不同转移潜能的卵巢癌裸鼠移植瘤和临床组织新生淋巴管形成与淋巴结转移的关系研究

发布时间：2018-05-15 13:31

本文选题：卵巢癌 + 绿色荧光蛋白　；参考：《广西医科大学》2014年硕士论文

【摘要】：目的：构建具有绿色荧光蛋白示踪的卵巢癌裸鼠爪垫移植瘤模型，动态观察卵巢癌淋巴结转移的过程。方法：利用慢病毒载体构建稳定表达绿色荧光蛋白（green fluorescent protein，GFP）的卵巢癌细胞SKOV3-GFP+细胞及具有定向淋巴道转移能力的卵巢癌细胞SKOV3-PM4-GFP+细胞。并分别接种于裸鼠后肢爪垫，建立爪垫荷瘤模型。利用小动物活体成像系统动态追踪肿瘤转移灶的出现时间、部位、路径。定期记录肿瘤体积的变化及体重变化。免疫组化法检测各组裸鼠淋巴结CA125的表达情况。结果：携带绿色荧光蛋白慢病毒载体转染细胞后，经流式细胞仪分选的SKOV3-GFP+细胞和SKOV3-PM4-GFP+细胞荧光强度强，且表达稳定。与接种SKOV3-GFP+细胞的裸鼠比较，接种了SKOV3-PM4-GFP+细胞的裸鼠出现转移灶的时间早，在相同时间点出现乆窝淋巴结肿大的裸鼠数量较多，淋巴结组织CA125为阳性的乆窝转移率较高。但两组裸鼠体重及瘤体体积无明显差异。结论：成功建立了绿色荧光蛋白示踪的卵巢癌定向淋巴道转移模型。目的：检测不同淋巴道转移潜能的上皮性卵巢癌裸鼠荷瘤模型及临床卵巢癌组织的新生淋巴管情况，确定影响淋巴管生成及转移的关键分子，明确瘤内新生淋巴管形成在卵巢癌淋巴结转移过程中的作用。方法：1、免疫组织化学法（IHC）检测SKOV3-PM4-GFP+组及SKOV3-GFP+组裸鼠移植瘤组织中D2-40标记的微淋巴管密度（LVD）、CD-34标记的微血管密度（MVD）及淋巴管生成相关分子VEGF-C、VEGF-D的表达情况。2、qRT-PCR技术检测裸鼠移植瘤中VEGF-C、VEGF-D、VEGFR-3基因mRNA的表达。3、透射电镜（TEM）观察不同淋巴道转移潜能细胞移植瘤内新生淋巴管的超微结构。4、结合临床病理资料分析卵巢癌组织中VEGF-C、VEGF-D的表达与LVD、MVD的关系，以及VEGF-C、VEGF-D、LVD、MVD的表达与卵巢癌淋巴结转移、大网膜转移等临床病理特征的相关性。结果：1、SKOV3-PM4-GFP+组裸鼠移植瘤内新生淋巴管密度(LVD)的表达较SKOV3-GFP+组高，分别为9.79±1.72、5.94±0.78（P0.05）；而两组裸鼠移植瘤内的血管密度（MVD）无统计学差异；2、裸鼠移植瘤内VEGF-C蛋白及mRNA的表达SKOV3-PM4-GFP+组均较SKOV3-GFP+组高，mRNA表达量分别为2.66±0.30、1.13±0.33（P0.05）；蛋白表达灰度值分别为253.18±23.6、104.22±17.4（P0.05）。而VEGF-D蛋白及mRNA的表达在两组移植瘤中无统计学差异。3、透射电镜超微结构（ultrastructure）显示，SKOV3-PM4-GFP+组形成明显淋巴管腔，淋巴管内皮细胞基底膜溶解，线粒体空泡化及染色质边集严重，细胞间连接较紧密。SKOV3-GFP+组淋巴管腔不明显，为条索状，基底膜部分断裂，线粒体嵴消失，细胞间连接疏松。4、在良性卵巢肿瘤29例，交界性卵巢肿瘤11例，，上皮性卵巢癌47例的卵巢肿瘤组织标本中VEGF-C、VEGF-D均有表达，VEGF-C阳性表达率分别为34.48%、54.50%、78.72%（P0.05）；VEGF-D阳性表达率在三组卵巢肿瘤组织中分别为24.13%、36.36%、57.44%（P0.05）。新生淋巴管密度（LVD）在恶性卵巢癌中最高，良性卵巢肿瘤中最低，三组组织中的LVD表达分别为5.3±2.49、10.2±1.13、14.2±2.26，三组间均有统计学差异（P0.05；而新生血管密度（MVD）在三组肿瘤组织间无差异。恶性卵巢癌中VEGF-C、LVD的表达均与临床FIGO分期、淋巴结转移、大网膜转移有关；而恶性卵巢癌中VEGF-D的表达仅与临床FIGO分期相关，MVD则与大网膜转移相关。VEGF-C在恶性卵巢癌中的表达与MVD、LVD呈显著正相关；VEGF-D的表达与MVD、LVD呈正相关。VEGF-C关联性最好。结论：卵巢癌新生淋巴管生成与淋巴道转移相关，SKOV3-PM4-GFP+细胞因其VEGF-C表达量较高及其对淋巴管内皮细胞超微结构的侵袭损伤，而具有更高的淋巴道转移潜能。VEGF-C/VEGFR-3信号轴可能为卵巢癌淋巴道转移的潜在治疗靶点。淋巴管在肿瘤转移过程中起着至关重要的作用。近年来发现在多种肿瘤组织内部也存在微淋巴管，肿瘤内淋巴管生成及淋巴管密度显著影响肿瘤转移。但肿瘤内淋巴管的研究有赖于特异淋巴管内皮标志物发现及淋巴管生成因子对肿瘤淋巴管生成的调控。针对V EGF-C/VEGF-D—VEGFR-3信号转导系统的抗淋巴管生成治疗,有望成为抗淋巴转移的一个有效途径，而其中每一个环节都可能成为控制肿瘤生长、转移以及治疗肿瘤的潜在靶点。
[Abstract]:Objective: to construct a nude mouse claw pad transplantation tumor model with green fluorescent protein (GFP), and to dynamically observe the process of lymph node metastasis of ovarian cancer. Methods: using the lentivirus vector to construct the SKOV3-GFP+ cells of ovarian cancer cells that express green fluorescent protein (GFP) and have the ability of directional lymphatic metastasis. SKOV3-PM4-GFP+ cells of ovarian cancer cells were inoculated in the hind paw pads of nude mice to establish a claw pad tumor model. The time, location and path of tumor metastasis were traced dynamically by the living body