HMGB1在卵巢癌血管生成中的作用研究
發(fā)布時(shí)間：2018-08-19 07:48
【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(guò)程中起關(guān)鍵作用，血管內(nèi)皮生長(zhǎng)因子（VEGF）是目前已知最強(qiáng)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(xì)胞外基質(zhì)的改變，從而促進(jìn)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(zhǎng)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(xì)胞釋放，誘導(dǎo)炎癥反應(yīng)，促進(jìn)組織修復(fù)和血管生成。因HMGB1可誘導(dǎo)血管內(nèi)皮細(xì)胞生長(zhǎng)、遷移及芽發(fā)而被定義為促血管生成因子。HMGB1誘導(dǎo)的細(xì)胞遷移要求激活經(jīng)典和非經(jīng)典的ＮＦ－κＢ通路，這樣會(huì)導(dǎo)致CXCL12基因的轉(zhuǎn)錄。HMGB1還可以保護(hù)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(dá)，而VEGF上調(diào)CXCL12表達(dá)，CXCR4-CXCL12和VEGF在促進(jìn)血管產(chǎn)生過(guò)程中有協(xié)同作用。HMGB1和CXCL12形成的異元復(fù)合體，僅通過(guò)CXCR4信號(hào)通路發(fā)揮炎癥細(xì)胞募集的作用。 因此，我們擬通過(guò)檢測(cè)HMGB1對(duì)卵巢癌血管內(nèi)皮生長(zhǎng)因子VEGF和趨化因子12（CXCL12）表達(dá)的影響及機(jī)制來(lái)了解是否對(duì)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(jī)制。 第一部分卵巢癌組織中HMGB1，CXCL12，VEGF的表達(dá)。 目的： 檢測(cè)卵巢癌組織中HMGB1，CXCL12，VEGF的表達(dá) 方法： 選取54例已確診為上皮性卵巢癌患者，對(duì)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(biāo)本20例，通過(guò)免疫組化方法，檢測(cè)卵巢癌組織中HMGB1、CXCL12、VEGF的表達(dá)情況。 結(jié)果： HMGB1在癌細(xì)胞、間質(zhì)細(xì)胞的細(xì)胞質(zhì)和細(xì)胞核中均有淡黃色至棕褐色顆粒；CXCL12在癌細(xì)胞和間質(zhì)細(xì)胞的細(xì)胞質(zhì)呈淡黃色至棕褐色；VEGF在癌細(xì)胞及間質(zhì)細(xì)胞的細(xì)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(dá)，且與淋巴結(jié)的轉(zhuǎn)移有關(guān)。 第二部分SKOV3，HUVECs細(xì)胞中HMGB1表達(dá) 目的： 檢測(cè)HMGB1在SKOV3，HUVECs細(xì)胞中具體表達(dá)情況。 方法： 分別提取SKOV3，HUVECs細(xì)胞總蛋白、胞漿及胞核蛋白，后利用westernblot檢測(cè)HMGB1在兩者細(xì)胞中的表達(dá)情況。 結(jié)果： HMGB1不僅在SKOV3細(xì)胞有蛋白表達(dá)，在HUVECs細(xì)胞中也有蛋白表達(dá)，但在SKOV3中蛋白表達(dá)高于HUVECs細(xì)胞中，HMGB1在SKOV3細(xì)胞胞漿的表達(dá)顯著高于HUVECs細(xì)胞胞漿表達(dá)（P=0.009）胞核的表達(dá)顯著高于HUVECs細(xì)胞胞核表達(dá)（P=0.000），SKOV3胞核中HMGB1蛋白的表達(dá)顯著高于其胞漿（P=0.000）。HUVECs胞核中HMGB1蛋白的表達(dá)顯著高于其胞漿表達(dá)（P=0.026）。 結(jié)論： MGB1在SKOV3，HUVECs細(xì)胞中均有表達(dá)，，可能與HMGB1所處狀態(tài)的不同有關(guān)。 第三部分HMGB1表達(dá)變化后對(duì)SKOV3血管生成的影響及作用機(jī)制研究 目的： 探討HMGB1在SKOV3血管生成中的作用及其機(jī)制 方法： 將構(gòu)建HMGB1真核表達(dá)質(zhì)粒和人工合成針對(duì)HMGB1基因的siRNA分別轉(zhuǎn)染SKOV3細(xì)胞，運(yùn)用Western blot檢測(cè)轉(zhuǎn)染前后HMGB1，CXCL12的表達(dá)變化，利用流式細(xì)胞術(shù)檢測(cè)SKOV3的凋亡。 結(jié)果： HMGB1過(guò)表達(dá)真核表達(dá)質(zhì)粒DNA轉(zhuǎn)染SKOV3細(xì)胞后SKOV3凋亡減少，而人工合成針對(duì)HMGB1基因的siRNA轉(zhuǎn)染SKOV3細(xì)胞后HMGB1，CXCL12，VEGF表達(dá)均減弱，SKOV3細(xì)胞凋亡增加，與對(duì)照組比差異顯著有統(tǒng)計(jì)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(xì)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(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R737.31
[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(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R737.31
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