缺氧卵巢癌细胞通过5-LOX代谢产物促进肿瘤相关巨噬细胞浸润

发布时间：2018-06-06 23:48

本文选题：5-脂氧合酶 + 卵巢癌　；参考：《山东大学》2014年博士论文

【摘要】：研究背景肿瘤相关巨噬细胞(TAM)是肿瘤间质中数量最多的炎症细胞群体,TAM的浸润在卵巢癌等许多肿瘤发展及转移中扮演着重要角色。TAM受肿瘤微环境影响,常表现为M2型,可分泌生长因子、蛋白水解酶、促血管生长因子及免疫抑制因子等。TAM可通过促血管和淋巴管形成、细胞外基质重建、抑制抗肿瘤免疫反应等促进肿瘤进展。研究表明,TAM与乳腺癌、前列腺癌、卵巢癌和宫颈癌等肿瘤的预后不良密切相关。TAM来源于外周血单核细胞,暴露在肿瘤微环境中,持续受到肿瘤微环境的影响。研究发现,TAM不仅富集在血管丰富的组织中,在缺氧的组织中也有较高的密度,这种现象在乳腺癌,膀胱癌等肿瘤中存在,但导致这种现象的机制还不清楚。5-LOX是脂氧合酶家族重要的一员,5-LOX代谢产物主要有5-HETE和白三烯等,对巨噬细胞有很强的趋化和促侵袭作用。本研究将探讨缺氧卵巢细胞促进肿瘤相关巨噬细胞浸润的机制,为卵巢癌提供新的治疗策略。目的肿瘤相关巨噬细胞在卵巢癌等许多肿瘤发展及转移中扮演着重要角色。肿瘤相关巨噬细胞不仅富集在血管丰富的组织中,在缺氧的组织中有较高的密度,但是导致这种现象的机制还不清楚。本研究将探讨缺氧卵巢细胞促进巨噬细胞浸润的机制。方法 1.通过免疫组化检测分析5-LOX、HIF-1α和CD68(巨噬细胞)在卵巢癌组织中的表达。 2.分析5-LOX、HIF-1α和CD68(巨噬细胞)的表达与患者临床病理学参数的相关性。 3.采用qRT-PCR, Western blotting、ELISA等方法检测缺氧对卵巢癌细胞5-LOX通路相关蛋白及代谢产物的影响。 4.采用transwell小室检测缺氧卵巢癌细胞5-LOX代谢产物对巨噬细胞迁移与侵袭的影响。 5.采用qRT-PCR、Western blotting、免疫荧光等方法检测5-LOX代谢产物对巨噬细胞MMP-7表达的影响。 6.采用Western blotting检测5-HETE/LTB4上调巨噬细胞MMP-7表达的信号通路。 7.采用ELISA方法检测5-LOX代谢产物对巨噬细胞分泌TNF-a和HB-EGF的影响。 8.采用裸鼠卵巢癌移植瘤模型,验证齐留通(Zileuton)对肿瘤相关巨噬细胞浸润和MMP-7表达的影响。采用免疫组化及免疫荧光方法检测移植瘤中F4/80、5-LOX、HIF-1α和MMP-7的表达。结果 1.人卵巢癌组织中5-LOX与HIF-la的表达水平与肿瘤相关巨噬细胞数量具有明显的相关性为了研究人卵巢癌组织中5-LOX与HIF-1α的表达与肿瘤相关巨噬细胞数量的相关性,我们用免疫组化方法检测了5-LOX、HIF-1α及CD68(肿瘤相关巨噬细胞)在人卵巢癌组织中的表达。通过分析发现,5-LOX的表达与HIF-1α的水平具有明显的相关性,CD68(肿瘤相关巨噬细胞)的水平与5-LOX、HIF-1α的表达具有明显的相关性。这些结果说明人卵巢癌组织中5-LOX与HIF-1α的表达及肿瘤相关巨噬细胞数量具有明显的相关性。在卵巢癌缺氧区域(即HIF-1α高表达区域),5-LOX呈高表达水平,并且肿瘤相关巨噬细胞数量较多。 2.5-LOX表达水平及肿瘤相关巨噬细胞的数量与卵巢癌分期和转移具有相关性为了研究人卵巢癌组织中5-LOX的表达水平及肿瘤相关巨噬细胞数量与卵巢分期和转移具有相关性,我们分析了人卵巢癌标本5-LOX、HIF-1α及CD68(肿瘤相关巨噬细胞)的表达水平与临床病理学参数的相关性。结果发现,HIF-1α、5-LOX和CD68的表达水平与卵巢癌Figo分期、转移具有明显的相关性,除此之外,CD68与卵巢癌淋巴结转移有明显相关性。这些结果表明人卵巢癌组织中5-LOX的表达水平及肿瘤相关巨噬细胞数量与卵巢癌分期、转移具有明显相关性。 3.缺氧上调卵巢癌细胞5-LOX通路相关蛋白及其代谢产物卵巢癌细胞5-LOX及HIF-1α表达水平具有明显相关性,提示缺氧可能提高卵巢癌细胞5-LOX表达水平并增加其代谢产物。通过细胞实验,我们检测了缺氧条件下培养的卵巢癌细胞5-LOX通路的变化。