可溶性HLA-G1抑制人自然杀伤细胞NK92 对猪内皮细胞PED的黏附

目的 研究可溶性HLA-G1(HLA-G5)对人自然杀伤细胞(NK细胞)黏附到猪血管内皮细胞(PED)的抑制作用,从而降低人NK细胞对猪血管内皮细胞杀伤杀伤活性.方法 利用核转染技术将pcDNA3-HLA-G5转入LCL721.221细胞株.以RT-PCR、Dot-ELISA技术分别在基因水平和蛋白水平检测HLA-G5的表达;以人类NK细胞系(NK92)效应细胞,猪内皮细胞系(PED)为靶细胞,检测NK92在静止和流动状态下对PED的黏附作用;四甲基偶氮唑盐(MTT)法检测HLA-G5抑制NK细胞的杀伤活性.结果 与未加入HLA-G5的对照组相比,NK92在静止和流动状态下对PED的黏附功能明显降低(P＜0.01),且对HLA-G5组PED的杀伤效率均有明显下降(P＜0.01).结论 HLA-G5可以通过抑制NK92对PED的黏附功能,来减轻NK92对PED的杀伤作用.     

α1,3-半乳糖基转移酶基因沉默对NK细胞介导的异种细胞毒作用的影响

探讨α1,3-半乳糖基转移酶（α1,3-GT）基因沉默对人自然杀伤（NK）细胞介导的异种细胞毒作用的影响。将培养的永生化猪血管内皮细胞系细胞株PED分为3组,转染组：PED转染了特异性α1,3-GT核糖核酸干扰分子（SiRNA-1）;错配组：PED转染了错配的SiRNA;空转染组：PED未转染SiRNA。     

食管鳞癌上皮细胞间质转化对NK细胞活化及其生物学作用的研究

目的 探讨食管鳞癌上皮细胞间质化（EMT）对自然杀伤（NK）细胞生物学活性的影响。方法 通过Western blot检测EMT化相关蛋白E-cadherin和Vimentin表达,从6株细胞系中筛选出 EMT化和非EMT化食管鳞癌细胞株;收集10例正常人外周血,负性分选NK细胞;根据共培养不同的条件培养基分为RPMI 1640正常培养基刺激-NK组（NC组）、EC9706细胞上清液刺激-NK组（EC9706组）与KYSE150细胞上清液刺激-NK组（KYSE150组）,将不同的条件培养基加入NK细胞共培养72 h,流式细胞术检测NK细胞表面/胞内分子的表达。10 ng/mL 转化生长因子-β（TGF-β）处理KYSE150细胞株7 d诱导EMT化后,收集KYSE150 EMT和KYSE150上清液,分别与NK细胞共培养,72 h后以流式细胞术检测NK细胞表面/胞内分子的表达、CD107a脱颗粒能力、靶细胞K562杀伤效应、NK细胞增殖及凋亡。结果 6株细胞系中筛选出2株EMT化和4株非EMT化的食管鳞癌细胞株,从中各选出一株（EC9706和KYSE150）用于后续实验;NK细胞负性筛选达到90%以上的纯度,T细胞<1%。不同的食管鳞癌细胞上清液刺激后,3组NK细胞表面活化型、抑制型受体,效应分子结果显示:与NC组和KYSE150组比较,EC9706组活化型受体NKP30、NKG2D、NKP44表达,颗粒酶释放均降低（ P<0.05）;抑制型受体NKG2A和CD158b表达增加（ P<0.05）;NKP46、CD226、CD16表达和穿孔素释放在3组间差异无统计学意义。与KYSE150上清刺激比较,KYSE150 EMT上清刺激后,NK细胞活化型受体NKP30、NKG2D、NKP44表达,穿孔素释放均降低;CD107a脱颗粒作用、靶细胞K562杀伤效应均减弱,NK细胞Ki67增殖指数下降（ P<0.05）;抑制型受体NKG2A和CD158b表达增加（ P<0.05）;NKP46、CD226、CD16表达,颗粒酶释放及NK细胞凋亡在两组间差异无统计学意义。结论 食管鳞癌细胞EMT化可能通过抑制NK细胞活化、减少NK细胞杀伤效应分子的分泌来降低NK细胞杀伤效应,从机体免疫监视中逃逸。     

