流式细胞仪分选侧群细胞条件的探索与优化

目的探讨不同浓度的荧光染料(Hoechst 33342)和抑制剂维拉帕米(verapamil)对流式细胞仪分选侧群(SP)细胞的影响。方法实验分为Hoechst 33342组(Hoechst 33342浓度分别为2.5、5.0和7.5μg/mL)和抑制组(Hoechst 33342+verapamil,verapamil浓度分别为50、100和150μmol/L),用流式细胞仪分选人肺腺癌细胞系A549中SP细胞,摸索Hoechst 33342和verapamil的最佳浓度,并用本实验室构建的一株肿瘤细胞系K1进行验证。结果在verapamil和Hoechst 33342浓度分别为150μmol/L和7.5μg/mL时,A549细胞染色充分,SP细胞亚群与非SP(non-SP)细胞亚群分群明显,SP细胞约为2.2%。K1细胞在verapamil为50μmol/L和Hoechst 33342为5μg/mL时,SP细胞亚群与non-SP细胞亚群分群明显,SP细胞亚群可被verapamil抑制,其比例约为1.3%。分选后的SP细胞和non-SP细胞可进一步扩增培养。结论 Hoechst 33342和verapamil可影响肿瘤细胞中SP细胞分选效率。     

左旋氧氟沙星对人肺腺癌多药耐药细胞株A_(549)/ADM的逆转作用

目的研究左旋氧氟沙星对人肺腺癌多药耐药细胞株A549/ADM的耐药逆转作用及机制。方法使用流式细胞仪分别检测耐药细胞系和敏感细胞系表面的多药耐药蛋白MRP和P-gp的表达;并通过流式细胞仪测定细胞内阿霉素的荧光强度来确定细胞的耐药程度以及左旋氧氟沙星对多药耐药的逆转作用。采用MTT方法检测耐药细胞系对肺癌几种常用的化疗药物的耐药程度及左旋氧氟沙星对其耐药逆转的效果。结果诱导的耐药细胞系与母细胞系相比:耐药细胞系A549/ADM表面的耐药蛋白MRP、P-GP表达分别达到74.8%和61.2%(P<0.01);耐药细胞系A549/ADM对ADM的耐药程度增加了14.29倍,对VCR、DDP、VP16均有不同程度的耐药,但对5-FU无耐药性;加用左旋氧氟沙星后,A549/ADM细胞内ADM的荧光强度明显增强,A549/ADM对ADM敏感性增加了2.55倍,对VCR、DDP及VP16敏感度分别增加了7.38倍、1.29倍和4.05倍。结论A549/ADM对肺癌的几种常用化疗药物表现出不同程度的耐药性,其耐药性可能与P-GP和MRP高表达有关;左旋氧氟沙星对A549/ADM的多药耐药有一定的逆转作用。     

从耐顺铂肺腺癌A549/DDP细胞系分离和鉴定肺癌干细胞的研究

目的从耐顺铂肺腺癌A549/DDP细胞系中分离侧群细胞(SP)以观察其是否富含肺癌干细胞(CSCs)样细胞,以期为肺癌CSCs样细胞生物学研究和干预建立基础。方法将A549/DDP细胞经过流式细胞术分选获得SP细胞并检测纯度,经过体外克隆形成试验检验其体外克隆形成能力。通过比较接种A549/DDP SP细胞和A549/DDP non-SP细胞的NOD/SCID鼠成瘤和肿瘤生长情况,观察其体内成瘤能力。结果流式细胞分析发现A549/DDP细胞中存在10%～17%的SP细胞。分离获得的SP细胞纯度>95%(平均97%)。SP细胞的体外克隆形成能力和non-SP细胞相比有统计学差异(克隆形成率的对数,即lgCFE:1.544±0.150vs.0.301±0.082,P<0.05)。接种A549/DDP SP细胞的NOD/SCID鼠的成瘤率较对照组存在明显差异。接种SP细胞的小鼠只需要5×103个SP细胞便足以成瘤,而接种non-SP细胞的小鼠却需要超过5×106个non-SP细胞才能在体内成瘤。A549/DDP SP成瘤能力是non-SP细胞成瘤能力的1000倍。结论耐顺铂肺腺癌A549/DDP细胞系中分离的SP细胞比例高且富含肺癌CSCs样细胞,可以用于肺癌CSCs样细胞生物学和干预治疗的研究。     