缺氧条件下,SKOV-3细胞的5-LOX、FLAP和LTA4H mRNA水平升高,此外,在OVCAR-3细胞中也发现了类似现象。5-LOX和FLAP在缺氧环境下蛋白表达也上升。缺氧卵巢癌细胞培养上清的5-HETE和LTB4较常氧的明显升高。5-LOX和FLAP的表达在缺氧环境下较常氧上升,卵巢癌细胞转染HIF-1α siRNA后,在缺氧条件下,HIF-1α siRNA组5-LOX和FLAP的蛋白表达水平较Con siRNA组下降。这些数据表明缺氧能够上调卵巢癌细胞5-LOX通路相关蛋白及增加代谢产物5-HETE和LTB4的产生。4.缺氧卵巢癌细胞5-LOX代谢产物增多能够促进巨噬细胞迁移与侵袭肿瘤相关巨噬细胞数量与卵巢癌细胞5-LOX表达水平密切相关,提示卵巢癌细胞的5-LOX代谢产物可能参与巨噬细胞的迁移和侵袭过程。在transwell小室迁移实验中,与常氧培养上清相比,缺氧培养上清能明显促进巨噬细胞的迁移。类似的,在transwell小室侵袭实验中,与常氧培养上清相比,缺氧培养上清能明显促进巨噬细胞的侵袭。因此,缺氧SKOV-3细胞分泌的一些介质可能促进了巨噬细胞的迁移及侵袭。为了进一步明确是否是缺氧卵巢癌细胞的5-LOX代谢产物促进了巨噬细胞迁移与侵袭,使用MK886(FLAP的抑制剂)阻断卵巢癌细胞的5-LOX代谢通路。与DMSO组的培养上清相比,MK886组迁移及侵袭的巨噬细胞数量减少。并且MK886组的缺氧上清与常氧上清中迁移及侵袭的巨噬细胞数量无明显差别。下层培养基加入5-HETE或LTB4后,迁移的巨噬细胞数量明显增多。加入5-HETE或LTB4培养巨噬细胞48h后,巨噬细胞的侵袭能力明显增强。类似于卵巢癌细胞系SKOV-3,与常氧培养上清相比,卵巢癌细胞系OVCAR-3缺氧培养上清能明显促进巨噬细胞的迁移与侵袭。这些实验结果说明,缺氧卵巢癌细胞5-LOX代谢产物增多能够促进巨噬细胞迁移与侵袭。5.5-LOX通路代谢产物通过上调MMP-7促进巨噬细胞侵袭 MMPs能够分解细胞外基质的组成部分从而促进巨噬细胞侵袭。与对照组相比,培养上清中巨噬细胞的MMP-2. MMP-7和MMP-9的转录水平明显升高。然而,与常氧培养上清相比,缺氧培养上清组只有MMP-7水平较高。与对照组相比,培养上清中巨噬细胞的侵袭能力明显增强。通过MMP-7中和抗体抑制MMP-7功能后,巨噬细胞的侵袭能力明显降低,缺氧上清与常氧上清中巨噬细胞的侵袭能力无明显差别。加入5-HETE和LTB4后,MMP-7的表达明显上升。加入5-HETE和LTB4培养巨噬细胞48h后,巨噬细胞的迁移能力明显增强,加入MMP-7中和抗体后,这种增强被抑制。此外,我们检测了人卵巢癌相关巨噬细胞MMP-7的表达,在5-LOX高表达的卵巢癌组织中,相关巨噬细胞的MMP-7表达较高。因此,5-LOX通路代谢产物是通过上调MMP-7促进巨噬细胞侵袭的。 6.5-HETE/LTB4通过p38通路上调巨噬细胞MMP-7的表达研究表明,许多信号通路例如PI3K/Akt、mTOR、SAPK/JNK、ERK1/2和p38通路等均可调节MMP-7的表达。使用多种信号通路抑制剂处理后,检测了5-HETE/LTB4对巨噬细胞MMP-7表达的影响。SB203580(p38通路抑制剂)明显抑制了5-HETE/LTB4对MMP-7的上调,提示5-HETE/LTB4可能通过p38通路上调巨噬细胞MMP-7的表达。5-HETE/LTB4能够明显激活p38通路。加入p38通路抑制剂SB203580后,5-HETE/LTB4对p38通路的激活被抑制。另外,5-HETE/LTB4不能明显激活PI3K/Akt、mTOR、SAPK/JNK (?)和ERK1/2通路。这些数据表明5-HETE/LTB4通过p38通路上调巨噬细胞MMP-7的表达。7.5-LOX通路代谢产物通过上调MMP-7促进TNF-α和HB-EGF的分泌 MMP-7在TNF-α和HB-EGF的剪切和释放过程中起重要作用.与常氧培养上清相比,缺氧培养上清组TNF-α和HB-EGF水平较高。与未阻断5-LOX代谢通路的培养上清组相比,阻断后的培养上清组中TNF-α和HB-EGF明显降低。