PDPN与CLEC-2促进血小板介导的小鼠黑色素瘤肺转移的实验研究

发布时间：2018-05-31 11:18

本文选题：黑色素瘤 + 平足蛋白　；参考：《吉林大学》2015年博士论文

【摘要】：肿瘤转移是大多数恶性肿瘤患者死亡的主要原因之一，但肿瘤转移的具体发生机制目前仍未完全明确。黑色素瘤是一种高度恶性、转移率高的肿瘤。黑色素瘤多发生于皮肤，大部分肿瘤在早期即可发生血行转移，肺转移是黑色素瘤最常见的血行转移部位。目前临床对于恶性黑色素瘤患者的治疗以手术联合放、化疗为主，但对于已发生转移的肿瘤患者仍然没有更好的治疗方法。肿瘤转移发生时，肿瘤细胞从原发部位病灶分离，通过降解周围组织细胞外基质而迁移并侵入血管和淋巴管，随循环到达远处脏器并在局部形成肿瘤转移灶。在肿瘤细胞转移过程中，受到体内多种因素的调节。肿瘤细胞诱导的血小板聚集（TCIPA）在肿瘤转移中起重要作用。血行转移发生时，由原发病灶进入到血液循环内的肿瘤细胞与血小板粘附聚集，激活血小板，形成血小板与肿瘤细胞聚集团块，使肿瘤细胞逃避宿主免疫系统捕获，同时活化的血小板释放各种生长因子和促血管生成因子，促进肿瘤细胞向新的转移部位迁移。平足蛋白（Podoplanin, PDPN）是一种I型唾液酸跨膜糖蛋白，由具有高度糖基化的胞外结构域，跨膜部分和短胞质尾区组成。PDPN选择性表达于淋巴内皮细胞，作为淋巴内皮细胞标志物已被广泛应用。除此之外，，PDPN还具有调节血管淋巴管的发育，调节细胞运动，促进肿瘤发生和转移的多种功能。在多种肿瘤细胞上可检测到PDPN的表达，包括小鼠及人黑色素瘤细胞上也可检测到PDPN的表达。有研究表明表达于某些肿瘤细胞表面上的PDPN通过促进肿瘤细胞侵袭和扩散促进肿瘤的发生及转移，但其具体作用机制尚不清楚。C型凝集素样受体2（CLEC-2）属于II型跨膜蛋白，表达于血小板表面，是PDPN的内源性受体。CLEC-2可通过与PDPN的相互作用诱导血小板激活、聚集。之前有研究表明，PDPN与CLEC-2相互作用诱导的血小板激活，是胚胎期血管淋巴管发育的重要影响因素。近年来，对于PDPN与CLEC-2相互作用诱导的血小板活化在免疫、肿瘤及感染等方面作用的研究越来越引起人们的重视。在本研究中，我们筛选分离PDPN表达水平不同的小鼠黑色素瘤细胞，建立CLEC-2基因缺陷小鼠模型并获得CLEC-2基因缺陷血小板。建立实验性小鼠黑色素瘤肺转移动物模型，通过进行体外和体内实验研究，探讨PDPN和CLEC-2在小鼠黑素瘤血行肺转移过程中对血小板激活的作用，以及二者间的相互作用对小鼠黑色素瘤肺转移的影响。并进一步研究Syk酪氨酸激酶信号传导通路在PDPN和CLEC-2激活血小板过程中及对小鼠黑色素瘤肺转移的作用。 1.流式细胞术分离小鼠黑色素瘤B16-F0PDPN+细胞及PDPN-细胞，建立骨髓移植CLEC-2基因缺陷小鼠模型。经抗PDPN抗体染色，流式细胞术筛选分离并培养B16-F0细胞，经流式细胞学及免疫印迹法检测证实PDPN+细胞及PDPN-细胞中PDPN表达情况。体外培养结果显示两种细胞体外生长增殖无差异。建立骨髓移植CLEC-2基因缺陷小鼠，并取外周血经流式细胞学检测证实小鼠体内CLEC-2基因清除率。 2. PDPN与CLEC-2在体外通过激活血小板促进肿瘤细胞与血小板的聚集。在体外将B16-F0PDPN+细胞及PDPN-细胞分别与野生型血小板及CLEC-2基因缺陷血小板相混合，通过流式细胞学检测、Western blot检测、IF及ICC法比较各组血小板与肿瘤细胞聚集情况。结果显示PDPN+细胞与野生型血小板混和组血小板与肿瘤细胞聚集明显高于其他组，并在镜下观察到明显的血小板与肿瘤细胞聚集团块。 3. PDPN与CLEC-2相互作用诱导体内血小板激活，通过血小板与肿瘤细胞聚集作用促进肺组织对肿瘤细胞的捕获。将以不同荧光染色剂染色的PDPN+细胞及PDPN-细胞等量混和后，经尾静脉注射给野生型小鼠建立实验性小鼠黑色素瘤肺转移模型。注射后30min，2h及6h处死小鼠并取小鼠肺组织，免疫荧光显微镜下观察到在野生型小鼠肺组织内被捕获的PDPN+肿瘤细胞数量明显高于PDPN-细胞。比较注射给野生型小鼠及CLEC-2基因缺陷小鼠30min后肺组织内肿瘤细胞被捕获情况，PDPN+在野生型小鼠肺组织内数量明显高于其他三组。将PDPN+细胞及PDPN-细胞分别注射给野生型小鼠，比较注射前后小鼠体内血小板水平的变化，注射PDPN+组小鼠体内血小板下降较PDPN-组明显。结果表明PDPN与CLEC-2相互作用诱导小鼠体内血小板激活，并引起体内血小板与肿瘤细胞的聚集。 4. PDPN与CLEC-2相互作用促进小鼠黑色素瘤肺转移的发生。比较PDPN+细胞、PDPN-细胞分别注射给野生型小鼠及CLEC-2缺陷小鼠体内引起小鼠黑色素瘤肺转移发生的情况。结果显示PDPN+肿瘤细胞在野生型小鼠体内肺转移率明显高于其他组。肺组织HE染色结果分析表明PDPN+细胞在野生型小鼠肺组织转移结节数量及转移结节面积均明显高于其他组。 5. Syk信号通路在PDPN与CLEC-2介导的血小板聚集及肿瘤转移过程中起着重要作用。用Syk酪氨酸激酶抑制剂R406在体内外干预血小板与肿瘤细胞介导的血小板激活及聚集。Western blot结果显示PDPN+细胞与野生型血小板作用激活Syk磷酸化，促进血小板的聚集作用。流式细胞术及免疫荧光染色分析结果表明R406在一定程度上阻断了体外PDPN+肿瘤细胞与血小板的聚集。