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

【摘要】：第一部分SLC8A2對(duì)膠質(zhì)瘤細(xì)胞株U87作用的體內(nèi)外實(shí)驗(yàn) 目的：構(gòu)建過表達(dá)基因SLC8A2的U87穩(wěn)定細(xì)胞株，通過體內(nèi)外實(shí)驗(yàn)觀察SLC8A2對(duì)膠質(zhì)瘤細(xì)胞株U87的功能影響。 方法：構(gòu)建攜帶有SLC8A2全基因的慢病毒載體Lenti-SLC8A2-IRES-EGFP和陰性對(duì)照空病毒載體Lenti-EGFP，轉(zhuǎn)染膠質(zhì)瘤細(xì)胞株U87，篩選獲得穩(wěn)定表達(dá)靶基因及報(bào)告基因EGFP的膠質(zhì)瘤細(xì)胞U87-SLC8A2（實(shí)驗(yàn)組）和僅穩(wěn)定表達(dá)EGFP的膠質(zhì)瘤細(xì)胞U87-NC（陰性對(duì)照組），并分別應(yīng)用熒光定量PCR和Western blot檢測(cè)SLC8A2在各組細(xì)胞中的表達(dá)；應(yīng)用Transwell小室及預(yù)鋪Matrigel基質(zhì)膠的Transwell小室檢測(cè)SLC8A2對(duì)U87細(xì)胞侵襲遷移能力的影響；應(yīng)用流式細(xì)胞儀技術(shù)檢測(cè)SLC8A2對(duì)U87細(xì)胞周期的影響；運(yùn)用CCK-8方法檢測(cè)SLC8A2對(duì)U87細(xì)胞增殖的影響；通過裸鼠皮下移植瘤實(shí)驗(yàn)檢測(cè)SLC8A2對(duì)U87細(xì)胞致瘤性的影響。 結(jié)果：成功構(gòu)建過表達(dá)SLC8A2的細(xì)胞株U87-SLC8A2；SLC8A2顯著抑制了U87細(xì)胞的侵襲和遷移能力，但對(duì)U87細(xì)胞的增殖及周期無顯著影響；裸鼠皮下移植瘤實(shí)驗(yàn)顯示，接種U87-SLC8A2細(xì)胞的裸鼠全都不致瘤，，而接種空白組與陰性對(duì)照組細(xì)胞的裸鼠全部致瘤成功。 結(jié)論：SLC8A2能顯著抑制膠質(zhì)瘤細(xì)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(guān)抑癌基因。 第二部分SLC8A2抑制膠質(zhì)瘤細(xì)胞株U87侵襲遷移能力的機(jī)制研究 目的：進(jìn)一步探索SLC8A2抑制膠質(zhì)瘤細(xì)胞株U87侵襲遷移能力的相關(guān)分子機(jī)制。 方法：應(yīng)用RT-PCR檢測(cè)各組細(xì)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(yīng)用Western blot檢測(cè)各組細(xì)胞中MMP-2蛋白表達(dá)水平的差異；運(yùn)用基質(zhì)金屬蛋白酶明膠酶譜法檢測(cè)各組細(xì)胞分泌至培養(yǎng)基中MMP-2酶活性的變化；應(yīng)用Western blot檢測(cè)各組細(xì)胞中NF-κB、MAPK及Akt通路中相關(guān)蛋白表達(dá)水平的差異。 結(jié)果：與陰性對(duì)照組細(xì)胞相比，U87-SLC8A2組細(xì)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(xì)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(duì)照組細(xì)胞相比，U87-SLC8A2組細(xì)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(hào)軸抑制了膠質(zhì)瘤細(xì)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(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.41

【參考文獻(xiàn)】

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

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