HMGB1在卵巢癌血管生成中的作用研究

发布时间：2018-08-19 07:48

【摘要】：侵袭和转移是卵巢癌患者死亡的重要因素，血管新生为侵袭和转移提供基础，所以在此过程中起关键作用，血管内皮生长因子（VEGF）是目前已知最强的血管通透剂，它能导致癌性腹水或肿瘤间质水肿，而且导致血浆、纤维等蛋白向血管外渗出，导致细胞外基质的改变，从而促进血管的形成，是重要的与恶性肿瘤侵袭、转移和腹水形成相关的生长因子。高迁移率族蛋白B1（HMGB1）是核内DNA结合蛋白由坏死细胞释放，诱导炎症反应，促进组织修复和血管生成。因HMGB1可诱导血管内皮细胞生长、迁移及芽发而被定义为促血管生成因子。HMGB1诱导的细胞迁移要求激活经典和非经典的ＮＦ－κＢ通路，这样会导致CXCL12基因的转录。HMGB1还可以保护CXCL12免受降解，诱导额外的CXCL12的分泌。 CXCL12在血管新生中起重要作用，CXCL12上调VEGF表达，而VEGF上调CXCL12表达，CXCR4-CXCL12和VEGF在促进血管产生过程中有协同作用。HMGB1和CXCL12形成的异元复合体，仅通过CXCR4信号通路发挥炎症细胞募集的作用。因此，我们拟通过检测HMGB1对卵巢癌血管内皮生长因子VEGF和趋化因子12（CXCL12）表达的影响及机制来了解是否对卵巢癌的血管新生有影响，初步探讨卵巢癌中血管新生的机制。第一部分卵巢癌组织中HMGB1，CXCL12，VEGF的表达。目的：检测卵巢癌组织中HMGB1，CXCL12，VEGF的表达方法：选取54例已确诊为上皮性卵巢癌患者，对照组为同期因其它妇科疾病行手术切除正常卵巢组织标本20例，通过免疫组化方法，检测卵巢癌组织中HMGB1、CXCL12、VEGF的表达情况。结果： HMGB1在癌细胞、间质细胞的细胞质和细胞核中均有淡黄色至棕褐色颗粒；CXCL12在癌细胞和间质细胞的细胞质呈淡黄色至棕褐色；VEGF在癌细胞及间质细胞的细胞质呈淡黄色至棕黄色。结论：HMGB1、VEGF和CXCL12在卵巢癌组织中均有表达，且与淋巴结的转移有关。第二部分SKOV3，HUVECs细胞中HMGB1表达目的：检测HMGB1在SKOV3，HUVECs细胞中具体表达情况。方法：分别提取SKOV3，HUVECs细胞总蛋白、胞浆及胞核蛋白，后利用westernblot检测HMGB1在两者细胞中的表达情况。结果： HMGB1不仅在SKOV3细胞有蛋白表达，在HUVECs细胞中也有蛋白表达，但在SKOV3中蛋白表达高于HUVECs细胞中，HMGB1在SKOV3细胞胞浆的表达显著高于HUVECs细胞胞浆表达（P=0.009）胞核的表达显著高于HUVECs细胞胞核表达（P=0.000），SKOV3胞核中HMGB1蛋白的表达显著高于其胞浆（P=0.000）。HUVECs胞核中HMGB1蛋白的表达显著高于其胞浆表达（P=0.026）。结论： MGB1在SKOV3，HUVECs细胞中均有表达，，可能与HMGB1所处状态的不同有关。第三部分HMGB1表达变化后对SKOV3血管生成的影响及作用机制研究目的：探讨HMGB1在SKOV3血管生成中的作用及其机制方法：将构建HMGB1真核表达质粒和人工合成针对HMGB1基因的siRNA分别转染SKOV3细胞，运用Western blot检测转染前后HMGB1，CXCL12的表达变化，利用流式细胞术检测SKOV3的凋亡。结果： HMGB1过表达真核表达质粒DNA转染SKOV3细胞后SKOV3凋亡减少，而人工合成针对HMGB1基因的siRNA转染SKOV3细胞后HMGB1，CXCL12，VEGF表达均减弱，SKOV3细胞凋亡增加，与对照组比差异显著有统计学意义（P0.05）结论： HMGB1的下降可能影响CXCL12，VEGF的下降，且可能与SKOV3细胞的凋亡也有关系。
[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.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31

【相似文献】