本文選題：5-脂氧合酶 + 卵巢癌��； 參考：《山東大學(xué)》2014年博士論文

【摘要】：研究背景 腫瘤相關(guān)巨噬細(xì)胞(TAM)是腫瘤間質(zhì)中數(shù)量最多的炎癥細(xì)胞群體,TAM的浸潤(rùn)在卵巢癌等許多腫瘤發(fā)展及轉(zhuǎn)移中扮演著重要角色。TAM受腫瘤微環(huán)境影響,常表現(xiàn)為M2型,可分泌生長(zhǎng)因子、蛋白水解酶、促血管生長(zhǎng)因子及免疫抑制因子等。TAM可通過促血管和淋巴管形成、細(xì)胞外基質(zhì)重建、抑制抗腫瘤免疫反應(yīng)等促進(jìn)腫瘤進(jìn)展。研究表明,TAM與乳腺癌、前列腺癌、卵巢癌和宮頸癌等腫瘤的預(yù)后不良密切相關(guān)。TAM來源于外周血單核細(xì)胞,暴露在腫瘤微環(huán)境中,持續(xù)受到腫瘤微環(huán)境的影響。研究發(fā)現(xiàn),TAM不僅富集在血管豐富的組織中,在缺氧的組織中也有較高的密度,這種現(xiàn)象在乳腺癌,膀胱癌等腫瘤中存在,但導(dǎo)致這種現(xiàn)象的機(jī)制還不清楚。5-LOX是脂氧合酶家族重要的一員,5-LOX代謝產(chǎn)物主要有5-HETE和白三烯等,對(duì)巨噬細(xì)胞有很強(qiáng)的趨化和促侵襲作用。本研究將探討缺氧卵巢細(xì)胞促進(jìn)腫瘤相關(guān)巨噬細(xì)胞浸潤(rùn)的機(jī)制,為卵巢癌提供新的治療策略。 目的 腫瘤相關(guān)巨噬細(xì)胞在卵巢癌等許多腫瘤發(fā)展及轉(zhuǎn)移中扮演著重要角色。腫瘤相關(guān)巨噬細(xì)胞不僅富集在血管豐富的組織中,在缺氧的組織中有較高的密度,但是導(dǎo)致這種現(xiàn)象的機(jī)制還不清楚。本研究將探討缺氧卵巢細(xì)胞促進(jìn)巨噬細(xì)胞浸潤(rùn)的機(jī)制。 方法 1.通過免疫組化檢測(cè)分析5-LOX、HIF-1α和CD68(巨噬細(xì)胞)在卵巢癌組織中的表達(dá)。 2.分析5-LOX、HIF-1α和CD68(巨噬細(xì)胞)的表達(dá)與患者臨床病理學(xué)參數(shù)的相關(guān)性。 3.采用qRT-PCR, Western blotting、ELISA等方法檢測(cè)缺氧對(duì)卵巢癌細(xì)胞5-LOX通路相關(guān)蛋白及代謝產(chǎn)物的影響。 4.采用transwell小室檢測(cè)缺氧卵巢癌細(xì)胞5-LOX代謝產(chǎn)物對(duì)巨噬細(xì)胞遷移與侵襲的影響。 5.采用qRT-PCR、Western blotting、免疫熒光等方法檢測(cè)5-LOX代謝產(chǎn)物對(duì)巨噬細(xì)胞MMP-7表達(dá)的影響。 6.采用Western blotting檢測(cè)5-HETE/LTB4上調(diào)巨噬細(xì)胞MMP-7表達(dá)的信號(hào)通路。 7.采用ELISA方法檢測(cè)5-LOX代謝產(chǎn)物對(duì)巨噬細(xì)胞分泌TNF-a和HB-EGF的影響。 8.采用裸鼠卵巢癌移植瘤模型,驗(yàn)證齊留通(Zileuton)對(duì)腫瘤相關(guān)巨噬細(xì)胞浸潤(rùn)和MMP-7表達(dá)的影響。采用免疫組化及免疫熒光方法檢測(cè)移植瘤中F4/80、5-LOX、HIF-1α和MMP-7的表達(dá)。 結(jié)果 1.