發(fā)布時(shí)間：2018-07-31 12:58

【摘要】：目的:通過觀察不同濃度雷帕霉素(Rapamycin,RAPA)和不同時(shí)間作用后Raji細(xì)胞:血管內(nèi)皮生長因子(Vascular endothelial growth factor,VEGF)、缺氧誘導(dǎo)因子-1α(Hypoxia-inducible factor-1α,HIF-1α)蛋白及其信使核糖核酸(Messenger RNA,m RNA);磷酸化的RAPA靶蛋白(Mammalian target of rapamycin,m TOR)及m RNA的表達(dá)水平。探討不同時(shí)間和不同濃度的RAPA對(duì)Raji細(xì)胞是否存在抑制細(xì)胞增殖及誘導(dǎo)凋亡,對(duì)m TOR、VEGF、HIF-1α基因及其蛋白表達(dá)的影響,并分析實(shí)驗(yàn)結(jié)果和相關(guān)作用機(jī)制,為雷帕霉素應(yīng)用于臨床治療Burkitt淋巴瘤(Burkitt lymphoma,BL)提供理論依據(jù)。方法:1細(xì)胞實(shí)驗(yàn)分組:選取處于對(duì)數(shù)生長期的人淋巴瘤Raji細(xì)胞。實(shí)驗(yàn)分組為:RAPA處理組、空白對(duì)照組�？瞻讓�(duì)照組使用僅含10%新生小牛血清(Newborn Bovine(super),NBS)的RP-MI1640培養(yǎng)。RAPA處理組用含不同濃度RAPA的10%新生牛血清RP-MI1640培養(yǎng)基培養(yǎng)。RAPA的終濃度分別為10n M、50n M、100n M、250n M、500n M。RAPA作用時(shí)間分別為:24h、48h、72h。2采用Cell Counting Kit-8(CCK-8)方法,觀察不同濃度RAPA不同時(shí)間作用后的Raji細(xì)胞增殖抑制情況。3使用流式細(xì)胞儀(Flow Cytometer,FCM)觀察觀察不同濃度RAPA和不同時(shí)間作用后的Raji細(xì)胞凋亡及細(xì)胞分期的影響。4應(yīng)用蛋白印跡(Western blot,WB)技術(shù)檢測RAPA作用后的Raji細(xì)胞,不同濃度和不同時(shí)間組細(xì)胞p-m TOR、VEGF及HIF-1α蛋白的表達(dá)情況。5應(yīng)用反轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)(Reverse transcription PCR,RT-PCR)方法檢測RAPA作用Raji細(xì)胞后,不同濃度和不同時(shí)間組細(xì)胞m TOR、VEGF及HIF-1α的m RNA表達(dá)情況。結(jié)果:1 CCK-8結(jié)果顯示:不同濃度(10n M、50n M、100n M、250n M、500n M)RAPA處理組Raji細(xì)胞的抑制率,24h時(shí)分別為(0.237±0.042)、(0.344±0.035)、(0.437±0.043)、(0.488±0.028)、(0.517±0.037);48h時(shí)分別為(0.216±0.051)、(0.331±0.046)、(0.439±0.047)、(0.545±0.039)、(0.670±0.032);72h時(shí)分別為(0.287±0.048)、(0.429±0.034)、(0.589±0.049)、(0.652±0.034)、(0.751±0.041)。與空白對(duì)照組相比,不同濃度RAPA作用后的Raji細(xì)胞在24h、48h、72h,結(jié)果顯示Raji細(xì)胞增殖均明顯受到抑制,不同濃度和不同時(shí)間組間有統(tǒng)計(jì)學(xué)意義(P0.01)。并且隨著RAPA作用時(shí)間的延長以及藥物劑量的增加,Raji細(xì)胞的抑制率也隨之升高,呈現(xiàn)出濃度和時(shí)間依賴性(P0.01)。Raji細(xì)胞在RAPA處理24h、48h、72h的IC50值分別為(317.064±2.739)n M、(156.276±1.523)n M、(64.762±1.102)n M。2 FCM檢測結(jié)果顯示:不同濃度(10n M、50n M、100n M、250n M、500n M)RAPA作用于Raji細(xì)胞:(1)24h結(jié)果顯示:不同濃度RAPA處理組細(xì)胞凋亡率分別為(12.54±2.25)%、(16.04±1.39)%、(16.22±1.29)%、(19.31±2.04)%、(20.49±1.54)%,空白對(duì)照組細(xì)胞的凋亡率為(4.88±1.93)%。不同濃度RAPA處理組與空白對(duì)照組相比,RAPA處理組Raji細(xì)胞的凋亡率明顯增加,均有統(tǒng)計(jì)學(xué)意義(P0.01),并且隨著RAPA濃度的增加,Raji細(xì)胞凋亡率隨之上升(P0.05)。Raji細(xì)胞周期G1期隨著RAPA濃度升高從40.71%增加到63.04%,不同濃度RAPA處理組與空白對(duì)照組比較具有統(tǒng)計(jì)學(xué)意義(P0.01),不同濃度RAPA處理組組間無統(tǒng)計(jì)學(xué)意義(P0.05)。(2)48h結(jié)果顯示:不同濃度RAPA處理組細(xì)胞凋亡率分別為(15.47±1.83)%、(18.82±1.52)%、(19.82±2.07)%、(20.56±1.81)%、(22.56±1.74)%,空白對(duì)照組細(xì)胞的凋亡率為(6.34±2.01)%。不同濃度RAPA處理組與空白對(duì)照組相比,RAPA處理組Raji細(xì)胞的凋亡率明顯增加,均具有統(tǒng)計(jì)學(xué)意義(P0.01),并且隨著RAPA濃度的增加,Raji細(xì)胞凋亡率隨之增加,RAPA處理組組間差異無統(tǒng)計(jì)學(xué)意義(P0.05)。Raji細(xì)胞周期G1期隨著RAPA濃度升高從37.54%增加到65.20%,不同濃度和不同時(shí)間的RAPA處理組與空白對(duì)照組比較有統(tǒng)計(jì)學(xué)意義(P0.01),不同濃度和不同時(shí)間的RAPA處理組組間無統(tǒng)計(jì)學(xué)意義(P0.05)。3 WB檢測結(jié)果顯示:RAPA作用Raji細(xì)胞后,空白對(duì)照組和不同濃度(10n M、50n M、100n M、250n M、500n M)RAPA處理組Raji細(xì)胞灰度比值為:(1)48h結(jié)果顯示:p-m TOR蛋白條帶相對(duì)灰度分別為(2.804±0.156)、(2.691±0.109)、(2.577±0.121)、(2.332±0.137)、(2.203±0.107)、(2.032±0.114);VEGF蛋白條帶相對(duì)灰度分別為(2.798±0.149)、(2.621±0.103)、(2.509±0.102)、(2.391±0.105)、(2.269±0.097)、(2.025±0.117);HIF-1α蛋白條帶相對(duì)灰度分別為(2.864±0.148)、(2.701±0.129)、(2.607±0.101)、(2.442±0.127)、(2.303±0.108)、(2.162±0.124)。