【摘要】：目的:研究沉默DJ-1對DADS抑制MGC803細(xì)胞增殖與遷移侵襲的影響。證實DJ-1是DADS抑制人胃癌細(xì)胞遷移與侵襲的作用靶點之一。方法:構(gòu)建DJ-1沉默質(zhì)粒,轉(zhuǎn)染MGC803細(xì)胞,建立穩(wěn)定DJ-1沉默MGC803細(xì)胞系。DADS處理MGC803細(xì)胞、空載體組細(xì)胞以及DJ-1沉默MGC803細(xì)胞后,采用MTT、平板克隆形成實驗、劃痕實驗與Transwell侵襲實驗、裸鼠成瘤實驗檢測DADS與DJ-1沉默對MGC803細(xì)胞增殖、遷移侵襲及裸鼠成瘤能力的影響。細(xì)胞免疫熒光、q RT-PCR、Western Blot檢測DADS處理前后MGC803細(xì)胞DJ-1的表達(dá)。Western Blot檢測DADS與DJ-1沉默對MGC803細(xì)胞PTEN、Akt、p-Akt表達(dá)的改變。結(jié)果:1.成功構(gòu)建穩(wěn)定DJ-1沉默MGC803細(xì)胞。將DJ-1干擾質(zhì)粒及陰性對照質(zhì)粒分別轉(zhuǎn)染MGC803細(xì)胞,通過Western Blot、q RT-PCR驗證質(zhì)粒A對DJ-1的干擾效果最明顯,轉(zhuǎn)染后用Puromycin篩選,建立穩(wěn)定DJ-1沉默MGC803細(xì)胞系。2.DJ-1沉默與DADS對MGC803細(xì)胞增殖與遷移侵襲能力的影響。MTT和平板克隆實驗結(jié)果表明,DJ-1沉默與DADS均能抑制MGC803細(xì)胞的增殖能力,并且DJ-1沉默可增強DADS對MGC803細(xì)胞增殖抑制的作用。劃痕實驗與Transwell侵襲實驗結(jié)果顯示,DJ-1沉默和DADS處理后MGC803細(xì)胞的遷移與侵襲能力減弱(P0.05),并且DJ-1沉默可增強DADS抑制腫瘤遷移與侵襲的能力。3.DADS處理前后各組MGC803細(xì)胞DJ-1表達(dá)的改變。細(xì)胞免疫熒光、q RT-PCR、Western Blot實驗結(jié)果顯示,DJ-1主要分布在細(xì)胞漿中,DADS處理各組MGC803細(xì)胞后較未處理組DJ-1、p-Akt表達(dá)明顯減少,PTEN的表達(dá)增加(P0.05)。4.DJ-1沉默與DADS對MGC803細(xì)胞裸鼠成瘤能力的影響裸鼠成瘤實驗表明,DJ-1沉默和DADS能明顯抑制MGC803細(xì)胞裸鼠體內(nèi)成瘤能力。免疫組化結(jié)果顯示,DJ-1沉默和DADS處理后,移植瘤組織中DJ-1陽性表達(dá)較對照組明顯減少,而PTEN的表達(dá)較對照組明顯增加。另外,DJ-1沉默及DADS處理后與對照組比較,移植瘤組織中Ki-67、CD-34、Vimentin陽性表達(dá)下降,而E-cadherin明顯增強。結(jié)論:1.DADS可下調(diào)DJ-1通過PTEN/Akt通路抑制MGC803細(xì)胞增殖與遷移侵襲。2.DJ-1沉默可抑制MGC803細(xì)胞的增殖與遷移侵襲,增強DADS對MGC803細(xì)胞的增殖與遷移侵襲的抑制作用。3.DADS與沉默DJ-1均可抑制人胃癌MGC803細(xì)胞的成瘤能力,并且兩者具有協(xié)同作用。
[Abstract]:Aim: to study the effect of silencing DJ-1 on the proliferation, migration and invasion of MGC803 cells inhibited by DADS. It is confirmed that DJ-1 is one of the targets of DADS in inhibiting migration and invasion of human gastric cancer cells. Methods: DJ-1 silencing plasmid was constructed and transfected into MGC803 cells. Stable DJ-1 silencing MGC803 cell line. Dads was established to treat MGC803 cells. After empty vector group and DJ-1 silencing MGC803 cells, MTT, flat clone formation test, scratch test and Transwell invasion test were used. The effects of DADS and DJ-1 silencing on the proliferation, migration and invasion of MGC803 cells and the tumorigenic ability of nude mice were detected by tumorigenic assay in nude mice. The expression of DJ-1 in MGC803 cells before and after DADS treatment. Western Blot was used to detect the changes of PTEN,Akt,p-Akt expression in MGC803 cells induced by DADS and DJ-1 silencing. The result is 1: 1. Stable DJ-1 silencing MGC803 cells were successfully constructed. DJ-1 interference plasmids and negative control plasmids were transfected into MGC803 cells respectively. Western Blot,q RT-PCR was used to verify the interference effect of plasmid A to DJ-1. After transfection, Puromycin was used to screen. Establishment of stable DJ-1 silencing MGC803 cell line. 2. The effects of DADS and DADS on the proliferation and invasion of MGC803 cells. The results showed that both DJ-1 silencing and DADS could inhibit the proliferation of MGC803 cells. DJ-1 silencing can enhance the inhibitory effect of DADS on the proliferation of MGC803 cells. The results of scratch test and Transwell invasion test showed that the migration and invasion ability of MGC803 cells was decreased after DJ-1 silencing and DADS treatment (P0.05), and DJ-1 silencing enhanced the ability of DADS to inhibit tumor migration and invasion. 3. The DJ-1 expression of MGC803 cells was increased before and after dads treatment. The results of cytoimmunofluorescence Q RT-PCR,Western Blot assay showed that DJ-1 mainly distributed in cytoplasm of MGC803 cells treated with dads significantly decreased the expression of DJ-1,p-Akt (P0.05) .4.The silencing of DJ-1 and the effect of DADS on the tumorigenic ability of MGC803 cells in nude mice Tumorigenic assay in nude mice showed that DJ-1 silencing and DADS could significantly inhibit the tumorigenesis of MGC803 cells in nude mice. The immunohistochemical results showed that the positive expression of DJ-1 was significantly decreased and the expression of PTEN was significantly increased in the transplanted tumor tissue after silencing and DADS treatment. In addition, compared with the control group, the positive expression of Ki-67,CD-34,Vimentin was decreased and E-cadherin was significantly increased after DJ-1 silencing and DADS treatment. Conclusion: 1. Dads can down-regulate the inhibition of MGC803 cell proliferation and migration invasion by DJ-1 via PTEN/Akt pathway. 2. DJ-1 silencing can inhibit the proliferation and migration of MGC803 cells. Enhancing the inhibitory effect of DADS on proliferation and migration and invasion of MGC803 cells. 3. Both dads and silencing DJ-1 can inhibit the tumorigenesis of MGC803 cells of human gastric cancer, and both of them have synergistic effects.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R735.2

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