鼠黑色素瘤眼内与皮下种植后血管生成拟态及其分子机制研究

The mechanisms of microenvironments influence on vasculogenic mimicry between introcular and subcutaneous melanoma

目的研究不同微环境对黑色素瘤局部循环模式的影响及其相关分子机制。方法实验研究。将60只C57BL／6J小鼠随机分皮下组和眼内组，每组30只，将B16细胞悬液分别接种到鼠鼷部皮下与视网膜下腔，接种后14d取材，检测移植瘤扩散与转移情况及瘤组织局部微循环模式，计数并比较血管生成拟态（MV）、马赛克血管（VM）、内皮依赖性血管（E）；检查肿瘤组织的扩散及转移情况。采用实时聚合酶链反应（RT—PCR）及免疫组化技术检测酪氨酸激酶受体-2（EphA2）、基质金属蛋白酶-2（MMP-2）及基质金属蛋白酶-2（MMP-9）蛋白及其mRNA表达。进一步分析移植瘤中VM与EphA2、MMP-2及MMP-9mRNA表达量的相关性。利用两组独立样本t检验分析眼内组与皮下组肿瘤组织各指标的差别，组内各指标之间的相关性用Pearson方法分析。结果黑色素瘤眼内与皮下移植瘤中3种微循环模式共存，并且眼内组VM较皮下组增多，两组比较具有统计学意义（t=4．188，P=0．000），而MV和E在皮下移植瘤中高于眼内移植瘤，但两组比较无统计学差异（t=1．473，1．805；P=0．146，0．076）。眼内组EphA2、MMP-2和MMP-9mRNA表达量显著增加，高于皮下组（t=3．642，8．109，9．357；P=0．002，0．001，0．001）。在EphA2表达增高的同时VM也相应增多，二者呈正相关（r=0．412，P=0．021）；VM增多与MMP-2和MMP-9表达增加也存在一定关系，但差异无统计学意义（P〉0．05）。眼内移植瘤中发现眶内扩散5例，肺转移6例。结论微环境变化可使黑色素瘤的微循环模式发生改变，眼内移植瘤细胞可通过增加EphA2、MMP-2和MMP-9表达使VM形成增加，使VM成为眼内移植瘤主要供血模式，从而使肿瘤更具有侵袭性。

Objective To investigate the influence of different microenvironments on melanoma vasculogenic mimicry, invasiveness and metastasis behavior. Methods It was an experimental study. Sixty C57BL/6J mice were randomly divided into two groups with 30 mice per group. Melanoma B16 cells were injected into the subretinal space and groin area of mice synchronously. The number of each type of microcirculation pattern was counted. The invasion and metastasis were observed. EphA2, MMP-2 and MMP-9 expression and their mRNA levels were detected by immunohistochemical staining and real time RTPCR and compared between two groups. Results Five invasions and six lung metastases were found in the subretinal group while no invasion and metastasis were found in the groin group. The number of VM channels was significantly higher in subretinal group （t =4. 188, P =0. 000）. However, no significant difference of mosaic vessel and endothelium-dependent vessel was observed between two groups （ t = 1. 473, 1. 805 ; P = 0. 146, 0. 076, respectively）. EphA2, MMP-2 and MMP-9 expression was significantly higher in the subretinal group （data not shown）. The mRNA levels of EphA2, MMP-2 and MMP-9 were rather higher in the subretinal tumor （t =3. 642, 8. 109,9. 357;P =0. 002, 0. 001 and 0. 001, respectively）. There was apositive association in melanoma cells of the VM between expression of EphA2 （ r = 0. 412, P = 0. 021 ） but no statistically significant correlation between VM and MMP-2 （ P 〉 0. 05 ）, nor between VM and MMP-9. Conclusions Different micmenvimnments affect invasiveness and blood supply patterns of melanoma. Melanoma cells in intraocular microenvimnment increased EphA2 expression which induced the formation of VM channels. Moreover, the expression of MMP-2 and MMP-9 in tumor tissue increased to enhance the invasiveness and metastasis behavior.