各組中內(nèi)參β-actin蛋白條帶無明顯差異(P=0.87)。不同濃度RAPA處理組的p-m TOR、VEGF和HIF-1α蛋白表達(dá)水平與空白對(duì)照組相比較均受到抑制(P0.05)。(2)72h結(jié)果顯示:p-m TOR蛋白條帶相對(duì)灰度分別為(2.924±0.149)、(2.781±0.099)、(2.647±0.111)、(2.493±0.112)、(2.023±0.114)、(1.833±0.104);VEGF蛋白條帶相對(duì)灰度分別為(2.932±0.156)、(2.721±0.169)、(2.507±0.141)、(2.303±0.132)、(1.903±0.134)、(1.733±0.124);HIF-1α蛋白條帶相對(duì)灰度分別為(2.912±0.148)、(2.711±0.158)、(2.518±0.139)、(2.343±0.140)、(1.943±0.144)、(1.763±0.144)。各組中內(nèi)參β-actin蛋白條帶無明顯差異(P=0.91)。不同濃度RAPA處理組的p-m TOR、VEGF和HIF-1α蛋白表達(dá)水平與空白對(duì)照組相比較均受到抑制(P0.05)。隨著RAPA濃度的增加,p-m TOR、VEGF和HIF-1α蛋白表達(dá)量呈減少趨勢,具有統(tǒng)計(jì)學(xué)意義(P0.05)。RAPA能抑制Raji細(xì)胞中p-m TOR、VEGF和HIF-1α蛋白表達(dá)。4 RT-PCR檢測結(jié)果顯示:不同濃度的(10n M、50n M、100n M、250n M、500n M)RAPA處理組與空白對(duì)照組Raji細(xì)胞m RNA相對(duì)表達(dá)水平:(1)24h組分別為:m TOR的m RNA相對(duì)表達(dá)水平分別為(0.94830±0.00986)、(0.81728±0.00714)、(0.73398±0.00962)、(0.62370±0.00875)、(0.52999±0.00227);VEGF的m RNA相對(duì)表達(dá)水平分別為(0.95162±0.01304)、(0.83276±0.00759)、(0.72035±0.00566)、(0.63304±0.04036)、(0.52864±0.00425);HIF-1α的m RNA相對(duì)表達(dá)水平分別為(0.85592±0.00949)、(0.71467±0.00931)、(0.61719±0.00651)、(0.52009±0.00998)、(0.34456±0.01020)。(2)48h組分別為:m TOR的m RNA相對(duì)表達(dá)水平分別為(0.90621±0.00666)、(0.75909±0.00624)、(0.68925±0.00792)、(0.55675±0.01345)、(0.41874±0.01487);VEGF的m RNA相對(duì)表達(dá)水平分別為(0.92884±0.00435)、(0.78852±0.00619)、(0.67400±0.01092)、(0.53642±0.00842)、(0.41374±0.01164);HIF-1α的m RNA相對(duì)表達(dá)水平分別為(0.79486±0.01025)、(0.69360±0.00717)、(0.59055±0.00917)、(0.48146±0.00545)、(0.26718±0.00938)。(3)72h組分別為:m TOR的m RNA相對(duì)表達(dá)水平分別為(0.84789±0.00563)、(0.69022±0.00870)、(0.60367±0.00396)、(0.49472±0.00814)、(0.36469±0.01018);VEGF的m RNA相對(duì)表達(dá)水平分別為(0.89231±0.00348)、(0.68937±0.00529)、(0.58713±0.00228)、(0.48321±0.00563)、(0.38016±0.00466);HIF-1α的m RNA相對(duì)表達(dá)水平分別為(0.70228±0.00695)、(0.59731±0.01057)、(0.48719±0.00457)、(0.38253±0.01126)、(0.16590±0.00932)。實(shí)驗(yàn)組中內(nèi)參β-actin的m RNA表達(dá)水平無明顯差異(P=0.93)。不同濃度RAPA與空白對(duì)照組相比較,RAPA處理后的m TOR、VEGF及HIF-1α的m RNA表達(dá)均明顯降低(P0.01)。隨著RAPA的濃度和作用時(shí)間增加,m TOR、VEGF和HIF-1α蛋白的m RNA表達(dá)水平明顯下降,具有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:1 RAPA具有明顯抑制Raji細(xì)胞增殖,誘導(dǎo)其凋亡的作用。隨著RAPA濃度及其作用時(shí)間的增加,Raji細(xì)胞抑制率及凋亡率逐漸升高,呈現(xiàn)出明顯的時(shí)間、劑量效應(yīng)依賴關(guān)系。2 RAPA具有抑制Raji細(xì)胞周期,使Raji細(xì)胞主要停滯G1期,但是隨著RAPA濃度及其作用時(shí)間的增加Raji細(xì)胞停滯G1期細(xì)胞率變化不明顯。3 RAPA處理組Raji細(xì)胞中p-m TOR、VEGF及HIF-1α蛋白表達(dá)水平明顯低于空白對(duì)照組,RAPA能明顯降低Raji細(xì)胞中p-m TOR、VEGF及HIF-1α蛋白表達(dá)水平。4 RAPA處理組Raji細(xì)胞中m TOR、VEGF及HIF-1α與空白對(duì)照組的m RNA相比,RAPA能明顯降低Raji細(xì)胞中m TOR、VEGF及HIF-1α的m RNA表達(dá)水平,同時(shí)表達(dá)減少的HIF-1α和VEGF呈正相關(guān)性,提示降低的HIF-1α可減少VEGF的轉(zhuǎn)錄與翻譯。5 RAPA能通過抑制Raji細(xì)胞VEGF和HIF-1α的m RNA表達(dá),從而抑制VEGF及HIF-1α蛋白的表達(dá),抑制Raji細(xì)胞的血管新生。