小分子Survivin干扰RNA逆转肺癌细胞耐药的研究

目的探讨小分子Survivin干扰RNA（siRNA-Survivin）逆转人肺癌耐药细胞（A549DDP）耐药性的研究。方法应用RT-PCR法及Western-blot方法证明Survivin基因在人肺腺癌耐药细胞系（A549DDP）细胞系呈现高表达;并采用脂质体为转染介质,将Survivin小片段RNA导入A549DDP细胞沉默Survivin基因;应用MTT法观察顺铂、多西他赛、吉西他滨、表阿霉素对沉默Survivin基因后的A549DDP细胞的细胞增殖抑制及半数抑制浓度的变化。结果 1,Sur-vivin-mRNA、Survivin蛋白在A549DDP细胞系表达水平均显著高于A549细胞系;2,顺铂、多西他赛、吉西他滨、表阿霉素对沉默Survivin后A549DDP细胞的细胞增殖抑制与对照组（A549DDP细胞s、iRNA-control A549DDP细胞）相比明显增加,半数抑制浓度（IC50）明显下降,差异显著（P〈0.01）。结论 Survivin参与了肺腺癌细胞耐药的形成;小分子Sur-vivin干扰RNA沉默Survivin可以提高耐药的肺癌细胞对化疗药物的敏感性。Survivin可能成为临床监测肿瘤耐药性变化,逆转肺癌耐药的一个新的靶点。     

5-氮杂-2′-脱氧胞苷联合SAHA对耐顺铂人肺腺癌细胞系A549/DDP的逆转作用及其机制

目的:探讨DNA甲基转移酶抑制剂5-氮杂-2′-脱氧胞苷(5-Aza-CdR)和组蛋白去乙酰化酶抑制剂(suberoylanilide hydroxamic acid,SAHA)对耐顺铂(DDP)的人肺腺癌细胞系A549/DDP的逆转作用及其可能的机制。方法:以A549、A549/DDP细胞为研究对象,采用四甲基偶氮唑蓝(MTT)法筛选出5-Aza-CdR和SAHA对A549/DDP细胞的无毒浓度(即对细胞抑制率<10%的药物浓度),并检测出顺铂与无毒浓度5-Aza-CdR和(或)SAHA联合对耐药细胞株的半数抑制浓度(IC50)和耐药指数;采用逆转录多聚酶链反应(RT-PCR)检测48h后各组细胞耐药相关基因MDRl、LRP mRNA的表达;采用流式细胞术检测各组细胞的凋亡情况。结果:5-Aza-CdR和SAHA对A549/DDP细胞的无毒浓度分别为5μmol/L和1μmol/L,两者均可使A549/DDP细胞对顺铂的IC50值下降,单独或联合应用时逆转倍数分别为1.6、1.8和4.6倍;经5-Aza-CdR和SAHA处理后各组细胞MDRl、LRP mRNA表达均明显减弱,凋亡明显增强,且两者联合具有协同作用(均P<0.05)。结论:5-Aza-CdR和SAHA可以逆转A549/DDP细胞的DDP耐药,且两者联合具有协同作用,其作用机制可能与下调耐药相关基因MDR1、LRPmRNA的表达,减弱肺癌细胞对化疗药物的外排作用有关。     