体内实验同样显示R406部分抑制了小鼠黑色素瘤的肺转移。 6. PDPN与CLEC-2的相互作用不影响小鼠黑色素瘤皮下肿瘤的生长将PDPN+细胞及PDPN-细胞分别注射给野生型小鼠皮下，建立小鼠黑色素瘤皮下移植肿瘤模型，结果显示PDPN+肿瘤及PDPN-肿瘤体内生长无明显差异。病理组织HE染色结果显示两种肿瘤在组织形态上无明显差异。综上，本研究利用FACS筛选PDPN表达不同的小鼠黑色素瘤细胞，并建立骨髓移植CLEC-2基因缺陷小鼠，通过肿瘤细胞与血小板体外实验研究及建立实验性小鼠黑色素瘤肺转移动物模型研究，明确了小鼠黑色素瘤细胞表达的PDPN与血小板CLEC-2相互作用，在体、内外均能够诱导血小板的活化，二者在体内诱导的血小板激活促进小鼠黑色素瘤肺转移的发生，其中血小板的活化是部分依赖于Syk酪氨酸激酶信号通路完成的。肿瘤细胞表达的PDPN及血小板CLEC-2有望成为预示黑色素瘤肿瘤转移发生及抗肿瘤转移治疗的新靶点。
[Abstract]:Tumor metastasis is one of the main causes of death in most malignant tumor patients, but the specific mechanism of tumor metastasis is still not completely clear. Melanoma is a highly malignant, high metastatic tumor. Melanoma occurs mostly in the skin, most of the tumor can occur in the early stage of blood metastasis, and lung metastasis is the most common melanoma. There is no better treatment for patients with malignant melanoma, but there is still no better treatment for cancer patients who have metastasize. When the metastasis occurs, the tumor cells are separated from the primary site and transferred and invaded through the degradation of the peripheral tissue extracellular matrix. Blood vessels and lymphatic vessels reach distant viscera and form tumor metastases locally. During the metastasis of tumor cells, many factors are regulated in the body. Tumor cell induced platelet aggregation (TCIPA) plays an important role in tumor metastasis. Blood metastasis occurs when the primary focus is entered into the blood circulation. Cell adhesion and aggregation, activating platelets, forming platelets and aggregation of tumor cells, causing tumor cells to escape from the host immune system, and the activated platelets release various growth factors and angiogenesis factors to promote the migration of tumor cells to the new metastatic sites.
Podoplanin (PDPN) is a kind of I type sialic acid transmembrane glycoprotein, which has a highly glycosylated extracellular domain, transmembrane part and short cytoplasmic tail region of.PDPN selectively expressed in lymphatic endothelial cells. It has been widely used as a marker of lymphatic endothelial cells. In addition, PDPN also regulates the development of vascular lymphatic vessels. A variety of functions that regulate cell movement and promote the occurrence