本文選題：卵巢癌 + 綠色熒光蛋白��； 參考：《廣西醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的：構(gòu)建具有綠色熒光蛋白示蹤的卵巢癌裸鼠爪墊移植瘤模型，動(dòng)態(tài)觀察卵巢癌淋巴結(jié)轉(zhuǎn)移的過程。方法：利用慢病毒載體構(gòu)建穩(wěn)定表達(dá)綠色熒光蛋白（green fluorescent protein，GFP）的卵巢癌細(xì)胞SKOV3-GFP+細(xì)胞及具有定向淋巴道轉(zhuǎn)移能力的卵巢癌細(xì)胞SKOV3-PM4-GFP+細(xì)胞。并分別接種于裸鼠后肢爪墊，建立爪墊荷瘤模型。利用小動(dòng)物活體成像系統(tǒng)動(dòng)態(tài)追蹤腫瘤轉(zhuǎn)移灶的出現(xiàn)時(shí)間、部位、路徑。定期記錄腫瘤體積的變化及體重變化。免疫組化法檢測(cè)各組裸鼠淋巴結(jié)CA125的表達(dá)情況。結(jié)果：攜帶綠色熒光蛋白慢病毒載體轉(zhuǎn)染細(xì)胞后，經(jīng)流式細(xì)胞儀分選的SKOV3-GFP+細(xì)胞和SKOV3-PM4-GFP+細(xì)胞熒光強(qiáng)度強(qiáng)，且表達(dá)穩(wěn)定。與接種SKOV3-GFP+細(xì)胞的裸鼠比較，接種了SKOV3-PM4-GFP+細(xì)胞的裸鼠出現(xiàn)轉(zhuǎn)移灶的時(shí)間早，在相同時(shí)間點(diǎn)出現(xiàn)乆窩淋巴結(jié)腫大的裸鼠數(shù)量較多，淋巴結(jié)組織CA125為陽(yáng)性的乆窩轉(zhuǎn)移率較高。但兩組裸鼠體重及瘤體體積無明顯差異。結(jié)論：成功建立了綠色熒光蛋白示蹤的卵巢癌定向淋巴道轉(zhuǎn)移模型。 目的：檢測(cè)不同淋巴道轉(zhuǎn)移潛能的上皮性卵巢癌裸鼠荷瘤模型及臨床卵巢癌組織的新生淋巴管情況，確定影響淋巴管生成及轉(zhuǎn)移的關(guān)鍵分子，明確瘤內(nèi)新生淋巴管形成在卵巢癌淋巴結(jié)轉(zhuǎn)移過程中的作用。方法：1、免疫組織化學(xué)法（IHC）檢測(cè)SKOV3-PM4-GFP+組及SKOV3-GFP+組裸鼠移植瘤組織中D2-40標(biāo)記的微淋巴管密度（LVD）、CD-34標(biāo)記的微血管密度（MVD）及淋巴管生成相關(guān)分子VEGF-C、VEGF-D的表達(dá)情況。2、qRT-PCR技術(shù)檢測(cè)裸鼠移植瘤中VEGF-C、VEGF-D、VEGFR-3基因mRNA的表達(dá)。3、透射電鏡（TEM）觀察不同淋巴道轉(zhuǎn)移潛能細(xì)胞移植瘤內(nèi)新生淋巴管的超微結(jié)構(gòu)。4、結(jié)合臨床病理資料分析卵巢癌組織中VEGF-C、VEGF-D的表達(dá)與LVD、MVD的關(guān)系，以及VEGF-C、VEGF-D、LVD、MVD的表達(dá)與卵巢癌淋巴結(jié)轉(zhuǎn)移、大網(wǎng)膜轉(zhuǎn)移等臨床病理特征的相關(guān)性。結(jié)果：1、SKOV3-PM4-GFP+組裸鼠移植瘤內(nèi)新生淋巴管密度(LVD)的表達(dá)較SKOV3-GFP+組高，分別為9.79±1.72、5.94±0.78（P0.05）；而兩組裸鼠移植瘤內(nèi)的血管密度（MVD）無統(tǒng)計(jì)學(xué)差異；2、裸鼠移植瘤內(nèi)VEGF-C蛋白及mRNA的表達(dá)SKOV3-PM4-GFP+組均較SKOV3-GFP+組高，mRNA表達(dá)量分別為2.66±0.30、1.13±0.33（P0.05）；蛋白表達(dá)灰度值分別為253.18±23.6、104.22±17.4（P0.05）。