[Abstract]:Objective: To observe Raji cells with different concentrations of Rapamycin (RAPA) and different time: Vascular endothelial growth factor (VEGF), the hypoxia inducible -1 alpha (Hypoxia-inducible factor-1 alpha, HIF-1 alpha) protein and its messenger ribonucleic acid (Vascular); The expression level of ammalian target of rapamycin, m TOR) and m RNA. The effect of RAPA on Raji cells at different time and at different concentrations to inhibit cell proliferation and induce apoptosis, and to investigate the effect of M TOR, gene and protein expression, and to analyze the experimental results and mechanism of action for the clinical treatment of rapamycin Burkitt lymphoma (BL) provides a theoretical basis. Methods: 1 cells experiment group: select the Raji cells in the logarithmic growth period of human lymphoma. The experiment group was divided into RAPA treatment group, blank control group. The control group used only 10% newborn calf serum (Newborn Bovine (super), NBS) RP-MI1640 culture.RAPA treatment group with different concentration. The final concentrations of.RAPA were 10N M, 50N M, 100N M and 250N M, respectively, for the 10% newborn bovine serum RP-MI1640 medium of RAPA. Er, FCM) observation and observation of the effect of different concentrations of RAPA and the effect of different time on the apoptosis and cell stage of Raji cells.4 application protein blot (Western blot, WB) technique to detect the Raji cells after RAPA, and the expression of P-M TOR in different concentrations and different time groups. Erse transcription PCR, RT-PCR) method to detect RAPA action Raji cells, m TOR in different concentrations and different time groups, VEGF and HIF-1 alpha m RNA expression. Results: 1 results showed that the inhibition rate was (0.237 + 0.042), (0.344 + 0.035), respectively, (0), (0 .437 + 0.043), (0.488 + 0.028), (0.517 + 0.037), 48h (0.216 + 0.051), (0.331 + 0.046), (0.439 + 0.047), (0.545 + 0.039), 72h respectively, (0.216), (0.216), compared to the blank control group, Raji cells after the action of different concentrations of RAPA, 48h, 48H H, the results showed that the proliferation of Raji cells was obviously inhibited, and there were statistical significance between different concentrations and different time groups (P0.01). And with the prolongation of the time of RAPA action and the increase of drug dosage, the inhibition rate of Raji cells also increased. The concentration and time showed the IC50 values of 24h, 48h, 72h, depending on the RAPA (P0.01).Raji cells in RAPA. (317.064 + 2.739) n M, (156.276 + 1.523) n M, and (64.762 + 1.102) n M.2 FCM detection results showed that different concentrations (1) showed that the cell withering rate of different concentrations was (12.54 + 2.25)%, (16.04 + 1.39)%, (16.22 + 1.29)%, (16.22 + 64.762%)%, .54%, the apoptosis rate of cells in the blank control group was (4.88 + 1.93)%. Compared with the blank control group, the apoptosis rate of Raji cells in the RAPA treatment group was significantly increased, with a statistical significance (P0.01), and with