本文選題：黑色素瘤 + siPGK1��； 參考：《山西醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:探討磷酸甘油酸激酶1(phosphoglycerate kinase 1,PGK1)在BRAFV600E突變型惡性黑色素瘤(Malignant melanoma,MM)細胞對Vemurafenib(Zelboraf?)的敏感性中的作用及其機制。方法:1)使用基因沉默技術(shù)(Small interfering RNA,siRNA),處理5株黑色素瘤細胞株(A375m,A375mR,1205LU,UACC903,C8161-C9)72小時,將每株分為PGK1基因沉默組(siPGK1組)和非沉默組(si-Non-Target,si NT組),siPGK1組沉默PGK1基因,siNT組不沉默PGK1基因。2)使用MTT(Thiazolyl Blue Tetrazolium Bromide,approx.98%TLC)實驗方法分別檢測5株黑色素瘤細胞株siPGK1組、siNT組細胞的存活能力,各組分別給以不同濃度的BRAF突變抑制劑vemurafenib(0,0.01,0.1,1,10μM)處理后測定細胞的存活能力,以及siPGK1組、siNT組,聯(lián)合vemurafenib處理后測定細胞的存活能力。3)使用Western Blot實驗方法檢測5株黑色素瘤細胞株P(guān)GK1基因的表達量,PGK1基因沉默效果,以及沉默PGK1基因后再給以vemurafenib(0,2μM)處理24小時后,PGK1的表達量變化和PARP蛋白表達量以及活性變化。4)使用克隆集落(Colongenic Assay)方法檢測培養(yǎng)14天后5株黑色素瘤細胞株siPGK1組和siNT組細胞形成集落個數(shù)、大小、增殖能力。以及聯(lián)合給以vemurafenib(A375m:0,0.25,0.5,1μM;A375mR,1205LU,UACC903,C8161-C9:0,2.5,5,10μM)處理后各組細胞形成集落個數(shù)、大小、增殖能力。5)使用流式細胞術(shù)(Flow Cytometry,FCM)檢測siPGK1組和siNT組聯(lián)合給以vemurafenib(A375m:0,0.25,0.5μM;A375mR,1205LU,UACC903,C8161-C9:0,2.5,5μM)處理24小時后5株黑色素瘤細胞株的凋亡情況,以及細胞早期凋亡和晚期凋亡的變化情況。結(jié)果:1)Western Blot結(jié)果顯示PGK1在4株BRAF突變型(A375m,A375mR,1205LU,UACC903)黑色素瘤細胞株中呈高表達,在野生型(C8161-C9)黑色素瘤細胞株中呈低表達。其中A375m PGK1的表達量約為C8161-C9的2.26倍(P0.01),A375mR PGK1的表達量約為C8161-C9的1.69倍(P0.05),1205LU PGK1的表達量約為C8161-C9的1.20倍,UACC903 PGK1的表達量約為C8161-C9的2.04倍(P0.01)。2)沉默PGK1基因后再給以BRAF突變型選擇性抑制劑vemurafenib,黑色素瘤細胞株的存活率明顯下降,并表現(xiàn)出一定的劑量依賴性。MTT Assay結(jié)果顯示:對野生型C8161-C9的抑制作用不明顯,A375mR在較低劑量濃度有一定的療效,但是在較大劑量濃度(1μM)時便出現(xiàn)耐藥性。但是對另外的3株BRAF突變型黑色素瘤細胞的作用效果明顯,對A375m的抑制作用效果最為明顯,10μM的vemurafenib對A375m的抑制率達到了78%(P0.05)。3)siPGK1增加黑色素瘤細胞對vemurafenib的敏感性,這一特征與激活細胞凋亡信號通路有關(guān)。以A375mR、UACC903的凋亡信號通路活化效果最為明顯,而C8161-C9的活化效果最差。4)1205LU、UACC903、C8161-C9細胞以早期凋亡為主,A375m、A375mR則以晚期凋亡為主。結(jié)論:沉默PGK1可能通過激活細胞凋亡通路增加黑色素瘤細胞對vemurafenib的敏感性,從而抑制黑色素瘤細胞的存活和增殖能力。
[Abstract]:Objective: to investigate the effect of phosphoglycerate kinase 1(phosphoglycerate kinase 1 (PGK1) in BRAFV600E mutant malignant melanoma cell line (MMMM1) against Vemurafenibia Zelborafa. The role and mechanism of the sensitivity. Methods using gene silencing technique, small interfering siRNAs were used to treat 5 melanoma cell lines: A375mRN 1205LUUUUACC903C8161-C9F72h. Each strain was divided into two groups: PGK1 gene silencing group (P < 0.05) and non-silencing PGK1 gene group (n = 5) PGK1 gene was not silenced by MTT(Thiazolyl Blue Tetrazolium Bromideapprox.98T) the viability of five melanoma cell lines, siPGK1 group, siNT group was detected by using MTT(Thiazolyl Blue Tetrazolium Bromideapprox.98T method. Each group was treated with different concentrations of BRAF mutagenic inhibitor vemurafenibbuterol (Vemurafenib0), and the viability of the cells was measured after treatment with 0. 01 渭 M and 0. 01 渭 M, respectively. The survival ability of the cells in the siPGK1 group was also determined, as well as in the siNT group. Western Blot assay was used to detect the expression of PGK1 gene in 5 melanoma cell lines and the silencing effect of PGK1 gene. PGK1 gene was silenced and then treated with vemurafenib0 2 渭 M for 24 hours. After 24 hours of treatment, the expression of PARP protein and the expression of PARP protein were changed. 4) Colongenic assay was used to detect 5 melanoma cell lines siPGK1 group and siNT group after 14 days of culture. The number of colony formation, Size, proliferation ability. 浠ュ強鑱斿悎緇欎互vemurafenib(A375m:0,0.25,0.5,1渭M;A375mR,1205LU,UACC903,C8161-C9:0,2.5,5,10渭M)澶勭悊鍚庡悇緇勭粏鑳?yōu)迮炴垚闆嗚惤涓暎?

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