本文選題：SLC8A2 + 膠質(zhì)瘤��； 參考：《蘇州大學》2014年碩士論文

【摘要】：第一部分SLC8A2對膠質(zhì)瘤細胞株U87作用的體內(nèi)外實驗 目的：構建過表達基因SLC8A2的U87穩(wěn)定細胞株，通過體內(nèi)外實驗觀察SLC8A2對膠質(zhì)瘤細胞株U87的功能影響。 方法：構建攜帶有SLC8A2全基因的慢病毒載體Lenti-SLC8A2-IRES-EGFP和陰性對照空病毒載體Lenti-EGFP，轉(zhuǎn)染膠質(zhì)瘤細胞株U87，篩選獲得穩(wěn)定表達靶基因及報告基因EGFP的膠質(zhì)瘤細胞U87-SLC8A2（實驗組）和僅穩(wěn)定表達EGFP的膠質(zhì)瘤細胞U87-NC（陰性對照組），并分別應用熒光定量PCR和Western blot檢測SLC8A2在各組細胞中的表達；應用Transwell小室及預鋪Matrigel基質(zhì)膠的Transwell小室檢測SLC8A2對U87細胞侵襲遷移能力的影響；應用流式細胞儀技術檢測SLC8A2對U87細胞周期的影響；運用CCK-8方法檢測SLC8A2對U87細胞增殖的影響；通過裸鼠皮下移植瘤實驗檢測SLC8A2對U87細胞致瘤性的影響。 結(jié)果：成功構建過表達SLC8A2的細胞株U87-SLC8A2；SLC8A2顯著抑制了U87細胞的侵襲和遷移能力，但對U87細胞的增殖及周期無顯著影響；裸鼠皮下移植瘤實驗顯示，接種U87-SLC8A2細胞的裸鼠全都不致瘤，，而接種空白組與陰性對照組細胞的裸鼠全部致瘤成功。 結(jié)論：SLC8A2能顯著抑制膠質(zhì)瘤細胞株U87的侵襲遷移能力和裸鼠致瘤性，表明SLC8A2有可能為膠質(zhì)瘤相關抑癌基因。 第二部分SLC8A2抑制膠質(zhì)瘤細胞株U87侵襲遷移能力的機制研究 目的：進一步探索SLC8A2抑制膠質(zhì)瘤細胞株U87侵襲遷移能力的相關分子機制。 方法：應用RT-PCR檢測各組細胞中MMP-2、MMP-3、MMP-7、MMP-9、MT1-MMP、uPA及uPAR轉(zhuǎn)錄水平的差異，并應用Western blot檢測各組細胞中MMP-2蛋白表達水平的差異；運用基質(zhì)金屬蛋白酶明膠酶譜法檢測各組細胞分泌至培養(yǎng)基中MMP-2酶活性的變化；應用Western blot檢測各組細胞中NF-κB、MAPK及Akt通路中相關蛋白表達水平的差異。 結(jié)果：與陰性對照組細胞相比，U87-SLC8A2組細胞中MMP-2、MMP-3、MMP-7、MMP-9、MT1-MMP、uPA及uPAR的轉(zhuǎn)錄水平及MMP-2蛋白水平被顯著下調(diào)（P＜0.01），且U87-SLC8A2細胞分泌至培養(yǎng)基中的MMP-2酶活性被顯著抑制（P＜0.01）；與陰性對照組細胞相比，U87-SLC8A2組細胞中MMPs和uPA/uPAR系統(tǒng)的上游調(diào)控通路NF-κB及ERK1/2活性被顯著抑制（P＜0.01）。 結(jié)論：SLC8A2可能是通過ERK1/2-NF-κB-MMPs/uPA/uPAR信號軸抑制了膠質(zhì)瘤細胞株U87的侵襲遷移能力。
[Abstract]:Part one: in vitro and in vivo effects of SLC8A2 on glioma cell line U87 Aim: to construct a stable U87 cell line with overexpression gene SLC8A2 and observe the effect of SLC8A2 on the function of U87 glioma cell line in vitro and in vivo. Methods: the lentivirus vector Lenti-SLC8A2-IRES-EGFP carrying the whole SLC8A2 gene and the negative control vector Lenti-EGFPwere constructed and transfected into glioma cell line U87. The glioma cells U87-SLC8A2 (experimental group) and U87-SLC8A2 (experimental group) with stable expression of target gene and reporter gene EGFP were obtained. The expression of SLC8A2 in U87-NCcells (negative control group) was detected by fluorescence quantitative PCR and Western blot, respectively. The effect of SLC8A2 on the invasion and migration of U87 cells was detected by Transwell chamber and Transwell chamber precoated with Matrigel matrix glue, the effect of SLC8A2 on U87 cell cycle was detected by flow cytometry, and the effect of SLC8A2 on the proliferation of U87 cells was detected by CCK-8 method. The effect of SLC8A2 on the tumorigenicity of U87 cells was detected by subcutaneous tumor transplantation in nude mice. Results: the cell line U87-SLC8A2SLC8A2 successfully constructed successfully inhibited the invasion and migration of U87 cells, but had no significant effect on the proliferation and cycle of U87 cells. All the nude mice inoculated with blank group and negative control group were successful in tumorigenesis. Conclusion WSLC8A2 can significantly inhibit the invasion and migration of glioma cell line U87 and the tumorigenicity of nude mice, suggesting that SLC8A2 may be a glioma-associated tumor suppressor gene. The Mechanism of SLC8A2 inhibiting invasion and Migration of Glioma Cell Line U87 Objective: to explore the molecular mechanism of SLC8A2 inhibiting invasion and migration of glioma cell line U87. Methods: RT-PCR was used to detect the transcription level of MMP-2, MMP-3, MMP-7, MMP-9, MT1-MMPUPA and uPAR, and Western blot was used to detect the expression of MMP-2 protein. The activity of MMP-2 was detected by matrix metalloproteinase gelatinase assay and the expression level of NF- 魏 B MMP-2 and related proteins in Akt pathway were detected by Western blot. Results: in U87-SLC8A2 group, the transcription levels of MMP-2, MMP-3, MMP-7, MMP-9, MT1-MMPUPA and MMP-2 protein were significantly down-regulated (P < 0.01), and the activity of MMP-2 enzyme secreted by U87-SLC8A2 cells into culture medium was significantly inhibited (P < 0.01), and compared with the negative control group, the activity of MMP-2 enzyme was significantly inhibited (P < 0.01), while that of MMPU UPA and MMPU UPA protein in U87-SLC8A2 group was significantly decreased than that in negative control group (P < 0.01). In U87-SLC8A2 group, the upstream regulatory pathway NF- 魏 B and ERK1/2 of MMPs and uPA/uPAR system were significantly inhibited (P < 0.01). Conclusion the invasion and migration ability of glioma cell line U87 may be inhibited by the ERK1 / 2-NF- 魏 B-MMPs/uPA/uPAR signal axis.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R739.41

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1 張傳海;STAT3活化介導肝細胞癌上皮間質(zhì)轉(zhuǎn)化機制研究[D];天津醫(yī)科大學;2011年

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