环孢素A联合苯基棕榈酰胺吗啡丙醇对K562/AO2细胞GCS基因表达的影响及对逆转多药耐药

目的 研究环孢素A(CsA)和苯基棕榈酰胺吗啡丙醇(PPMP)联合应用对人白血病多药耐药细胞株K562/AO2的葡萄糖神经酰胺合酶(GCS)基因表达的影响及对白血病多药耐药的逆转作用,探索逆转白血病细胞耐药的策略.方法 应用Alamar BlueTM多功能细胞染色法测定阿霉素(ADR)对K562和K562/AO2细胞的半数抑制浓度(IC50)及判断CSA的非细胞毒性剂量;采用RT-PCR法检测GCS基因和mdr1基因的mRNA表达.结果 CsA浓度低于4.5μg/ml对K562/AO2细胞无细胞毒性.ADR对K562和K562/AO2细胞的IC50分别为(0.84±0.04)μg/ml和(89.24±1.27)μg/ml;当1μg/ml CsA和25μmol/L PPMP单用或联合作用于K562/AO2细胞时,ADR的IC50分别为(7.81±0.74)、(17.49±0.53)、(2.82±0.07)μg/ml;而低浓度CsA(0.01μg/ml)和PPMP(10 μmol/1)联合作用时IC50为(16.08±1.25)μg/ml.CsA联合PPMP可明显降低GCS基因的mRNA表达,低浓度CsA和PPMP联用也有此作用.结论 CsA联合PPMP可通过抑制K562/AO2细胞的GCS基因mRNA表达有效逆转其耐药,低浓度CsA联合PPMP在逆转其耐药的同时降低了细胞毒性.     

多药耐药相关蛋白-2在白血病耐药细胞株K562/A02的表达

目的:研究多药耐药相关蛋白-2（MRP-2）与白血病细胞耐药的关系,并探讨其在白血病多药耐药中的意义。方法：应用RT-PCR方法检测白血病细胞株K562与耐药细胞株K562/A02中MRP-2 mRNA的差异表达,然后将MRP-2反义及正义寡核苷酸片段分别用脂质体转染至耐药细胞株K562/A02,采用MTT法、流式细胞术及RT-PCR法检测转染后细胞内阿霉素的荧光强度、耐药指数及MRP-2 mRNA的表达情况。结果：耐药细胞株K562/A02中MRP-2 mRNA表达水平明显高于白血病细胞株K562（P<0.05）。MRP-2反义寡核苷酸片段转染后耐药细胞株K562/A02细胞内的阿霉素浓度升高,耐药指数较未转染细胞明显降低（P<0.05）;转染后耐药细胞株K562/A02细胞MRP-2 mRNA表达水平较未转染细胞下降了67.5%,两者比较差异具有显著性（P<0.05）。结论：MRP-2的高表达导致白血病耐药细胞内化疗药物浓度降低,可能是其导致白血病多药耐药的机制之一。     

HZ08冻干脂质体的制备及其逆转肿瘤多药耐药的研究

制备了HZ08冻干脂质体,考察脂质体冻干前后的包封率、粒径和长期放置稳定性等指标。通过K562/A02细胞逆转阿霉素耐药性实验,考察了HZ08冻干脂质体对逆转阿霉素耐药性作用。结果表明：制备的HZ08脂质体冻干前后包封率、粒径等指标基本相同。12周的放置实验,HZ08冻干脂质体的药物渗漏率为未冻干的25.92%,且脂质体粒径的增幅仅为未冻干的12.32%,冻干后的脂质体稳定性显著提高。10,3,1 μmol/L HZ08脂质体冻干粉对K562/A02细胞具有不同程度的逆转阿霉素的耐药性作用,其逆转倍数分别为50.87、17.75和2.05。分别于3个月、6个月和12月时,取样测定批量生产的HZ08冻干脂质体的各项指标,各项指标无明显变化。     

