肾癌树突状细胞疫苗的体外活性研究

目的 探讨负载人肾癌细胞抗原肽制备树突状细胞(DC)疫苗体外杀伤肾癌细胞的作用.方法 利用细胞膜酸洗脱法获得人肾透明细胞癌细胞株786-0细胞表面抗原肽.外周血单个核细胞在体外经人粒细胞一巨噬细胞集落刺激因子,白细胞介素4和脂多糖诱导获得成熟的DC,并负载分离到的抗原肽制备DC疫苗.利用疫苗体外诱导出特异性细胞毒性T淋巴细胞(CTL)作为实验组.同时设置4个对照组,对照组1用未负载抗原肽的DC和单个核细胞混合培养,对照组2用单个核细胞进行培养,对照组3用抗原肽与单个核细胞混合培养,对照组4用未负载抗原肽的DC单独培养.4个对照组也加入同实验组相同的各种因子进行培养.51Cr释放法检测特异性CTL的杀伤活性.结果 疫苗诱导的特异性CTL对肾癌细胞的杀伤活性为(31.93±5.05)%,与对照组(5.88±2.26)%、(8.03±6.70)%、(9.70±2.09)%、(9.35±3.58)%相比差异有统计学意义(P<0.05).结论 负载抗原肽的DC疫苗体外试验有高效的抗肾癌细胞活性.     

裸鼠人肾癌SOI模型的建立及高转移亚株的筛选

目的：建立人肾癌裸鼠转移模型，并分离肺转移亚株。方法；用自建肾癌细胞系RCC-9863建立裸鼠SOI(surgical orthotopic implantation)模型。从裸鼠皮下移植瘤中取组织块，手术包埋于裸鼠左肾实质内。取肺结节，组织块法体外培养。癌细胞行左肾细胞悬液原位接种，重复1次，克隆筛选肺转移亚株。结果；15d左右裸鼠左肾区可触及包块，质硬，有结节。55d裸鼠出现恶液质。SOI模型肿瘤生长快，呈浸润性，肺脏、淋巴结、肝脏可见转移灶。转移株细胞增殖周期短，软琼脂集落形成率高，裸鼠皮下接种后，成瘤潜伏期短，瘤体大，并可以形成广泛肺转移。获得的高转移株命名为MRCC，其IV型胶原酶呈强阳性，增殖细胞核抗原（PCNA）高表达，nm23表达率与RCC-9863差别无显著性意义。结论：肾癌SOI模型的建立并筛选出一株肺脏高转移性细胞亚株，为肾癌高转移性研究提供了有效手段。     

人表皮干细胞的体外分离培养和鉴定

目的:探讨人表皮干细胞的体外快速分离培养及鉴定方法。方法:中性蛋白酶和胰蛋白酶两步法从手术切除的人包皮组织中分离表皮层和真皮层,并获得表皮单细胞悬液,采用Ⅳ型胶原铺板选择性粘附、分离和角质形成细胞无血清培养基(K-SFM)培养表皮干细胞。倒置显微镜下观察培养细胞的生长状况,检测细胞克隆形成率,免疫组化染色观察表皮干细胞标志物β1整合素和角蛋白19(K19)的表达;以角质形成细胞作为对照。结果:组织学观察显示,培养24h后细胞呈克隆状生长;所分离、培养细胞的克隆形成率高于对照角质形成细胞组;免疫组化染色显示,培养细胞β1整合素及Kl9均呈阳性表达。结论:运用Ⅳ型胶原粘附结合K-SFM培养可以实现人表皮干细胞的体外快速分离和培养。     

