發(fā)布時間：2019-05-30 03:57

【摘要】：目的：觀察Ⅰ類組蛋白脫乙酰化酶(HDAC)抑制劑4SC-202體內(nèi)外抗結腸癌細胞的作用,旨在為結腸癌的分子靶向治療進行新的探索。方法：1)MTT法、臺盼藍染色法、細胞集落試驗檢測不同濃度的4SC-202處理不同作用時間后對結腸癌細胞存活、細胞死亡及細胞增殖的影響；2) Histone DNA ELISA法、Caspase-3/-9試劑盒方法、Annexin V-PI流式細胞儀法檢測4SC-202對結腸癌細胞凋亡的作用；4SC-202處理后對其抗凋亡蛋白Bcl-2的表達進行檢測,運用的方法為Western Blotting;3) PI流式細胞儀法觀察4SC-202處理結腸癌細胞后,細胞周期的變化。4)用藥理學和shRNA等手段抑制結腸癌細胞內(nèi)AKT的活性,觀察抑制后4SC-202體外抗結腸癌細胞活性的變化。5)MTT法、Histone DNA ELISA法等方法檢測比較奧沙利鉑組、4SC-202組以及奧沙利鉑聯(lián)合4SC-202組對結腸癌細胞的存活和凋亡的影響。6)建立裸鼠荷瘤模型,觀察奧沙利鉑和/或4SC-202在體內(nèi)抗結腸癌細胞活性。結果：1)4SC-202顯著抑制結腸癌細胞(原代人結腸癌細胞和HT-29,HCT-116,HT-15,DLD-1細胞株)的存活和增殖,其抑制作用呈時間和濃度依賴性。2)4SC-202誘導結腸癌細胞凋亡。4SC-202可以抑制抗凋亡蛋白Bcl-2的表達。3)當4SC-202濃度不斷的增大時,結腸癌細胞的G1和S期所占比例逐漸減少,G2-M期比例增加。4)添加AKT選擇性抑制劑perifosine、MK-2206或shRNA敲減AKT1/2顯著增加4SC-202誘導的體外抗結腸癌細胞的活性。而導入持續(xù)活化的AKT1(CA-AKT1)則抑制4SC-202抗結腸癌細胞的作用。5)奧沙利鉑誘導的體外抗結腸癌細胞的活性在有低濃度4SC-202存在時明顯增強。兩者協(xié)同給藥誘導的抗結腸癌細胞的活性要顯著強于單獨給藥。6)在裸鼠荷瘤模型中,4SC-202 (100mg/kg, Q2D)口服給藥顯著抑制HT-29移植瘤的在體生長。而同時腹腔注射奧沙利鉑(5.0mg/kg, Q3D)則進一步增強4SC-202在體抗癌活性。結論：1)4SC-202濃度及時間依賴性的抑制人結腸癌細胞存活、增殖,并誘導癌細胞凋亡；2)4SC-202誘導結腸癌細胞G2-M周期停滯；3)抑制AKT能增強結腸癌細胞對4SC-202的敏感性；4)4SC-202增強奧沙利鉑體外抗結腸癌細胞的作用；5)4SC-202和奧沙利鉑協(xié)同抑制HT-29移植瘤的在體生長。
[Abstract]:Aim: to observe the effect of class I histone deacetylase (HDAC) inhibitor 4SC-202 on colon cancer cells in vitro and in vivo in order to explore the molecular targeting therapy of colon cancer. Methods: 1) MTT assay, trypan blue staining and cell colony test were used to detect the effects of different concentrations of 4SC-202 on the survival, cell death and cell proliferation of colon cancer cells. 2) Histone DNA ELISA assay, Caspase-3/-9 kit method and Annexin V-PI flow cytometry were used to detect the effect of 4SC-202 on apoptosis of colon cancer cells. After 4SC-202 treatment, the expression of anti-apoptosis protein Bcl-2 was detected by Western Blotting;. 3) PI flow cytometry was used to observe the changes of cell cycle in colon cancer cells treated with 4SC-202. 4) Pharmacology and shRNA were used to inhibit the activity of AKT in colon cancer cells. To observe the changes of anti-colon cancer cell activity of 4SC-202 in vitro after inhibition. 5) MTT, Histone DNA ELISA assay was used to detect and compare the activity of oxaliplatinum group. The effects of 4SC-202 group and oxaliplatin combined with 4SC-202 group on the survival and apoptosis of colon cancer cells. 6) the tumor-bearing model of nude mice was established to observe the anti-colon cancer cell activity of oxaliplatin and / or 4SC-202 in vivo. Results: 1) 4SC-202 significantly inhibited the survival and proliferation of colon cancer cells (primary human colon cancer cells and HT-29,HCT-116,HT-15,DLD-1 cell lines). The inhibitory effect was time-and concentration-dependent. 2) 4SC-202 induced apoptosis in colon cancer cells. 4SC-202 could inhibit the expression of anti-apoptosis protein Bcl-2. 3) when the concentration of 4SC-202 increased, The proportion of G _ 1 and S phase of colon cancer cells decreased gradually, while the proportion of G _ 2-M phase increased. 4) the addition of AKT selective inhibitor perifosine,MK-2206 or shRNA knockdown AKT1/2 significantly increased the activity of 4SC-202-induced anti-colon cancer cells in vitro. However, the introduction of continuously activated AKT1 (CA-AKT1) inhibited the anti-colon cancer cell effect of 4SC-202. 5) the activity of oxaliplatin induced anti-colon cancer cells in vitro was significantly enhanced in the presence of low concentration of 4SC-202. The activity of anti-colon cancer cells induced by co-administration was significantly stronger than that induced by single administration. 6) in nude mice tumor-bearing model, oral administration of 4SC-202 (100mg / kg, Q2D) significantly inhibited the growth of HT-29 transplantable tumor in vivo. At the same time, intraperitoneal injection of oxaliplatin (5.0 mg 路kg / kg, Q3D) further enhanced the anticancer activity of 4SC-202 in vivo. Conclusion: 1) 4SC-202 can inhibit the survival, proliferation and apoptosis of human colon cancer cells in a concentration-and time-dependent manner, 2) 4SC-202 can induce the arrest of G2 / M cycle of colon cancer cells. 3) inhibition of AKT could enhance the sensitivity of colon cancer cells to 4SC-202, 4) 4SC-202 enhanced the anti-colon cancer cell effect of oxaliplatin in vitro, and 5) 4SC-202 and oxaliplatin combined with oxaliplatin could inhibit the growth of HT-29 transplantation tumor in vivo.
【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R96

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