病毒灭活血浆对人自然杀伤细胞功能影响的体外实验研究

目的：探讨病毒灭活血浆对人自然杀伤细胞（ natural killer cell ,NK细胞）生长、细胞表型以及体外杀伤活性的影响。方法采用病毒灭活血浆和新鲜冰冻血浆分别培养人NK细胞,观察2组培养体系中NK细胞的扩增情况;用流式细胞仪（FCM）检测NK细胞CD3-CD56＋表达以及NK细胞穿孔素、颗粒酶B和CD107a的含量变化;用乳酸脱氢酶法测定NK细胞杀伤SGC-7901细胞活性。结果病毒灭活血浆与新鲜冰冻血浆比较,在促进NK细胞的增殖以及CD3-CD56＋的表达上无明显差异（ P＞0．05）;检测培养10天的NK细胞,病毒灭活血浆培养组细胞穿孔素、颗粒酶B和CD107a以及杀伤活性与新鲜冰冻血浆培养组也无明显差异（P＞0．05）。结论病毒灭活血浆对人NK细胞的增殖、细胞表型和体外杀伤功能均无明显影响。     

NK92细胞的胞吐效应与SNAP-23蛋白转位相关

研究SNAP-23蛋白在NK细胞系NK92细胞胞吐效应中的作用，探讨NK细胞胞吐的分子机制。利用已糖氨酶(β-hexosaminidase)释放实验检测NK细胞的胞吐作用，通过RT-PCR和Western blot显示NK92细胞中SNAF-23a mRNA和蛋白的表达，利用激光共聚焦显微镜扫描术确定靶细胞触发前后SNAP-23a在NK92细胞中的定位。结果显示：NK细胞的胞吐作用在靶细胞刺激后2h明显，4～6h达高峰；NK92细胞组成性表达SNAP-23a mRNA和蛋白，靶细胞刺激前后蛋白表达量虽无明显差异，但其定位发生了显著改变，激光共聚焦显微镜扫描结果显示在静息NK92中SNAP-23a主要表达在细胞质内的颗粒上，经靶细胞刺激后逐渐向细胞膜发生转位，2h明显转位，4～6h几乎完全转位。结果提示：NK细胞的胞吐效应与SNAP-23蛋白转位相关。     

IL-18联合IL-2诱导NK92细胞的实验研究

目的：探讨白细胞介素18（IL 18）联合白细胞介素2（IL 2）对自然杀伤细胞系NK92抗卵巢肿瘤作用的影响。方法：用不同浓度的IL 18联合IL 2（5?U/ml）体外诱导刺激NK92细胞,流式细胞仪检测细胞周期变化。乳酸脱氢酶释放试验观察刺激前后NK92细胞及培养上清对SKOV3体外生长的抑制作用。ELISA测定NK92细胞杀伤活性最大时IFN γ、TNF α含量。结果：IL 18（25?ng/ml）+IL 2（5?U/ml）刺激后的NK92细胞与对照组比较,S期细胞增多,G0/G1期减少（P＜0.05）。效靶比为10∶1时,诱导后NK92细胞对SKOV3细胞作用4?h杀伤活性最高,IL 18+IL 2刺激前后的NK92细胞杀伤率分别为（42.3±4.82）%、（77.63±5.27）%。NK92培养上清36?h对SKOV3杀伤作用达到最高峰,刺激前后上清杀伤率分别为（21.2±3.49）%、（37.14±2.69）%。NK92细胞杀伤活性最大时,上清中IFN γ、TNF α水平明显升高（P＜0.05）。结论：IL 18联合IL 2可提高NK92细胞的抗肿瘤活性,为临床应用NK92细胞免疫治疗卵巢肿瘤提供了实验依据。     