and metastasis of tumors. The expression of PDPN can be detected on a variety of tumor cells, including the expression of PDPN on mice and human melanoma cells. Studies have shown that PDPN expressed on the surface of certain tumor cells promotes the occurrence of tumor by promoting tumor cells invasion and diffusion. However, it is not clear that the.C type lectin like receptor 2 (CLEC-2) belongs to the II type transmembrane protein, which is expressed on the surface of the platelets. It is the endogenous receptor.CLEC-2 of PDPN, which can induce platelet activation and aggregation through the interaction with PDPN. Previous studies showed that the platelet activation induced by the interaction of PDPN and CLEC-2 is the embryo. In recent years, more and more attention has been paid to the role of platelet activation induced by the interaction of PDPN and CLEC-2 in immunization, tumor and infection.
In this study, we screened the mouse melanoma cells with different levels of PDPN expression, established a CLEC-2 gene deficient mouse model and obtained the CLEC-2 gene defective platelets. The experimental mice model of pulmonary metastasis of melanoma was established. In vitro and in vivo experiments were carried out to explore the blood line lung of PDPN and CLEC-2 in murine melanoma. The effect of platelet activation in the process of metastasis, and the effect of the interaction between the two groups on the lung metastasis of melanoma in mice, and the effect of Syk tyrosine kinase signal transduction pathway on the activation of platelets in PDPN and CLEC-2 and the pulmonary metastasis of melanoma in mice were further studied.
1. mouse melanoma B16-F0PDPN+ cells and PDPN- cells were isolated by flow cytometry, and CLEC-2 gene deficient mice model was established.
B16-F0 cells were isolated and cultured by flow cytometry by flow cytometry. The expression of PDPN in PDPN+ cells and PDPN- cells was confirmed by flow cytometry and immunoblotting. In vitro culture results showed that there was no difference in growth and proliferation of two kinds of cells in vitro. A bone marrow transplant CLEC-2 gene defect mouse was established and the peripheral blood flow cytometry was taken. Cytology test confirmed the clearance rate of CLEC-2 gene in mice.
