RSRC2影响三阴性乳腺癌细胞增殖迁移及侵袭的初步研究

目的:探究富含精氨酸/丝氨酸卷曲螺旋2（RSRC2）对三阴性乳腺癌细胞生物学行为的影响。方法:将RSRC2过表达、降表达和空载体质粒通过慢病毒包装转染至三阴性乳腺癌细胞MDA-MB-231中,通过Western blot检测转染效果,通过Transwell 迁移实验、侵袭实验、划痕实验检测RSRC2对MDA-MB-231细胞迁移、侵袭能力的影响,通过平板克隆形成实验检测RSRC2对MDA-MB-231细胞增殖能力的影响。结果:Western blot结果显示RSRC2过表达和降表达质粒成功转染至MDA-MB-231细胞中,Transwell迁移实验、侵袭实验、划痕实验、平板克隆实验显示,RSRC2过表达的MDA-MB-231细胞其迁移、侵袭及增殖能力减弱,RSRC2降表达的MDA-MB-231细胞其迁移、侵袭及增殖能力增强（均P<0.05）。结论:RSRC2在三阴性乳腺癌细胞增殖、迁移及侵袭过程中起着重要的负调控作用。     

miR-99a对乳腺癌细胞侵袭和迁移的负向调控机制研究

目的探讨miR-99a对乳腺癌细胞侵袭及迁移的影响,并初步分析其影响乳腺癌细胞侵袭及迁移的可能分子机制。方法利用荧光实时定量PCR检测乳腺癌细胞系MDA-MB-231和MCF-7中miR-99a的表达。运用脂质体介导的转染方法分别将miR-99a模拟物（miR-99amimics）、miR-99a抑制物（miR-99ainhibitors）以及相应对照miRNA转染MDA-MB-231和MCF-7细胞,通过Transwell侵袭实验检测细胞的侵袭力;采用Transwell迁移实验及划痕实验检测细胞的迁移能力;利用生物信息学方法预测miR-99a的靶基因,并对靶基因进行验证。结果（1）高转移潜能的MDA-MB-231细胞中miR-99a表达明显低于低转移潜能的MCF-7细胞,划痕实验中转染miR-99amimics的与转染controlmimics的MDA-MB-231细胞比较迁移能力显著减弱（P＜0.05）,而MCF-7细胞转染miR-99ainhibitors后迁移能力明显增强（P＜0.01）。（2）Transwell的侵袭及迁移实验显示,转染miR-99amimics后MDA-MB-231细胞的侵袭和迁移能力明显减弱（P＜0.01）;MCF-7细胞转染miR-99ainhibitors后迁移能力增强（P＜0.01）,而侵袭能力基本不变（P＞0.05）。（3）生物信息学方法预测微管相关蛋白（MTMR3）是miR-99a的靶点,实时定量PCR和3′UTR荧光素酶报告基因实验验证了该靶点。（4）干扰了MTMR3后MDA-MB-231细胞的迁移和侵袭能力明显减弱。结论（1）miR-99a对乳腺癌细胞的侵袭及迁移发挥负向调控作用。（2）miR-99a可能通过靶向于MTMR3发挥其对乳腺癌细胞迁移和侵袭的调控作用。     

miRNA-4465过表达对乳腺癌 MDA-MB-231细胞迁移侵袭的影响及其机制

目的：探讨 miRNA-4465的过表达对乳腺癌 MDA-MB-231细胞迁移侵袭能力的影响及机制。方法将 miR-NA-4465的模拟物（minics）转染入 MDA-MB-231细胞,通过实时定量 PCR 检测 miRNA-4465的表达变化;运用 Transwell实验检测转染后细胞迁移和侵袭能力的改变。采用生物信息学技术预测 miRNA-4465的潜在靶基因,并通过荧光报告载体实验及 Western blot 加以证实。结果与阴性对照组相比,转染 miRNA-4465 minics 组 miRNA-4465表达水平明显升高（P ＝0．001）。Transwell 迁移实验结果显示转染 miR-NA-4465 minics 组细胞迁移能力减弱（P ＝0．001）;Transwell侵袭实验结果显示转染 miRNA-4465 minics 组细胞侵袭能力降低（P ＝0．010）。通过生物信息学技术预测了 EZH2为miRNA-4465的潜在靶基因;荧光报告载体实验结果显示转染 miRNA-4465 minics ＋psiCHECK2-EZH23’UTR（野生型）后荧光素酶活性较转染阴性对照序列（NC）＋psiCHECK2-EZH23’UTR（野生型）降低66％（P ＝0．001）。此外将 miR-NA-4465转染入 MDA-MB-231细胞后 EZH2蛋白表达降低。结论 miRNA-4465能降低 MDA-MB-231细胞的迁移和侵袭能力,这个过程可能是通过调控 EZH2基因的表达实现的。     

