肿瘤热休克蛋白70-抗原肽复合物抗移植瘤的实验研究

目的 研究肺腺癌有效的治疗方法。方法 采用提纯小鼠肺腺癌LA795细胞热休克蛋白抗原肽复合物，对小鼠背部LA795肺腺癌移植瘤进行治疗。结果 免疫组和免疫 化疗组移植瘤重量明显减少，小鼠生存期明显延长。结论 热休克蛋白70抗原肽复合物有强大的抗肿瘤免疫原性，保护实验动物免受肿瘤细胞的攻击，抑制肿瘤细胞的生长，延长动物生存期。     

梁金菇多糖对荷瘤鼠的抗肿瘤作用和免疫功能影响

目的 :研究梁金菇多糖的抗肿瘤效果及其对荷瘤小鼠免疫功能的调节作用。方法 :建立荷瘤小鼠模型 ,观察梁金菇多糖体内的抑瘤作用 ,采用MTT法检测小鼠的脾淋巴细胞转化、NK杀伤活性及LAK杀伤活性的影响。结果 :梁金菇多糖能够明显抑制荷瘤小鼠的肿瘤生长 ,能够促进正常和荷瘤小鼠的脾淋巴细胞的转化 ,提高刺激指数 ,提高NK杀伤活性及LAK杀伤活性。结论 :梁金菇多糖提高荷瘤小鼠的免疫功能是其掏肿瘤效应的机制之一。     

脐血来源树突状细胞负载热休克蛋白70-肿瘤抗原肽复合物的细胞表型鉴定及其特异性杀伤效应

目的:观察从脐血中提取的树突状细胞负载热休克蛋白70-肿瘤抗原肽复合物的细胞表面表型特点及其所激活的细胞毒T淋巴细胞的杀伤活性。方法:实验于2003-10/2005-12在解放军军事医学院兽医研究所完成。①脐血来源于长春市妇产医院足月顺产的非高危妊娠产妇,要求无急慢性感染及血液系统疾病,产妇及其家属均签署知情同意书。热休克蛋白70由本室制备纯化。人骨肉瘤细胞Saos-2(北京肿瘤研究所,批号222RXB013538)。②封闭式采集脐带血,体外分离脐血单个核细胞,调整浓度为1×109L-1,接种于24孔细胞培养板,每孔均加入100μg/L人重组粒-巨噬细胞集落刺激因子、50μg/L白细胞介素4、10μg/L肿瘤坏死因子α。培养14d后,鉴定细胞表面CD1a、HLA-DR的百分率。③用含胎牛血清的IMDM培养Saos-2骨肉瘤细胞至对数生长期,胰酶消化后调整细胞数为5×107L-1。集传20代生长的Saos-2,冻融2次,低渗震荡,离心,上清用盐酸调pH至7.0,过滤后即为肿瘤抗原肽,与热休克蛋白70体外结合。④另取脐血40mL,分离T淋巴细胞,诱导分化至第4天,加入热休克蛋白70-肿瘤抗原肽100μL,促使T淋巴细胞活化为细胞毒T淋巴细胞。⑤效应细胞(细胞毒T淋巴细胞)与靶细胞(Saos-2)按效靶比50∶1,25∶1,12.5∶1,6.25∶1,3.125∶1梯度,四甲基偶氮唑盐法检测细胞毒T淋巴细胞杀伤活性。结果:①细胞形态学观察:细胞因子诱导后第3天,镜下脐血细胞呈均匀散布的细胞聚体,胞体拉长;第7天可见不明显突起;第14天出现具有典型树枝状突起的树突状细胞。②细胞因子诱导后脐血单核细胞表面表型鉴定结果:经细胞因子诱导后,脐血单核细胞CD1a与HLA-DR表达率均较诱导前明显升高[0,(18.43±3.26)%;(4.03±1.66)%,(59.69±8.47)%;P均<0.01]。③细胞毒T淋巴细胞杀伤活性检测结果:培养72h后,细胞毒T淋巴细胞与靶细胞Saos-2效靶比为50∶1时可杀伤决大部分肿瘤细胞,杀伤率为(97.50±11.24)%,效靶比从50∶1至3.125∶1可使靶细胞Saos-2生长呈梯度受到不同程度的抑制。单纯的树突状细胞及未经激活的T淋巴细胞对Saos-2细胞仅有微弱的生长抑制作用。结论:脐血来源的树突状细胞经过热休克蛋白70-肿瘤抗原肽复合物负载致敏后,激活的细胞毒T淋巴细胞对人骨肉瘤细胞Saos-2具有极强的杀伤能力。     

