肿瘤来源的血管内皮生长因子动员循环内皮祖细胞参与了肾细胞癌的血管发生

　　杨斌，顾闻宇，郭长城，孙伟，车建平，刘敏，姚旭东，郑军华

　　同济大学附属上海市第十人民医院泌尿外科

　　通讯作者：郑军华教授 同济大学附属上海市第十人民医院泌尿外科

　　背景和目的：循环内皮祖细胞（CEPCs）是骨髓来源的内皮细胞的前体细胞，具有分化成为成熟内皮细胞的能力。目前研究提示CEPCs可以通过血管发生途径在新生血管形成中起重要作用。然而，CEPCs在肾细胞癌（RCC）中的作用还未明确。因此，本实验的目的在于研究CEPC是否通过血管发生途径参与了肾细胞癌的新生血管形成，并且明确CEPC动员出骨髓的潜在驱动力。

　　材料和方法：本研究纳入86例RCC患者和50例健康对照（HCs）。通过PCR、WB和免疫荧光技术分别检测了接受肾癌根治术患者中配对的肾癌肿瘤组织（TT）、癌旁组织（AT）和正常组织（NT）中CD34+ /VEGFR-2+的CEPCs。统计不同组织中微血管密度（MVD），并且统计CEPC+MVD/total MVD为CEPC对于肿瘤组织血管的贡献。ELISA法检测RCC患者和HCs中血清血管内皮生长因子（VEGF）的含量。RT-PCR、WB和免疫组化技术检测血清VEGF的来源。在20例RCC患者中培养并鉴定CEPC。通过细胞生物学方法，评估来自RCC或HCs的浓缩血清中的VEGF在调控CEPC增殖、迁移、划痕愈合和成管中的作用。

　　结果：RCC患者中平均CEPC水平显著高于HCs（P<0.001）。AT和TT中CD34、VEGF-R2和VEGF的水平显著高于NT（both P<0.05）。并且，这三个指标在TT中的表达显著高于AT（P<0.05）。免疫荧光染色在TT中发现大量CEPCs（CD34+ /VEGFR-2+ 细胞），而在NT或AT中仅发现散在CEPCs。在TT中CEPC+MVD/total MVD的平均值为28.8 %， 显著高于AT和NT（8.43% vs 2.39%， both P<0.01）。这些结果提示了RCC患者外周血和组织中CEPC含量升高，并且CEPC参与了RCC新生血管形成。RCC患者中外周血清VEGF的含量显著高于HCs（P<0.001）。RCC患者和HCs外周单个核细胞VEGF的表达含量无明显差异。然而，VEGF在TT中的表达含量显著高于AT和NT（both P <0.01）。这些结果进一步提示了血清VEGF来源中TT组织。体外培养结果发现CEPC呈现典型鹅卵石样克隆细胞团，并且培养的CEPC表达eNOS、VE-cadherin 和内皮细胞特异性抗体，具有摄取Ac-LDL、结合lectin 和类似内皮细胞一样的体外成管能力。细胞生物学实验发现，RCC浓缩血清促进CEPC增殖、迁移、划痕愈合和成管的能力可以被VEGF中和抗体显著消除。

　　结论：本研究发现了肿瘤来源的VEGF动员CEPCs，通过血管发生途径参与了RCC的新生血管形成，提示了骨髓来源的CEPCs是RCC治疗的一个潜在靶点。

肾细胞癌患者循环内皮祖细胞的计数、培养和鉴定

　　杨斌，顾闻宇，孙伟，郭长城，姚旭东，郑军华

　　同济大学附属上海市第十人民医院泌尿外科

　　背景和目的：尽管目前大量研究证实循环内皮祖细胞（CEPCs）在肿瘤新生血管形成中起重要作用。然而，在新生血管异常丰富的肾细胞癌患者中，CEPCs的水平及其与血清血管内皮生长因子（VEGF）的关系目前还未明确。肾细胞癌患者中的CEPC还没有被成功的培养和鉴定。