HER3影响辐照诱导的Luminal A型乳腺癌细胞的迁移和侵袭

目的：探讨敲低人表皮生长因子受体?3（human epidermal growth factor receptor?3,HER3）表达及联合辐照对Luminal A型乳腺癌细胞迁移和侵袭能力的影响及分子机制。方法：利用短发夹RNA（shRNA）稳定转染乳腺癌细胞MCF?7和ZR75?1,敲低HER3表达。蛋白免疫印迹实验（Western blot）检测细胞转染效率。将转染空病毒的对照组（shRNA?Control）与HER3敲低组（shRNA?HER3）细胞进行6?Gy X线辐照,细胞划痕实验及侵袭实验（Transwell assay）分析各组乳腺癌细胞迁移和侵袭能力。Western blot检测紧密连接蛋白Claudin?1的表达变化。结果：敲低HER3后,乳腺癌细胞MCF?7和ZR75?1内HER3蛋白表达水平明显降低。单纯辐照的乳腺癌细胞,迁移和侵袭能力增强,Claudin?1蛋白表达增加;而敲低HER3及联合辐照后,乳腺癌细胞迁移和侵袭能力降低,Claudin?1蛋白表达明显降低。结论：敲低HER3表达可以降低辐照诱导的luminal A型乳腺癌细胞的迁移和侵袭能力,其机制可能通过下调紧密连接蛋白Claudin?1的表达而实现。     

ALG3通过JAK/STAT通路促进三阴性乳腺癌增殖、侵袭和迁移

目的 探讨ALG3对乳腺癌细胞增殖、侵袭和迁移以及JAK/STAT信号通路的影响。方法 Western blot检测正常人乳腺上皮细胞HBL-100和乳腺癌细胞株SKBr3、MCF-7、MDA-MB-231中ALG3的表达情况。Western blot检测过表达和敲减序列的转染效率。先将实验分为control组(Lip 2000)、NC-KD组(Lip 2000+si-NC)和ALG3-KD组(Lip 2000+ALG3-siRNA),CCK-8,集落克隆和体内裸鼠成瘤实验检测乳腺癌细胞增殖能力；划痕实验和Transwell实验检测乳腺癌细胞侵袭和迁移能力；Western blot检测JAK/STAT通路相关蛋白JAK2/p-JAK2、STAT3/p-STAT3、MMP2、Cyclin D1、Bax表达情况。再将实验分为NC-OE组(Lip 2000+mimic-NC)、ALG3-OE组(Lip 2000+ALG3-mimic)和ALG3-OE+WP1066组(Lip 2000+ALG3-mimic+通路抑制剂WP1066),Western blot检测WP1066对JAK/STAT通...     

DTX2通过Notch2/Akt轴促进结直肠癌细胞的迁移和侵袭

目的 探讨DTX2对结直肠癌（CRC）细胞迁移侵袭的影响及作用机制。方法 利用基因工具干预CRC细胞,分为敲低组（DTX2-shRNA）、敲低空载组（neg-shRNA）、未转染组（con）、过表达空载组（pcDNA）及过表达组（pcDNA-DTX2）,利用qRTPCR及Western blotting法检测转染效率。采用划痕和Transwell侵袭实验检测DTX2基因的表达改变对CRC细胞迁移侵袭能力的影响,并通过Western blotting检测各组中Notch2、NICD、Akt、p-AKT、MMP-2及MMP-9蛋白的表达水平。CRC细胞共转染pcDNA-DTX2和Notch2 siRNA,检测回复实验对CRC细胞迁移侵袭能力的影响。结果 CRC细胞中DTX2 mRNA和蛋白表达量,敲低组中明显降低（P<0.01）,过表达组中明显升高（P<0.01）。划痕和Transwell侵袭实验提示,CRC细胞迁移侵袭能力,DTX2 shRNA显著低于con和neg-shRNA(P<0.01);pcDNA-DTX2显著高于con和pcDNA(P<0.01)。Wes...     

miR⁃588在乳腺癌中的表达及对乳腺癌细胞增殖和侵袭的作用

目的：探讨miR?588在乳腺癌中的表达及对乳腺癌细胞增殖、迁移和侵袭能力的影响。方法：用化学合成的miR?588成熟模拟物（miR?588 mimic）过表达miR?588,并验证miR?588过表达率;采用MTT、克隆形成实验检测miR?588对乳腺癌细胞MCF7和MDA?MB?231增殖的影响;采用划痕愈合实验和Transwell侵袭实验检测miR?588对MDA?MB?231细胞迁移和侵袭的影响。结果：miR?588在乳腺癌组织及细胞中的相对表达均低于癌旁正常组织及乳腺正常上皮细胞;过表达miR?588能明显抑制MCF7和MDA?MB?231细胞的增殖;上调miR?588可显著抑制MDA?MB?231细胞的迁移和侵袭能力。结论：miR?588在乳腺癌中低表达,过表达miR?588可抑制乳腺癌细胞的增殖和侵袭。     