热休克蛋白70激发树突状细胞抗肝癌作用的实验研究

肿瘤的治疗除了手术、放疗、化疗外,另一个不可忽视的疗法就是免疫治疗,近年来它在肿瘤治疗中的作用越来越受到重视。本实验通过在体外用脐带血诱导树突状细胞（DC）,并用由肝癌细胞SMMC-7721提取的热休克蛋白70-肿瘤肽复合物（HSP70-PC）负载DC,检测其对肝癌细胞的杀伤作用,证明此活化DC对肿瘤细胞的特异性杀伤活性,为临床应用提供实验依据。     

肝癌HSP70-肽复合物抗肿瘤研究

目的 研究肝癌HSP70-肽复合物激活的T淋巴细胞的抗肿瘤作用,探讨其用于临床治疗的可行性.方法 取肝癌手术标本,用凝胶梯度洗脱法从手术切取肝癌组织中提取HSP70-肽复合物,并激活T淋巴细胞,观察激活T淋巴细胞对肝癌细胞的杀伤率.结果 未经肝癌HSP70-肽复合物激活的T淋巴细胞对肝癌细胞的杀伤率明显低于HSP70-肽复合物激活T淋巴细胞对肝癌细胞的杀伤率(P＜0.01).结论 肝癌组织中或细胞中提取的HSP70-肽复合物作用于T淋巴细胞在体外对肝癌细胞能产生高效而特异的杀伤作用,在肿瘤免疫中起重要作用.     

MUC1-2VNTR基因免疫治疗多发性骨髓瘤荷瘤小鼠的实验研究

目的:明确粘蛋白1两串联重复区(MUC1-2VNTR)基因疫苗对多发性骨髓瘤荷瘤BALB/c小鼠的治疗效果。方法:用转染pcDNA3.1-MUC1的P3X63Ag8.653小鼠骨髓瘤细胞皮下接种以建立BALB/c荷瘤小鼠模型。用含MUC1-2VNTR的重组质粒pcDNA3.1-2VNTR/myc-hisB肌肉注射免疫小鼠,采用乳酸脱氢酶法检测细胞毒性T细胞(CTL)反应活性,CCK-8法检测脾淋巴细胞增殖活性。观察小鼠的抑瘤率,肿瘤质量。结果:用重组质粒免疫BALB/c荷瘤小鼠25 d后,重组质粒组肿瘤质量明显轻于空质粒对照组(0.5605±0.2065 g vs.1.521±0.6985g)(P0.01)。重组质粒肌肉注射组CTL、NK细胞活性显著高于对照组;小鼠脾淋巴细胞得到增殖,与空质粒对照组比较,差异非常显著(P0.01)。MUC1-2VNTR基因免疫能诱发小鼠抗肿瘤作用。结论:MUC1-2VNTR基因疫苗可针对多发性骨髓瘤荷瘤BALB/c小鼠诱导产生特异性细胞及体液免疫应答,能诱发小鼠抗肿瘤效应,对多发性骨髓瘤的治疗具有潜在的应用价值。     

人乳腺癌组织中热休克蛋白70-肽复合物（HSP70-PC）的分离纯化

[目的]建立人肿瘤组织中热休克蛋白-70(hsp70)的分离鉴定方法。[方法]用手术切除的人乳腺癌组织为材料来源，制备匀浆，经100，000r／min超速离心处理后，依次上样于肝素-琼脂糖凝胶、ADP-琼脂糖凝胶柱进行亲和层析，所得蛋白经过7．5％与3．4％双层不连续SDS-聚丙烯酰胺凝胶电泳，银染色后经凝胶成像系统分析确定其分子量，再用特异性抗体及碱性磷酸酶标记的二抗做Westem Blot进行蛋白质性质与抗原性鉴定。[结果]经肝素-琼脂糖亲和层析后，在离子浓度0．03-0．23mol／L的梯度洗脱范围内得到富含HSP70的组分，其进一步经ADP-琼脂糖亲和层析，洗脱不结合组分，结合组分经鉴定确系相对分子量约为70000Da的HSP70蛋白。[结论]用上述肝素-琼脂糖凝胶、ADP-琼脂糖凝胶柱进行蛋白质亲和层析法可分离获得纯度高、并保留肿瘤抗原信息的HSP70。     

