肠癌细胞分化过程中NDRG2表达显著升高,提示NDRG2是参与结肠癌细胞分化的相关基因．     

突变的大肠癌线粒体DNA转染NIH3T3及LST细胞的研究

日的了解大肠癌细胞株（SW480，LOVO，HT29）线粒体DNA的突变，克隆突变的大肠癌线粒体DNA（mtDNA）基因，构建pcDNA3．1（＋）-mtDNA真核表达重组体，并导人NIH3T3及LST细胞。以探讨线粒体基因突变与肿瘤发生的关系。方法提取大肠癌细胞株（SW480，LOVO,HT29）mtDNA，扩增D-LOOP区，产物用DNA自动测序法进行序列分析。利用DNA重组技术将其定向插人真核表达质粒pcDNA3．1（＋）。并用脂质体法导人NIH3T3及LST细胞。用MitoCapture Mitochondrial Apoptosis Detection Kit试剂盒染色后用流式细胞仪及荧光显微镜检测转染细胞的凋亡情况。扩增并测序分析转染细胞的D-LOOP区突变特点。结果检测出大肠癌细胞株SW480、LOVO和HT29细胞mtDNAD-LOOP分别有10、9和8个突变位点。转染前后，各组间细胞凋亡无明显变化。转染细胞的核基因组可扩增出目的基因及Neo基因。4株NIH3T3转染细胞mtDNA D-环区分别检测到9、11、8和4个突变点，并相应有3、4、3和2个多态性变化。结论转染突变的大肠癌细胞mtDNA后转染细胞的mtDNA均可发生多处的突变位点；通过转染后突变的外源性的mtDNA可以整合到核基因组内；突变的mtDNA转染LST细胞及NIH3T3细胞后。不影响转染细胞的凋亡改变；mtDNA的突变可能通过影响体细胞mtDNA的突变和通过外源性mtDNA在核内的整合从而影响癌基因或抑癌基因的表达异常，从而参与肿瘤的发生发展。     

MIF对结直肠癌细胞周期和侵袭力的影响

目的探讨巨噬细胞迁移抑制因子(MIF)对人结肠腺癌细胞株LS174T、人结肠癌细胞SW480细胞周期及侵袭力的影响。方法采用聚合酶链反应、凝胶电泳进行LS174T细胞、SW480细胞MIF mRNA检测,MIF干扰剂细胞增殖抑制率应用MTT法检测,细胞生长周期及细胞凋亡率用流式细胞仪分析,FAS的表达变化、肿瘤组织细胞中E-cadherin及N-cadherin表达水平的变化采用Western blot法。结果 LS174T细胞MIF mRNA相对表达水平为(1.128±0.103)、SW480细胞为(1.036±0.078),MIF干扰剂与LS174T细胞共孵育后MIF mRNA相对表达水平为(0.502±0.087)、SW480细胞为(0.419±0.028),差异均有统计学意义(t=6.849、7.193,P均0.05)。干扰剂浓度≥40μmol/L,LS174T细胞、SW480细胞G0/G1期、S期细胞数所占百分比及凋亡率与未经处理的空白对照组相比差异均有统计学意义(P0.05),其中G0/G1期细胞显著高于未经处理的空白对照组(P0.05),S期细胞显著低于未经处理的空白对照组(P0.05)。在SW480细胞培养液中加入抑制剂之后,E-cadherin的表达水平由(0.95±0.12)上调为(1.26±0.24),而N-cadherin的表达水平由(0.92±0.17)下调为(0.63±0.12),差异均有统计学意义(t=2.039、10.204,P均0.05)。LS174T、SW480细胞经MIF干扰剂处理后FAS蛋白表达量降低,且随着MIF干扰剂浓度的加大而减低,差异有统计学意义(F=8.024、12.419,P均0.05)。结论抑制MIF能抑制LS174T、SW480细胞增殖和促进其凋亡,可能是结直肠癌治疗的新靶点。     

