人CD38分子基因的克隆及其在真核细胞中的表达

目的：克隆并表达人CD38抗原分子的全长cDNA。方法：采用RT－PCR法，从高表达CD38抗原的Daudi细胞系中克隆出CD38全长cDNA并将其插入pGEM-T载体中，重新设计引物，引入酶切 位点，二次PCR；从重组pGEMT载体上扩增CD38抗原分子的cDNA编码序列，再亚克隆至真核表达载体pcDNA3.1( )，用脂质体法转染COS7细胞。用免疫荧光及A－PAAP方法检测CD38抗原的表达。结果：克隆的CD38抗原的全长cDNA，经酶切鉴定及序列分析表明，其序列与文献报道完全一致。免疫荧光及APAAP方法检测表明，CD38抗原分子在COS7细胞中获得表达。结论：成功构建了CD38真核表达载体，并在COS7细胞中获得表达，对研究CD38分子的功能具有重要的意义。     

调节性T细胞在宫颈癌外周血及肿瘤组织中的检测及其相关性研究

目的：探讨CD4^＋CD25^＋ CD127^low 调节性T细胞（Treg）在宫颈癌患者外周血及肿瘤组织中的表达特点及临床意义。方法：采用流式细胞技术及免疫组化SP法,检测30例宫颈癌、60例宫颈非典型增生（CIN）患者及25例正常人群志愿者外周血及肿瘤组织中Treg细胞表达率。结果：宫颈癌患者外周血及肿瘤组织中Treg细胞表达率明显高于宫颈CINⅠ～Ⅱ组、正常对照组（P〈0．05）,且CINⅢ、宫颈癌患者外周血中的Treg细胞表达率与肿瘤组织中Treg细胞表达率呈正相关（rs分别为0．453、0．702,P〈0．05）。结论：Treg细胞在宫颈癌外周血及肿瘤组织中高表达,是一个较好反映宫颈癌患者免疫功能状态的参考指标,外周血中Treg细胞的表达率可间接反映宫颈组织肿瘤病变状况。     

小细胞肺癌细胞系H446中肿瘤干细胞的分选与鉴定

 目的 明确能否以表面黏附分子CD133为干细胞标志物,使用免疫磁珠分选（magnetic cell separation,MACS）法分离小细胞肺癌细胞系H446细胞中的肿瘤干细胞。方法 以CD133为特异性分子标志物,使用MACS方法分选H446细胞,比较CD133阳性（CD133+）细胞与CD133阴性（CD133-）细胞在集落形成、自我更新、增殖分化、侵袭性、耐药性和成瘤性方面的不同。结果 CD133+细胞和CD133-细胞在集落形成能力、自我更新能力、增殖能力、分化能力、侵袭能力和耐药性方面基本相同。集落形成和成瘤性实验结果显示：CD133+或CD133-细胞中40%以上的细胞都能够形成含有100个细胞以上的大集落,增殖成为与未分选细胞相同的细胞群,并能在裸鼠身上成瘤。结论 表面黏附分子CD133不能作为分选小细胞肺癌H446细胞系中肿瘤干细胞的分子标志物,分选后的CD133+与CD133-细胞中含有相同的肿瘤干细胞。     

真核双基因表达载体pBud-TRAIL-IL-24的构建及其在宫颈癌细胞中的表达

目的：构建肿瘤坏死因子相关凋亡诱导配体(Tumor necrosis factor related apoptosis inducing ligand,TRAIL)基因和白细胞介素24(Interleutin-24,IL-24)基因的真核双基因表达载体,并观察其对宫颈癌细胞生长的影响情况。方法:采用分子克隆技术,将IL-24基因插入真核表达载体 pBud-TRAIL中,构建双表达质粒 pBud-TRAIL-IL-24,经酶切和测序鉴定后,转染宫颈癌HeLa细胞。荧光显微镜和免疫荧光双标技术检测TRAIL和IL-24在HeLa细胞中的表达。MTT法检测重组质粒对HeLa细胞生长状况的影响。结果:成功构建了真核双基因表达载体pBud-TRAIL-IL-24,并在宫颈癌细胞中表达;转染48 h后可见重组质粒对宫颈癌细胞生长有明显抑制作用(P<0.05)。结论:构建的双表达载体pBud-TRAIL-IL-24能在宫颈癌细胞中表达,并可有效抑制宫颈癌HeLa细胞增殖,为进一步研究TRAIL和IL-24联合应用于宫颈癌的基因治疗奠定了基础。     