混合性热休克蛋白/肽疫苗与白细胞介素-12联合诱导特异性细胞毒T淋巴细胞产生的实验研究

目的研究肿瘤组织来源混合热休克蛋白（mHSPs）／肽疫苗诱导产生特异性细胞毒性T淋巴细胞（CTL）的作用及其抗肿瘤免疫效应，为应用其治疗人类恶性肿瘤提供实验依据。方法利用细胞培养、流式细胞技术、蛋白质的分离纯化技术、电泳技术、Western blot检测方法、T淋巴细胞的诱导及体外扩增、乳酸脱氢酶法等。测定肿瘤多肽复合物诱导活化小鼠体内CTL杀伤效应。结果mHSPS免疫组和mHSPs＋Cy＋IL-12免疫组体外诱生的CTL反应活性均比对照组的CTL活性有所升高，尤其mHSPs＋Cy＋IL-12免疫组升高明显，且随效靶比的上升而上升；与正常小鼠（CD4^＋46％、CD8^＋18％）比较。对照组、mHSPs免疫组及mHSPs＋Cy＋IL-12免疫组小鼠CD4^＋亚群的比例分别为38％、46％、54％。CD8^＋亚群的比例分别为26％、22％、16％。结论混合热休克蛋白／肽可诱发强大的抗肿瘤免疫效应，当与IL-12、低剂量Cy者联合应用后免疫功能增强最为明显。     

反义核酸封闭H-2Kd基因对鼠淋巴因子激活杀伤细胞活性的影响

目的 观察反义寡核苷酸封闭鼠淋巴细胞H-2Kd基因后,对其表达的影响及淋巴因子激活杀伤细胞(LAK)的活性改变,探讨淋巴细胞Ⅰ类主要组织相容性抗原(MHC-Ⅰ)在肿瘤免疫中的作用。方法 将人工合成的H-2Kd起始区反义寡核苷酸作用于小鼠脾LAK,流式细胞仪检测作用前后H-2Kd的表达率。噻唑蓝法检测H-2Kd表达降低的LAK对肿瘤杀伤活性。结果 反义寡核苷酸(15μmoL/l)组H-2Kd蛋白的表达率由90.1％±4.4％下降至79.8％±2.5％(P<0.01),H-2Kd降低的LAK杀伤活性明显降低,对K562的杀伤活性由82.3％±3.1％降到60.2％±6.7％(P<0.01),对H22细胞株的杀伤活性由67.4％±2.8％降到53.2％±8.1％(P<0.01)。结论 反义寡核苷酸能够抑制小鼠LAK表面H-2Kd的表达,但不影响LAK增殖活性,因而可导致鼠LAK对同种及异种肿瘤细胞的杀伤活性降低。     

特异性细胞毒T淋巴细胞与脑荷瘤鼠生存期延长的研究

目的 :探索过继性免疫与脑荷瘤鼠的生存期。方法 :瘤苗致敏小鼠的脾细胞在体外再次经瘤细胞和重组白细胞介素 -2(rIL -2)刺激后 ,过继免疫治疗荷瘤鼠。结果 :局部注射细胞毒T淋巴细胞(CTL) rIL -2治疗荷瘤鼠的生存期明显延长。结论 :上述方法获得的效应细胞对G422瘤细胞具有特异性杀伤,局部注射CTL rIL -2能有效的治疗脑胶质瘤并延长其生存期。     

Determination of creatine kinase isoenzyme MB activity in serum using immunological inhibition of creatine kinase M subunit activity. Activity kinetics and diagnostic significance in myocardial infarction.

This is a new method for the determination of creatine kinase isoenzyme MB activity in serum. The method uses direct activity measurement of creatine kinase B subunit activity after blocking of CK-M subunit activity by inhibiting antibodies. The test takes no longer than 15 min. The method yields an intra-serial C.V. of 2.0-12.9%, and a C.V. from day to day of 5.5%. The detection limit is 3.4 U/l creatine kinase MB. In the 95 cases with proven myocardial infarction several types of creatine kinase MB activity kinetics could be determined. The percentage of creatine kinase MB of peak CK-total is 6-25%, with a mean of 11.1%. The amount of creatine kinase MB with respect to total CK activity after reinfarction is higher than the amount after initial infarction.     