RalB在多种肿瘤细胞中的表达及意义

目的探讨RalB（v-ral simian leukemia viral oneogene homolog B，ras related；GTP binding protein）蛋白在多种肿瘤细胞中的表达及意义。方法以人胚肾293T细胞为正常细胞对照，选用8种不同组织的肿瘤细胞，采用抗RalB的单克隆抗体通过Western blot方法检测其RalB蛋白的表达。结果RalB在293T细胞中表达明显降低，在乳腺癌细胞株MCF-7、卵巢癌细胞株H08910、宫颈癌细胞株Hela、肺癌细胞株、结肠癌细胞株LOVO、SW480中均有表达，其中RalB蛋白在结肠癌细胞株SW480中表达明显增高。结论RalB可能以组织特异性的方式在不同肿瘤细胞中表达。     

选择性及非选择性COX-2抑制剂对结肠癌细胞生长的影响

目的探讨选择性及非选择性环氧合酶-2（cylooxygenase-2,COX-2）抑制剂对体外培养的结肠癌细胞生长的影响及凋亡的诱导作用。方法体外培养HT-29、SW480及LS174-T三种结肠癌细胞,分别加入不同浓度的非选择性COX-2抑制剂阿司匹林（aspirin）、选择性COX-2抑制剂塞来昔布（celecoxib）及美洛昔康（meloxicam）作用于HT-29及SW480细胞。采用MTT法检测结肠癌细胞增殖;用免疫细胞化学法及免疫印迹技术分别检测结肠癌细胞增殖细胞核抗原（PCNA）及细胞COX-2的表达;流式细胞技术分析对结肠癌细胞周期的影响;用Annexin V／PI染色结合流式细胞术检测细胞凋亡。结果aspirin、celecoxib及meloxicam均能有效抑制体外培养的HT-29、SW480结肠癌细胞生长,并具有良好的量-效关系。Western blot表明,HT-29细胞表达COX-2,而SW480细胞不表达COX-2。Aspirin及celecoxib处理组细胞PCNA表达较对照组明显下调。10mmol／L的aspirin及50μmol／L的celecoxib能诱导HT-29及SW480细胞凋亡,凋亡率分别为4．8％,17．7％;5．1％,20．4％。结论选择性及非选择性COX-2抑制剂均能有效抑制结肠癌细胞生长,这种作用亦存在于COX-2阴性表达的结肠癌细胞。     

过表达CCR9对SW480结肠癌细胞侵袭的影响

目的初步探讨人CCR9基因对SW480结肠癌细胞侵袭功能的影响。方法取人外周血单个核细胞提取总RNA逆转录为c DNA,扩增提取CCR9的CDS序列,连接p LVX-puro质粒,Lipo2000感染人SW480结肠癌细胞,嘌呤霉素筛选并建立稳定转染细胞株。免疫印迹证实CCR9基因在SW480结肠癌细胞高效表达。Transwell方法检测SW480结肠癌细胞侵袭。结果免疫印迹证实成功构建p LVX-puro-CCR9质粒,相比SW480细胞组,CCR9过表达SW480结肠癌细胞中CCR9显著表达[(26.0±10.7)vs(10.0±4.5),n=8,P=0.0078],且在体外能够促进SW480结肠癌细胞侵袭(P=0.0136)。结论成功构建CCR9稳定表达结肠癌细胞株,并能够在体外显著促进其侵袭功能。     