　　材料和方法：本实验纳入53例肾细胞癌（RCC）患者， 33例肾脏良性肿瘤患者（BRT）和40例健康对照（HCs），鉴定外周循环中CD45-/VEGF-R2+ +/CD34+ 的细胞为CEPCs，结果表示为CEPCs占外周血单个核细胞的百分比。用ELISA法检测血清VEGF的含量。同时从上述纳入对象中收集33例RCC和30例HCs外周血，分离单个核细胞并进行CEPCs培养。

　　结果：RCC患者中平均CEPC的水平为0.281%，显著高于BRT和HCs（分别为0.073% 和0.076%，P值均 <0.001）。RCC患者中平均血清VEGF的含量显著高于BRT和HCs（分别为315.5 vs 34.6 and 26.9 pg/ml，P值均 <0.001）。在RCC患者中，术前CEPC的水平和血清VEGF的水平呈正相关（r =0.710， P<0.001）。体外培养发现，RCC患者中CEPC克隆出现的时间早于HCs（P<0.001）；RCC患者CEPCs培养成功率高于于HCs（87.8% vs. 40.0%， P<0.001）；RCC患者CEPCs克隆数多于健康对照（均数 10.6个vs. 1.83个， P<0.001）。 RCC患者和HCs培养的CEPCs在细胞增殖、免疫表型、摄取Ac-LDL和结合lectin、和体外成管方面均无显著差异。然而，RCC患者培养的CEPCs中VEGF-R2+ CEPCs数要显著多于HCs（21.1% vs. 13.4%， P<0.001）。

　　结论：RCC患者中CEPCs的水平显著升高，且和血清VEGF呈正相关。 RCC患者CEPCs克隆出现的时间早，克隆数多，培养的成功率高于HCs。两组培养的CEPCs具有基本相同的免疫表型和体外成管功能。结果还提示了VEGF-R2+  的CEPCs在RCC中可能起重要作用。

杨斌

　　医学博士，上海市第十人民医院泌尿外科主治医师，擅长前列腺癌，膀胱癌和肾癌的早期诊断与微创根治术。从事泌尿系统肿瘤的转化研究，主持多项科研基金，其中包括国家自然科学基金2项，入选上海市浦江人才培养计划，医院攀登人才培养计划等。在国内外一流学术刊物上以第一作者和通讯作者发表科学论文20余篇，其中SCI收录论文12篇。

研究摘要

220：Tumor-Derived Vascular Endothelial Growth Factor Mobilizes Circulating Endothelial Progenitor Cells and Contributes to Vasculogenesis of Renal Cell Carcinoma

Introduction & Objectives: Circulating Endothelial Progenitor Cells (CEPCs)， bone marrow (BM) derived precursors， have the capacity of differentiation into endothelial cells. Accumulated evidence indicates that CEPCs play an important role in formation of new vessels through a process known as “vasculogenesis”. However， the role of CEPCs in renal cell carcinoma (RCC) hasn’t been well documented. Therefore， the aim of this study was to investigate the contribution of CEPCs to vasculogenesis of RCC and the mechanism by which CEPCs mobilize from BM.

Material & Methods: CEPC levels in blood were quantified by flow cytometry in 86 RCC patients and in 50 healthy controls (HC). CEPCs (CD34+/VEGFR-2+ cells) were detected by real time PCR， WB， and immunofluorescence analyses of the matched tumor， adjacent and normal tissues (TT， AT， NT) from RCC patients receiving radical nephroectomy. The microvessel density (MVD) was recorded and the contribution of CEPC in tissue vasculature was calculated as CEPC+MVD/total MVD. Serum VEGF was quantified by ELISA analysis in RCC patients and HCs. The origination of serum VEGF was analyzed by real time PCR， WB， and immunohistochemistry. Early CEPCs from 20 RCC patients were cultured and characterized. The roles of serum VEGF for CEPC proliferation， migration， wound healing and tube formation were evaluated by stimulating the early CEPCs with concentrated serum from either RCC patient or HCs without or with neutralizing antibody against human VEGF.

