SphK1基因沉默对结肠癌RKO细胞化疗敏感性的影响

发布时间：2018-02-01 07:34

  本文关键词： 鞘氨醇激酶1(SphK1) 结肠癌细胞 顺铂(DDP) 化疗敏感性 细胞增殖 细胞凋亡　出处：《广西医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的探讨通过慢病毒介导人结肠腺癌RKO细胞中的鞘氨醇激酶-1(Sphingosine kinase-1,SphK1)基因沉默后对化疗药物顺铂(cisplatin,DDP)敏感性的影响及其可能的作用机制。方法通过重组DNA技术,设计和合成特异性针对SphK1基因的pLenti6.3-shRNA-SphK1慢病毒表达载体,同时无义序列pLenti6.3-EGFP慢病毒载体作为阴性对照,感染人结肠腺癌RKO细胞株,然后采用杀稻瘟菌素(blasticidin,BSD)来筛选,以获得稳定不变低表达SphK1基因的单一克隆RKO稳转细胞株。根据是否转染,以及转染的处理方式分为SphK1敲低组(shSphK1组)、阴性转染对照组(NC组),同时将未予转染处理的原始RKO细胞记作空载对照组(Control组)。为验证基因的沉默效率,在荧光倒置显微镜下检测转染后各组细胞的EGFP(绿色荧光)的表达情况,采用实时荧光定量PCR(quantitative real-time PCR,RT-qPCR)法检测Sph K1的mRNA水平,以及Western blot法检测SphK1的蛋白表达水平。根据是否暴露于DDP,将实验分为非DDP组和DDP组两个大组。非DDP组包括shSphK1组、Control组和NC组;DDP组则记为shSphK1+组、Control+组和NC+组。然后以不同剂量(0、2、4、8、16、32μg/ml)的DDP分别作用于细胞24、48、72小时后,利用MTT法检测各组细胞的增殖活性。采用原位末端转移酶标记技术(TdT-mediated dUTP-biotin nick end labeling,TUNEL)检测细胞凋亡。Western blot法检验增殖标志蛋白Ki-67以及凋亡标记蛋白Bcl-2、Caspase-9和Caspase-3蛋白的表达变化程度。结果在荧光倒置显微镜下,shSphK1组和NC组可观看到多量的egfp(绿色荧光)的表达。rt-qpcr法和westernblot法均表明shsphk1组的sphk1表达显著低于control组及nc组(p0.05)。mtt结果显示shsphk1组、control组和nc组细胞24h的增殖活性分别为(1.1063±0.0316)、(1.2821±0.0463)、(1.2508±0.0365),以上三组细胞48h的增殖活性分别为(1.1937±0.0347)、(1.5733±0.0554)、(1.5314±0.0572),三组细胞72h的增殖活性分别为(1.2288±0.0495)、(1.6907±0.0785)、(1.6667±0.0658);shsphk1组24、48、72h的细胞增殖活力显著比control组及nc组减小(p0.05),这提示沉默sphk1基因能够降低rko细胞的增殖活力。mtt结果还显示以2μg/ml的ddp处理shsphk1+组、control+组和nc+组细胞24h后的细胞增殖活性分别为(0.7587±0.0422)、(0.9379±0.0535)、(0.9189±0.0512),48h的细胞增殖活性分别为(0.6675±0.0429)、(0.8151±0.0704)、(0.7868±0.0893),72h的细胞增殖活性分别为(0.5224±0.0535)、(0.7510±0.0632)、(0.7456±0.0605);以4μg/ml的ddp处理细胞24h后的细胞增殖活性分别为(0.6332±0.0303)、(0.8561±0.0697)、(0.8364±0.0714),48h的细胞增殖活性分别为(0.5236±0.0219)、(0.7121±0.0253)、(0.6971±0.0536),72h的细胞增殖活性分别为(0.4035±0.0709)、(0.6495±0.0375)、(0.6002±0.0218);以8μg/ml的ddp处理细胞24h后的细胞增殖活性分别为(0.5447±0.0399)、(0.7683±0.0402)、(0.7409±0.0622),48h的细胞增殖活性分别为(0.4343±0.0529)、(0.5938±0.0701)、(0.5576±0.0365),72h的细胞增殖活性分别为(0.2781±0.0195)、(0.4725±0.0427)、(0.4543±0.0594);以16μg/ml的ddp处理细胞24h后的细胞增殖活性分别为(0.4575±0.0622)、(0.6387±0.0899)、(0.6193±0.0323),48h的细胞增殖活性分别为(0.2519±0.0353)、(0.4163±0.0532)、(0.3952±0.0413),72h的细胞增殖活性分别为(0.1472±0.0477)、(0.3061±0.0319)、(0.2707±0.0242);以32μg/ml的ddp处理细胞24h后的细胞增殖活性分别为(0.2867±0.0746)、(0.4519±0.0698)、(0.4298±0.0706),48h的细胞增殖活性分别为(0.1120±0.0493)、(0.2194±0.0389)、(0.1888±0.0439),72h的细胞增殖活性分别为(0.0478±0.0073)、(0.1399±0.0231)、(0.1040±0.0365)。以上结果显示ddp呈时间和剂量依赖性抑制各组细胞的增殖,并且在同一浓度的ddp作用相同时间的条件下,shsphk1+组细胞的增殖能力显著低于control+组和nc+组(p0.05)。tunel法结果显示未予ddp处理的shsphk1组细胞凋亡率(13.54±2.19)%明显高于control组(5.04±1.05)%和nc组(6.15±1.56)%(p0.05)。予8μg/ml的ddp处理shsphk1+组、control+组和nc+组细胞24h后的细胞凋亡率则分别为(25.91±3.01)%、(18.68±3.16)%、(19.28±2.97)%;予16μg/ml的ddp处理以上三组细胞24h后的细胞凋亡率分别为(55.26±4.71)%、(35.82±3.97)%、(37.52±3.51)%;予32μg/ml的ddp处理三组细胞24h的细胞凋亡率分别为(76.04±4.52)%、(59.60±4.87)%、(62.28±3.11)%;以上结果说明ddp呈浓度依赖性诱导rko细胞凋亡,并且shsphk1+组细胞的凋亡率明显高于control+组和nc+组(p0.05)。westernblot法检测沉默sphk1后,shsphk1组、control组和nc组ki-67蛋白的相对表达量分别为(0.9410±0.0437)、(1.1592±0.0753)、(1.1543±0.0981),bcl-2蛋白的相对表达量分别为(0.7317±0.0374)、(0.9180±0.0633)、(0.9205±0.0495),caspase-9蛋白的相对表达量分别为(0.4367±0.0757)、(0.2317±0.0593)、(0.2495±0.0435),caspase-3蛋白的相对表达量分别为(0.6479±0.0762)、(0.3989±0.0499)、(0.3942±0.0614)。以16μg/ml的ddp处理shsphk1+组、control+组和nc+组细胞24h后,ki-67的相对表达量分别为(0.3105±0.0367)、(0.8049±0.0684)、(0.8138±0.0596),bcl-2的相对表达量分别为(0.2329±0.0389)、(0.5203±0.0312)、(0.5270±0.0497),caspase-9蛋白的相对表达量分别为(1.8941±0.1025)、(0.8627±0.0624)、(0.8785±0.0858),caspase-3蛋白的相对表达量分别为(2.5849±0.1586)、(0.8955±0.0735)、(0.8872±0.0942)。结果显示敲低SphK1后,Ki-67和Bcl-2的表达减弱,Caspase-9和Caspase-3的表达增强;经DDP处理后,shSphK1+组的Ki-67和Bcl-2蛋白表达水平较Control+组和NC+组明显减少(P0.05);shSphK1+组的Caspase-9和Caspase-3蛋白表达水平与Control+组和NC+组相比显著增强(P0.05)。结论沉默SphK1基因可以抑制结肠癌RKO细胞的增殖活力,下调SphK1基因可能通过抑制Bcl-2的水平和激活Caspase 9/3的表达促进RKO细胞的凋亡,抑制SphK1基因可能通过激活Caspase-9/3依赖的细胞凋亡信号通路增强结肠癌RKO细胞对DDP的化疗敏感性。
