本文選題：多發(fā)性骨髓瘤 + RPMI8226　； 參考：《川北醫(yī)學(xué)院》2017年碩士論文

【摘要】：研究目的:1.人多發(fā)性骨髓瘤細(xì)胞株中DJ-1 mRNA及蛋白質(zhì)的表達(dá)情況;2.初步探明DJ-1是否對多發(fā)性骨髓瘤細(xì)胞增殖、細(xì)胞周期及凋亡有調(diào)節(jié)作用;3.初步探明DJ-1是否影響藥物(硼替佐米、地塞米松)誘導(dǎo)的骨髓瘤細(xì)胞凋亡。研究方法:1.實時熒光定量PCR(realtime quantitative PCR,RT-qPCR)檢測骨髓瘤細(xì)胞系(human myeloma cell lines,HMCLs)RPMI8226、U266及正常人外周血單個核細(xì)胞DJ-1 mRNA水平,免疫印跡雜交(Western Blot,WB)檢測DJ-1蛋白表達(dá)水平;2.分別用無關(guān)序列shRNA病毒液(Control shRNA Lentiviral Particles-A)及靶向下調(diào)DJ-1的shRNA病毒液(DJ-1 shRNA Lentiviral Particles)感染RPMI8226、U266細(xì)胞,使用含適當(dāng)濃度的嘌呤霉素持續(xù)篩選穩(wěn)定轉(zhuǎn)染細(xì)胞,運用RT-qPCR和WB檢測穩(wěn)定轉(zhuǎn)染細(xì)胞中DJ-1的表達(dá)情況以驗證轉(zhuǎn)染效果;3.慢病毒轉(zhuǎn)染RPMI8226和U266細(xì)胞下調(diào)DJ-1表達(dá)后,采用CCK8比色法、流式細(xì)胞術(shù)檢測細(xì)胞增殖、自發(fā)凋亡、細(xì)胞周期,并與對照組細(xì)胞比較;4.轉(zhuǎn)染慢病毒下調(diào)RPMI8226、U266細(xì)胞DJ-1表達(dá)后,給予硼替佐米、地塞米松處理細(xì)胞,采用CCK8比色法檢測細(xì)胞增殖、流式細(xì)胞術(shù)檢測細(xì)胞周期及細(xì)胞凋亡情況,并與對照組細(xì)胞比較。研究結(jié)果:1.rt-qpcr及wb檢測發(fā)現(xiàn),與正常人外周血單個核細(xì)胞相比,多發(fā)性骨髓瘤細(xì)胞株rpmi8226、u266中dj-1mrna和蛋白均呈高表達(dá)狀態(tài)。2.dj-1shrna慢病毒干擾載體(shdj-1)轉(zhuǎn)染多發(fā)性骨髓瘤細(xì)胞株rpmi8226、u266,并用含嘌呤霉素的完全培養(yǎng)基持續(xù)培養(yǎng),篩選出穩(wěn)定轉(zhuǎn)染細(xì)胞。經(jīng)驗證,shdj-1轉(zhuǎn)染的骨髓瘤細(xì)胞中,dj-1mrna和蛋白質(zhì)水平明顯低于空白對照組及shcontrol組,表明我們成功構(gòu)建了dj-1下調(diào)的骨髓瘤細(xì)胞模型。3.轉(zhuǎn)染shdj-1后,除種板48hrpmi8226兩組細(xì)胞od值無明顯差異(p=0.0779),其余各時間點檢測發(fā)現(xiàn),與shcontorl對照組相比,細(xì)胞增殖減慢(p0.05);細(xì)胞周期中g(shù)1期細(xì)胞比例減少,s期細(xì)胞比例上升,細(xì)胞阻滯于s期(p0.05);細(xì)胞早期凋亡率顯著升高(p0.05)。4.分別使用終濃度為0、2.0、4.0、6.0、8.0、10.0nmol/l的硼替佐米,0、0.5、1.0、5.0、10.0、20.0μmol/l的地塞米松處理各組細(xì)胞24h,cck8法檢測各組細(xì)胞od值,并計算細(xì)胞增殖抑制率,結(jié)果顯示,隨著藥物濃度升高,細(xì)胞增殖抑制率逐漸升高。比較同一藥物濃度處理下的各組細(xì)胞發(fā)現(xiàn),除0.5、1.0μmol/l地塞米松處理u266各組細(xì)胞的增殖抑制率差異無統(tǒng)計學(xué)意義外,其余各濃度藥物處理下,shdj-1組細(xì)胞增殖抑制率較shcontrol組細(xì)胞顯著升高(p0.05)。5.使用終濃度為2.0nmol/l的硼替佐米、1.0μmol/l的地塞米松處理骨髓瘤細(xì)胞株rpmi8226各組細(xì)胞48h,流式細(xì)胞術(shù)檢測細(xì)胞周期,結(jié)果顯示,與shControl組相比,shDJ-1組G1期細(xì)胞比例降低,S期細(xì)胞比例升高,細(xì)胞阻滯于S期(P0.05)。6.分別用不同濃度的硼替佐米(終濃度為0、2.5、5.0、10.0nmol/L)、地塞米松(終濃度為0、0.1、1.0、10.0μmol/L)處理各組細(xì)胞,24h后收集細(xì)胞FITC Annexin V/PI染色,采用流式細(xì)胞術(shù)檢測細(xì)胞凋亡,結(jié)果發(fā)現(xiàn),隨著藥物濃度增加,細(xì)胞早期凋亡率升高;同一濃度藥物處理下,除5.0nmol/L硼替佐米、0.1μmol/L地塞米松處理U266細(xì)胞差異無統(tǒng)計學(xué)意義外(P=0.0505;0.051),其余shDJ-1組細(xì)胞凋亡率較shControl組均明顯升高(P0.05),差異具有統(tǒng)計學(xué)意義。結(jié)論:1.在兩種常見的人多發(fā)性骨髓瘤細(xì)胞株RPMI8226、U266中,DJ-1mRNA及蛋白質(zhì)呈異常高表達(dá)。2.DJ-1參與調(diào)節(jié)RPMI8226、U266細(xì)胞增殖、細(xì)胞周期及凋亡,抑制DJ-1增強細(xì)胞對硼替佐米和地塞米松的敏感性,但具體作用機制有待進(jìn)一步研究。3.DJ-1可能是多發(fā)性骨髓瘤潛在的治療靶點,值得進(jìn)一步研究。
[Abstract]:Objective: To investigate the expression of DJ-1 mRNA and protein in 1. human myeloma cell lines; 2. preliminarily explore whether DJ-1 can regulate the proliferation, cell cycle and apoptosis of multiple myeloma cells; 3. preliminarily explore whether DJ-1 affects the apoptosis of myeloma cells induced by bortezomizomi and dexamethasone. Method: 1. real-time fluory PCR (realtime quantitative PCR, RT-qPCR) was used to detect the myeloma cell line (human myeloma cell lines, HMCLs) RPMI8226, U266 and normal human peripheral blood mononuclear cells were level, and the level of protein expression was detected by immunoblotting hybridization. 2. Icles-A) and DJ-1 shRNA Lentiviral Particles (DJ-1 shRNA Lentiviral Particles) infected RPMI8226, U266 cells, using a suitable concentration of purinamycin to continuously screen the stable transfected cells, using RT-qPCR and WB to detect the expression of DJ-1 in the stable transfected cells to verify the transfection effect. 