the increase of RAPA concentration, the apoptosis rate of Raji cells increased (P0.05) the G1 period of.Raji cell cycle increased with RAPA concentration. From 40.71% to 63.04%, the RAPA treatment group with different concentrations had statistical significance compared with the blank control group (P0.01), and there was no statistical significance between different concentrations of RAPA treatment group (P0.05). (2) 48h results showed that the apoptosis rate of different concentrations of RAPA treatment group was (15.47 + 1.83)%, (18.82 + 1.52)%, (19.82 + 2.07)%, (20.56 + 1.81)%, (22.56 + 1.7). 4)%, the apoptosis rate of the cells in the blank control group was (6.34 + 2.01)%. The apoptosis rate of Raji cells in the RAPA treatment group was significantly increased compared with the blank control group (P0.01), and the apoptosis rate of Raji cells increased with the increase of RAPA concentration, and there was no significant difference between the RAPA treatment group and the RAPA treatment group (P0.05). The G1 phase of Raji cell cycle increased from 37.54% to 65.20% with the increase of RAPA concentration. The RAPA treatment group with different concentrations and different times had statistical significance (P0.01). There was no statistical significance between the RAPA treatment groups at different concentrations and different times (P0.05).3 WB test results showed: RAPA action Raji cells, blank control group The grayscale ratio of the Raji cells in the RAPA treatment group (10N M, 50N M, 100N M, 250N M, 500N M) was as follows: (1) the results showed that the relative gray level of the protein strips was (2.804 + 0.156), (2.691 + 0.109), (2.577 + 0.121), (2.332 + 0.137), (2.203 + 0.107), (2.032 + 0.114); (103), (2.509 + 0.102), (2.391 + 0.105), (2.269 + 0.097), (2.025 + 0.117), the relative gray level of HIF-1 alpha protein band is respectively (2.864 + 0.148), (2.701 + 0.129), (2.701 + 2.269), (P=0.87). There is no obvious difference in the band of beta -actin protein bands in each group (P=0.87). The p-m TOR, VEGF and HI of different concentration RAPA treatment groups The expression level of F-1 alpha protein was inhibited compared with that in the blank control group (P0.05). (2) 72h results showed that the relative gray level of P-M TOR protein bands was (2.924 + 0.149), (2.781 + 0.099), (2.647 + 0.111), (2.493 + 0.112), (2.023 + 0.114), (1.833 + 0.104), and VEGF protein bands respectively (141), (2.303 + 0.132), (1.903 + 0.134), (1.733 + 0.124), the relative gray level of HIF-1 alpha protein bands was (2.912 + 0.148), (2.711 + 0.158), (2.518 + 0.139), (2.518 + 0.134), and there was no significant difference in the band of beta -actin protein bands (P=0.91) in each group. The p-m TOR, VEGF and HIF-1 alpha protein table of different concentrations of RAPA treatment group With the increase of RAPA concentration, the expression of P-M TOR, VEGF and HIF-1 alpha showed a decreasing trend, which was statistically significant (P0.05).RAPA could inhibit P-M TOR in Raji cells. 