siRNA干扰整合素β4的表达对MDA-MB-231乳腺癌细胞侵袭和迁移的影响

目的采用RNA干扰（Small interference RNA,siRNA）技术抑制MDA-MB-231乳腺癌细胞中整合素β4（integrinβ4）基因表达,观察其对细胞侵袭和迁移能力的影响。方法选用MDA-MB-231乳腺癌细胞为研究对象,应用siRNA技术沉默癌细胞Integrinβ4 mRNA的表达,Real-time PCR和Western blotting方法分别检测未转染Integrinβ4 siRNA的乳腺癌细胞组（简称空白对照组）及转染Integrinβ4 siRNA的乳腺癌细胞组（简称Integrinβ4 RNA干扰组）的Integrinβ4 mRNA和蛋白表达水平;Transwell实验检测两组细胞的侵袭及迁移能力。结果与空白对照组相比,Integrinβ4 RNA干扰组Integrinβ4的mRNA及蛋白表达水平分别降低了86%（P〈0.01）和89%（P〈0.01）,细胞侵袭和迁移能力下降（P〈0.05）。结论应用siRNA干扰技术抑制Integrinβ4表达可使MDA-MB-231细胞迁移及侵袭能力降低,提示Integrinβ4在乳腺癌侵袭转移中发挥重要作用。     

瞬时受体电位通道蛋白6在人乳腺癌细胞中的表达及其对乳腺癌细胞侵袭能力的影响

目的：探讨瞬时受体电位通道蛋白6（TRPC6）在人乳腺癌细胞中的表达,阐明TRPC6与乳腺癌细胞侵袭能力的关联性。方法：常规培养的不同侵袭能力乳腺癌细胞株分为MCF-7（低侵袭组）和MDA-MB-231（高侵袭组）,采用Western blotting法和RT-PCR法检测2组中TRPC6蛋白和mRNA的表达;将MDA-MB-231细胞分为空白对照组和SKF96365组,不同浓度SKF96365（5、25和40 μmol/L）预处理MDA-MB-231细胞,阻滞TRPC6通道,采用Transwell实验和细胞划痕实验检测细胞侵袭能力。结果：Western blotting和RT-PCR法检测,高侵袭组MDA-MB-231细胞中TRPC6蛋白及mRNA表达水平均高于低侵袭组MCF-7细胞（P<0.05);划痕实验, 5、25和40　 μmol/LSKF96365组迁移细胞数(76.24±7.54、45.33±4.50和25.12±1.57)均少于空白对照组(130.48±9.55)（P<0.05);Transwell体外侵袭实验,不同浓度SKF96365（5、25和40 μmol/L）组迁移细胞数明显低于对照组(P<0.05)。 结论：高侵袭乳腺癌细胞MDA-MB-231可通过上调TRPC6表达提高乳腺癌细胞的侵袭能力,提示TRPC6在乳腺癌转移中可能起作用。     

c-Ski癌基因对肿瘤相关成纤维细胞活化的影响

目的：研究癌基因c-Ski对乳腺癌相关成纤维细胞（cancer-associated fibroblasts,CAFs）活化的影响。方法：应用基因重组技术,将c-Ski基因克隆到载体pBABE-puro,转染至永生化的正常乳腺成纤维细胞（normal fibroblasts,NFs）,嘌呤霉素药物筛选稳定转染细胞株;Western blot检测转染后c-Ski、α-平滑肌肌动蛋白（α-smooth muscle actin,α-SMA）和成纤维细胞活化蛋白（fibroblast activation protein,FAP）的表达;通过MTT法、流式细胞术、细胞划痕实验及Transwell细胞侵袭实验研究转染前、后成纤维细胞增殖、迁移、侵袭能力的差异;收集细胞上清制备条件培养基,用Transwell细胞侵袭实验分析转染后成纤维细胞对MD-MB-231细胞侵袭能力的影响。结果：成功构建重组质粒pBABE-puro-Ski;Western blot检测结果显示,pBABE-puro-Ski转染组细胞中c-Ski、α-SMA和FAP表达明显高于空白对照组细胞（P=0.000,P=0.001,P=0.002）;MTT法结果显示转染组细胞增殖能力高于空白对照组细胞（P=0.011）;转染组细胞的S期含量（38.58±1.28）明显高于空载体组（25.05±0.52）和空白对照组（26.01±0.86）,差异具有统计学意义（P=0.000）;细胞划痕实验及Transwell细胞侵袭实验结果表明转染组细胞的迁移、侵袭能力均高于空载体组和空白对照组细胞;转染组细胞条件培养基能明显促进MD-MB-231细胞的侵袭能力。结论：c-Ski癌基因表达上调能促进NFs活化为CAFs,并进一步促进乳腺癌细胞MD-MB-231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.     

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.