人表皮干细胞的体外分离与培养

目的 探索人表皮干细胞（epidermal stem cells，ESCs）的分离方法和培养体系。方法 用Ⅳ型胶原纯化、富集ESCs，将黏附细胞（实验组）和未黏附细胞（对照组）分别接种在Ⅳ型胶原摹质（实验组为A1，对照组为A2）和3T3细胞滋养层（实验照组为B1，对照组为B2），培养体系为：低糖无钙DMEM培养基（添加10％胎牛血清、表皮生长因子10μg／L、氯化钙0．05mmol／L、氢化可的松0．8mg／L），观察细胞能否呈克隆状生长，用流式细胞仪和免疫细胞化学染色，对ECSs周期和表型进行分析。结果 实验组细胞呈克隆状生长，G0／G1期细胞和α6^briCD71 dim细胞百分率明显高于对照组，差异有统计学意义（P〈0．05），实验组角蛋白19免疫细胞化学染色呈阳性，对照组呈刚性。结论 人ESCs可通过Ⅳ型胶原快速黏附分选，并可在适当的培养体系里扩增。     

人胆囊癌SGC996细胞株侧群细胞的分离与体外培养

目的 分选人胆囊癌细胞株SGC996的侧群细胞,并行体外培养,初步验证其干细胞的特性.方法 应用流式细胞仪分选人胆囊癌细胞株SGC996的侧群细胞和主群细胞,将两群细胞分别在有血清和添加干细胞因子的无血清培养液条件下培养.应用噻唑蓝(MTT)法检测两群细胞在血清培养条件下的增殖能力.结果 人胆囊癌细胞株SGC996的侧群细胞分选比例仅为1.0％,在维拉帕米阻断后降至0.1％.侧群细胞在无血清培养液中能形成干细胞样“肿瘤球”,但主群细胞在该条件下生长能力较差,甚至死亡.MTT结果显示,在有血清培养条件下,侧群细胞在第5、6、7天的吸光度值分别为1.26±0.15、1.73±0.18、2.21±0.17,较主群细胞的0.95±0.06、1.31±0.10、1.86±0.11,各组差异均有统计学意义(t=3.30、3.54、3.03,P＜0.05).结论 人胆囊癌细胞株SGC996中存在侧群细胞,这群细胞在体外培养能形成干细胞样“肿瘤球”,表现出更强的体外增殖能力,为胆囊癌干细胞进一步研究提供理论基础.     

肾癌的树突状细胞治疗

肾癌是泌尿系最常见的恶性肿瘤之一,以往以手术治疗为主.但大多数患者就诊时肿瘤已发生转移,对这部分患者尽管采用手术治疗,往往不能取得满意的疗效.近年来,随着分子生物学、免疫学的研究进展,以树突状细胞为基础的免疫治疗在肾癌的治疗中发挥着重要的作用,本文就最近几年其在肾癌患者中的应用作一综述.     

人骨膜细胞生物学特性的实验研究

目的 探讨人骨膜细胞体外培养的生物学特性. 方法 以胫骨骨膜组织为材料,采用组织块法分离细胞,完全培养基培养,通过倒置显微镜观察细胞形态学,锥虫蓝染色计数法检侧细胞增殖能力;流式细胞学分析细胞表面抗原.随机分为3组,成骨实验组和成软骨实验组分别加入不同的定向培养剂,对照组加入完全培养基,采用碱性磷酸酶染色、Von Kossa染色、甲苯胺蓝染色、Ⅱ型胶原免疫组化染色检测各组细胞的成骨和成软骨分化指标. 结果 骨膜细胞在体外培养条件下呈贴壁生长,细胞生长曲线证实骨膜细胞传至第9代仍保持良好的增殖能力,流式细胞仪检测证实细胞表面抗原CD90及CD105呈阳性;组织化学染色检测证实成骨实验组分化后细胞碱性磷酸酶及钙结节呈阳性,成软骨实验组分化后细胞蛋白聚糖及Ⅱ型胶原呈阳性,对照组各项指标均生阴性.结论 骨膜细胞体外培养细胞增殖能力强,具有间充质干细胞的特性和良好的成骨和成软骨分化潜能,其定向诱导分化的成骨细胞和软骨细胞均具有各自明显的细胞功能表达.     