提示缺氧卵巢癌细胞的5-LOX通路代谢产物促进巨噬细胞TNF-α和HB-EGF的分泌。加入5-HETE/LTB4后,TNF-α和HB-EGF水平明显上升。使用MMP-7中和抗体抑制MMP-7功能后,TNF-α和HB-EGF水平明显降低。这些数据表明5-LOX通路代谢产物通过上调MMP-7促进TNF-α和HB-EGF的分泌。 8.在动物模型中齐留通(Zileuton)抑制肿瘤相关巨噬细胞的浸润和MMP-7的表达裸鼠成瘤后,给予Zileuton灌胃,每周测量两次小鼠肿瘤大小。采用免疫组化及免疫荧光方法检测HIF-1α、5-LOX、F4/80(鼠巨噬细胞)和MMP-7在小鼠移植瘤中的表达。免疫组化结果表明5-LOX的表达与HIF-1α相关。巨噬细胞的数量与5-LOX和HIF-1α的表达相关。加入Zileuton后,肿瘤的缺氧区域中的巨噬细胞减少。免疫组化及免疫荧光结果表明巨噬细胞MMP-7的表达与肿瘤细胞5-LOX的表达相关。并且,Zileuton (?)能够抑制肿瘤的生长。这些数据表明Zileuton能够抑制肿瘤相关巨噬细胞的浸润和MMP-7的表达。结论综上所述,这些体内和体外实验首次证明缺氧卵巢癌细胞通过5-LOX产物上调肿瘤相关巨噬细胞的MMP-7表达从而促进肿瘤相关巨噬细胞浸润,为卵巢癌的治疗提供新的策略,具有积极的意义。
[Abstract]:Research background
Tumor related macrophage (TAM) is the largest number of inflammatory cells in the tumor interstitial. The infiltration of TAM plays an important role in the development and metastasis of ovarian cancer, such as.TAM, which is often expressed as M2, secreting growth factor, protein hydrolase, promoting vascular growth factor and immunosuppressive factor, and so on. TAM is closely related to the poor prognosis of breast cancer, prostate cancer, ovarian cancer, and cervical cancer, and.TAM is derived from peripheral blood mononuclear cells, exposed to the tumor microenvironment and sustained by the tumor microenvironment. The study found that TAM is not only enriched in vascular rich tissues, but also has a high density in anoxic tissues. This phenomenon exists in breast and bladder cancers, but the mechanism that causes this phenomenon is not clear that.5-LOX is an important member of the lipoxygenase family, and the main metabolites of 5-LOX are 5-HETE and leukotrienes. Cells have strong chemotaxis and invasiveness. This study will explore the mechanism of hypoxic ovarian cells to promote tumor related macrophage infiltration and provide a new therapeutic strategy for ovarian cancer.
