siPGK1增加黑色素瘤细胞对vemurafenib敏感性及其机制的初步探索

发布时间：2018-05-18 22:15

  本文选题：黑色素瘤 + siPGK1　； 参考：《山西医科大学》2017年硕士论文

【摘要】：目的:探讨磷酸甘油酸激酶1(phosphoglycerate kinase 1,PGK1)在BRAFV600E突变型恶性黑色素瘤(Malignant melanoma,MM)细胞对Vemurafenib(Zelboraf?)的敏感性中的作用及其机制。方法:1)使用基因沉默技术(Small interfering RNA,siRNA),处理5株黑色素瘤细胞株(A375m,A375mR,1205LU,UACC903,C8161-C9)72小时,将每株分为PGK1基因沉默组(siPGK1组)和非沉默组(si-Non-Target,si NT组),siPGK1组沉默PGK1基因,siNT组不沉默PGK1基因。2)使用MTT(Thiazolyl Blue Tetrazolium Bromide,approx.98%TLC)实验方法分别检测5株黑色素瘤细胞株siPGK1组、siNT组细胞的存活能力,各组分别给以不同浓度的BRAF突变抑制剂vemurafenib(0,0.01,0.1,1,10μM)处理后测定细胞的存活能力,以及siPGK1组、siNT组,联合vemurafenib处理后测定细胞的存活能力。3)使用Western Blot实验方法检测5株黑色素瘤细胞株PGK1基因的表达量,PGK1基因沉默效果,以及沉默PGK1基因后再给以vemurafenib(0,2μM)处理24小时后,PGK1的表达量变化和PARP蛋白表达量以及活性变化。4)使用克隆集落(Colongenic Assay)方法检测培养14天后5株黑色素瘤细胞株siPGK1组和siNT组细胞形成集落个数、大小、增殖能力。以及联合给以vemurafenib(A375m:0,0.25,0.5,1μM;A375mR,1205LU,UACC903,C8161-C9:0,2.5,5,10μM)处理后各组细胞形成集落个数、大小、增殖能力。5)使用流式细胞术(Flow Cytometry,FCM)检测siPGK1组和siNT组联合给以vemurafenib(A375m:0,0.25,0.5μM;A375mR,1205LU,UACC903,C8161-C9:0,2.5,5μM)处理24小时后5株黑色素瘤细胞株的凋亡情况,以及细胞早期凋亡和晚期凋亡的变化情况。结果:1)Western Blot结果显示PGK1在4株BRAF突变型(A375m,A375mR,1205LU,UACC903)黑色素瘤细胞株中呈高表达,在野生型(C8161-C9)黑色素瘤细胞株中呈低表达。其中A375m PGK1的表达量约为C8161-C9的2.26倍(P0.01),A375mR PGK1的表达量约为C8161-C9的1.69倍(P0.05),1205LU PGK1的表达量约为C8161-C9的1.20倍,UACC903 PGK1的表达量约为C8161-C9的2.04倍(P0.01)。2)沉默PGK1基因后再给以BRAF突变型选择性抑制剂vemurafenib,黑色素瘤细胞株的存活率明显下降,并表现出一定的剂量依赖性。MTT Assay结果显示:对野生型C8161-C9的抑制作用不明显,A375mR在较低剂量浓度有一定的疗效,但是在较大剂量浓度(1μM)时便出现耐药性。但是对另外的3株BRAF突变型黑色素瘤细胞的作用效果明显,对A375m的抑制作用效果最为明显,10μM的vemurafenib对A375m的抑制率达到了78%(P0.05)。3)siPGK1增加黑色素瘤细胞对vemurafenib的敏感性,这一特征与激活细胞凋亡信号通路有关。以A375mR、UACC903的凋亡信号通路活化效果最为明显,而C8161-C9的活化效果最差。4)1205LU、UACC903、C8161-C9细胞以早期凋亡为主,A375m、A375mR则以晚期凋亡为主。结论:沉默PGK1可能通过激活细胞凋亡通路增加黑色素瘤细胞对vemurafenib的敏感性,从而抑制黑色素瘤细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. 浠ュ強鑱斿悎缁欎互vemurafenib(A375m:0,0.25,0.5,1渭M;A375mR,1205LU,UACC903,C8161-C9:0,2.5,5,10渭M)澶勭悊鍚庡悇缁勭粏鑳炲舰鎴愰泦钀戒釜鏁，

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