【摘要】：侵襲和轉(zhuǎn)移是卵巢癌患者死亡的重要因素，血管新生為侵襲和轉(zhuǎn)移提供基礎(chǔ)，所以在此過程中起關(guān)鍵作用，血管內(nèi)皮生長因子（VEGF）是目前已知最強的血管通透劑，它能導(dǎo)致癌性腹水或腫瘤間質(zhì)水腫，而且導(dǎo)致血漿、纖維等蛋白向血管外滲出，導(dǎo)致細胞外基質(zhì)的改變，從而促進血管的形成，是重要的與惡性腫瘤侵襲、轉(zhuǎn)移和腹水形成相關(guān)的生長因子。 高遷移率族蛋白B1（HMGB1）是核內(nèi)DNA結(jié)合蛋白由壞死細胞釋放，誘導(dǎo)炎癥反應(yīng)，促進組織修復(fù)和血管生成。因HMGB1可誘導(dǎo)血管內(nèi)皮細胞生長、遷移及芽發(fā)而被定義為促血管生成因子。HMGB1誘導(dǎo)的細胞遷移要求激活經(jīng)典和非經(jīng)典的ＮＦ－κＢ通路，這樣會導(dǎo)致CXCL12基因的轉(zhuǎn)錄。HMGB1還可以保護CXCL12免受降解，誘導(dǎo)額外的CXCL12的分泌。 CXCL12在血管新生中起重要作用，CXCL12上調(diào)VEGF表達，而VEGF上調(diào)CXCL12表達，CXCR4-CXCL12和VEGF在促進血管產(chǎn)生過程中有協(xié)同作用。HMGB1和CXCL12形成的異元復(fù)合體，僅通過CXCR4信號通路發(fā)揮炎癥細胞募集的作用。 因此，我們擬通過檢測HMGB1對卵巢癌血管內(nèi)皮生長因子VEGF和趨化因子12（CXCL12）表達的影響及機制來了解是否對卵巢癌的血管新生有影響，初步探討卵巢癌中血管新生的機制。 第一部分卵巢癌組織中HMGB1，CXCL12，VEGF的表達。 目的： 檢測卵巢癌組織中HMGB1，CXCL12，VEGF的表達 方法： 選取54例已確診為上皮性卵巢癌患者，對照組為同期因其它婦科疾病行手術(shù)切除正常卵巢組織標本20例，通過免疫組化方法，檢測卵巢癌組織中HMGB1、CXCL12、VEGF的表達情況。 結(jié)果： HMGB1在癌細胞、間質(zhì)細胞的細胞質(zhì)和細胞核中均有淡黃色至棕褐色顆粒；CXCL12在癌細胞和間質(zhì)細胞的細胞質(zhì)呈淡黃色至棕褐色；VEGF在癌細胞及間質(zhì)細胞的細胞質(zhì)呈淡黃色至棕黃色。 結(jié)論：HMGB1、VEGF和CXCL12在卵巢癌組織中均有表達，且與淋巴結(jié)的轉(zhuǎn)移有關(guān)。 第二部分SKOV3，HUVECs細胞中HMGB1表達 目的： 檢測HMGB1在SKOV3，HUVECs細胞中具體表達情況。 方法： 分別提取SKOV3，HUVECs細胞總蛋白、胞漿及胞核蛋白，后利用westernblot檢測HMGB1在兩者細胞中的表達情況。 結(jié)果： HMGB1不僅在SKOV3細胞有蛋白表達，在HUVECs細胞中也有蛋白表達，但在SKOV3中蛋白表達高于HUVECs細胞中，HMGB1在SKOV3細胞胞漿的表達顯著高于HUVECs細胞胞漿表達（P=0.009）胞核的表達顯著高于HUVECs細胞胞核表達（P=0.000），SKOV3胞核中HMGB1蛋白的表達顯著高于其胞漿（P=0.000）。HUVECs胞核中HMGB1蛋白的表達顯著高于其胞漿表達（P=0.026）。 結(jié)論： MGB1在SKOV3，HUVECs細胞中均有表達，，可能與HMGB1所處狀態(tài)的不同有關(guān)。 第三部分HMGB1表達變化后對SKOV3血管生成的影響及作用機制研究 目的： 探討HMGB1在SKOV3血管生成中的作用及其機制 方法： 將構(gòu)建HMGB1真核表達質(zhì)粒和人工合成針對HMGB1基因的siRNA分別轉(zhuǎn)染SKOV3細胞，運用Western blot檢測轉(zhuǎn)染前后HMGB1，CXCL12的表達變化，利用流式細胞術(shù)檢測SKOV3的凋亡。 結(jié)果： HMGB1過表達真核表達質(zhì)粒DNA轉(zhuǎn)染SKOV3細胞后SKOV3凋亡減少，而人工合成針對HMGB1基因的siRNA轉(zhuǎn)染SKOV3細胞后HMGB1，CXCL12，VEGF表達均減弱，SKOV3細胞凋亡增加，與對照組比差異顯著有統(tǒng)計學(xué)意義（P0.05） 結(jié)論： HMGB1的下降可能影響CXCL12，VEGF的下降，且可能與SKOV3細胞的凋亡也有關(guān)系。
[Abstract]:Invasion and metastasis are important factors in the death of ovarian cancer patients. Angiogenesis provides the basis for invasion and metastasis. Vascular endothelial growth factor (VEGF) plays a key role in this process. Vascular endothelial growth factor (VEGF) is the strongest known vasodilator. It can lead to malignant ascites or interstitial edema, and lead to plasma, fibrin and other proteins outside the blood vessels. Exudation, which results in the alteration of extracellular matrix and thus promotes angiogenesis, is an important growth factor associated with invasion, metastasis and ascites formation of malignant tumors.
