慢病毒介导的双自杀基因诱导胃癌细胞凋亡的研究

目的 观察慢病毒介导的KDR启动子驱动的CD/TK双自杀基因体系对胃癌细胞SGC-7901的体外杀伤作用.方法 用重组慢病毒FGW-KDRP-CD/TK体外感染SGC-7901细胞.噻唑蓝(MTT)比色法检测前药对细胞的杀伤效应;透射电镜及流式细胞术观察细胞凋亡及细胞内DNA含量.结果 慢病毒的感染率随病毒滴度的增高而递增.当前药更昔洛韦(GCV)为100 mg/L、5-氟胞嘧啶(5-FC)为320 mg/L时,对未转基因及转基因细胞的抑制率分别为18%、94%,两者筹异有统计学意义(P<0.01).电镜下用药组出现细胞凋亡.流式细胞仪检测显示,随着药物浓度的增加,凋亡细胞所占的比例逐渐增加.结论 KDR启动子可调控双自杀基因系统选择性杀伤SGC-7901细胞,并且该体系可以诱导胃癌细胞凋亡.     

双自杀基因重组腺病毒对胰腺癌细胞特异性杀伤作用

目的 研究腺病毒介导的KDR启动子驱动CD/TK融合基因系统(Ad-KDR-CDTK)对胰腺癌细胞Capan-2特异性的杀伤作用.方法 重组腺病毒体外感染表达KDR的Capaw2细胞株,用不表达KDR的肝癌细胞HepG2做对照.观察其感染效率并以RT-PCR方法 检测转基因细胞CDTK的表达,然后给予不同浓度的前药更昔洛韦(ganciclovir,GCV)和5-氟胞嘧啶(5-fluorocy-tosine,5-FC),MTT法观察该体系对Capan-2和HepG2细胞生长增殖的影响及其旁观者效应;电镜观察细胞的病变;流式细胞仪检测细胞周期的变化和DNA含量的变化.建立Capan-2裸鼠皮下移植瘤模型,瘤内注射Ad-KDR-CD/TK,腹腔注射前药GCV(50 mg·kg-1·d-1)和5-FC(500 mg·kg-1·d-1)14 d,观察肿瘤生长抑制效应.结果 腺病毒对两种细胞株的感染率相似,其感染率随腺病毒滴度的增高而递增.RT-PCR方法 检测发现转染Ad-KDR-CDTK的Capan-2细胞有目的 基因表达.MTT法检测显示前药呈剂量依赖性抑制Capan-2生长,而不表达KDR的肝癌细胞HepG2对前药不敏感,且观察到该体系对Capan-2明显的旁观者效应.电镜下可见Capan-2有凋亡改变.用流式细胞仪测定用药组出现典型的凋亡峰;细胞周期分析显示治疗后细胞G0-G1期比率增多,G2-M及S期细胞减少.在Capan-2裸鼠移植瘤模型中,该双自杀基因系统能够显著抑制肿瘤的生长.结论 KDR启动子可调控双自杀基因体系选择性杀伤胰腺癌细胞Capan-2,诱导胰腺癌细胞凋亡,并可显著抑制人胰腺癌裸鼠移植瘤的生长.     

腺病毒驱动KDR启动子介导CD/TK融合基因系统对血管内皮细胞的靶向杀伤作用

目的　探讨腺病毒介导KDR启动子驱动的融合基因体系,对人脐血管内皮细胞ECV30 4的选择性杀伤作用。方法　将质粒pAdEasy -KDR- CDglyTK在2 93细胞内包装、扩增后,体外感染表达KDR的ECV30 4细胞株和不表达KDR的LS174T细胞株,并给予不同浓度的前药5 - 氟胞嘧啶(5 -fuorocytosine ,5 -FC)和/或丙氧鸟苷(ganciclovir,GCV) ,观察该体系对不同细胞株的杀伤效应及其旁观者效应;并以流式细胞仪检测细胞周期的变化,电镜观察细胞的病变。结果　所得病毒对两种细胞细胞的感染率相似,其感染率随腺病毒滴度的递增而增加;表达KDR的ECV30 4细胞对前药的具有较高的敏感性,不表达KDR的LS174T细胞对前药不敏感(P均<0 . 0 1) ;融合基因的疗效优于任一单自杀基因(P均<0 . 0 1) ;且观察到该体系明显的旁观者效应。流式细胞术检测治疗后ECV30 4细胞G1期比率增多及S期细胞减少(P均<0 . 0 1) ,同时,电镜下可见ECV30 4有凋亡和坏死改变。结论　KDR基因启动子可调控融合基因体系选择性杀伤人血管内皮细胞。     