细胞粘附分子CD44的变异表达与肾癌的关系

目的探讨变异型细胞粘附分子（ＣＤ４４ｖ）表达与肾细胞癌临床行为的关系。方法采用逆转录聚合酶链反应（ＲＴＰＣＲ）方法分析了ＣＤ４４ｖ在３１例肾癌及１８例正常肾组织标本中的表达情况。结果３１例肾癌中有１８例ＣＤ４４ｖ表达阳性,作为对照的１８例正常肾组织中无一例呈阳性表达（Ｐ＜０００１）。进一步结合病理资料,发现在有转移或病理分期较高的肾癌中ＣＤ４４ｖ的表达率亦较高。结论ＣＤ４４ｖ的表达与肾癌的恶性表型及浸润转移密切相关,可能作为评价肾癌临床行为的一个新的指标     

基质金属蛋白酶与肾癌侵袭转移关系的研究进展

浸润与转移是恶性肿瘤的主要生物学特征.基质金属蛋白酶(MMPs)是一组含Zn2+的蛋白水解酶,其不仅能降解ECM,还参与了原发肿瘤的形成,肿瘤血管的新生,以及诱导肿瘤细胞免疫耐受.在肿瘤侵袭转移的复杂过程中,MMPs起着重要的作用.本文综述了MMPs的分类、特征及功能,并分析其与肾癌的关系.     

多房性囊性肾癌与肾癌出血坏死囊性变的临床比较

目的:探讨多房性囊性肾癌(MCRCC)与肾癌出血坏死囊性变(NCRCC)的临床特点,提高两种疾病的诊治水平。方法:对MCRCC及NCRCC各10例的临床资料进行对比研究,并对其囊壁增厚、强化特点等CT征象以及病理分级进行统计学分析。结果:两组患者无特异性,NCRCC组最重要的CT征象为囊壁及分隔局部结节或肿块,与MCRCC组相比差异有统计学意义(P<0.01)。MCRCC组病理分级低,与NCRCC组相比差异有统计学意义(P<0.05)。结论:MCRCC与NCRCC是两种生物学行为不同的肾癌类型,应注意鉴别,鉴别主要依靠影像学检查。MCRCC恶性程度低,预后较好。     

Effects of cytokines on growth in vitro of primary human renal cell carcinoma

Summary In clinical trials different haematopoietic active cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) have been proven to alleviate myelosuppressive side effects of intensive chemotherapy in different non-urological malignancies. On the other hand, these cytokines can directly stimulate the proliferation of cells originating from some non-urological tumours. To clarify the impact of these cytokines on the proliferative behaviour of human renal cell carcinoma (RCC), 29 previously untreated RCC tumours were prepared for culturing in vitro using the cell cluster technique. The success rate for growth in vitro was 82.8% (24/29). The malignant renal cells were treated with different cytokines (GM-CSF, G-CSF and interleukin-3) in different dosages. Cell number and proliferation rates detected by immunostaining were used for treatment evaluation. A dosage-dependent stimulation of cell growth could not be observed compared to untreated cells. From the data presented in this study, proliferative stimulation of RCC by administering colony-stimulating factors in clinical trials cannot be assumed.     

Current pathology keys of renal cell carcinoma

Context

Renal cell carcinoma (RCC) in adults comprises a heterogeneous group of tumours with variable clinical outcomes that range from indolent to overtly malignant. The application of molecular genetic techniques to the study of renal neoplasms has resulted in an improved classification of these entities and a better understanding of the biologic mechanisms responsible for tumour development and progression. The current 2004 World Health Organisation classification of adult renal epithelial neoplasms has expanded rapidly with new categories recently incorporated.

Objective

To review and evaluate the evidence implicating pathologic features and classification of RCC in adults as a tool to approach patients’ prognosis and modulate current therapy.

Evidence acquisition

Members of Committee 3: Pathology, under the auspices of the International Consultation on Urological Diseases and the European Association of Urology (ICUD-EAU) International Consultation on Kidney Cancer, performed a systematic review using PubMed. Participating pathologists discussed pathologic categories and diagnostic features of RCC in adults.

Evidence synthesis

We reviewed and discussed articles and the personal experiences of participating uropathologists.