3. After DJ-1 expression, CCK8 colorimetric method was used to detect cell proliferation, spontaneous apoptosis and cell cycle, and compared with the control group; 4. transfected with lentivirus and RPMI8226, U266 cell DJ-1 expression, the cells were treated with bortezomizomi and dexamethasone, cell proliferation was detected by CCK8 colorimetry, cell cycle was detected by flow cytometry, and cell cycle was detected by flow cytometry. The results of apoptosis were compared with those in the control group. The results of 1.rt-qpcr and WB detection showed that the multiple myeloma cell line RPMI8226, dj-1mrna and protein in U266 were higher than those of normal human peripheral blood mononuclear cells..2.dj-1shrna lentivirus vector (shdj-1) transfected with multiple myeloma cell line RPMI8226, U266, The stable transfection cells were screened using the complete medium of purinamycin, and the level of dj-1mrna and protein in shdj-1 transfected myeloma cells was obviously lower than that of the blank control group and the shcontrol group. It showed that we successfully constructed the DJ-1 down-regulated myeloma cell model.3. transfected shdj-1, except the 48hrpmi8226 two group of the seed plate. The cell OD value was not significantly different (p=0.0779). The cell proliferation slowed down (P0.05) compared with the shcontorl control group, the proportion of G1 cells in the cell cycle decreased, the proportion of cells in the S phase increased, the cells blocked in the S phase (P0.05), and the early apoptosis rate of the cells was significantly increased (P0.05).4. use final concentration was 0,2.0,4.0,6.0,8.0,10.. 0nmol/l's Boron for Zomi, 0,0.5,1.0,5.0,10.0,20.0 mol/l dexamethasone treated each group of cells 24h, CCK8 method was used to detect the OD value of each cell, and the cell proliferation inhibition rate was calculated. The results showed that the cell proliferation inhibition rate increased with the increase of drug concentration. Compared with the same drug concentration, all the cells were found in 0.5,1.0 mu mol/l. There was no significant difference in the proliferation inhibition rate of U266 cells in each group. The inhibition rate of cell proliferation in group shdj-1 was significantly higher than that in group shcontrol (P0.05).5. using bortezomizomi at the final concentration of 2.0nmol/l, and 48h in each group of myeloma cell line RPMI8226 by 1 mu mol/l. The cell cycle was detected by flow cytometry. The results showed that compared with the shControl group, the proportion of G1 phase cells in group shDJ-1 decreased, the proportion of S cells increased, and the cells blocked in S phase (P0.05).6. with different concentrations of bortel (final concentration 0,2.5,5.0,10.0nmol/L), and dexamethasone (the final concentration of 0,0.1,1.0,10.0 micron mol/L) treated each group of cells, after 24h. The cell apoptosis was detected by flow cytometry, and cell apoptosis was detected by flow cytometry. The results showed that the early apoptosis rate increased with the increase of drug concentration. Under the same concentration of drug treatment, there was no significant difference in the difference of U266 cells between 0.1 mol/L dexamethasone except 5.0nmol/L boron for Zomi and 0.1 Mu mol/L dexamethasone (P=0.0505; 0.051), and the other shDJ-1 group cells. The apoptosis rate was significantly higher than that in the shControl group (P0.05), and the difference was statistically significant. Conclusion: 1. in the two common human multiple myeloma cell lines, RPMI8226, U266, DJ-1mRNA and protein are abnormal high expression of.2.DJ-1 involved in regulating RPMI8226, U266 cell proliferation, cell cycle and apoptosis, and inhibiting the DJ-1 enhanced cells to bortezomizomi and the ground The sensitivity of amethasone, but the specific mechanism remains to be further studied..3.DJ-1 may be a potential therapeutic target for multiple myeloma and deserves further study.
【學(xué)位授予單位】：川北醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R733.3

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