KDR启动子驱动的双自杀基因对实体瘤细胞及血管内皮细胞的靶向杀伤作用

目的腺病毒介导KDR启动子驱动的CDglyTK融合基因(AdKDRCDglyTK)体系对实体肿瘤细胞及血管内皮细胞靶向杀伤作用。方法分别将KDR和CMV启动子融合基因腺病毒体外感染表达KDR的ECV304、MCF7、MGC803及SW620细胞株和不表达KDR的LS174T细胞株,并给予不同浓度的前药5FC和/或GCV,观察其对各细胞株的杀伤效应及其旁观者效应。结果两种腺病毒对各细胞的感染率随腺病毒滴度的递增而增加,当MOI为200时,各细胞株均达约100%感染;感染AdCMVCDglyTK的各组细胞和感染AdKDRCDglyTK的ECV304、MCF7、MGC803及SW620对前药具有较高的敏感性,且其敏感性差异无统计学意义(P均>0.05);与其他细胞相比,感染AdKDRCDglyTK的LS174T细胞对前药不敏感(P<0.01);且观察到该体系明显的旁观者效应。结论KDR基因启动子调控的CDglyTK融合基因体系可选择性杀伤实体肿瘤细胞及血管内皮细胞。     

FasL、B7-1联合基因修饰的胃癌细胞诱导抗胃癌主动免疫的研究

目的 探讨FasL、B7-1联合基因转移是否具有协同的抗肿瘤效应。方法 采用重组腺病毒载体将FasL、B7-1基因导入人胃癌细胞SGC-7901,G418阳性克隆筛选,流式细胞分析,逆转录-聚合酶链反应(RT-PCR),显示FasL和B7-1的表达,并且通过Hoechst33342染色检测胃癌细胞凋亡;将携带 FasL和 B7-1基因的胃癌细胞(命名为 SGC-7901/FB-11)接种于 C57BL/6小鼠背部皮下,观测其致瘤性能;SGC-7901/FB-11致敏的小鼠对野生型瘤细胞是否具有免疫保护作用;用SGC-7901和 SGC-7901/FB-11细胞分别经腹腔免疫小鼠,得到腹腔浸润淋巴细胞及致敏脾细胞,MTT法检测其体外杀伤实验。结果 FasL和B7-1基因在胃癌细胞中获得高表达并可诱导胃癌细胞的凋亡,双基因转染的胃癌细胞的致瘤性明显下降;SGC-7901/FB-11诱导的细胞毒T淋巴细胞(CTL)对SGC-7901的杀伤活性显著高于野生型SGC-7901诱导的CTL对相同靶细胞的杀伤活性(P<0.05);SGC-7901/FB-11诱导的 CTL对 SGC-7901/FB-11的杀伤率显著高于对野生型 SGC-7901的杀伤率(P<0.05)。结论 FasL促进胃癌细胞凋亡,B7-1促进抗胃癌CTL的增殖、活化,在效应阶段两基因发挥着重要的协同作用。     