[Abstract]:Objective to explore the lentivirus mediated human colon adenocarcinoma RKO cells by sphingosine kinase -1 (Sphingosine kinase-1 SphK1) gene silencing on cisplatin (cisplatin, DDP) sensitivity and its possible mechanism. Methods by recombinant DNA technology, the design and synthesis of specific targeting SphK1 gene pLenti6.3-shRNA-SphK1 lentiviral expression at the same time the carrier, the antisense sequence of pLenti6.3-EGFP lentiviral vector as a negative control, the infection of human colon adenocarcinoma cell line RKO, and then by blasticidin (blasticidin, BSD) to screen, to obtain a stable low expression of SphK1 gene single clone RKO stably transfected cell lines. According to whether the transfection and transfection treatment divided into SphK1 knockdown group (shSphK1 group), negative transfection control group (NC group), while the original RKO cells were not treated as no-load transfection control group (group Control). In order to verify the base Because of the silencing efficiency under fluorescence microscope after transfection was detected by cell EGFP (green fluorescent protein), by using real-time quantitative PCR (quantitative real-time PCR, RT-qPCR Sph K1) method was used to detect the levels of mRNA, Western and SphK1 expression was detected by blot protein levels. According to whether exposure to DDP, the divided into non DDP group and DDP group two groups. DDP group including shSphK1 group, Control group and NC group; DDP group was labeled as shSphK1+ group, Control+ group and NC+ group. Then at different doses (0,2,4,8,16,32 g/ml) DDP cells were stimulated by 24,48,72 hours after the cells were detected by MTT the activity of proliferation. Using TUNEL (TdT-mediated dUTP-biotin nick end labeling, TUNEL).Western blot test method to detect apoptosis proliferation marker protein Ki-67 and apoptosis marker proteins Bcl-2, Caspase-9 and Ca The change degree of the expression of spase-3 protein. Results under fluorescence microscope, shSphK1 group and NC group were more impressive to see the amount of EGFP (green fluorescence) expression of.Rt-qpcr method and Westernblot method showed that the expression of shsphk1 in group SphK1 was significantly lower than that of group control and group NC (P0.05).Mtt showed that shsphk1 group, control group and NC group cell proliferation activity of 24h were (1.1063 + 0.0316), (1.2821 + 0.0463), (1.2508 + 0.0365), more than three groups of cell proliferation activity of 48h were (1.1937 + 0.0347), (1.5733 + 0.0554), (1.5314 + 0.0572), three groups of cell proliferation in 72h respectively (1.2288 + 0.0495), (1.6907 + 0.0785), (1.6667 + 0.0658); cell proliferation activity of shsphk1 24,48,72h group was significantly decreased than control group and NC group (P0.05), suggesting that SphK1 gene silencing can reduce the proliferation of RKO cells..mtt results also showed that in the DDP treatment group shsphk1+ 2 g/ml, contr ol+缁勫拰nc+缁勭粏鑳，

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