组织芯片检测淋巴瘤中活化的细胞毒性细胞

目的：运用组织芯片及常规病理切片检测活化的细胞毒性细胞在各型淋巴瘤中的表达分布情况，为临床治疗和判断预后提供依据。方法：运用免疫组化方法检测60份淋巴瘤标本制成的组织芯片中穿孔素、颗粒酶B的表达和分布情况，同时选择10份鼻自然杀伤细胞（NK）／T细胞淋巴瘤常规标本与组织芯片进行比较研究，10份淋巴组织反应性增生标本作为对照。结果：在60份淋巴瘤组织芯片中，发生在淋巴结内者48份，淋巴结外者12份。B细胞淋巴瘤42份，T细胞淋巴瘤16份（外周T细胞淋巴瘤10份、NK／T细胞淋巴瘤2份、T淋巴母细胞瘤2份及问变性大细胞淋巴瘤2份），2份霍奇金淋巴瘤。42份B细胞淋巴瘤的瘤细胞均不表达穿孔素和颗粒酶B。10份外周T细胞淋巴瘤中8份表达穿孔素，9份表达颗粒酶B，阳性表达细胞均为非肿瘤细胞。芯片中的2份和常规10份鼻NK／T细胞淋巴瘤中均见穿孔素和颗粒酶B过度表达。T、B细胞淋巴瘤与NK／T细胞淋巴瘤比较有显著性差异（P〈0．01）。结论：穿孔素和颗粒酶B是鉴定活化的细胞毒性细胞的免疫标志物，可作为NK／T细胞淋巴瘤的诊断性标志物；其在T细胞淋巴瘤中的表达反映了机体的抗肿瘤免疫反应机制。组织芯片技术具有高信息量、简捷、高效、实验误差小、重复性好等优点，可作为研究淋巴瘤的有用工具。     

姜黄素对CIK细胞杀伤胃癌细胞株SGC-7901作用的影响及其机制的研究

目的探讨姜黄素对细胞因子诱导的杀伤细胞（CIK细胞）杀伤胃癌细胞株SGC-7901作用的影响及其机制。方法10名健康者外周血单个核细胞（PBMC）在体外经多种细胞因子诱导为CIK细胞;收集培养第5天的CIK细胞,给予不同浓度的姜黄素诱导,37℃、5%CO2条件下继续培养72 h,LDH法检测CIK细胞对SGC-7901细胞的杀伤活性;FACS法检测CIK细胞表型CD3＋CD8＋、CD3＋CD56＋含量及穿孔素、颗粒酶B水平。结果①姜黄素诱导后,CIK细胞杀伤胃癌SGC-7901的活性明显提高,在10μmol/L时杀伤活性显著高于对照组（P〈0.05）。②姜黄素诱导后,CIK细胞内穿孔素和颗粒酶B的水平明显提高（P〈0.05）。③姜黄素能够显著增加CIK细胞的CD3＋CD56＋表达（P〈0.05）,降低其CD3＋CD8＋表达（P〈0.05）。结论姜黄素能提高CIK细胞对胃癌细胞株SGC-7901的杀伤活性,其机制可能与其增加CIK细胞穿孔素和颗粒酶含量以及增加CD3＋CD56＋表达有关。     

穿孔素和颗粒酶B在急性期登革病毒患者体内的表达水平及调控机制初探

目的 登革病毒(Dengue virus,DENV)感染引起的登革热和登革出血热已给世界带来极大危害,明确其感染引起的免疫病理损伤机制尤为关键。方法 以DENV感染急性期患者样本为研究对象,抽取静脉血,利用密度梯度法提取外周单个核细胞（PBMCs）,流式细胞术检测PBMCs各亚群表达穿孔素和颗粒酶B的水平,进而探讨DENV急性期患者T细胞亚群和NK细胞亚群免疫功能的差异。结果 DENV患者CD8⁺T细胞和NK细胞内穿孔素和颗粒酶B的表达水平发生显著上调,细胞脱颗粒水平也显著增加;同时表达穿孔素或颗粒酶B的NK细胞比例显著高于CD8⁺T细胞。另外,本研究还发现三个重要miRNAs （miR-27a*、miR-30e和miR-378）在DENV患者体内的表达量明显降低,已有的研究表明其可能靶向调控穿孔素或/和颗粒酶B的表达。结论 DENV感染可诱导CD8⁺T细胞和NK细胞发挥效应功能,并依赖穿孔素和颗粒酶途径杀伤被病毒感染的靶细胞;DENV患者体内PRF1和GrzB的表达很可能受到miR-27a*、miR-30e和miR-378的靶向调控。通过上述研究为阐明T细胞和NK细胞在DENV感染过程中所发挥的免疫效应功能提供更多的理论基础。     

双重酶免疫组化法识别淋巴细胞亚群中穿孔素的分布

0 引 言穿孔素(perforin)又称成孔蛋白(pore-forming protein,PFP),是一种相对分子质量为67 000,存在于激活的细胞毒T淋巴细胞(CTL)和自然杀伤(NK)细胞胞质杀伤颗粒中的糖蛋白,当CTL和NK细胞与靶细胞相遇时,释放PFP,在Ca2+存在下,于靶细胞膜上形成一个520 nm的活性孔道,使靶细胞渗透压改变而溶解.杀伤颗粒中的颗粒酶等其他成分通过此孔道进入靶细胞,也可诱导靶细胞凋亡［1］.CTL和NK细胞介导的细胞毒作用在抗感染、抗肿瘤免疫和器官移植排斥反应中发挥着重要作用［24］.本文介绍采用双重酶免疫组化染色法检测CD3+T淋巴细胞和CD5…     