KDR启动子介导胸苷激酶体外靶向杀伤血管内皮细胞

目的: 构建含以KDR为启动子的HSV-tk重组腺病毒，体外评估其对血管内皮细胞的特异性杀伤效能。方法: 采用pAdeasy系统，构建受KDR启动子或巨细胞病毒(CMV)启动子调控并可表达HSV-tk基因的AdKDR-tk和AdCMV-tk，在293细胞中包装、扩增后，体外分别感染表达KDR的人脐静脉血管内皮细胞系（HUVEC）和不表达KDR的肝癌细胞系HepG2，用丙氧鸟苷(GCV)处理受染细胞，并以MTT法检测其细胞增殖情况。结果:病毒滴度均为1×1010pfu/mL。在感染复数（MOI）为100的条件下， 当细胞培养液中加入的GCV浓度由0增至50μg/mL时，感染含AdKDR-tk的HUVEC细胞和HepG2细胞其存活率由100%分别下降至(28.94±5.67)%和(75.45±2.91)%（P<0.01），而感染含AdCMV-tk的HUVEC细胞和HepG2细胞其存活率分别下降至(17.56±2.48)%和(23.15±5.72)%（P>0.05）。结论:KDR启动子介导的 HSV-tk具有特异性杀伤血管内皮细胞的作用。     

腺病毒驱动KDR启动子介导CD/TK融合基因系统对肝癌细胞的靶向杀伤作用

目的 研究腺病毒介导的KDR启动子驱动CD/TK双自杀基因系统（AdKDR- CDglyTK）对肝癌细胞选择性杀伤作用。方法 将质粒pAdEasy-KDR-CDglyTK在293细胞内包装、扩增后，体外感染表达KDR的BEL-7402细胞株和对照组不表达KDR的LS174T细胞株，并给予不同浓度的前药5-FC和/或GCV，观察该体系对不同细胞株的杀伤效应及其旁观者效应。结果 所得病毒滴度为2.5×10^12pfu/ml。两种细胞的感染率相似，其感染率随腺病毒滴度的递增而增加。RT-PCR方法 检测发现：感染AdKDR-CDglyTK的BEL-7402有目的 基因CDglyTK的表达，感染AdKDR- CDglyTK的LS174T细胞无目的 基因表达。表达KDR的BEL-7402细胞对前药具有较高的敏感性，不表达KDR的LS174T细胞对前药不敏感（F=750.03，P〈0.001）。融合基因的疗效优于任一单自杀基因（F=275.89，P〈0.05）。将感染腺病毒的细胞与未感染细胞以不同比例混合培养，观察到该体系明显的旁观者效应。结论 KDR基因启动子可调控双自杀基因系统选择性杀伤表达KDR的肝癌细胞。     

热CO2气腹对胃癌细胞杀伤作用的实验研究

目的:体外研究热CO2气腹对人胃癌细胞SGC-7901的杀伤作用,探讨临床上腹腔镜胃癌手术使用热CO2气腹抑制腹膜转移和戳孔种植的可行性。方法:建立体外热CO2气腹实验模型,人胃癌细胞SGC-7901经热CO2气腹(42℃、43℃,1～3 h)处理后,WST-8法检测细胞毒作用,流式细胞术定量分析、Hoechst 33342/PI荧光显微镜观察细胞形态,RT-PCR检测Bax、bcl-2基因mRNA的表达。结果:WST-8法检测显示热CO2气腹能显著抑制人胃癌细胞SGC-7901增殖,43℃作用3 h后,细胞存活率降至(65.83±2.40)%。流式细胞术及细胞形态学结果显示,诱导胃癌细胞凋亡是热CO2气腹作用的主要方式,与温度和作用时间呈正相关。RT-PCR检测结果显示,实验组(43℃,3 h)Bax基因mRNA表达水平显著高于对照组(25℃,3 h)差异有统计学意义(P<0.05);但对bcl-2的表达无明显影响。结论:热CO2气腹通过诱导细胞凋亡对人胃癌细胞有显著的增殖抑制作用,发生机制可能是通过上调Bax的表达促进细胞凋亡。     