miR-126在人结肠癌细胞中的表达及其对结肠癌细胞生物学行为的影响

目的探讨MicroRNA-126(miR-126)在人结肠癌细胞中的表达以及对结肠癌细胞生物学行为的影响。方法应用荧光PCR定量检测60例结肠癌患者的结肠癌组织以及癌旁组织中miR-126的表达,共60对,再测定5株结肠癌细胞(SW480、HT29、HCT116、LOVO、SW620)中的表达情况;CCK-8法、划痕实验和Transwell实验检测细胞的增殖、迁移和侵袭能力,并进一步通过体外实验研究miR-126对结肠癌细胞生物学行为的影响。结果与癌旁组织细胞比较,miR-126在结肠癌细胞中表达下降(95.0%vs.55.0%,P<0.05),发生转移者与未发生转移者比较,差异有统计学意义(25.0%vs.75.0%,P<0.05)。miR-126可以抑制SW480细胞的生长及其侵袭转移,有基质胶Tran—swell实验中,转染组及空白对照组细胞迁移数分别为(10.0±4.3)个和(258.0±30.6)个;无基质胶的Transwell实验中,分别为(84.0±13.2)个和(335.0±27.3)个,差异均有统计学意义(P<0.05)。miR-126高表达可以增强癌细胞对奥沙利铂的敏感性。结论miR-126可明显抑制结肠癌细胞的发生发展,影响多种结肠癌细胞株生物学行为。     

iASPP真核表达载体构建及其功能鉴定

目的构建P53凋亡刺激蛋白(ASPP)家族的抑制成员iASPP的真核表达载体,并将其通过脂质体转染到结肠癌细胞株SW480及Lovo中,观察转染前后iASPP表达变化及其对细胞凋亡的影响。方法将解放军第十六医院检验科经测序鉴定的pMD19-T-iASPP质粒亚克隆至真核表达质粒pcDNA3.1(+),构建重组真核表达质粒pcDNA3.1(+)-iASPP,测序鉴定后用脂质体将重组质粒转染至结肠癌细胞株SW480及Lovo中,用逆转录聚合酶链反应(RT-PCR)检测iASPP的表达以及用流式细胞仪检测细胞凋亡的变化情况。结果重组表达质粒pcDNA3.1(+)-iASPP,经酶切测序与GenBank上记录的人iASPP cDNA序列(gi 60457962)完全一致。经pcDNA3.1(+)-iASPP质粒转染的结肠癌细胞株SW480及Lovo的iASPP mRNA表达增高,细胞凋亡率下降。结论成功构建了重组表达质粒pcDNA3.1(+)-iASPP,并成功在结肠癌细胞株SW480及Lovo中获得了表达,细胞株的凋亡率下降,提示抑制iASPP高表达有可能成为恢复P53抑癌功能的新策略。     

ETV4在结肠癌中的表达及其对结肠癌侵袭能力的影响

目的 分析E26转录因子变异体4(ETV4)的表达与结肠癌临床病理参数间的关系,并探讨其对结肠癌细胞侵袭能力的作用及分子机制.方法 通过GEPIA和R2在线生物信息学分析并挖掘结肠癌中高表达基因.免疫组化法检测结肠癌组织及配对癌旁组织中ETV4蛋白表达情况,并分析ETV4表达与结肠癌病理参数之间的关系.RT-qPCR检测结肠癌细胞系和永生化正常人结肠上皮细胞中ETV4的mRNA表达情况.在LOVO细胞中沉默或过表达ETV4后,用Transwell法观察细胞侵袭能力的改变.用生物信息学分析预测ETV4的下游基因,并通过Western blot进行验证.结果 GEPIA和R2在线生物信息学分析结果显示,ETV4和MMP7在结肠癌癌组织中高表达.免疫组化结果显示,与配对癌旁组织比较,ETV4在癌组织中表达升高,且其表达水平与肿瘤的T、N、M分期正相关.RT-qPCR结果显示,结肠癌细胞系HCT116、LS174T、SW480、LOVO、SW620和RKO中ETV4 mRNA的表达水平明显高于永生化正常人结肠上皮细胞FHC.Transwell实验结果显示,ETV4促进LOVO细胞的侵袭能力.R2在线分析发现ETV4和MMP7呈正相关.Western blot结果显示,在LOVO细胞中ETV4对MMP7具有正向调控作用;同时恢复实验显示,沉默MMP7逆转了ETV4过表达对LOVO细胞侵袭能力的促进作用.结论 ETV4在结肠癌组织及细胞系中表达升高,并通过上调MMP7基因促进结肠癌的侵袭能力.     

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.     

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.     

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.