Results:The mean CEPC level was significantly higher in patients with RCC than in HCs (p<0.001). Compared with NT samples， the levels of CD34， VEGFR-2， and VEGF expression were significantly increased in AT and TT samples (both p<0.05). Results also demonstrated the levels of the three markers were significantly higher in TT samples than in AT samples (p<0.05). Immunofluorescent staining identified abundant CEPCs (CD34+/VEGFR-2+ cells) in TT samples， while scarce cells in either NT or AT samples. The mean percentage of CEPC+MVD/ total MVD in TT samples was 28.8 %， significantly higher than in AT and NT samples (8.43% and 2.39%， respectively， both p <0.01). The results suggest increased mobilization of CEPC in blood and tumor tissue in RCC patients and the CEPCs contribute to the formation tumor vasculature. Mean serum VEGF in patients with RCC was higher than healthy controls (p<0.001). No statistical difference was found when comparing the VEGF level in peripheral blood mononuclear cells (PBMNCs) between RCC patients and HCs. However， The expression of VEGF in TT was significantly higher than in AT samples and NT samples (both p <0.01). These Results further suggest that serum VEGF mainly originate from tumor tissue. The CEPCs demonstrated a well-circumscribed monolayer of cobblestone-shape in cuture， expressed of eNOS， VE-cadherin and endothelial cell-surface antigens， were ability to uptake Ac-LDL， bind lectin and form capillary tubes in vitro. The results demonstrated that the enhanced cellular activities of CEPCs to concentrated serum from RCC patients can be substantially abrogated by neutralizing antibody against human VEGF.

Conclusions: Our results indicate that tumor-derived VEGF mobilizes CEPCs and the CEPCs contribute to vasculogenesis of RCC， supporting BM-derived CEPC as a potential therapy target in RCC patients.

219: Quantification， Culture and Characterization of Circulating Endothelial Progenitor Cells in Patients with Renal Cell Carcinoma

Introduction & Objectives: Although accumulated evidence indicates that circulating endothelial progenitor cells (CEPCs) contribute to tumor neovascularization， to our knowledge the level of these cells and its correlation with serum vascular endothelial growth factor (VEGF) has not been well reported in patient with renal cell carcinoma (RCC). The culture and characterization of CEPC have not yet been described in these patients with highly angiogenic cancers.

Material & Methods: The CEPC level (percent of CD45-CD34+VEGF-R2+ cells in total peripheral blood mononuclear cells) was quantified in in 53 patients with RCC， 33 with benign renal tumors (BRT) and 40 healthy controls (HC). Serum VEGF was quantified. Peripheral blood mononuclear cells were isolated from 33 patients with RCC and 30 HC to culture and characterize the CEPCs.

Results: The mean CEPC level in patients with RCC was 0.281%， significantly higher than in patients with BRT and HC (0.073% and 0.076%， respectively， each p <0.001). Mean serum VEGF in patients with RCC was higher than in patients with BRT and HC (315.5 vs 34.6 and 26.9 pg/ml， respectively， each p<0.001). The level of preoperative CEPC positively correlated with serum VEGF in patients with RCC (r =0.710， p <0.001). In vitro culture， a colony of CEPC first emerged significantly earlier in RCC patients than in HC (p<0.001). Moreover， the culture success rate (87.8% vs 40.0% of participants) and the number of colonies (10.06 vs 1.83) were significantly greater for RCC patients than for HCs (each p <0.001). Cells cultured from RCC patients and HC showed a similar growth pattern， immunophenotype， ability to uptake Ac-LDL and bind lectin， and form capillary tubes in vitro. However， significantly more VEGF-R2+ CEPCs were found in RCC patients than from HC (21.1% vs 13.4%， p <0.001).

Conclusion: A high CEPC level was found in patients with RCC， which positively correlated with serum VEGF. Furthermore， earlier emergence of circulating endothelial progenitor cell colonies， a higher cell culture success rate and more colonies were found for patients with RCC than for HC. Results indicate the important significance of VEGF-R2+ CEPCs in patients with RCC.