5-氮杂-2'-脱氧胞苷联合SAHA对耐顺铂人肺腺癌细胞系A549/DDP的逆转作用及其机制

目的:探讨DNA甲基转移酶抑制剂5-氮杂-2′-脱氧胞苷(5-Aza-CdR)和组蛋白去乙酰化酶抑制剂(suberoylanilide hydroxamic acid,SAHA)对耐顺铂(DDP)的人肺腺癌细胞系A549/DDP的逆转作用及其可能的机制.方法:以A549、A549/DDP细胞为研究对象,采用四甲基偶氮唑蓝(MTT)法筛选出5-Aza-CdR和SAHA对A549/DDP细胞的无毒浓度(即对细胞抑制率＜10％的药物浓度),并检测出顺铂与无毒浓度5-Aza-CdR和(或)SAHA联合对耐药细胞株的半数抑制浓度(IC50)和耐药指数;采用逆转录多聚酶链反应(RT-PCR)检测48 h后各组细胞耐药相关基因MDR1、LRP mRNA的表达;采用流式细胞术检测各组细胞的凋亡情况.结果:5-Aza-CdR和SAHA对A549/DDP细胞的无毒浓度分别为5μmol/L和1μmol/L,两者均可使A549/DDP细胞对顺铂的IC50值下降,单独或联合应用时逆转倍数分别为1.6、1.8和4.6倍;经5-Aza-CdR和SAHA处理后各组细胞MDR1、LRP mRNA表达均明显减弱,凋亡明显增强,且两者联合具有协同作用(均P＜0.05).结论:5-Aza-CdR和SAHA可以逆转A549/DDP细胞的DDP耐药,且两者联合具有协同作用,其作用机制可能与下调耐药相关基因MDR1、LRPmRNA的表达,减弱肺癌细胞对化疗药物的外排作用有关.     

ABCG2-SiRNA转染前后Hep-2细胞系侧群细胞含量变化的研究

目的探讨腺苷三磷酸结合区转运蛋白G超家族成员一2（ABCG2）基因失活前后hep-2细胞系中侧群细胞含量的差别。方法体外培养hep-2细胞,应用荧光染料Hoechst33342让对数期hep-2细胞进行染色,对照组加入荧光染料Hoechst33342及MDRI（多重耐药基因multipledrugresistance）抑制剂维拉帕米,流式细胞仪检测SP细胞亚群含量;ABCG2．SiRNA转染hep-2细胞系后,应用荧光染料Hoechst33342对转染后细胞进行染色,对照组加入荧光染料Hoechst33342及维拉帕米,流式细胞仪检测SP细胞亚群含量,比较转染前后SP细胞亚群含量变化。结果ABCG2-SiRNA转染前hep-2细胞Hoechst33342组sP细胞含量（3．15±0．32）％,Hoechst33342＋维拉帕米组SP细胞含量（0．4±0．11）％;ABCG2-SiRNA转染后hep-2细胞Hoechsd3342组SP细胞含量（1．15±0．22）％,Hoechst33342＋维拉帕米组sP细胞含量（0．0±0．09）％,转染后sP细胞亚群含量明显降低（P〈0．05）。结论ABCG2基因在喉癌sP细胞的发生发展过程中起重要作用,可能成为喉癌靶向治疗的潜在靶点。     

丙戊酸逆转人肺癌细胞对顺铂的耐药性

目的:探讨丙戊酸(valproic acid,VPA)对人肺癌耐顺铂细胞株A549/DDP耐药性的逆转作用。方法:MTT法分析VPA单用以及联合顺铂对A549/DDP细胞增殖的影响,流式细胞术检测VPA和DDP联合诱导细胞凋亡和对细胞周期的影响。结果:VPA对A549/DDP细胞的增殖有明显抑制作用,其IC50为8mmol。VPA和顺铂联合干预A549/DDP细胞结果显示,低剂量VPA和顺铂联合对A549/DDP细胞生长有明显的抑制作用,显示VPA逆转A549/DDP细胞对顺铂的耐药作用。细胞周期结果显示,VPA联合顺铂作用36 h可见细胞周期受阻,G2/M期细胞比例明显增加,48 h时可见明显的细胞凋亡。结论:VPA不仅具有抑制人肺癌耐顺铂细胞增殖的作用,而且能够明显提高A549/DDP对顺铂的敏感性,有效地逆转A549/DDP细胞对顺铂的耐药性,其作用机制与阻滞细胞周期有关。     