人卵巢癌組織中5-LOX與HIF-la的表達(dá)水平與腫瘤相關(guān)巨噬細(xì)胞數(shù)量具有明顯的相關(guān)性 為了研究人卵巢癌組織中5-LOX與HIF-1α的表達(dá)與腫瘤相關(guān)巨噬細(xì)胞數(shù)量的相關(guān)性,我們用免疫組化方法檢測(cè)了5-LOX、HIF-1α及CD68(腫瘤相關(guān)巨噬細(xì)胞)在人卵巢癌組織中的表達(dá)。通過分析發(fā)現(xiàn),5-LOX的表達(dá)與HIF-1α的水平具有明顯的相關(guān)性,CD68(腫瘤相關(guān)巨噬細(xì)胞)的水平與5-LOX、HIF-1α的表達(dá)具有明顯的相關(guān)性。這些結(jié)果說明人卵巢癌組織中5-LOX與HIF-1α的表達(dá)及腫瘤相關(guān)巨噬細(xì)胞數(shù)量具有明顯的相關(guān)性。在卵巢癌缺氧區(qū)域(即HIF-1α高表達(dá)區(qū)域),5-LOX呈高表達(dá)水平,并且腫瘤相關(guān)巨噬細(xì)胞數(shù)量較多。 2.5-LOX表達(dá)水平及腫瘤相關(guān)巨噬細(xì)胞的數(shù)量與卵巢癌分期和轉(zhuǎn)移具有相關(guān)性 為了研究人卵巢癌組織中5-LOX的表達(dá)水平及腫瘤相關(guān)巨噬細(xì)胞數(shù)量與卵巢分期和轉(zhuǎn)移具有相關(guān)性,我們分析了人卵巢癌標(biāo)本5-LOX、HIF-1α及CD68(腫瘤相關(guān)巨噬細(xì)胞)的表達(dá)水平與臨床病理學(xué)參數(shù)的相關(guān)性。結(jié)果發(fā)現(xiàn),HIF-1α、5-LOX和CD68的表達(dá)水平與卵巢癌Figo分期、轉(zhuǎn)移具有明顯的相關(guān)性,除此之外,CD68與卵巢癌淋巴結(jié)轉(zhuǎn)移有明顯相關(guān)性。這些結(jié)果表明人卵巢癌組織中5-LOX的表達(dá)水平及腫瘤相關(guān)巨噬細(xì)胞數(shù)量與卵巢癌分期、轉(zhuǎn)移具有明顯相關(guān)性。 3.缺氧上調(diào)卵巢癌細(xì)胞5-LOX通路相關(guān)蛋白及其代謝產(chǎn)物 卵巢癌細(xì)胞5-LOX及HIF-1α表達(dá)水平具有明顯相關(guān)性,提示缺氧可能提高卵巢癌細(xì)胞5-LOX表達(dá)水平并增加其代謝產(chǎn)物。通過細(xì)胞實(shí)驗(yàn),我們檢測(cè)了缺氧條件下培養(yǎng)的卵巢癌細(xì)胞5-LOX通路的變化。缺氧條件下,SKOV-3細(xì)胞的5-LOX、FLAP和LTA4H mRNA水平升高,此外,在OVCAR-3細(xì)胞中也發(fā)現(xiàn)了類似現(xiàn)象。5-LOX和FLAP在缺氧環(huán)境下蛋白表達(dá)也上升。缺氧卵巢癌細(xì)胞培養(yǎng)上清的5-HETE和LTB4較常氧的明顯升高。5-LOX和FLAP的表達(dá)在缺氧環(huán)境下較常氧上升,卵巢癌細(xì)胞轉(zhuǎn)染HIF-1α siRNA后,在缺氧條件下,HIF-1α siRNA組5-LOX和FLAP的蛋白表達(dá)水平較Con siRNA組下降。這些數(shù)據(jù)表明缺氧能夠上調(diào)卵巢癌細(xì)胞5-LOX通路相關(guān)蛋白及增加代謝產(chǎn)物5-HETE和LTB4的產(chǎn)生。4.