0n M) RAPA treatment group and blank control group Raji cell m RNA relative expression level: (1) 24h group: m TOR m RNA relative expression level is respectively (0.94830 + 0.00986), (0.81728 + 0.00714), (0.73398 + 0.00962), (0.62370 + 0.00875), (0.52999 + 0.00227), respectively (0.95162 + 0.01304), respectively (0.95162 + 0.01304), respectively. ) (0.72035 + 0.00566), (0.63304 + 0.04036), (0.52864 + 0.00425), and the relative expression level of M RNA of HIF-1 alpha is (0.85592 + 0.00949), (0.71467 + 0.00931), (0.61719 + 0.00651), (0.52009 + 0.52864). The relative expression level of M RNA in M TOR is respectively 25 + 0.00792), (0.55675 + 0.01345), (0.41874 + 0.01487), the relative expression level of M RNA in VEGF is respectively (0.92884 + 0.00435), (0.78852 + 0.00619), (0.67400 + 0.01092), and (0.53642 + +), and the relative table of M RNA of HIF-1 alpha is respectively. (45) (0.26718 + 0.00938). (3) the relative expression level of M RNA in M TOR is respectively (0.84789 + 0.00563), (0.69022 + 0.00870), (0.60367 + 0.00396), (0.49472 + 0.00814), (0.36469 +), and the relative expression level of VEGF's M RNA The relative expression level of M RNA in HIF-1 alpha was (0.70228 + 0.00695), (0.59731 + 0.01057), (0.48719 + 0.00457), (0.38253 + 0.01126), (0.38253 + 0.01126), (0.16590 + 0.00932). There was no significant difference in the m RNA expression level of the internal reference beta -actin in the experimental group (P=0.93). The different concentration RAPA was compared with the blank control group, m TOR, VEGF, and alpha after RAPA treatment. The expression of M RNA decreased significantly (P0.01). As the concentration and time of RAPA increased, the expression level of M RNA of M TOR, VEGF and HIF-1 a protein decreased significantly, with statistical significance (P0.05). Conclusion: 1 RAPA has a significant inhibition of cell proliferation and induction of apoptosis. The rate and apoptosis rate gradually increased, showing a clear time. The dose effect dependence.2 RAPA had the inhibition of the Raji cell cycle and the Raji cells mainly stagnated the G1 stage. But with the RAPA concentration and the time of action, the cell rate of the Raji cell stagnation at G1 stage was not obvious in.3 RAPA processing group Raji cells. The level of RAPA was significantly lower than that in the blank control group, and the expression level of P-M TOR in Raji cells, VEGF and HIF-1 alpha protein expression level in.4 RAPA treatment group was significantly lower than that of the blank control group. Positive correlation suggests that reduced HIF-1 alpha can reduce the transcription of VEGF and translation of.5 RAPA by inhibiting the m RNA expression of VEGF and HIF-1 alpha in Raji cells, thus inhibiting the expression of VEGF and HIF-1 alpha protein and inhibiting angiogenesis in the Raji cells.
【學(xué)位授予單位】：承德醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R733.1

【參考文獻(xiàn)】

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