姜黄素调控LncRNA MEG3/miR-21/PTEN通路促HPV阳性宫颈癌细胞凋亡

目的：探究姜黄素杀伤HPV阳性宫颈癌细胞的作用机制。方法：采用细胞活性检测实验和细胞凋亡检测实验分析姜黄素对HPV阳性宫颈癌细胞活性以及细胞凋亡的影响;采用实时定量PCR实验和蛋白质印迹（Western blot）法检测姜黄素促进HPV阳性宫颈癌细胞凋亡过程中关键信号通路因子LncRNA MEG3、miR-21、PTEN和活化的caspase3的变化水平。结果：姜黄素显著促进了HPV阳性宫颈癌细胞凋亡,升高了LncRNA MEG3、PTEN和活化的caspase3的表达水平,下调了miR-21的表达（P＜0.05）;利用siRNA敲低LncRNA MEG3的表达,能够逆转姜黄素对miR-21、PTEN和caspase3的调控（P＜0.05）。结论：姜黄素可能通过上调LncRNA MEG3而降低miR-21的表达,从而上调PTEN的表达,最终促进HPV阳性宫颈癌细胞的凋亡。     

CD158a表达调节胃癌患者CTL细胞功能的体外研究

目的 研究CD158a表达对胃癌CTL细胞体外功能的影响.方法 利用免疫磁珠法分选培养表达与不表达CD158a的两型细胞,MTT法测定其体外杀伤胃癌MNK45细胞的活性,Elispot试验检测其分泌细胞因子的变化,RT-PCR方法 检测其mRNA的表达差异及CD158a受体激发后(Trigerring)的细胞毒T细胞凋亡情况.结果 CD158a+ CTL几乎不杀伤人胃癌MNK45细胞,但阻断相应CD158a分子或靶细胞MHC-1分子后,杀伤活性得到一定程度恢复.CD158a- CTL以分泌γ-IFN为主,参与Th1反应,而CD158a+ CTL主要分泌IL-4,可能介导Th2反应.3例胃癌患者的CD158a+ CTL细胞均可扩增出300 bp的P58.2 cDNA特征性条带.CD158a分子激发后,CD158a+ CTL凋亡细胞具有显著的形态学特征,DNA电泳有典型的"ladder"梯型条带.结论 CD158a表达抑制胃癌CTL体外杀瘤活性,影响其细胞因子的分泌,并在激发后诱导CTL细胞凋亡.     

人子宫颈癌PD-L1的表达及其与肿瘤内浸润T细胞的相关性研究

目的 观察负性免疫调节分子程序性死亡配体1(programmed death receptor-ligand 1,PD-L1)在人子宫颈癌的表达及其与肿瘤内T淋巴细胞浸润的关系.方法 免疫组织化学染色观察5例正常宫颈组织、7例高级别宫颈上皮内瘤变(cervical intraepithelial neoplasia,CINⅡ-Ⅲ)组织和67例子宫颈癌组织中PD-L1的表达和淋巴细胞PD-1的表达,免疫荧光染色观察相应组织内浸润的CD4+T和CD8+T细胞数量,TUNEL法检测淋巴细胞凋亡.结果 正常子宫颈上皮不表达PD-L1;宫颈瘤变上皮不表达或弱阳性表达PD-L1,70%(47/67)的宫颈癌表达PD-L1.肿瘤内浸润的部分淋巴细胞表达PD-1.子宫颈瘤变上皮与子宫颈鳞状细胞癌PD-L1表达存在明显差异(P<0.01),浅表浸润的子宫颈鳞状细胞癌PD-L1 的表达略低于深部浸润的鳞状细胞癌,但无显著差异;PD-L1阳性病例肿瘤局部CD8+T细胞数量显著减少,且PD-L1 表达与CD8+T细胞数量明显负相关(r=-0.82,P<0.01),但与肿瘤局部CD4+T细胞数量无明显相关性(r=-0.05,P>0.05);部分PD-L1阳性的子宫颈癌局部浸润的淋巴细胞存在凋亡现象.结论 子宫颈癌细胞表达PD-L1,且PD-L1的表达与肿瘤局部浸润的CD8+T细胞数量减少有关,但与浸润的CD4+T细胞数量无关.癌细胞表达PD-L1可能促进了肿瘤内浸润的淋巴细胞凋亡.     