抗原致敏树突细胞增强细胞因子诱导杀伤细胞抗胃癌的效应

目的 观察抗原致敏树突细胞(DC)与细胞因子诱导的杀伤细胞(CIK)共培养体内外抗胃癌(SCC-7901)的效应.方法 取健康供者外周血体外诱导培养DC和CIK,应用噻唑蓝(MTT)法测定比较(每组n=3)单纯CIK(CIK组)、DC诱导CIK(DC-CIK组)和经胃癌抗原致敏的DC诱导CIK(抗原-DC-CIK组)体外对SGC-7901的杀伤活性,并利用SGC-7901建立胃癌裸鼠模型,设立PBS对照组,比较各组(每组n=5)瘤体体积、抑瘤率和肿瘤坏死面积评分.结果 成熟DC与CIK共培养第7天时,DC-CIK扩增倍数为17.8±2.0,约为单纯CIK扩增倍数(10.9±1.8)的1.63倍(P＜0.05);抗原-DC-CIK组、DC-CIK组、CIK组,体外实验中效靶比为20:1时对SGC-7901的杀伤活性分别为:(72.3±0.5)%、(53.9±0.7)%、(46.4±0.4)%(P均＜0.01);体内动物实验中抑瘤率分别为30.02%、18.11%、15.94%(P均＜0.01).结论 SGC-7901抗原致敏DC能增加CIK的扩增数量,增强CIK对SGC-7901的杀伤作用.     

索拉菲尼对人胃癌细胞SGC-7901增殖、凋亡和P-ERK表达的影响

目的：探讨多分子靶向药物索拉菲尼（sorafenib）对体外人胃癌细胞SGC-7901增殖、凋亡的影响及对癌细胞P-ERK表达的影响,并探讨其可能机制。 方法：以MTT法检测索拉菲尼对SGC-7901细胞的杀伤抑制作用;免疫细胞化学法检测胃癌细胞内P-ERK蛋白的表达;流式细胞仪检测胃癌细胞凋亡的变化情况。 结果：索拉菲尼对胃癌细胞生长增殖具有抑制作用,随药物浓度的增加作用也增强,呈剂量—时间双效应关系(P<0.05);经索拉菲尼处理的SGC-7901细胞的P-ERK表达明显下降(P<0.05);细胞凋亡率增高(P<0.05)。 结论：sorafenib在体外对SGC-7901细胞具有明显的抑制作用,主要机制为抑制其P-ERK表达,从而抑制其增殖和促进凋亡。     

姜黄素对人肾癌ACHN细胞放疗增敏作用的实验研究

目的研究姜黄素对人肾癌ACHN细胞放射增敏作用并探讨其作用机制。方法不同浓度姜黄素作用于ACHN人肾癌细胞24h后,MTT法检测姜黄素药物毒性;克隆形成实验观察其对放射敏感性的影响;流式细胞术检测姜黄素诱导细胞的凋亡率、细胞周期分布。结果姜黄素对人肾癌ACHN细胞有明显的抑制作用,可引起细胞的凋亡,且存在剂量和时间依赖;较低浓度的姜黄素（5μmol和10μmol）即可降低放射后ACHN细胞的克隆形成率,其放射增敏比分别为1．61及2．36;姜黄素及姜黄素联合辐射组的细胞周期阻滞在辐射敏感时相G2／M期;结论低剂量的姜黄素对人肾癌ACHN细胞有放射增敏作用,其机制可能与其引起细胞的凋亡增加,细胞周期阻滞有关。     

Islet cell proteins defined by monoclonal islet cell antibody HISL-19

A monoclonal islet cell antibody, HISL-19, reactive with human, bovine, and porcine pancreatic islets has been used to identify and characterize a novel group of islet cell proteins (p120, p69, p67, and p56). Besides the islets, HISL-19-reactive antigenic determinants are also expressed on selected cell types, namely, gut endocrine cells, thyroid parafollicular cells (p120), anterior pituitary cells (p40 and p24), specific hypothalamic neuroendocrine cells, and a single layer of large pyramidal cells of the cerebral cortex, thus defining a new family of neuroendocrine molecules.     