Dissociable correlates of two classes of retrieval processing in prefrontal cortex

Although substantial evidence suggests that the prefrontal cortex (PFC) implements processes that are critical for accurate episodic memory judgments, the specific roles of different PFC subregions remain unclear. Here, we used event-related functional magnetic resonance imaging to distinguish between prefrontal activity related to operations that (1) influence processing of retrieval cues based on current task demands, or (2) are involved in monitoring the outputs of retrieval. Fourteen participants studied auditory words spoken by a male or female speaker and completed memory tests in which the stimuli were unstudied foil words and studied words spoken by either the same speaker at study, or the alternate speaker. On "general" test trials, participants were to determine whether each word was studied, regardless of the voice of the speaker, whereas on "specific" test trials, participants were to additionally distinguish between studied words that were spoken in the same voice or a different voice at study. Thus, on specific test trials, participants were explicitly required to attend to voice information in order to evaluate each test item. Anterior (right BA 10), dorsolateral prefrontal (right BA 46), and inferior frontal (bilateral BA 47/12) regions were more active during specific than during general trials. Activation in anterior and dorsolateral PFC was enhanced during specific test trials even in response to unstudied items, suggesting that activation in these regions was related to the differential processing of retrieval cues in the two tasks. In contrast, differences between specific and general test trials in inferior frontal regions (bilateral BA 47/12) were seen only for studied items, suggesting a role for these regions in post-retrieval monitoring processes. Results from this study are consistent with the idea that different PFC subregions implement distinct, but complementary processes that collectively support accurate episodic memory judgments.     

俯卧位通气对高海拔地区肺复张治疗无效急性呼吸窘迫综合征患者氧合的影响

目的 探讨俯卧位通气对高海拔地区肺复张术(RM)治疗无效急性呼吸窘迫综合征(ARDS)患者的治疗作用.方法 从海拔2260m的地区医院筛选RM治疗无效的41例ARDS患者[平均氧合指数( PaO2/FiO2)较RM前升高＜20％视为RM无效],依不同病因分为肺内源性ARDS组(ARDSp组)和肺外源性ARDS组(ARDSexp组),每组再按信封法随机分为俯卧位组和仰卧位组,即ARDSp俯卧位组(11例)、ARDSp仰卧位组(9例)、ARDSexp俯卧位组(10例)、ARDSexp仰卧位组(11例).在通气前及通气1、2、3、4h监测动脉血氧分压( PaO2)、PaO2/FiO2、静态顺应性(Cst)、气道阻力(Raw)的变化.结果 通气lh时,ARDSexp俯卧位组PaO2/FiO2( mm Hg,l mm Hg=0.133 kPa)即较通气前显著升高(157.4±40.6比129.3±48.7,P＜0.05),并随通气时间延长呈持续增高趋势,4h达峰值(219.1 ±41.1);且ARDSexp俯卧位组通气3h内PaO2/FiO2较其他3组显著增高,另3组间则差异无统计学意义.ARDSp俯卧位组、ARDSexp俯卧位组通气4h时PaO2/FiO2均较相应仰卧位组显著增高(208.8±39.7比127.4±47.1,219.1±41.1比124.9±50.8,均P＜0.05).4组通气前后Cst无显著改变,各组间差异也无统计学意义.ARDSp俯卧位组通气4h时Raw(cmH2O·L-1·s-1)较通气前显著降低(6.8±1.7比10.7±1.8,P＜0.05),且明显低于其他3组;其他3组各时间点Raw组内及组间比较差异均无统计学意义.结论 俯卧位通气作为ARDS机械通气重要策略之一,可以改善RM无效高原ARDS患者的氧合,为抢救患者赢得宝贵的时间.     

