【摘要】：目的:研究靶向抑制PI3K亞型對(duì)體內(nèi)外前列腺癌轉(zhuǎn)移的作用及其分子機(jī)理,初步闡明在前列腺癌轉(zhuǎn)移中起關(guān)鍵作用的PI3K亞型,為前列腺癌治療提供新的思路。方法:1.利用Transwell小室遷移實(shí)驗(yàn)和小室侵襲實(shí)驗(yàn)研究PI3K各亞型特異性抑制劑對(duì)PC3和DU145細(xì)胞遷移能力以及侵襲能力的影響;2.利用Western blot技術(shù)研究PI3Kβ及d亞型特異性抑制劑對(duì)PC3和DU145細(xì)胞中遷移和侵襲相關(guān)信號(hào)蛋白Akt及其下游蛋白Integrinβ1的表達(dá)以及磷酸化水平的影響;3.利用si RNA技術(shù)分別對(duì)PC3和DU145細(xì)胞中PI3Kβ及d亞型降表達(dá),進(jìn)一步證明兩種亞型在前列腺癌細(xì)胞遷移中的作用;4.建立前列腺癌骨轉(zhuǎn)移模型,結(jié)合micro CT檢測(cè)技術(shù)研究PI3Kβ及d亞型特異性抑制劑對(duì)體內(nèi)前列腺癌轉(zhuǎn)移裸鼠骨量的影響;5.利用HE染色、TRAP染色及Macneal’s染色等方法研究PI3Kβ及d亞型特異性抑制劑對(duì)體內(nèi)前列腺癌轉(zhuǎn)移的效果。結(jié)果:1.PI3K各亞型特異性抑制劑對(duì)PC3和DU145細(xì)胞的遷移和侵襲有不同程度的抑制活性,與酶水平抑制活性的IC50值相比,PI3Kβ及d亞型特異性抑制劑活性強(qiáng),PI3Kα及γ亞型特異性抑制劑活性弱;2.PI3Kβ及d亞型特異性抑制劑可顯著降低PC3和DU145細(xì)胞Akt及下游蛋白Integrinβ1的磷酸化水平,呈一定的劑量依賴性;3.采用si RNA的方法對(duì)PI3Kβ及d亞型降表達(dá)能顯著降低PC3和DU145細(xì)胞Akt的磷酸化水平,且抑制PC3和DU145細(xì)胞遷移;4.PI3Kβ及d亞型特異性抑制劑均能有效提升前列腺癌骨轉(zhuǎn)移裸鼠的骨量,增加骨密度;5.PI3Kβ及d亞型特異性抑制劑均能抑制前列腺癌細(xì)胞的骨轉(zhuǎn)移,減少骨中破骨細(xì)胞數(shù)目,有增加成骨細(xì)胞數(shù)目的趨勢(shì)。結(jié)論:1.PI3K四種亞型特異性抑制劑中,β及d亞型特異性抑制劑在較低濃度下有顯著的抑制前列腺癌細(xì)胞遷移和侵襲的能力,表現(xiàn)出較強(qiáng)的體外抗腫瘤轉(zhuǎn)移活性;2.PI3Kβ及d亞型特異性抑制劑及si RNA在產(chǎn)生抗前列腺癌細(xì)胞遷移的濃度下可顯著降低前列腺癌細(xì)胞中Akt的磷酸化水平,提示其可能通過PI3K/Akt信號(hào)通路來抑制前列腺癌細(xì)胞的遷移和侵襲等;3.PI3Kβ及d亞型特異性抑制劑可顯著抑制前列腺癌細(xì)胞體內(nèi)骨轉(zhuǎn)移,并通過減少破骨細(xì)胞骨吸收來改善骨微環(huán)境,提示這兩種亞型在前列腺癌轉(zhuǎn)移中起關(guān)鍵作用。
[Abstract]:Objective: to study the effect of targeting inhibition of PI3K subtype on prostate cancer metastasis in vivo and in vitro and its molecular mechanism, and to clarify the PI3K subtype which plays a key role in prostate cancer metastasis, so as to provide a new idea for the treatment of prostate cancer. Methods: 1. The effects of specific inhibitors of PI3K subtypes on the migration and invasion of PC3 and DU145 cells were studied by Transwell cell migration assay and ventricular invasion assay. 2. Western blot technique was used to study the effects of PI3K 尾 and d subtype specific inhibitors on the expression and phosphorylation level of Akt and its downstream protein Integrin 尾 1 in PC3 and DU145 cells. The expression of PI3K 尾 and d subtypes in PC3 and DU145 cells were down-regulated by si RNA technique, which further demonstrated the role of the two subtypes in the migration of prostate cancer cells. 4. The bone metastasis model of prostate cancer was established and the effects of PI3K 尾 and d subtype specific inhibitors on bone mass of nude mice with prostate cancer metastasis were studied by micro CT assay. HE staining, TRAP staining and Macneal's staining were used to study the effect of PI3K 尾 and d subtype specific inhibitors on prostate cancer metastasis in vivo. Results: the specific inhibitors of various subtypes of 1.PI3K inhibited the migration and invasion of PC3 and DU145 cells to varying degrees, and PI3K 尾 and d subtype specific inhibitors were stronger than the IC50 values of inhibition activity at enzyme level. The activity of PI3K 偽 and 緯 subtype specific inhibitors was weak. 2.PI3K 尾 and d subtype specific inhibitors significantly decreased the phosphorylation level of Akt and downstream protein Integrin 尾 1 in PC3 and DU145 cells in a dose-dependent manner. The down-regulated expression of PI3K 尾 and d subtype by si RNA could significantly reduce the phosphorylation level of Akt in PC3 and DU145 cells, and inhibit the migration of PC3 and DU145 cells. Both 4.PI3K 尾 and D-subtype specific inhibitors could effectively increase bone mass and bone density in nude mice with prostate cancer bone metastasis. Both 5.PI3K 尾 and D-subtype specific inhibitors could inhibit bone metastasis of prostate cancer cells and decrease the number of osteoclasts in the bone, with the tendency of increasing the number of osteoblasts. Conclusion: among the four subtype specific inhibitors of 1.PI3K, 尾 and d subtype specific inhibitors can significantly inhibit the migration and invasion of prostate cancer cells at lower concentrations and exhibit strong anti-metastasis activity in vitro. 2.PI3K 尾 and D-subtype specific inhibitors and si RNA significantly reduced the level of Akt phosphorylation in prostate cancer cells at the concentration of anti-prostate cancer cell migration. These results suggest that PI3K/Akt signaling pathway may inhibit the migration and invasion of prostate cancer cells. 3.PI3K 尾 and D-subtype specific inhibitors can significantly inhibit bone metastasis in prostate cancer cells and improve bone microenvironment by reducing osteoclast bone resorption, suggesting that these two subtypes play a key role in prostate cancer metastasis.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.25

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