Headache caused by giant cell arteritis

Opinion statement Giant cell arteritis (GCA) is the most common primary systemic vasculitis in older adults. Patients usually are older than 50 years and have an erythrocyte sedimentation rate (Westergren) greater than 50 mm/h. Headache is a common symptom, occurring in approximately 90% of patients. However, the most serious complications of GCA, blindness and stroke, may occur in the absence of headache. Nonspecific constitutional symptoms such as weight loss, fever, and malaise may dominate the clinical presentation. Currently, corticosteroids are the mainstay of therapy for GCA. Treatment is initiated at 0.7 to 1 mg/kg mg of prednisone (or equivalent) per day as soon as the diagnosis is suspected. The medication is tapered based on laboratory parameters and symptoms. Relapse is common, especially during the first year of therapy. Side effects from steroids in the elderly are common and often serious. Steroid resistance (manifesting as continued high dose requirements after 3 to 6 months) may complicate therapy and place patients at increased risk of side effects. Methotrexate and azathioprine have been used as steroid-sparing agents based on anecdotal evidence. More recently, evidence is emerging that antitumor necrosis factor-α agents may be efficacious and act as steroid-sparing agents. New-onset headache or worsening headache in a patient older than 50 years should raise the possibility of GCA and appropriate therapeutic and diagnostic measures should be begun promptly.     

Germ cell tumors complicated by pneumothorax

Spontaneous pneumothorax is an unusual consequence of pulmonary metastases. This complication occurs mainly in children and adolescents with primary osseous sarcomas and is extremely rare in patients with pulmonary metastases secondary to carcinomas. Germ cell tumors have a proclivity to metastasize to the lung, but few instances of pneumothorax have been recorded. The authors present 3 cases of pneumothorax secondary to pulmonary metastases from germ cell tumors, both gonadal and extragonadal in type.     

Angiogenesis by endothelial cell transplantation.

PURPOSE: Myocardial angiogenesis may improve regional perfusion and perhaps function after cardiac injury. We evaluated the effect of endothelial cell transplantation into a myocardial scar on angiogenesis and ventricular function, as an alternative to angiogenic gene or protein therapy.Methods and Results: A transmural myocardial scar was created in the left ventricular free wall of rat hearts by cryoinjury. Allogeneic aortic endothelial cells were injected into the scar 2 weeks after cryoinjury. A cluster of transplanted cells was identified at the site of injection 1 day and 1 week after transplantation, but not after 2 weeks. The size of this cluster of transplanted cells decreased as vascular density in the transplanted scar tissue increased with time. Six weeks after transplantation, vascular density was significantly greater in transplanted hearts than in control hearts. Regional blood flow, by microsphere analysis, was greater in the transplanted rats. Systolic and diastolic ventricular function was similar between groups. In a second series of experiments, syngeneic aortic endothelial cells labeled with bromodeoxyuridine were transplanted 2 weeks after cryoinjury. Vascular density in the transplanted scar was greater than in controls. Labeled transplanted endothelial cells were identified forming part of the newly developed blood vessels. No difference in vascular density was found between allogeneic and syngeneic cell transplantation. Vascular endothelial growth factor was not expressed at greater levels in the transplanted cells or the myocardial scar. CONCLUSION: Transplanted endothelial cells stimulated angiogenesis, were incorporated into the new vessels, and increased regional perfusion in myocardial scar tissue, but did not improve global function in this cryoinjury rat model.     

Membranous nephropathy complicated by renal cell carcinoma

We experienced a patient with membranous nephropathy (MN) and renal cell carcinoma (RCC). Although MN is known to be commonly associated with malignant tumors, association with RCC is rare. In our patient, no malignant focus was found in the major organs examined, except for a small lesion in the right kidney. Diagnosis of a small RCC (1.6 × 1.6cm), which was made by renal biopsy and histology, was impossible by other, imaging, methods. As surgical treatment for the patient, laparoscopic partial nephrectomy was performed successfully, alleviating the symptoms of nephrotic syndrome for up to 21 months.