Digital imaging among medical facilities

The Department of Veterans Affairs (VA) in the USA operates a network of 172 medical centres which all utilize a hospital information system (HIS) which has been developed and is currently maintained by the VA. During the past several years, an image management and communication module has been developed, installed and clinically utilized at the Washington DC and Maryland VA Medical Centres. This image management and communication system, referred to as the decentralized hospital computer program (DHCP) imaging system, is fully integrated with a commercial picture archiving and communication system (PACS). The system is utilized to capture, archive, and display all images generated within the hospital including radiology, nuclear medicine, pathology, endoscopy, bronchoscopy, and dermatology, intraoperative photographs, ECG data, and a limited number of paper documents. The ultimate goal of the project is to have all patient text and image data available at any clinical workstation to any authorized user anywhere within the network of medical centres. Clinical requirements for an imaging workstation include ease of use, rapid and reliable access to the complete set of patient information, and images which are of acceptable quality to meet the requirements of the user and the subspecialty. Patient confidentiality and data security must be safeguarded at all times. Integration of the images with the remainder of the patient's database was found to be critical to the success of the project. The experience at the Washington and Maryland facilities suggests that an imaging system that is successfully integrated with a hospital information system can provide substantial clinical and economic benefits both within and among medical centres. Clinical acceptance and utilization of the system has been excellent, particularly in diagnostic radiology where DHCP Imaging has been interfaced to a commercial PAC system. Based upon this initial experience, the VA has begun to deploy the system throughout its large network of medical centres.     

Myocardial elastography at both high temporal and spatial resolution for the detection of infarcts

Myocardial elastography is a novel method for noninvasively assessing regional myocardial function, with the advantages of high spatial and temporal resolution and high signal-to-noise ratio (SNR). In this paper, in-vivo experiments were performed in anesthetized normal and infarcted mice (one day after left anterior descending coronary artery [LAD] ligation) using a high-resolution (30 MHz) ultrasound system (Vevo 770, VisualSonics Inc., Toronto, ON, Canada). Radiofrequency (RF) signals of the left ventricle (LV) in longitudinal (long-axis) view and the associated electrocardiogram (ECG) were simultaneously acquired. Using a retrospective ECG gating technique, 2-D full field-of-view RF frames were acquired at an extremely high frame rate (8 kHz) that resulted in high-quality incremental displacement and strain estimation of the myocardium. The incremental results were further accumulated to obtain the cumulative displacements and strains. Two-dimensional and M-mode displacement images and strain images (elastograms), as well as displacement and strain profiles as a function of time, were compared between normal and infarcted mice. Incremental results clearly depicted cardiac events including LV contraction, LV relaxation and isovolumetric phases in both normal and infarcted mice, and also evidently indicated reduced motion and deformation in the infarcted myocardium. The elastograms indicated that the infarcted regions underwent thinning during systole rather than thickening, as in the normal case. The cumulative elastograms were found to have higher elastographic SNR (SNR(e)) than the incremental elastograms (e.g., 10.6 vs. 4.7 in a normal myocardium, and 6.0 vs. 2.4 in an infarcted myocardium). Finally, preliminary statistical results from nine normal (m = 9) and seven infarcted (n = 7) mice indicated the capability of the cumulative strain in differentiating infracted from normal myocardia. In conclusion, myocardial elastography could provide regional strain information at simultaneously high temporal (>/=0.125 ms) and spatial ( approximately 55 microm) resolution as well as high precision ( approximately 0.05 microm displacement). This technique was thus capable of accurately characterizing normal myocardial function throughout an entire cardiac cycle, at the same high resolution, and detecting and localizing myocardial infarction in vivo.     

Clinical Pharmacokinetics of Morphine

Morphine, the most widely used mu-opioid analgesic for acute and chronic pain, is the standard against which new analgesics are measured. A thorough understanding of the pharmacokinetics of morphine is required in order to safely and effectively use this analgesic in a wide variety of patients with different levels of organ function. A MEDLINE search was conducted to identify literature published between 1966 and January 2002 relevant to the pharmacokinetics of morphine. These publications were reviewed and the literature summarized regarding unique and clinically important elements of morphine disposition relative to its parenteral administration (including intravenous, intramuscular, subcutaneous, epidural and intrathecal administration), absorption profile (immediate release, controlled release, and sublingual/buccal, and rectal administration), distribution, and its metabolism/ excretion. Special populations, including infants, elderly, and those with renal/liver failure, have a unique morphine pharmacokinetic profile that must be taken into account in order to maximize analgesic efficacy and reduce the risk of adverse events.