本文關(guān)鍵詞： 鞘氨醇激酶1(SphK1) 結(jié)腸癌細(xì)胞 順鉑(DDP) 化療敏感性 細(xì)胞增殖 細(xì)胞凋亡　出處：《廣西醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的探討通過(guò)慢病毒介導(dǎo)人結(jié)腸腺癌RKO細(xì)胞中的鞘氨醇激酶-1(Sphingosine kinase-1,SphK1)基因沉默后對(duì)化療藥物順鉑(cisplatin,DDP)敏感性的影響及其可能的作用機(jī)制。方法通過(guò)重組DNA技術(shù),設(shè)計(jì)和合成特異性針對(duì)SphK1基因的pLenti6.3-shRNA-SphK1慢病毒表達(dá)載體,同時(shí)無(wú)義序列pLenti6.3-EGFP慢病毒載體作為陰性對(duì)照,感染人結(jié)腸腺癌RKO細(xì)胞株,然后采用殺稻瘟菌素(blasticidin,BSD)來(lái)篩選,以獲得穩(wěn)定不變低表達(dá)SphK1基因的單一克隆RKO穩(wěn)轉(zhuǎn)細(xì)胞株。根據(jù)是否轉(zhuǎn)染,以及轉(zhuǎn)染的處理方式分為SphK1敲低組(shSphK1組)、陰性轉(zhuǎn)染對(duì)照組(NC組),同時(shí)將未予轉(zhuǎn)染處理的原始RKO細(xì)胞記作空載對(duì)照組(Control組)。為驗(yàn)證基因的沉默效率,在熒光倒置顯微鏡下檢測(cè)轉(zhuǎn)染后各組細(xì)胞的EGFP(綠色熒光)的表達(dá)情況,采用實(shí)時(shí)熒光定量PCR(quantitative real-time PCR,RT-qPCR)法檢測(cè)Sph K1的mRNA水平,以及Western blot法檢測(cè)SphK1的蛋白表達(dá)水平。根據(jù)是否暴露于DDP,將實(shí)驗(yàn)分為非DDP組和DDP組兩個(gè)大組。非DDP組包括shSphK1組、Control組和NC組;DDP組則記為shSphK1+組、Control+組和NC+組。然后以不同劑量(0、2、4、8、16、32μg/ml)的DDP分別作用于細(xì)胞24、48、72小時(shí)后,利用MTT法檢測(cè)各組細(xì)胞的增殖活性。采用原位末端轉(zhuǎn)移酶標(biāo)記技術(shù)(TdT-mediated dUTP-biotin nick end labeling,TUNEL)檢測(cè)細(xì)胞凋亡。Western blot法檢驗(yàn)增殖標(biāo)志蛋白Ki-67以及凋亡標(biāo)記蛋白Bcl-2、Caspase-9和Caspase-3蛋白的表達(dá)變化程度。結(jié)果在熒光倒置顯微鏡下,shSphK1組和NC組可觀看到多量的egfp(綠色熒光)的表達(dá)。rt-qpcr法和westernblot法均表明shsphk1組的sphk1表達(dá)顯著低于control組及nc組(p0.05)。mtt結(jié)果顯示shsphk1組、control組和nc組細(xì)胞24h的增殖活性分別為(1.1063±0.0316)、(1.2821±0.0463)、(1.2508±0.0365),以上三組細(xì)胞48h的增殖活性分別為(1.1937±0.0347)、(1.5733±0.0554)、(1.5314±0.0572),三組細(xì)胞72h的增殖活性分別為(1.2288±0.0495)、(1.6907±0.0785)、(1.6667±0.0658);shsphk1組24、48、72h的細(xì)胞增殖活力顯著比control組及nc組減小(p0.05),這提示沉默sphk1基因能夠降低rko細(xì)胞的增殖活力。mtt結(jié)果還顯示以2μg/ml的ddp處理shsphk1+組、control+組和nc+組細(xì)胞24h后的細(xì)胞增殖活性分別為(0.7587±0.0422)、(0.9379±0.0535)、(0.9189±0.0512),48h的細(xì)胞增殖活性分別為(0.6675±0.0429)、(0.8151±0.0704)、(0.7868±0.0893),72h的細(xì)胞增殖活性分別為(0.5224±0.0535)、(0.7510±0.0632)、(0.7456±0.0605);以4μg/ml的ddp處理細(xì)胞24h后的細(xì)胞增殖活性分別為(0.6332±0.0303)、(0.8561±0.0697)、(0.8364±0.0714),48h的細(xì)胞增殖活性分別為(0.5236±0.0219)、(0.7121±0.0253)、(0.6971±0.0536),72h的細(xì)胞增殖活性分別為(0.4035±0.0709)、(0.6495±0.0375)、(0.6002±0.0218);以8μg/ml的ddp處理細(xì)胞24h后的細(xì)胞增殖活性分別為(0.5447±0.0399)、(0.7683±0.0402)、(0.7409±0.0622),48h的細(xì)胞增殖活性分別為(0.4343±0.0529)、(0.5938±0.0701)、(0.5576±0.0365),72h的細(xì)胞增殖活性分別為(0.2781±0.0195)、(0.4725±0.0427)、(0.4543±0.0594);以16μg/ml的ddp處理細(xì)胞24h后的細(xì)胞增殖活性分別為(0.4575±0.0622)、(0.6387±0.0899)、(0.6193±0.0323),48h的細(xì)胞增殖活性分別為(0.2519±0.0353)、(0.4163±0.0532)、(0.3952±0.0413),72h的細(xì)胞增殖活性分別為(0.1472±0.0477)、(0.3061±0.0319)、(0.2707±0.0242);以32μg/ml的ddp處理細(xì)胞24h后的細(xì)胞增殖活性分別為(0.2867±0.0746)、(0.4519±0.0698)、(0.4298±0.0706),48h的細(xì)胞增殖活性分別為(0.1120±0.0493)、(0.2194±0.0389)、(0.1888±0.0439),72h的細(xì)胞增殖活性分別為(0.0478±0.0073)、(0.1399±0.0231)、(0.1040±0.0365)。以上結(jié)果顯示ddp呈時(shí)間和劑量依賴性抑制各組細(xì)胞的增殖,并且在同一濃度的ddp作用相同時(shí)間的條件下,shsphk1+組細(xì)胞的增殖能力顯著低于control+組和nc+組(p0.05)。tunel法結(jié)果顯示未予ddp處理的shsphk1組細(xì)胞凋亡率(13.54±2.19)%明顯高于control組(5.04±1.05)%和nc組(6.15±1.56)%(p0.05)。予8μg/ml的ddp處理shsphk1+組、control+組和nc+組細(xì)胞24h后的細(xì)胞凋亡率則分別為(25.91±3.01)%、(18.68±3.16)%、(19.28±2.97)%;予16μg/ml的ddp處理以上三組細(xì)胞24h后的細(xì)胞凋亡率分別為(55.26±4.71)%、(35.82±3.97)%、(37.52±3.51)%;予32μg/ml的ddp處理三組細(xì)胞24h的細(xì)胞凋亡率分別為(76.04±4.52)%、(59.60±4.87)%、(62.28±3.11)%;以上結(jié)果說(shuō)明ddp呈濃度依賴性誘導(dǎo)rko細(xì)胞凋亡,并且shsphk1+組細(xì)胞的凋亡率明顯高于control+組和nc+組(p0.05)。westernblot法檢測(cè)沉默sphk1后,shsphk1組、control組和nc組ki-67蛋白的相對(duì)表達(dá)量分別為(0.9410±0.0437)、(1.1592±0.0753)、(1.1543±0.0981),bcl-2蛋白的相對(duì)表達(dá)量分別為(0.7317±0.0374)、(0.9180±0.0633)、(0.9205±0.0495),caspase-9蛋白的相對(duì)表達(dá)量分別為(0.4367±0.0757)、(0.2317±0.0593)、(0.2495±0.0435),caspase-3蛋白的相對(duì)表達(dá)量分別為(0.6479±0.0762)、(0.3989±0.0499)、(0.3942±0.0614)。以16μg/ml的ddp處理shsphk1+組、control+組和nc+組細(xì)胞24h后,ki-67的相對(duì)表達(dá)量分別為(0.3105±0.0367)、(0.8049±0.0684)、(0.8138±0.0596),bcl-2的相對(duì)表達(dá)量分別為(0.2329±0.0389)、(0.5203±0.0312)、(0.5270±0.0497),caspase-9蛋白的相對(duì)表達(dá)量分別為(1.8941±0.1025)、(0.8627±0.0624)、(0.8785±0.0858),caspase-3蛋白的相對(duì)表達(dá)量分別為(2.5849±0.1586)、(0.8955±0.0735)、(0.8872±0.0942)。結(jié)果顯示敲低SphK1后,Ki-67和Bcl-2的表達(dá)減弱,Caspase-9和Caspase-3的表達(dá)增強(qiáng);經(jīng)DDP處理后,shSphK1+組的Ki-67和Bcl-2蛋白表達(dá)水平較Control+組和NC+組明顯減少(P0.05);shSphK1+組的Caspase-9和Caspase-3蛋白表達(dá)水平與Control+組和NC+組相比顯著增強(qiáng)(P0.05)。結(jié)論沉默SphK1基因可以抑制結(jié)腸癌RKO細(xì)胞的增殖活力,下調(diào)SphK1基因可能通過(guò)抑制Bcl-2的水平和激活Caspase 9/3的表達(dá)促進(jìn)RKO細(xì)胞的凋亡,抑制SphK1基因可能通過(guò)激活Caspase-9/3依賴的細(xì)胞凋亡信號(hào)通路增強(qiáng)結(jié)腸癌RKO細(xì)胞對(duì)DDP的化療敏感性。
[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+緇勫拰nc+緇勭粏鑳，

本文編號(hào)：1481315