肺腺癌细胞A549及其顺铂耐药细胞株A549/DDP中GSK-3β蛋白磷酸化水平的差异

目的研究人肺腺癌细胞系A549和其顺铂耐药细胞系A549/DDP中GSK-3β的磷酸化和胞内分布以探讨GSK-3β在顺铂耐药中的作用。方法蛋白质免疫印迹法检测A549/DDP和A549细胞质和细胞核中总GSK-3β、p-GSK-3βser9和p-GSK-3βtyr6的表达。MTT法、流式细胞术分别检测顺铂耐药性、肺癌细胞凋亡率。结果 A549/DDP细胞胞质中p-GSK-3βser9水平明显高于A549细胞（P〈0.01）,顺铂的处理增加了A549/DDP细胞中p-GSK-3βser9的水平（P〈0.01）,相反却减少了A549细胞中p-GSK-3βser9的水平（P〈0.01）。A549/DDP细胞质中p-GSK-3βtyr6水平明显低于A549细胞（P〈0.01）,顺铂的处理减少了A549/DDP细胞中p-GSK-3βtyr6的水平（P〈0.01）,却增加了A549细胞中p-GSK-3βtyr6的水平（P〈0.01）。结论细胞质中GSK-3β活性受抑可能是非小细胞肺癌顺铂耐药的原因。     

肠癌细胞系SW-620中肿瘤干细胞相关SP亚群分析

背景:在某些恶性肿瘤中,SP细胞具有肿瘤干细胞的特性,如人脑瘤、乳腺癌等肿瘤干细胞的研究就是采用了这种方法.目前关于肠癌干细胞的研究尚处于探索阶段,国内外尚无肠癌干细胞分离、鉴定成功的报道.目的:分析肠癌细胞系SW-620中是否包含肿瘤干细胞相关的SP亚群.方法:制备SW-620细胞悬液,经Hoechst33342和PI染色,实验组加入Hoechst33342至终浓度为5 mg/L,维拉帕米组同时加入维拉帕米全终浓度5 mg/L,流式细胞仪分析SP亚群,共选取3次实验的结果.结果与结论:通过Hoechest33342蓝光和红光双参图,实验组SP细胞亚群位于左下角两种荧光均阴性或很弱的区域,经3次检测,肠癌细胞株SW620 中SP比例分别为0.1%,1.0%和0.5%,经维拉帕米阻断后SP细胞比例均为0.提示人肠癌细胞系SW620中存在SP亚群细胞,即提示肠癌干细胞存在的可能;维拉帕米可抑制染料外从排而明显减少SP细胞的比例.     

Clear cell renal cell carcinoma

Clear cell RCC is the most common type of RCC that occurs in adults. It has the worst prognosis among the common epithelial tumors of the kidney. Histologically, a wide range of morphologic patterns can be encountered. Those cases with a multi-locular cystic architecture are considered to be a distinct subtype because of the clinicopathologic features.     

Tumor cell growth and cell kinetics

Cancer cells differ from normal cells in many ways, but most importantly by not responding to normal growth-control mechanisms. Whereas the growth and division of normal cells is carefully regulated to meet the needs of the body, tumor cells proliferate autonomously and continually, eventually interfering with and destroying the functions of normal tissue. Knowledge of molecular cell biology has grown exponentially over the last decade. Yet much remains to be understood before there can be a significant impact on our ability to design more effective therapeutic strategies for cancer patients, thereby decreasing mortality.     