2. PDPN and CLEC-2 promote platelet aggregation by activating platelets in vitro.
In vitro, B16-F0PDPN+ cells and PDPN- cells were mixed with wild type platelets and CLEC-2 gene defective platelets, respectively, by flow cytometry, Western blot detection, IF and ICC methods to compare the aggregation of platelets and tumor cells in each group. The results showed that the platelet and tumor cells in the mixed group of PDPN+ cells and wild type platelets were clustered with the tumor cells. The aggregation was obviously higher than that of other groups, and obvious aggregation of platelet and tumor cells was observed under microscope.
3. the interaction between PDPN and CLEC-2 induces platelet activation in vivo, and promotes the acquisition of tumor cells by the aggregation of platelets and tumor cells.
After being mixed with PDPN+ cells and PDPN- cells stained with different fluorescent stains, an experimental mouse model of pulmonary metastasis was established by injecting the tail vein to the wild mice. After injection, 30min, 2h and 6h were killed in mice and the lung tissues were taken and the PDPN+ captured in the lung tissue of wild mice was observed under the immunofluorescence microscope. The number of tumor cells was significantly higher than that of PDPN- cells. The tumor cells in the lung tissues of the wild type and CLEC-2 gene deficient mice were compared. The number of PDPN+ in the lung tissues of the wild type mice was significantly higher than that of the other three groups. The PDPN+ and PDPN- cells were injected to the wild type mice respectively, and the mice were compared before and after the injection. The changes in the level of internal platelets in the PDPN+ group were significantly lower than that in the PDPN- group. The results showed that the interaction between PDPN and CLEC-2 induced the activation of platelets in the mice and the aggregation of platelets and tumor cells in the body.
4. the interaction of PDPN and CLEC-2 promotes the occurrence of pulmonary metastasis of melanoma in mice.
The results showed that the lung metastasis rate of the PDPN+ tumor cells in the wild type mice was significantly higher than that of the other groups in the wild type mice and the CLEC-2 deficient mice. The results of HE staining in the lung tissue showed that the PDPN+ cells were transferred to the lung tissue of the wild type mice in the lung tissue. The results of the HE staining of the lung tissue showed that the PDPN+ cells were transferred to the lung tissue of the wild type mice. The number of nodules and the area of metastatic nodules were significantly higher than those of other groups.
5. the Syk signaling pathway plays an important role in platelet aggregation and tumor metastasis mediated by PDPN and CLEC-2.
The effect of Syk tyrosine kinase inhibitor R406 on platelet activation and aggregation of platelets mediated by platelet and tumor cells in vitro and in vivo.Western blot results showed that the action of PDPN+ cells and wild type platelets activated Syk phosphorylation and promoted platelet aggregation. The results of flow cytometry and immunofluorescence staining showed that R406 was to a certain extent. The aggregation of PDPN+ tumor cells and platelets in vitro was blocked. In vivo experiments also showed that R406 partially inhibited the metastasis of melanoma in mice.
6. the interaction between PDPN and CLEC-2 does not affect the growth of melanoma subcutaneous tumor in mice.
PDPN+ cells and PDPN- cells were injected subcutaneously into the subcutaneous tissue of wild type mice. The tumor model of subcutaneous melanoma in mice was established. The results showed that there was no significant difference in the growth of PDPN+ tumor and PDPN- tumor. The pathological tissue HE staining showed that there was no significant difference in the tissue morphology between the two kinds of tumor.
To sum up, this study uses FACS to screen PDPN to express different murine melanoma cells, and to establish a mouse with CLEC-2 gene defect in bone marrow transplantation. Through the study of the tumor cells and platelets in vitro and the establishment of experimental model of experimental mouse melanoma lung metastasis, the PDPN and platelet CLEC-2 expressed in the melanoma cells of the rat are clearly defined. Interaction, both in vivo and in vivo, can induce platelet activation, and the activation of platelets induced by two in vivo promotes the occurrence of lung metastases in murine melanoma, in which the activation of platelets is partly dependent on the Syk tyrosine kinase signaling pathway. The PDPN and platelet CLEC-2 expressed by the tumor cells are expected to be a prediction of melanoma. Tumor metastasis and new target of anti-tumor metastasis therapy.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R739.5

【共引文献】