High mobility group protein B1 (HMGB1) is an intranuclear DNA binding protein released by necrotic cells, which induces inflammation, promotes tissue repair and angiogenesis. HMGB1 is defined as an angiogenic factor because it induces the growth, migration and germination of vascular endothelial cells. HMGB1-induced cell migration requires activation of classical and non-classical NF-kappa B channels. HMGB1 also protects CXCL12 from degradation and induces extra CXCL12 secretion.
CXCL12 plays an important role in angiogenesis. CXCL12 up-regulates the expression of VEGF, while VEGF up-regulates the expression of CXCL12. CXCR4-CXCL12 and VEGF play a synergistic role in promoting angiogenesis. Heterogeneous complexes formed by HMGB1 and CXCL12 play a role in inflammatory cell recruitment only through CXCR4 signaling pathway.
Therefore, we intend to investigate the effect of HMGB1 on the expression of vascular endothelial growth factor (VEGF) and chemokine 12 (CXCL12) in ovarian cancer and its mechanism.
The first part is the expression of HMGB1, CXCL12 and VEGF in ovarian cancer.
Objective:
Detection of HMGB1, CXCL12 and VEGF expression in ovarian cancer tissues
Method:
The expression of HMGB1, CXCL12 and VEGF in 54 patients with epithelial ovarian cancer and 20 normal ovarian tissues were detected by immunohistochemistry.
Result:
HMGB1 had pale yellow to brown granules in cytoplasm and nucleus of cancer cells, stromal cells, CXCL12 had pale yellow to brown cytoplasm of cancer cells and stromal cells, and VEGF had pale yellow to brown cytoplasm of cancer cells and stromal cells.
Conclusion: HMGB1, VEGF and CXCL12 are all expressed in ovarian cancer tissues, and are related to lymph node metastasis.
The second part is the expression of HMGB1 in SKOV3 and HUVECs cells.
Objective:
The specific expression of HMGB1 in SKOV3 and HUVECs cells was detected.
Method:
SKOV3 and HUVECs cell total protein, cytoplasmic and nuclear protein were extracted respectively, and the expression of HMGB1 was detected by Western blot.
Result:
HMGB1 was expressed not only in SKOV3 cells, but also in HUVECs cells. However, the expression of HMGB1 in SKOV3 cells was higher than that in HUVECs cells. The expression of HMGB1 in the cytoplasm of SKOV3 cells was significantly higher than that in the cytoplasm of HUVECs cells (P = 0.009), and the expression of HMGB1 in the nucleus of SKOV3 cells was significantly higher than that in the nucleus of HUVECs cells (P = 0.000). The expression of HMGB1 protein in the nucleus of HUVECs was significantly higher than that in the cytoplasm (P = 0.026).
Conclusion:
MGB1 is expressed in SKOV3 and HUVECs cells, which may be related to the different state of HMGB1.
The third part is the effect of HMGB1 expression on SKOV3 angiogenesis and its mechanism.
Objective:
The role and mechanism of HMGB1 in SKOV3 angiogenesis
Method:
SKOV3 cells were transfected with HMGB1 eukaryotic expression plasmid and synthesized siRNA targeting HMGB1 gene. The expression of HMGB1 and CXCL12 was detected by Western blot. The apoptosis of SKOV3 cells was detected by flow cytometry.
Result:
The apoptosis of SKOV3 cells was decreased after transfection of HMGB1 overexpression plasmid DNA into SKOV3 cells, while the expression of HMGB1, CXCL12 and VEGF were decreased after transfection of SKOV3 cells with synthetic siRNA targeting HMGB1 gene, and the apoptosis of SKOV3 cells was increased significantly compared with the control group (P 0.05).
Conclusion:
The decrease of HMGB1 may affect the decrease of CXCL12 and VEGF, and may be related to the apoptosis of SKOV3 cells.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.31

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