Conclusions

The conclusions reached by the ICUD-EAU 2010 International Consultation on Kidney Cancer emphasise the appropriate pathologic diagnosis of RCC in adults as a tool to approach patients’ prognosis and modulate current therapy. Further emphasis should be placed on defining risk groups of RCC and diagnostic features of unusual tumours such as familial RCC, translocation RCC, and tubular mucinous and spindle cell carcinoma. A number of recently described entities and morphologic variants of classical categories deserves recognition because they can be important in differential diagnosis and therapy.     

The use of prognostic factors in metastatic renal cell carcinoma

BackgroundOver the last decade, the treatment landscape of metastatic renal cell carcinoma (mRCC) has evolved tremendously. The outcome of patients with mRCC has been improved since the advent of targeted therapy.ObjectiveIn this review, we address the use of prognostic schema in the era of targeted treatment. This article summarizes the current available prognostic models and the evidence to support their use in clinical settings.ConclusionPrognostic models can help guide clinicians in their decision making, as they have been validated in the first- and second-line targeted therapy settings as well as in non–clear cell mRCC. Prognostic factors are important in patient counseling, clinical trial stratification, and therapy planning. Very selected favorable-risk patients with minimal bulk and slow-growing disease could potentially be observed before needing treatment. Patients with poor-risk disease may be eligible for treatment with temsirolimus. Patients with a very poor prognosis may not be suitable candidates for cytoreductive nephrectomy. New biomarkers are on the horizon, though their roles need to be validated and their additive contribution to improve existing prognostic models examined.     

In vitro stimulation of renal tubular p-aminohippurate transport by dexamethasone in rat kidneys and in intact kidney tissue of patients suffering from renal cell carcinoma

The aim of this study was to test whether or not the accumulation of p-aminohippurate (PAH) can be increased in intact human renal cortical slices obtained from tumor-bearing kidneys of patients suffering from renal cell carcinoma (RCC). Tissue slices were incubated for 24 h in Williams medium E containing 0.01–50 μM dexamethasone. Thereafter slices were placed in PAH-containing Cross-Taggart medium and PAH uptake into kidney tissue was measured for 2 h. In both rat and human renal tissue slices, PAH uptake capacity increased significantly in a concentration-dependent manner after 24 h of incubation in dexamethasone-containing medium (rat, 136%; man, 156%). The stimulatory effect was already significant after 12 h of incubation. In additional experiments it was shown that incubation in triiodothyronine (T₃)-containing medium has different effects: in man, T₃ does not influence the PAH accumulation capacity of renal cortical slices whereas in rats PAH accumulation is significantly lower after 24 h of incubation with T₃. Thus stimulation of tubular transport capacity can be performed in vitro in human renal cortical slices. Discrepancies between the effects of dexamethasone and T₃ indicate different modes of action of the two hormones at the cellular level. Received: 4 August 1997 / Accepted: 28 October 1997     

Synchronous ipsilateral conventional renal cell and transitional cell carcinoma

Simultaneous occurrence of renal cell carcinoma (RCC) and transitional cell carcinoma (TCC) in the same kidney is unusual. We report a 61-year-old man with ipsilateral synchronous renal adenocarcinoma and renal pelvic TCC. He was referred to our department for gross hematuria and right flank pain. CT and MRI studies revealed a 57 × 50 mm irregular and infiltrative upper right kidney mass with necrotic components. A right radical nephrectomy was done. Pathological diagnosis was a high grade tumor originating from just beneath the intact urothelium of renal pelvis and infiltrating through the parenchyma showing solid and occasional tubular growth patterns. A second tumor in close proximity to the first was reported as well differentiated RCC. This is a rare case of combined renal malignancies.     