objective
Tumor related macrophages play an important role in the development and metastasis of many tumors, such as ovarian cancer. Tumor related macrophages are not only enriched in vascular rich tissues, but also have high density in anoxic tissues, but the mechanism that causes this phenomenon is not clear. This study will explore the promotion of macrophage leaching by hypoxic ovarian cells. The mechanism of moistening.
Method
1. the expression of 5-LOX, HIF-1 and CD68 (macrophages) in ovarian cancer tissues was analyzed by immunohistochemistry.
2. to analyze the correlation between the expression of 5-LOX, HIF-1 alpha and CD68 (macrophages) and clinicopathological parameters.
3. qRT-PCR, Western blotting and ELISA were used to detect the effects of hypoxia on the protein and metabolites of 5-LOX pathway in ovarian cancer cells.
4. Transwell chamber was used to detect the effects of 5-LOX metabolites on macrophage migration and invasion in anoxia ovarian cancer cells.
5. qRT-PCR, Western blotting and immunofluorescence were used to detect the effects of 5-LOX metabolites on macrophage MMP-7 expression.
6. Western blotting was used to detect 5-HETE/LTB4 signaling pathway that upregulated MMP-7 expression in macrophages.
7. ELISA method was used to detect the effects of 5-LOX metabolites on TNF-a and HB-EGF secretion by macrophages.
8. the effect of Zileuton on the infiltration of macrophages and the expression of MMP-7 in tumor related macrophages was verified by the model of xenograft tumor of nude mice ovarian cancer. The expression of F4/80,5-LOX, HIF-1 A and MMP-7 in the transplanted tumor was detected by immunohistochemistry and immunofluorescence.
Result
There was a significant correlation between the expression of 5-LOX and HIF-la and the number of tumor associated macrophages in 1. human ovarian cancer tissues.
In order to study the correlation between the expression of 5-LOX and HIF-1 alpha in human ovarian cancer tissues and the number of tumor related macrophages, we detected the expression of 5-LOX, HIF-1 A and CD68 (tumor related macrophages) in human ovarian cancer tissues by immunohistochemistry. It was found that the expression of 5-LOX was significantly related to the level of HIF-1 alpha, CD6, CD6. The level of 8 (tumor related macrophages) was significantly correlated with the expression of 5-LOX, HIF-1 alpha. These results suggest that the expression of 5-LOX and HIF-1 alpha in human ovarian cancer is significantly correlated with the number of tumor related macrophages. In the anoxic region of the ovarian cancer (that is, the high expression area of HIF-1 a), 5-LOX is highly expressed, and the tumor phase The number of macrophages is more.
The level of 2.5-LOX expression and the number of tumor associated macrophages are correlated with the stage and metastasis of ovarian cancer.