缺氧卵巢癌細(xì)胞5-LOX代謝產(chǎn)物增多能夠促進(jìn)巨噬細(xì)胞遷移與侵襲 腫瘤相關(guān)巨噬細(xì)胞數(shù)量與卵巢癌細(xì)胞5-LOX表達(dá)水平密切相關(guān),提示卵巢癌細(xì)胞的5-LOX代謝產(chǎn)物可能參與巨噬細(xì)胞的遷移和侵襲過程。在transwell小室遷移實(shí)驗(yàn)中,與常氧培養(yǎng)上清相比,缺氧培養(yǎng)上清能明顯促進(jìn)巨噬細(xì)胞的遷移。類似的,在transwell小室侵襲實(shí)驗(yàn)中,與常氧培養(yǎng)上清相比,缺氧培養(yǎng)上清能明顯促進(jìn)巨噬細(xì)胞的侵襲。因此,缺氧SKOV-3細(xì)胞分泌的一些介質(zhì)可能促進(jìn)了巨噬細(xì)胞的遷移及侵襲。為了進(jìn)一步明確是否是缺氧卵巢癌細(xì)胞的5-LOX代謝產(chǎn)物促進(jìn)了巨噬細(xì)胞遷移與侵襲,使用MK886(FLAP的抑制劑)阻斷卵巢癌細(xì)胞的5-LOX代謝通路。與DMSO組的培養(yǎng)上清相比,MK886組遷移及侵襲的巨噬細(xì)胞數(shù)量減少。并且MK886組的缺氧上清與常氧上清中遷移及侵襲的巨噬細(xì)胞數(shù)量無明顯差別。下層培養(yǎng)基加入5-HETE或LTB4后,遷移的巨噬細(xì)胞數(shù)量明顯增多。加入5-HETE或LTB4培養(yǎng)巨噬細(xì)胞48h后,巨噬細(xì)胞的侵襲能力明顯增強(qiáng)。類似于卵巢癌細(xì)胞系SKOV-3,與常氧培養(yǎng)上清相比,卵巢癌細(xì)胞系OVCAR-3缺氧培養(yǎng)上清能明顯促進(jìn)巨噬細(xì)胞的遷移與侵襲。這些實(shí)驗(yàn)結(jié)果說明,缺氧卵巢癌細(xì)胞5-LOX代謝產(chǎn)物增多能夠促進(jìn)巨噬細(xì)胞遷移與侵襲。5.5-LOX通路代謝產(chǎn)物通過上調(diào)MMP-7促進(jìn)巨噬細(xì)胞侵襲 MMPs能夠分解細(xì)胞外基質(zhì)的組成部分從而促進(jìn)巨噬細(xì)胞侵襲。與對(duì)照組相比,培養(yǎng)上清中巨噬細(xì)胞的MMP-2. MMP-7和MMP-9的轉(zhuǎn)錄水平明顯升高。然而,與常氧培養(yǎng)上清相比,缺氧培養(yǎng)上清組只有MMP-7水平較高。與對(duì)照組相比,培養(yǎng)上清中巨噬細(xì)胞的侵襲能力明顯增強(qiáng)。通過MMP-7中和抗體抑制MMP-7功能后,巨噬細(xì)胞的侵襲能力明顯降低,缺氧上清與常氧上清中巨噬細(xì)胞的侵襲能力無明顯差別。加入5-HETE和LTB4后,MMP-7的表達(dá)明顯上升。加入5-HETE和LTB4培養(yǎng)巨噬細(xì)胞48h后,巨噬細(xì)胞的遷移能力明顯增強(qiáng),加入MMP-7中和抗體后,這種增強(qiáng)被抑制。此外,我們檢測(cè)了人卵巢癌相關(guān)巨噬細(xì)胞MMP-7的表達(dá),在5-LOX高表達(dá)的卵巢癌組織中,相關(guān)巨噬細(xì)胞的MMP-7表達(dá)較高。因此,5-LOX通路代謝產(chǎn)物是通過上調(diào)MMP-7促進(jìn)巨噬細(xì)胞侵襲的。 6.5-HETE/LTB4通過p38通路上調(diào)巨噬細(xì)胞MMP-7的表達(dá) 研究表明,許多信號(hào)通路例如PI3K/Akt、mTOR、SAPK/JNK、ERK1/2和p38通路等均可調(diào)節(jié)MMP-7的表達(dá)。