保留肾单位手术治疗肾癌(附46例报告)

目的:探讨保留肾单位手术治疗肾癌的安全性和疗效。方法:对1993年2月～2006年10月共46例采用保留肾单位手术的肾癌患者的临床资料进行回顾性分析,其中双侧肾癌4例,孤立肾癌3例,对侧肾有病变或潜在功能受损的肾癌25例,对侧肾正常的肾癌14例。结果:46例患者术前均未发现转移灶。术后组织病理学结果示肾透明细胞癌36例,颗粒细胞癌6例,混合性细胞癌4例。术后42例(91%)获随访,随访时间6～160个月,平均随访65个月。5、10年生存率分别为94%、86%。3例术后出现局部复发和远处转移。结论:保留肾单位手术治疗肾癌安全有效,手术指征可扩展至对侧肾脏正常的患者。     

Familial renal cell carcinoma from the Swedish Family-Cancer Database

Background

Reliable data on familial risks are important for clinical counselling and cancer genetics.

Objective

To evaluate familial risks for renal cell carcinomas (RCC) through parental and sibling probands in the largest available dataset.

Design, setting, and participants

This study examined the Swedish Family-Cancer Database on 12.2 million individuals, which contains families with parents and offspring. Cancer data were retrieved from the Swedish Cancer Registry for the years 1961-2008, including 8513 patients with RCC.

Measurements

Familial risk for offspring was defined through standardised incidence ratios (SIRs) and adjusted for many variables, including a proxy for smoking and obesity.

Results and limitations

The familial risk for RCCs was 1.75 when a parent and 2.61 when a sibling was diagnosed with any kidney cancer. Also, RCCs were shown to be associated with prostate cancer (PCa) when parents or parents and siblings were diagnosed with PCa. Among siblings, the associations of RCC with melanoma, non-Hodgkin's lymphoma, and urinary bladder and papillary thyroid tumours were found. None of the results differed significantly after excluding the families with cancer pathognomonic of a von Hippel-Lindau (VHL) disease. Limitations of this study include the small number of familial cases (229 familial cases).

Conclusions

The present analysis showed a high familiarity for RCC, and recessive effects may be important for familial aggregation of RCC. As a novel association, offspring RCC was in excess when parents or parents and siblings were diagnosed with PCa. There is familial clustering beyond VHL and the recent low-risk gene that probably explains a small proportion of the observed familial clustering.     

三氧化二砷抑制肾癌细胞系786-0增殖及诱导凋亡的研究

目的探讨三氧化二砷(As2O3)对人肾癌细胞系786-0的增殖抑制和诱导凋亡的作用及可能机制。方法采用细胞增殖检测、形态学观察、琼脂糖凝胶电泳和肿瘤克隆形成等方法观察As2O3对786-0细胞的增殖抑制和诱导凋亡的作用,以免疫组织化学和逆转录-聚合酶链反应(RT-PCR)技术等探讨As2O3的作用机制。结果2μmol/L以上浓度As2O3可显著抑制786-0细胞的增殖、降低细胞核分裂指数并出现凋亡的形态学改变和DNA片段化,经2.0μmol/LAs2O3处理72h后,其抑制786-0细胞增殖率为69.13%(P<0.01),使其核分裂指数由6.00降低至1.80(P<0.01),肿瘤细胞克隆形成抑制率达到100.00%。As2O3使786-0细胞内bcl-2、PCNA和Ki-67基因表达降低(P<0.01),其作用随药物浓度升高和时间延长而增加。结论As2O3对786-0细胞具有显著的增殖抑制作用,并具有浓度和时间依赖性特点,可能通过下调其PCNA和Ki-67基因表达抑制增殖,抑制bcl-2基因的表达诱导细胞凋亡。     

肾癌的肾外表现分析

目的：探讨肾癌的肾外表现在肾癌早期诊断及预后评估中的临床意义。方法：对1988—2001年经手术和病理检查证实为肾癌的372例临床资料进行回顾性分析。结果：发现253例(68．0％)有各种肾外表现,主要表现为血沉快,血压高,食欲不振,消瘦,乏力,贫血,发热,肝功能紊乱,高钙血症及红细胞增多症,其中82例(32．4％)是以肾外表现为首发症状。结论：肾外表现有助于肾癌的早期诊断。肾癌的治疗以手术切除为主,肾癌的肾外表现不能作为手术禁忌证。