Basal cell and squamous cell carcinoma

OBJECTIVES: To describe the clinical and histologic subtypes, pathophysiology, recognition, and treatment options for basal cell and squamous cell carcinoma, and the molecular biology of sunlight-induced carcinogenesis. DATA SOURCES: Journal and review articles, research studies, textbooks, and clinical practice. CONCLUSIONS: Basal cell and squamous cell carcinoma will occur in more than one million cases annually in the United States, and are highly curable when detected and treated early. During the last decade, significant progress has been made in elucidating the molecular basis of skin carcinogenesis and in identifying newer approaches for the management and treatment of these keratinocyte cancers. IMPLICATIONS FOR NURSING PRACTICE: Nurses can play crucial roles in decreasing the morbidity and mortality from the skin cancer epidemic by identifying and referring patients with lesions suspicious for basal cell and squamous cell carcinomas.     

White cell related red cell antibodies

Occasional sera react weakly with a few red cells in the antiglobulin phase but without a recognizable pattern. We sought to identify the nature of such antibodies in 27 samples referred to our HLA laboratory for lymphocytotoxin testing. All samples were tested against a panel of 15 red cells by a capillary tube antiglobulin technique developed to conserve sera. This technique correlates well with tube antiglobulin tests, and can be performed with either fresh or thawed red cells. Of 27 sera, 14 contained anti-HLA B7, B17, or A28, since they reacted only with red cells from donors whose lymphocytes were B7, B17, or A28. Eight further sera probably contained anti-B7, -B17, or -A28, but reacted with one or two additional red cells. Two samples agglutinated all panel red cells so the presence of anti-B7, B17, or A28 could not be determined. In three additional sera, lymphocytotoxin testing suggested that specificity other than anti-B7, B17, or A28 was present. Of 27 sera containing weak unidentified red cell antibodies, 22 (81%) contain definite or probable anti-B7, -B17, or -A28. The identity of these troublesome antibodies can be determined by maintaining red cell panels of donors whose HLA phenotypes are known.     

Red blood cell storage and cell morphology

Aim: In this study, we performed weekly assessment of morphology‐related parameters through monitoring of CPD‐SAGM leuco‐filtered erythrocyte concentrates from blood withdrawal until the 42nd day of storage. Background: Liquid storage of red blood cells (RBCs) delivers a blood‐derived therapeutic, which is safe, available, effective and affordable for most patients who need transfusion therapy in developed countries. However, a growing body of accumulating controversial evidences, from either biochemical or retrospective clinical studies, prompted safety concerns about longer stored RBCs. Methods: Statistical image analysis through scanning electron microscope was coupled to osmotic fragility and erythrocyte sedimentation rate. Results: We could observe that by day 21 more than 50% of RBCs displayed non‐discocyte phenotypes. This observation was related to an increase in osmotic fragility, which was totally overlapped in day 0 controls and day 7 RBCs while only slightly augmented in day 14 samples. Cation dysregulation (pH internal/external alteration and potassium) might both reflect and trigger a negative feedback loop with metabolic fluxes and membrane cation pumps. Conclusion: Morphology parameters suggest that significant alterations to RBC morphology over storage duration occur soon after the 14th day of storage, as to become significant enough within the 21st day.     

Human mononuclear cell modulation of endothelial cell proliferation

Endothelial cell proliferation is a histologic characteristic of several forms of nephritis characterized by infiltration of the glomerulus with mononuclear cells. To investigate the mechanism mediating this event, human endothelial cells isolated from umbilical veins and cultured in vitro were incubated with supernatants of cultured human mononuclear cells. Supernatants from mononuclear cells exerted a dose-dependent stimulatory effect on endothelial cell proliferation. The stimulatory effect of supernatant was almost entirely removed by prior depletion of mononuclear cells of monocytes by adherence, suggesting that a monocyte product was responsible for the activity. To investigate the nature of the ligand responsible, partially purified human interleukin I added to endothelial cell cultures was found to stimulate cellular proliferation.     