imaging system of small animals. The changes of tumor volume and weight change were recorded regularly. The expression of CA125 in nude mice lymph nodes was detected by immunohistochemical method. Results: after the transfection of the green fluorescent protein lentivirus vector to the cells, the fluorescence intensity and expression of the SKOV3-GFP+ and SKOV3-PM4-GFP+ cells were strong and stable. Compared with the nude mice inoculated with SKOV3-GFP+ cells, the nude mice inoculated with SKOV3-PM4-GFP+ cells were early and appeared at the same time point. The number of nude mice with swollen lymph nodes was more and the metastatic rate of CA125 was higher in the lymph nodes. However, there was no significant difference in body weight and tumor volume between the two groups. Conclusion: the model of directional lymphatic metastasis of ovarian cancer with green fluorescent protein tracer was successfully established.
Objective: to detect the tumor model of epithelial ovarian cancer in nude mice with different lymphatic metastasis potential and the new lymphoid tube in the clinical ovarian cancer tissue, to determine the key molecules affecting the formation and metastasis of lymphatic vessels, and to determine the role of the neoplastic lymphatic formation in the lymph node metastasis of ovarian cancer. Methods: 1, immunohistochemical method (IHC) D2-40 labeled microlymphatic vessel density (LVD), CD-34 labeled microvascular density (MVD) and lymphoduction related molecules VEGF-C, VEGF-D expression in SKOV3-PM4-GFP+ group and SKOV3-GFP+ group nude mice were detected by CD-34 The ultrastructure of new lymphoid tube in different lymphatic metastatic potential cell transplantation tumor (.4), combined with clinicopathological data, the relationship between the expression of VEGF-C, VEGF-D and LVD, MVD, and the correlation between the expression of VEGF-C, VEGF-D, LVD, MVD and the clinicopathological features of ovarian cancer lymph node metastasis and large mesh membrane metastasis were analyzed. Results: 1, SKOV3-P The expression of nascent lymphatic density (LVD) in the transplanted tumor of M4-GFP+ group was higher than that in group SKOV3-GFP+, 9.79 + 1.72,5.94 + 0.78 (P0.05), and there was no statistical difference in the density of vascular density (MVD) in the two groups of nude mice. 2, the SKOV3-PM4-GFP+ group of VEGF-C protein and mRNA in the transplanted tumor of nude mice was higher than the SKOV3-GFP+ group, and the mRNA expression was respectively The expression of the protein expression was 2.66 + 0.30,1.13 + 0.33 (P0.05), the expression of protein was 253.18 + 23.6104.22 + 17.4 (P0.05), while the expression of VEGF-D protein and mRNA had no statistical difference in the two groups of