恶性造血细胞中P-gp 170表达的比较研究

目的:通过研究恶性造血细胞中P-糖蛋白(P-gp)170的表达,为骨髓增生异常综合征(MDS)治疗提供P-gp 170低表达的体外细胞株模型。方法:用含0.1 mmol.L-1阿霉素的RPM I 1640分别培养MDS细胞株MUTZ-1和多药耐药(MDR)红白血病细胞株K562/A02。用MTT法检测细胞的增殖抑制作用,光镜和电镜技术分别观察与阿霉素共同培养后的MUTZ-1细胞和K562/A02细胞形态和超微结构,流式细胞仪分别测定MUTZ-1细胞和K562/A02细胞P-gp 170表达水平。结果:0.1 mmol.L-1阿霉素对MUTZ-1细胞增殖呈时间依赖性抑制作用,而对K562/A02细胞的增殖没有影响;MUTZ-1细胞出现典型的凋亡形态学改变,K562/A02细胞表面呈现出许多长的微绒毛和许多微孔;K562/A02细胞与MUTZ-1细胞相比高表达P-gp 170。结论:P-gp 170在恶性造血细胞中表达有显著性差异,P-gp 170低表达的MUTZ-1细胞株可用于MDS治疗的体外研究模型,P-gp 170高表达的K562/A02细胞株可用于MDR逆转的体外研究模型。     

SiRNA联合asODN靶向逆转白血病细胞多药耐药的研究

目的:探讨靶向多药耐药基因(MDR-1)的siRNA和asODN联合应用逆转人白血病耐药细胞K562/A02的效果。方法:设计并合成针对MDR-1基因同一序列的siRNA和asODN及阴性对照siRNA,采用转染试剂lipofectin2 000分别转染人白血病耐药细胞K562/A02;利用RT-PCR检测MDR-1mRNA和Western Blot检测MDR-1蛋白质的表达;采用罗丹明123外排实验检测P-糖蛋白(P-gp)的转运功能,MTT法检测K562/A02细胞对阿霉素的耐药逆转效果。结果:siRNA、asODNa、sODN和siRNA联合应用均能降低MDR-1mRNA和蛋白质的表达,提高P-gp的转运功能,对阿霉素的敏感性明显恢复,asODN和siRNA联合应用效果明显提高(P<0.05),低浓度的siRNA(200 nmol/L)比高浓度的asODN(5μmol/L)的效果强(P<0.05)。结论:siRNA、asODN能有效地逆转人白血病耐药细胞K562/A02的多药耐药,asODN和siRNA联合应用效果明显加强。     

慢性粒细胞白血病细胞株伊马替尼耐药与MDR1基因表达相关性探讨

目的 探讨慢性粒细胞白血病细胞株伊马替尼耐药与MDR1基因表达相关性.方法 采用Real-time PCR检测MDR1 mRNA表达,Western-blot检测P-gp蛋白表达,MTS法检测细胞药物敏感性.结果 耐伊马替尼细胞株K562/G01MDR1的mRNA表达明显上调,P-gp表达明显上调,维拉帕米能部分逆转K562/G01细胞株对伊马替尼的耐药.结论 MDR1基因参与伊马替尼耐药机制,P-gp抑制剂能改善伊马替尼临床疗效.     

葡萄糖神经酰胺合酶基因在多药耐药肿瘤细胞株K562/AO2中的表达及其与白血病多药耐药的关系

目的观察葡萄糖神经酰胺合酶（GCS）基因在人红白血病多药耐药细胞株K562/AO2的表达及其与肿瘤多药耐药性（MDR）的关系。方法运用Alamar Blue^TM多功能细胞染色法证实K562/AO2的多药耐药性;采用RT-PCR技术检测K562/AO2及其亲本K562细胞株的GCS基因、MDR1基因、bcl-2基因、bax基因的表达水平及其差异。结果（1）阿霉素（ADR）对K562/AO2细胞和K562细胞的IC50分别为75μg/ml和0.65μg/ml,耐药指数为115;长春新碱（VCR）对K562/AO2细胞和K562细胞的IC50分别为8μg/ml和0.22μg/ml,耐药指数为36。（2）K562/AO2细胞GCS基因和MDR1基因的表达明显强于K562细胞;K562/AO2细胞bcl-2基因表达强于K562细胞,而bax基因表达弱于K562细胞。结论GCS基因可能在白血病多药耐药中起着重要的作用,而细胞凋亡基因的表达异常可能是它导致肿瘤多药耐药的主要分子病理机制之一。     