Dendritic cell vaccine strategies for renal cell carcinoma

Dendritic cell (DC) vaccines are an important experimental immunotherapy for renal cell carcinomas. DC vaccines have proven safe, but only minimal clinical efficacy has been observed to date. DC vaccine strategies reflect the continually evolving understanding of DC biology. The use of mature DCs is particularly important to avoid the induction of regulatory T cells. Better defined sources of immunizing antigens and more efficient antigen-loading will contribute to DC vaccines of better quality. Improved clinical efficacy may also be achieved using DCs that secrete biologically active IL-12, which fosters innate immunity and polarizes T helper type 1 responses that contribute to optimal antitumor immunity. Furthermore, combination therapies that treat systemic immune suppression will be crucial for obtaining improved clinical responses to DC vaccines in patients with advanced disease.     

胰岛细胞移植过程中细胞受损的提示

背景:胰岛移植后可能发生有害的组织不相容性反应,影响细胞的存活及功能.目的:探讨胰岛细胞移植中早期胰岛细胞的损害程度及原因.方法:采用脑死亡自愿捐赠器官供者的胰腺,采用胶原酶P进行消化分离胰岛细胞,测定不同冷缺血时间下胰岛细胞损害程度.将胰岛细胞与血液进行分组培养,HLA匹配组:受者全血+胰岛细胞,受者全血+胰岛细胞+肝素:错配组:受者全血+胰岛细胞,受者全血+胰岛细胞+肝素;对照组:受者伞血+RPMI1640.观察移植早期可能出现的胰岛细胞损害.结果与结论:胰腺切取顺利,在冷缺血5 h以内胰岛细胞活性率都在80%以上,超过8 h活性胰岛细胞数量只有19%甚至更低.人胰岛暴露于未经抗凝的人血液中,胰岛将诱发一个迅速血细胞消耗.血小板、中性粒细胞和单核细胞计数显示,无论HLA错配还是匹配与对照组相比较血细胞都发牛明显的消耗:加入肝素后HLA错配组及HLA匹配组血细胞消耗反应明显减轻;HLA匹配组胰岛细胞体外培养24 h活性胰岛细胞数量高丁HLA错配组(P<0.05),说明良好组织相容性有利于胰岛细胞存活.结果提示冷缺血时间对胰岛细胞活性的影响很大,在冷缺血时间小于5 h的情况下获取的胰腺可以用于临床胰岛细胞移植的胰腺获取;移植到血液的胰岛细胞会有普遍性的炎症性损害及HLA相关性损害.     

CEUS鉴别诊断肾透明细胞癌和嫌色细胞癌

目的 探讨CEUS对肾透明细胞癌（CCRCC）和嫌色细胞癌（ChRCC）的鉴别诊断价值。方法 收集接受肾脏CEUS检查并经术后病理证实为CCRCC的患者75例及ChRCC的患者26例。观察CCRCC和ChRCC的增强方式、增强程度、增强形态、假包膜征及病灶对局部淋巴结、肾包膜及肾静脉的侵犯情况,并绘制时间-强度曲线,获得校正的始增时间（ΔAT）、达峰时间（ΔTTP）和峰值强度（ΔPI）,进行统计学分析。结果 CCRCC多表现高增强（41/75,54.67%）、弥漫性增强（54/75,72.00%）和不均匀增强（58/75,77.33%）,56.00%（42/75）有假包膜征。ChRCC多表现为低增强（19/26,73.08%）、向心性增强（14/26,53.85%）和均匀增强（17/26,65.38%）,61.54%（16/26）有假包膜征。CCRCC与ChRCC增强程度、增强方式及增强形态的差异均有统计学意义（P均<0.05）,假包膜征检出率的差异无统计学意义（P>0.05）。CCRCC的ΔAT和ΔTTP与ChRCC比较,差异均无统计学意义（P均>0.05）,而CCRCC的ΔPI明显高于ChRCC（P<0.001）。以ΔPI=0.05%为阈值鉴别诊断CCRCC和ChRCC的准确率最高,其敏感度为82.70%,特异度为100%,ROC曲线下面积为0.969。CCRCC出现肾周和（或）肾窦脂肪受累和肾门和（或）腹膜后淋巴结转移的百分率均高于ChRCC（P均<0.05）。结论 CCRCC和ChRCC具有不同的CEUS特征,有助于二者的鉴别诊断。