transplanted tumors. The ultrastructure of the transmission electron microscope (ultrastructure) showed that the SKOV3-PM4-GFP+ group formed the obvious lymphatic cavity, the basal membrane of the lymphatic endothelium was dissolved, and the line was dissolved. The granular vacuolation and chromatin side collection were serious, and the intercellular connection was not obvious in the.SKOV3-GFP+ group. It was stripe like, the basal membrane partially ruptured, the mitochondrial crista disappeared, the intercellular connection was loose.4, 29 cases of benign ovarian tumors, 11 borderline ovarian tumors, and 47 ovarian tumor tissue specimens of epithelial ovarian cancer, VEGF-C, VEGF-D The positive expression rates of VEGF-C were 34.48%, 54.50%, 78.72% (P0.05), and the positive expression rate of VEGF-D was 24.13%, 36.36%, 57.44% (P0.05) in the three groups of ovarian tumors. The newborn lymphatic density (LVD) was the highest in the malignant ovarian cancer, the lowest in the benign ovarian tumor, and the LVD expression in the three group was 5.3 + 2.49,10.2 + 1.13, respectively. 14.2 + 2.26, there were statistical differences between the three groups (P0.05; and the new vascular density (MVD) was no difference between the three groups. The expression of VEGF-C and LVD in malignant ovarian cancer was related to the clinical FIGO stage, lymph node metastasis and greater omentum metastasis; and the expression of VEGF-D in malignant ovarian cancer was only associated with the clinical FIGO staging, and MVD with greater omentum. The expression of.VEGF-C in malignant ovarian cancer was significantly correlated with MVD and LVD, and the expression of VEGF-D was positively associated with MVD, LVD and.VEGF-C. Conclusion: lymphangiogenesis in ovarian cancer is associated with lymphatic metastasis, and SKOV3-PM4-GFP+ cells are higher in VEGF-C expression and the invasion of lymphatic endothelial cell ultrastructure. .VEGF-C/VEGFR-3 signal axis may be a potential therapeutic target for lymphatic metastasis of ovarian cancer.
Lymphatic vessels play a vital role in the process of tumor metastasis. In recent years, there have been microlymphatics in various tumor tissues. Lymphangiogenesis and lymphatic density in tumors have a significant influence on tumor metastasis. However, the study of lymphatics depends on the discovery of specific lymphatic endothelial markers and the swelling of lymphangiogenic factors. The regulation of tumor lymphangiogenesis is an effective way to resist lymphatic metastasis for V EGF-C/VEGF-D VEGFR-3 signal transduction system, and every link may be a potential target for controlling tumor growth, metastasis and treatment of tumor.

【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31

【参考文献】