使用多種信號(hào)通路抑制劑處理后,檢測(cè)了5-HETE/LTB4對(duì)巨噬細(xì)胞MMP-7表達(dá)的影響。SB203580(p38通路抑制劑)明顯抑制了5-HETE/LTB4對(duì)MMP-7的上調(diào),提示5-HETE/LTB4可能通過p38通路上調(diào)巨噬細(xì)胞MMP-7的表達(dá)。5-HETE/LTB4能夠明顯激活p38通路。加入p38通路抑制劑SB203580后,5-HETE/LTB4對(duì)p38通路的激活被抑制。另外,5-HETE/LTB4不能明顯激活PI3K/Akt、mTOR、SAPK/JNK (?)和ERK1/2通路。這些數(shù)據(jù)表明5-HETE/LTB4通過p38通路上調(diào)巨噬細(xì)胞MMP-7的表達(dá)。7.5-LOX通路代謝產(chǎn)物通過上調(diào)MMP-7促進(jìn)TNF-α和HB-EGF的分泌 MMP-7在TNF-α和HB-EGF的剪切和釋放過程中起重要作用.與常氧培養(yǎng)上清相比,缺氧培養(yǎng)上清組TNF-α和HB-EGF水平較高。與未阻斷5-LOX代謝通路的培養(yǎng)上清組相比,阻斷后的培養(yǎng)上清組中TNF-α和HB-EGF明顯降低。提示缺氧卵巢癌細(xì)胞的5-LOX通路代謝產(chǎn)物促進(jìn)巨噬細(xì)胞TNF-α和HB-EGF的分泌。加入5-HETE/LTB4后,TNF-α和HB-EGF水平明顯上升。使用MMP-7中和抗體抑制MMP-7功能后,TNF-α和HB-EGF水平明顯降低。這些數(shù)據(jù)表明5-LOX通路代謝產(chǎn)物通過上調(diào)MMP-7促進(jìn)TNF-α和HB-EGF的分泌。 8.在動(dòng)物模型中齊留通(Zileuton)抑制腫瘤相關(guān)巨噬細(xì)胞的浸潤(rùn)和MMP-7的表達(dá) 裸鼠成瘤后,給予Zileuton灌胃,每周測(cè)量?jī)纱涡∈竽[瘤大小。采用免疫組化及免疫熒光方法檢測(cè)HIF-1α、5-LOX、F4/80(鼠巨噬細(xì)胞)和MMP-7在小鼠移植瘤中的表達(dá)。免疫組化結(jié)果表明5-LOX的表達(dá)與HIF-1α相關(guān)。巨噬細(xì)胞的數(shù)量與5-LOX和HIF-1α的表達(dá)相關(guān)。加入Zileuton后,腫瘤的缺氧區(qū)域中的巨噬細(xì)胞減少。免疫組化及免疫熒光結(jié)果表明巨噬細(xì)胞MMP-7的表達(dá)與腫瘤細(xì)胞5-LOX的表達(dá)相關(guān)。并且,Zileuton (?)能夠抑制腫瘤的生長(zhǎng)。這些數(shù)據(jù)表明Zileuton能夠抑制腫瘤相關(guān)巨噬細(xì)胞的浸潤(rùn)和MMP-7的表達(dá)。 結(jié)論 綜上所述,這些體內(nèi)和體外實(shí)驗(yàn)首次證明缺氧卵巢癌細(xì)胞通過5-LOX產(chǎn)物上調(diào)腫瘤相關(guān)巨噬細(xì)胞的MMP-7表達(dá)從而促進(jìn)腫瘤相關(guān)巨噬細(xì)胞浸潤(rùn),為卵巢癌的治療提供新的策略,具有積極的意義。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.31

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