藤黄酸对 K562 细胞 hERG 钾通道蛋白的调控作用

目的 探讨藤黄酸对白血病细胞系 K562 增殖、凋亡、细胞周期的影响,并观察藤黄酸对 hERG 钾通道蛋白的调控作用。方法 K562 细胞以不同浓度藤黄酸 (0.125～8.0 μmol/L) 处理 0～72 h 后,MTT 法观察藤黄酸对 K562 细胞生长抑制的情况,Annexin-V/PI 双标法及透射电镜检测细胞凋亡,PI 单染法检测细胞周期分布,Western blotting、RT-PCR 法分别检测藤黄酸对 K562 细胞内 hERG 通道的调控作用。结果 藤黄酸能明显抑制 K562 细胞增殖,具有时间-剂量依赖性,其 24 h 的 IC₅₀ 为 (2.637±0.208) μmol/L。此外,藤黄酸以浓度依赖性方式诱导 K562 细胞凋亡,并伴随明显的凋亡细胞形态学改变,而藤黄酸的凋亡诱导效应可能与其诱导 K562 细胞周期阻滞于 G₀/G₁ 期有关。hERG 钾通道蛋白在人白血病 K562 细胞中表达量较高,藤黄酸对 hERG 钾通道蛋白及其表达水平均有不同程度的抑制作用,该抑制作用呈明显的量效关系 (P＜0.01)。结论 藤黄酸可通过下调 hERG 钾通道蛋白的表达发挥较强的抗白血病效应,hERG 钾通道有望成为白血病诊治的新靶标。     

汉防己甲素逆转白血病细胞株K562/A02耐药的机制

目的:研究汉防己甲素（TTD）对白血病细胞株K562/A02多药耐药(MDR)逆转的机理。方法：以白血病细胞系K562及其耐药细胞系K562/A02为TTD作用的靶细胞。细胞水平检测实验分5组（K562组、K562/A02组、K562+ADM组、K562/A02+ADM组和K562/A02+TTD+ADM组）,采用MTT法检测TTD对K562和K562/A02细胞的非细胞毒性剂量,流式细胞术检测细胞内阿霉素(ADM)的浓度,基因、酶学、蛋白水平检测实验分3组（K562组、K562/A02组和K562/A02+TTD组）,采用RT-PCR法检测mdr1 mRNA的表达,免疫细胞化学方法检测谷胱甘肽S转移酶π（GST-π）和拓扑异构酶Ⅱ（Topo Ⅱ）的表达水平,Western-blotting法检测P-糖蛋白（P-gp）和bcl-2表达。结果：1.562 5 mg•L^-1的TTD处理K562/A02细胞后,细胞内ADM的浓度较单用ADM组明显提高（P<0.01）;与空白对照组比较,K562/A02细胞内mdr1 mRNA/P-gp的表达量减少（P<0.01）;GST-π和TopoⅡ表达无明显变化;凋亡抑制基因bcl-2的表达量减少（P<0.01）。结论:TTD主要通过增加细胞内ADM浓度,下调mdr1/P-gp和bcl-2表达逆转耐药。     

和厚朴酚对U937/ADR细胞系耐药逆转作用及其机制研究

目的 探讨和厚朴酚（HNK）对白血病细胞U937阿霉素（ADR）耐药细胞系U937/ADR多药耐药逆转作用及其机制。方法 以大剂量（IC₅₀）ADR短时间诱导方法,构建U937/ADR细胞系。以低浓度HNK（IC₂₀）与不同化疗药物联合作用于U937/ADR细胞系,检测其对不同化疗药物耐药逆转倍数。罗丹明123检测药物外排功能;荧光定量PCR（FQ-PCR）和Western blotting检测不同浓度HNK对U937/ADR细胞系核转录因子-κB(NF-κB)和耐药相关基因MDR1及其蛋白P-糖蛋白（P-gp）表达的影响;ELISA法检测NF-κB亚单位p65（NF-κB p65）的DNA结合活性。结果 成功构建了白血病多药耐药细胞系U937/ADR,对ADR耐药指数为亲代U937细胞的11倍。6.5 μg/mL HNK可以逆转U937/ADR对ADR的耐药,逆转倍数为2.20倍。HNK能够浓度依赖性地下调U937/ADR细胞NF-κB、MDR1 mRNA和P-gp表达,抑制NF-κB p65的活性。结论 HNK能有效逆转U937/ADR多药耐药,其机制可能与抑制NF-κB p65活性、下调MDR1和P-gp表达有关。     

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.