In order to study the correlation between the expression level of 5-LOX in human ovarian cancer tissues and the number of tumor related macrophages with ovarian staging and metastasis, we analyzed the correlation between the expression level of 5-LOX, HIF-1 A and CD68 (tumor related macrophages) in human ovarian cancer specimens and the correlation of the clinicopathological parameters. The results showed that the expression of HIF-1, 5-LOX and CD68 was expressed. There was a significant correlation between the level of Figo and the metastasis of ovarian cancer. In addition, there was a significant correlation between CD68 and lymph node metastasis in ovarian cancer. These results showed that the expression level of 5-LOX and the number of tumor related macrophages in the human ovarian cancer tissues were significantly correlated with the stages and metastasis of ovarian cancer.
3. hypoxia increases upregulated 5-LOX pathway related proteins and their metabolites in ovarian cancer cells.
The expression level of 5-LOX and HIF-1 alpha in ovarian cancer cells has a significant correlation, suggesting that hypoxia may increase the level of 5-LOX expression and increase its metabolites in ovarian cancer cells. Through cell experiments, we detected the changes in the 5-LOX pathway of ovarian cancer cells cultured under hypoxia conditions. The 5-LOX, FLAP and LTA4H mRNA levels of SKOV-3 cells under hypoxia conditions. In addition, the expression of.5-LOX and FLAP in the OVCAR-3 cells was also found in the hypoxia environment. The 5-HETE and LTB4 in the culture supernatant of the anoxic ovarian cancer cell culture were significantly higher than that of the normal oxygen. The expression of.5-LOX and FLAP in the hypoxia environment was higher than that in the hypoxia environment, and the ovarian cancer cells transfected to HIF-1 a siRNA, under the condition of hypoxia, HIF. The protein expression level of 5-LOX and FLAP in -1 alpha siRNA group is lower than that in Con siRNA group. These data suggest that hypoxia can up regulate the 5-LOX pathway related proteins and increase the production of 5-HETE and LTB4 metabolites of ovarian cancer cells, and the increase of 5-LOX metabolites in.4. hypoxia ovarian cancer cells can promote macrophage migration and invasion.
The number of tumor related macrophages is closely related to the level of 5-LOX expression in ovarian cancer cells, suggesting that the 5-LOX metabolites of ovarian cancer cells may be involved in the migration and invasion process of macrophages. In the Transwell compartment migration experiment, the hypoxia culture supernatant can significantly promote the migration of macrophages. Similar, in t In the ranswell chamber invasion experiment, the hypoxia culture supernatant obviously promoted the invasion of macrophages compared with the oxygen culture supernatant. Therefore, some mediators secreted by hypoxia SKOV-3 cells may promote the migration and invasion of macrophages. In order to further clarify whether the 5-LOX metabolites of anoxic ovarian cancer cells promote macrophage Migration and invasion, using MK886 (FLAP inhibitor) blocking the 5-LOX metabolic pathway of ovarian cancer cells. Compared with the culture supernatant of the DMSO group, the number of macrophages migrated and invaded in the MK886 group decreased, and there was no significant difference in the number of macrophages migrated and attacked in the hypoxia supernatant and the normal oxygen supernatant of the MK886 group. The subculture medium was added to the 5-HETE or the 5-HETE or the subculture medium. After LTB4, the number of migratory macrophages increased significantly. After adding 5-HETE or LTB4 to the macrophage 48h, the invasion ability of macrophages was obviously enhanced. It was similar to the ovarian cancer cell line SKOV-3. Compared with the normal oxygen culture supernatant, the ovarian cancer cell line OVCAR-3 hypoxia culture supernatant could obviously promote the migration and invasion of macrophages. The results suggest that the increase of 5-LOX metabolites in anoxic ovarian cancer cells can promote macrophage migration and invasion of.5.5-LOX pathway metabolites by up regulation of MMP-7 to promote macrophage invasion
MMPs could decompose the components of the extracellular matrix to promote the invasion of macrophages. Compared with the control group, the MMP-2. MMP-7 and MMP-9 transcriptional levels of macrophages in the culture supernatant were significantly higher. However, the level of MMP-7 was higher in the hypoxia culture supernatant group than in the aerobic culture supernatant. The invasion ability of the cell was obviously enhanced. The invasion ability of macrophages was obviously decreased after the inhibition of MMP-7 function by MMP-7 neutralization antibody. There was no significant difference in the invasion ability of macrophages from hypoxia supernatant and normoxic supernatant. After adding 5-HETE and LTB4, the expression of MMP-7 was obviously increased. After adding 5-HETE and LTB4 to culture macrophage 48h, macrophage In addition, we detected the expression of MMP-7 in human ovarian cancer related macrophages. In the ovarian cancer tissue with high expression of 5-LOX, the expression of MMP-7 in macrophages was higher in 5-LOX high expression of ovarian cancer. Therefore, the metabolite of 5-LOX pathway promoted the invasion of macrophages by up regulation of MMP-7.