而VEGF-D蛋白及mRNA的表達(dá)在兩組移植瘤中無統(tǒng)計(jì)學(xué)差異。3、透射電鏡超微結(jié)構(gòu)（ultrastructure）顯示，SKOV3-PM4-GFP+組形成明顯淋巴管腔，淋巴管內(nèi)皮細(xì)胞基底膜溶解，線粒體空泡化及染色質(zhì)邊集嚴(yán)重，細(xì)胞間連接較緊密。SKOV3-GFP+組淋巴管腔不明顯，為條索狀，基底膜部分?jǐn)嗔眩�粒體嵴消失，細(xì)胞間連接疏松。4、在良性卵巢腫瘤29例，交界性卵巢腫瘤11例，，上皮性卵巢癌47例的卵巢腫瘤組織標(biāo)本中VEGF-C、VEGF-D均有表達(dá)，VEGF-C陽(yáng)性表達(dá)率分別為34.48%、54.50%、78.72%（P0.05）；VEGF-D陽(yáng)性表達(dá)率在三組卵巢腫瘤組織中分別為24.13%、36.36%、57.44%（P0.05）。新生淋巴管密度（LVD）在惡性卵巢癌中最高，良性卵巢腫瘤中最低，三組組織中的LVD表達(dá)分別為5.3±2.49、10.2±1.13、14.2±2.26，三組間均有統(tǒng)計(jì)學(xué)差異（P0.05；而新生血管密度（MVD）在三組腫瘤組織間無差異。惡性卵巢癌中VEGF-C、LVD的表達(dá)均與臨床FIGO分期、淋巴結(jié)轉(zhuǎn)移、大網(wǎng)膜轉(zhuǎn)移有關(guān)；而惡性卵巢癌中VEGF-D的表達(dá)僅與臨床FIGO分期相關(guān)，MVD則與大網(wǎng)膜轉(zhuǎn)移相關(guān)。VEGF-C在惡性卵巢癌中的表達(dá)與MVD、LVD呈顯著正相關(guān)；VEGF-D的表達(dá)與MVD、LVD呈正相關(guān)。VEGF-C關(guān)聯(lián)性最好。結(jié)論：卵巢癌新生淋巴管生成與淋巴道轉(zhuǎn)移相關(guān)，SKOV3-PM4-GFP+細(xì)胞因其VEGF-C表達(dá)量較高及其對(duì)淋巴管內(nèi)皮細(xì)胞超微結(jié)構(gòu)的侵襲損傷，而具有更高的淋巴道轉(zhuǎn)移潛能。VEGF-C/VEGFR-3信號(hào)軸可能為卵巢癌淋巴道轉(zhuǎn)移的潛在治療靶點(diǎn)。 淋巴管在腫瘤轉(zhuǎn)移過程中起著至關(guān)重要的作用。近年來發(fā)現(xiàn)在多種腫瘤組織內(nèi)部也存在微淋巴管，腫瘤內(nèi)淋巴管生成及淋巴管密度顯著影響腫瘤轉(zhuǎn)移。但腫瘤內(nèi)淋巴管的研究有賴于特異淋巴管內(nèi)皮標(biāo)志物發(fā)現(xiàn)及淋巴管生成因子對(duì)腫瘤淋巴管生成的調(diào)控。針對(duì)V EGF-C/VEGF-D—VEGFR-3信號(hào)轉(zhuǎn)導(dǎo)系統(tǒng)的抗淋巴管生成治療,有望成為抗淋巴轉(zhuǎn)移的一個(gè)有效途徑，而其中每一個(gè)環(huán)節(jié)都可能成為控制腫瘤生長(zhǎng)、轉(zhuǎn)移以及治療腫瘤的潛在靶點(diǎn)。
[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.

【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.31

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