APPLICATION OF LORNOXICAM TO PATIENT-CONTROLLED ANALGESIA IN PATIENTS UNDERGOING ABDOMINAL SURGERIES

FOR anesthesiologis s ,treatingpostoperativepainhas alwaysbeen a problem.Althoughopioidshave been provedtobe effective,theirsideeffectscouldnotbeignored.With thedevelopmentofscienceand pharmacology,many drugs with aspectsof satisfactoryanalgesicefficacyand couldbe welltoleratedby patientshave been developed.And lornoxicamisone of them, which isa non-steroidalanti-inflammatorydrug (NSAID ), with analgesic, anti-infl-ammatory,andantipyreticproperties.Itseliminationhalf-time(3 to 5 hours) isle…     

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.     

Acupuncture Combined with Spinal Tui Na for Treatment of Primary Dysmenorrhea in 30 Cases

Objective: To observe the therapeutic effects in acupunture treatment of primary dysmenorrhea combined with spinal Tui Na, and study its mechanism. Methods: Thirty cases of the treatment group were treated by acupuncture combined with spinal Tui Na, and thirty cases in the control group were treated by routine acupuncture. Results: The total effective rate was 93.3% in the treatment group, and 73.3% in the control group, with a significant difference between the two groups (P<0.05). Conclusions: Acupuncture combined with spinal Tui Na has good prospects for treatment of primary dysmenorrhea.     

Luo Lingjie's Experience in Treating Chronic Nephropathy

In treating chronic nephropathy,Luo Lingjie,a chief physician,pays attention to regulating the balance between yin and yang,treating infection if present,and removing pathogenic factors.He prescribes gentle drugs and uses carefully strongly warming-tonifying ones,emphasizes the importance of persuading the patient to persist in treatment with medication and nurse one's health for recuperation,and is good at combined use of TCM and western medicine therapy and brings the merits of various therapies into full play,with obvious theraoeutic effects.     

Dr.Zhang Ren's Experience in the Acupuncture Treatment of Different Diseases with the Same Therapeutic Principle

Dr.Zhang Ren,the chief physician,is the chairman of Shanghai Acupuncture and Moxibustion Association.Having been engaged in medicine for about 40 years,he is experienced in treating various intractable diseases.In his long years of clinical practice,he advocates taking the TCM differentiation as the basis to seek for the acupuncture method for treatment of modern intractable diseases.The author of this essay had the fortune to follow Dr.Zhang in study.The following is a summary of Dr.Zhang's experience in the acupuncture treatment for different intractable diseases with the same therapeutic principle.     

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.     

猪肺磷脂注射液联合经鼻持续气道正压通气对呼吸衰竭早产儿的临床疗效及肌酸激酶同工酶活性的影响

目的 探讨猪肺磷脂注射液联合经鼻持续气道正压通气(NCPAP)对呼吸衰竭早产儿的临床疗效及肌酸激酶同工酶活性(CK-MB)的影响.方法 选取呼吸衰竭早产儿80例,分为观察组和对照组各40例.对照组采用NCPAP给氧治疗,观察组给予NCPAP给氧联合猪肺磷脂气管内给药.观察两组患儿治疗前及治疗12h、24 h后PaO2、PaCO2、血氧饱和度(SaO2)、pH的变化情况,检测治疗前及治疗5d后血清CK-MB水平;评估两组患儿的临床治疗效果.结果 两组患儿PaO2、PaCO2、SaO2、pH比较,差异均有统计学意义(P＜0.05),其中观察组治疗后的PaO2、SaO2、pH均高于对照组,PaCO2则低于对照组.两组的PaO2、SaO2、pH均随观察时间延长而升高(P＜0.05),PaCO2均随观察时间的延长而降低(P＜0.05).观察组治疗有效率为87.5％,显著高于对照组的70.0％ (P ＜0.05).治疗5d后两组患儿血清CK-MB水平均较前降低(P＜0.05),且观察组明显低于对照组(P＜0.05).结论 猪肺磷脂注射液气管内给药联合NCPAP可以显著降低呼吸衰竭早产儿CK-MB的含量,提高治疗有效率,起到很好的呼吸循环支持作用.