靶向抑制PI3K亚型抗前列腺癌转移的作用及机理研究

发布时间：2019-02-17 13:52

【摘要】：目的:研究靶向抑制PI3K亚型对体内外前列腺癌转移的作用及其分子机理,初步阐明在前列腺癌转移中起关键作用的PI3K亚型,为前列腺癌治疗提供新的思路。方法:1.利用Transwell小室迁移实验和小室侵袭实验研究PI3K各亚型特异性抑制剂对PC3和DU145细胞迁移能力以及侵袭能力的影响;2.利用Western blot技术研究PI3Kβ及d亚型特异性抑制剂对PC3和DU145细胞中迁移和侵袭相关信号蛋白Akt及其下游蛋白Integrinβ1的表达以及磷酸化水平的影响;3.利用si RNA技术分别对PC3和DU145细胞中PI3Kβ及d亚型降表达,进一步证明两种亚型在前列腺癌细胞迁移中的作用;4.建立前列腺癌骨转移模型,结合micro CT检测技术研究PI3Kβ及d亚型特异性抑制剂对体内前列腺癌转移裸鼠骨量的影响;5.利用HE染色、TRAP染色及Macneal’s染色等方法研究PI3Kβ及d亚型特异性抑制剂对体内前列腺癌转移的效果。结果:1.PI3K各亚型特异性抑制剂对PC3和DU145细胞的迁移和侵袭有不同程度的抑制活性,与酶水平抑制活性的IC50值相比,PI3Kβ及d亚型特异性抑制剂活性强,PI3Kα及γ亚型特异性抑制剂活性弱;2.PI3Kβ及d亚型特异性抑制剂可显著降低PC3和DU145细胞Akt及下游蛋白Integrinβ1的磷酸化水平,呈一定的剂量依赖性;3.采用si RNA的方法对PI3Kβ及d亚型降表达能显著降低PC3和DU145细胞Akt的磷酸化水平,且抑制PC3和DU145细胞迁移;4.PI3Kβ及d亚型特异性抑制剂均能有效提升前列腺癌骨转移裸鼠的骨量,增加骨密度;5.PI3Kβ及d亚型特异性抑制剂均能抑制前列腺癌细胞的骨转移,减少骨中破骨细胞数目,有增加成骨细胞数目的趋势。结论:1.PI3K四种亚型特异性抑制剂中,β及d亚型特异性抑制剂在较低浓度下有显著的抑制前列腺癌细胞迁移和侵袭的能力,表现出较强的体外抗肿瘤转移活性;2.PI3Kβ及d亚型特异性抑制剂及si RNA在产生抗前列腺癌细胞迁移的浓度下可显著降低前列腺癌细胞中Akt的磷酸化水平,提示其可能通过PI3K/Akt信号通路来抑制前列腺癌细胞的迁移和侵袭等;3.PI3Kβ及d亚型特异性抑制剂可显著抑制前列腺癌细胞体内骨转移,并通过减少破骨细胞骨吸收来改善骨微环境,提示这两种亚型在前列腺癌转移中起关键作用。
[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 伪 and 纬 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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.25

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