發(fā)布時間：2018-02-01 07:34

  本文關鍵詞： 鞘氨醇激酶1(SphK1) 結腸癌細胞 順鉑(DDP) 化療敏感性 細胞增殖 細胞凋亡　出處：《廣西醫(yī)科大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的探討通過慢病毒介導人結腸腺癌RKO細胞中的鞘氨醇激酶-1(Sphingosine kinase-1,SphK1)基因沉默后對化療藥物順鉑(cisplatin,DDP)敏感性的影響及其可能的作用機制。方法通過重組DNA技術,設計和合成特異性針對SphK1基因的pLenti6.3-shRNA-SphK1慢病毒表達載體,同時無義序列pLenti6.3-EGFP慢病毒載體作為陰性對照,感染人結腸腺癌RKO細胞株,然后采用殺稻瘟菌素(blasticidin,BSD)來篩選,以獲得穩(wěn)定不變低表達SphK1基因的單一克隆RKO穩(wěn)轉細胞株。根據(jù)是否轉染,以及轉染的處理方式分為SphK1敲低組(shSphK1組)、陰性轉染對照組(NC組),同時將未予轉染處理的原始RKO細胞記作空載對照組(Control組)。為驗證基因的沉默效率,在熒光倒置顯微鏡下檢測轉染后各組細胞的EGFP(綠色熒光)的表達情況,采用實時熒光定量PCR(quantitative real-time PCR,RT-qPCR)法檢測Sph K1的mRNA水平,以及Western blot法檢測SphK1的蛋白表達水平。根據(jù)是否暴露于DDP,將實驗分為非DDP組和DDP組兩個大組。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分別作用于細胞24、48、72小時后,利用MTT法檢測各組細胞的增殖活性。采用原位末端轉移酶標記技術(TdT-mediated dUTP-biotin nick end labeling,TUNEL)檢測細胞凋亡。Western blot法檢驗增殖標志蛋白Ki-67以及凋亡標記蛋白Bcl-2、Caspase-9和Caspase-3蛋白的表達變化程度。結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。rt-qpcr法和westernblot法均表明shsphk1組的sphk1表達顯著低于control組及nc組(p0.05)。mtt結果顯示shsphk1組、control組和nc組細胞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細胞的增殖活力。mtt結果還顯示以2μg/ml的ddp處理shsphk1+組、control+組和nc+組細胞24h后的細胞增殖活性分別為(0.7587±0.0422)、(0.9379±0.0535)、(0.9189±0.0512),48h的細胞增殖活性分別為(0.6675±0.0429)、(0.8151±0.0704)、(0.7868±0.0893),72h的細胞增殖活性分別為(0.5224±0.0535)、(0.7510±0.0632)、(0.7456±0.0605);以4μg/ml的ddp處理細胞24h后的細胞增殖活性分別為(0.6332±0.0303)、(0.8561±0.0697)、(0.8364±0.0714),48h的細胞增殖活性分別為(0.5236±0.0219)、(0.7121±0.0253)、(0.6971±0.0536),72h的細胞增殖活性分別為(0.4035±0.0709)、(0.6495±0.0375)、(0.6002±0.0218);以8μg/ml的ddp處理細胞24h后的細胞增殖活性分別為(0.5447±0.0399)、(0.7683±0.0402)、(0.7409±0.0622),48h的細胞增殖活性分別為(0.4343±0.0529)、(0.5938±0.0701)、(0.5576±0.0365),72h的細胞增殖活性分別為(0.2781±0.0195)、(0.4725±0.0427)、(0.4543±0.0594);以16μg/ml的ddp處理細胞24h后的細胞增殖活性分別為(0.4575±0.0622)、(0.6387±0.0899)、(0.6193±0.0323),48h的細胞增殖活性分別為(0.2519±0.0353)、(0.4163±0.0532)、(0.3952±0.0413),72h的細胞增殖活性分別為(0.1472±0.0477)、(0.3061±0.0319)、(0.2707±0.0242);以32μg/ml的ddp處理細胞24h后的細胞增殖活性分別為(0.2867±0.0746)、(0.4519±0.0698)、(0.4298±0.0706),48h的細胞增殖活性分別為(0.1120±0.0493)、(0.2194±0.0389)、(0.1888±0.0439),72h的細胞增殖活性分別為(0.0478±0.0073)、(0.1399±0.0231)、(0.1040±0.0365)。以上結果顯示ddp呈時間和劑量依賴性抑制各組細胞的增殖,并且在同一濃度的ddp作用相同時間的條件下,shsphk1+組細胞的增殖能力顯著低于control+組和nc+組(p0.05)。tunel法結果顯示未予ddp處理的shsphk1組細胞凋亡率(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+組細胞24h后的細胞凋亡率則分別為(25.91±3.01)%、(18.68±3.16)%、(19.28±2.97)%;予16μg/ml的ddp處理以上三組細胞24h后的細胞凋亡率分別為(55.26±4.71)%、(35.82±3.97)%、(37.52±3.51)%;予32μg/ml的ddp處理三組細胞24h的細胞凋亡率分別為(76.04±4.52)%、(59.60±4.87)%、(62.28±3.11)%;以上結果說明ddp呈濃度依賴性誘導rko細胞凋亡,并且shsphk1+組細胞的凋亡率明顯高于control+組和nc+組(p0.05)。westernblot法檢測沉默sphk1后,shsphk1組、control組和nc組ki-67蛋白的相對表達量分別為(0.9410±0.0437)、(1.1592±0.0753)、(1.1543±0.0981),bcl-2蛋白的相對表達量分別為(0.7317±0.0374)、(0.9180±0.0633)、(0.9205±0.0495),caspase-9蛋白的相對表達量分別為(0.4367±0.0757)、(0.2317±0.0593)、(0.2495±0.0435),caspase-3蛋白的相對表達量分別為(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+組細胞24h后,ki-67的相對表達量分別為(0.3105±0.0367)、(0.8049±0.0684)、(0.8138±0.0596),bcl-2的相對表達量分別為(0.2329±0.0389)、(0.5203±0.0312)、(0.5270±0.0497),caspase-9蛋白的相對表達量分別為(1.8941±0.1025)、(0.8627±0.0624)、(0.8785±0.0858),caspase-3蛋白的相對表達量分別為(2.5849±0.1586)、(0.8955±0.0735)、(0.8872±0.0942)。結果顯示敲低SphK1后,Ki-67和Bcl-2的表達減弱,Caspase-9和Caspase-3的表達增強;經(jīng)DDP處理后,shSphK1+組的Ki-67和Bcl-2蛋白表達水平較Control+組和NC+組明顯減少(P0.05);shSphK1+組的Caspase-9和Caspase-3蛋白表達水平與Control+組和NC+組相比顯著增強(P0.05)。結論沉默SphK1基因可以抑制結腸癌RKO細胞的增殖活力,下調(diào)SphK1基因可能通過抑制Bcl-2的水平和激活Caspase 9/3的表達促進RKO細胞的凋亡,抑制SphK1基因可能通過激活Caspase-9/3依賴的細胞凋亡信號通路增強結腸癌RKO細胞對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：1481315