6.5-HETE/LTB4 upregulated the expression of MMP-7 in macrophages through p38 pathway.
Studies have shown that many signal pathways, such as PI3K/Akt, mTOR, SAPK/JNK, ERK1/2 and p38 pathway, can regulate the expression of MMP-7. The effect of 5-HETE/LTB4 on the expression of MMP-7 in macrophages (p38 pathway inhibitor) has been detected by a variety of signal pathway inhibitors. The expression of.5-HETE/LTB4 in the macrophage MMP-7 through the p38 pathway can activate the p38 pathway. The activation of p38 pathway is inhibited by the addition of the p38 pathway inhibitor SB2035 80. Moreover, 5-HETE/LTB4 can not activate PI3K/Akt, mTOR, SAPK/JNK (?) and the pathway. Phagocyte MMP-7 expression.7.5-LOX pathway metabolites promote the secretion of TNF- and HB-EGF through up regulation of MMP-7.
MMP-7 plays an important role in the shear and release of TNF- alpha and HB-EGF. Compared with the oxygen culture supernatant, the level of TNF- alpha and HB-EGF is higher in the hypoxia culture supernatant group. The TNF- alpha and HB-EGF in the cultured supernatant group after the blocking of the 5-LOX metabolic pathway are significantly lower than that in the culture supernatant group. The metabolites promoted the secretion of TNF- alpha and HB-EGF in macrophages. After adding 5-HETE/LTB4, the level of TNF- alpha and HB-EGF increased significantly. The level of TNF- A and HB-EGF decreased significantly after the use of MMP-7 neutralizing antibodies to inhibit MMP-7 function. These data showed that the 5-LOX pathway metabolites promoted TNF- alpha and secretion by up MMP-7 up.
8. in animal models, Zileuton inhibited the infiltration of tumor associated macrophages and the expression of MMP-7.
After tumor formation in nude mice, the tumor size was measured two times a week by Zileuton gavage. Immunohistochemistry and immunofluorescence were used to detect the expression of HIF-1 alpha, 5-LOX, F4/80 (rat macrophage) and MMP-7 in the transplanted tumor of mice. The immunohistochemical results showed that the expression of 5-LOX was related to HIF-1 alpha. The number of macrophages and the expression of 5-LOX and HIF-1 alpha Correlation. After Zileuton, macrophages in the anoxic region of the tumor were reduced. Immunohistochemical and immunofluorescence results showed that the expression of MMP-7 in macrophages was associated with the expression of 5-LOX in the tumor cells. And Zileuton (?) could inhibit the growth of the tumor. These data suggest that Zileuton can inhibit the infiltration of tumor related macrophages and MMP-7 Expression.
conclusion
In summary, these in vivo and in vitro experiments have proved for the first time that anoxic ovarian cancer cells increase the MMP-7 expression of tumor related macrophages through 5-LOX products to promote tumor related macrophage infiltration and provide a new strategy for the treatment of ovarian cancer, which is of positive significance.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.31

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