宫颈癌中影响预后的生物分子及免疫细胞浸润

目的 探讨预测宫颈癌预后的潜在生物标志物和免疫浸润细胞,为宫颈癌治疗和预后研究提供理论依据和研究方向。方法 基因表达综合数据库下载宫颈癌组织的芯片数据集(GSE9750),R的“limma”包鉴定差异表达基因,使用R的“clusterProfiler”包进行基因本体论和京都基因与基因组百科全书富集分析。用STRING构建蛋白-蛋白互作网络。利用基因表达谱交互式分析平台验证宫颈癌肿瘤组织和正常样品中抑制素βA(inhibin beta A,INHBA)的表达水平并预测预后。在R中经“e1071”、“parallel”和“preprocessCore”包用“CIBERSORT”软件获得宫颈癌组织样本的免疫浸润细胞表达情况。结果 最终鉴定出与宫颈癌预后高度相关的关键基因INHBA,宫颈癌组织中INHBA的表达水平明显升高,高表达组INHBA的生存周期明显短于低表达组。与正常宫颈组织样本相比,宫颈癌样本中初始CD4⁺ T细胞、未活化的自然杀伤细胞、M0和M1巨噬细胞均升高;而未活化的CD4⁺记忆性T细胞、单核细胞、未活化的树突状细胞在宫颈癌样本中表达...     

CD90作为卵巢癌干细胞标志物的相关研究

目的 研究 CD90 作为分选卵巢癌干细胞标志物的可行性。方法 选择笔者实验室制备的人卵巢癌顺铂耐药细胞株 SKOV3-MD3,采用 CD90 单抗染色,应用流式细胞仪分选获得 CD90⁺ 细胞及 CD90-细胞。体外实验检测两个不同亚群细胞的体外悬浮球形成能力(自我更新能力)、分化潜能、表达干细胞标志物的差异,体内动物实验检测不同亚群细胞皮下成瘤能力是否有差异。结果 人卵巢癌细胞 SKOV3-MD3 中存在具有干性特征的 CD90⁺ 细胞,CD90⁺ 细胞比CD90- 细胞具有更高的体外悬浮球形成能力(自我更新能力)及分化潜能,表达更高水平的干细胞标志物,以及具有更强的皮下成瘤能力,差异有统计学意义。结论 CD90 可能是分选卵巢癌干细胞的分子学标志物。     

宫颈组织中Th、树突状细胞及自然杀伤细胞的表达及与免疫功能的关系

目的 探讨正常宫颈、CIN、宫颈癌组织的局部免疫功能差异及HPV感染与宫颈局部免疫功能的关系.方法 采用免疫组化和图像分析技术,通过CD4、S-100、CD57抗体测定正常宫颈组织、CIN宫颈组织、宫颈癌组织中Th细胞、树突状细胞、NK细胞的形态、数量、分布及表达强度.结果 在癌前病变中,CD4+ Th细胞、S-100+ DC细胞和CD57+ NK细胞随着病变加重而逐渐增多(P<0.05),表达强度增大;而在发生癌变之后,宫颈局部的免疫细胞减少(P<0.05),表达强度下降.结论 人宫颈癌前病变组织局部的免疫功能比较活跃,而癌变的组织免疫功能降低.     

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.