雷帕霉素抑制人淋巴瘤Raji细胞增殖、血管新生相关性研究
发布时间：2018-07-31 12:58
【摘要】：目的:通过观察不同浓度雷帕霉素(Rapamycin,RAPA)和不同时间作用后Raji细胞:血管内皮生长因子(Vascular endothelial growth factor,VEGF)、缺氧诱导因子-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的表达水平。探讨不同时间和不同浓度的RAPA对Raji细胞是否存在抑制细胞增殖及诱导凋亡,对m TOR、VEGF、HIF-1α基因及其蛋白表达的影响,并分析实验结果和相关作用机制,为雷帕霉素应用于临床治疗Burkitt淋巴瘤(Burkitt lymphoma,BL)提供理论依据。方法:1细胞实验分组:选取处于对数生长期的人淋巴瘤Raji细胞。实验分组为:RAPA处理组、空白对照组。空白对照组使用仅含10%新生小牛血清(Newborn Bovine(super),NBS)的RP-MI1640培养。RAPA处理组用含不同浓度RAPA的10%新生牛血清RP-MI1640培养基培养。RAPA的终浓度分别为10n M、50n M、100n M、250n M、500n M。RAPA作用时间分别为:24h、48h、72h。2采用Cell Counting Kit-8(CCK-8)方法,观察不同浓度RAPA不同时间作用后的Raji细胞增殖抑制情况。3使用流式细胞仪(Flow Cytometer,FCM)观察观察不同浓度RAPA和不同时间作用后的Raji细胞凋亡及细胞分期的影响。4应用蛋白印迹(Western blot,WB)技术检测RAPA作用后的Raji细胞,不同浓度和不同时间组细胞p-m TOR、VEGF及HIF-1α蛋白的表达情况。5应用反转录聚合酶链式反应(Reverse transcription PCR,RT-PCR)方法检测RAPA作用Raji细胞后,不同浓度和不同时间组细胞m TOR、VEGF及HIF-1α的m RNA表达情况。结果:1 CCK-8结果显示:不同浓度(10n M、50n M、100n M、250n M、500n M)RAPA处理组Raji细胞的抑制率,24h时分别为(0.237±0.042)、(0.344±0.035)、(0.437±0.043)、(0.488±0.028)、(0.517±0.037);48h时分别为(0.216±0.051)、(0.331±0.046)、(0.439±0.047)、(0.545±0.039)、(0.670±0.032);72h时分别为(0.287±0.048)、(0.429±0.034)、(0.589±0.049)、(0.652±0.034)、(0.751±0.041)。与空白对照组相比,不同浓度RAPA作用后的Raji细胞在24h、48h、72h,结果显示Raji细胞增殖均明显受到抑制,不同浓度和不同时间组间有统计学意义(P0.01)。并且随着RAPA作用时间的延长以及药物剂量的增加,Raji细胞的抑制率也随之升高,呈现出浓度和时间依赖性(P0.01)。Raji细胞在RAPA处理24h、48h、72h的IC50值分别为(317.064±2.739)n M、(156.276±1.523)n M、(64.762±1.102)n M。2 FCM检测结果显示:不同浓度(10n M、50n M、100n M、250n M、500n M)RAPA作用于Raji细胞:(1)24h结果显示:不同浓度RAPA处理组细胞凋亡率分别为(12.54±2.25)%、(16.04±1.39)%、(16.22±1.29)%、(19.31±2.04)%、(20.49±1.54)%,空白对照组细胞的凋亡率为(4.88±1.93)%。不同浓度RAPA处理组与空白对照组相比,RAPA处理组Raji细胞的凋亡率明显增加,均有统计学意义(P0.01),并且随着RAPA浓度的增加,Raji细胞凋亡率随之上升(P0.05)。Raji细胞周期G1期随着RAPA浓度升高从40.71%增加到63.04%,不同浓度RAPA处理组与空白对照组比较具有统计学意义(P0.01),不同浓度RAPA处理组组间无统计学意义(P0.05)。(2)48h结果显示:不同浓度RAPA处理组细胞凋亡率分别为(15.47±1.83)%、(18.82±1.52)%、(19.82±2.07)%、(20.56±1.81)%、(22.56±1.74)%,空白对照组细胞的凋亡率为(6.34±2.01)%。不同浓度RAPA处理组与空白对照组相比,RAPA处理组Raji细胞的凋亡率明显增加,均具有统计学意义(P0.01),并且随着RAPA浓度的增加,Raji细胞凋亡率随之增加,RAPA处理组组间差异无统计学意义(P0.05)。Raji细胞周期G1期随着RAPA浓度升高从37.54%增加到65.20%,不同浓度和不同时间的RAPA处理组与空白对照组比较有统计学意义(P0.01),不同浓度和不同时间的RAPA处理组组间无统计学意义(P0.05)。3 WB检测结果显示:RAPA作用Raji细胞后,空白对照组和不同浓度(10n M、50n M、100n M、250n M、500n M)RAPA处理组Raji细胞灰度比值为:(1)48h结果显示:p-m TOR蛋白条带相对灰度分别为(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蛋白条带相对灰度分别为(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蛋白条带相对灰度分别为(2.864±0.148)、(2.701±0.129)、(2.607±0.101)、(2.442±0.127)、(2.303±0.108)、(2.162±0.124)。各组中内参β-actin蛋白条带无明显差异(P=0.87)。不同浓度RAPA处理组的p-m TOR、VEGF和HIF-1α蛋白表达水平与空白对照组相比较均受到抑制(P0.05)。(2)72h结果显示:p-m TOR蛋白条带相对灰度分别为(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蛋白条带相对灰度分别为(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蛋白条带相对灰度分别为(2.912±0.148)、(2.711±0.158)、(2.518±0.139)、(2.343±0.140)、(1.943±0.144)、(1.763±0.144)。各组中内参β-actin蛋白条带无明显差异(P=0.91)。不同浓度RAPA处理组的p-m TOR、VEGF和HIF-1α蛋白表达水平与空白对照组相比较均受到抑制(P0.05)。随着RAPA浓度的增加,p-m TOR、VEGF和HIF-1α蛋白表达量呈减少趋势,具有统计学意义(P0.05)。RAPA能抑制Raji细胞中p-m TOR、VEGF和HIF-1α蛋白表达。4 RT-PCR检测结果显示:不同浓度的(10n M、50n M、100n M、250n M、500n M)RAPA处理组与空白对照组Raji细胞m RNA相对表达水平:(1)24h组分别为:m TOR的m RNA相对表达水平分别为(0.94830±0.00986)、(0.81728±0.00714)、(0.73398±0.00962)、(0.62370±0.00875)、(0.52999±0.00227);VEGF的m RNA相对表达水平分别为(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相对表达水平分别为(0.85592±0.00949)、(0.71467±0.00931)、(0.61719±0.00651)、(0.52009±0.00998)、(0.34456±0.01020)。(2)48h组分别为:m TOR的m RNA相对表达水平分别为(0.90621±0.00666)、(0.75909±0.00624)、(0.68925±0.00792)、(0.55675±0.01345)、(0.41874±0.01487);VEGF的m RNA相对表达水平分别为(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相对表达水平分别为(0.79486±0.01025)、(0.69360±0.00717)、(0.59055±0.00917)、(0.48146±0.00545)、(0.26718±0.00938)。(3)72h组分别为:m TOR的m RNA相对表达水平分别为(0.84789±0.00563)、(0.69022±0.00870)、(0.60367±0.00396)、(0.49472±0.00814)、(0.36469±0.01018);VEGF的m RNA相对表达水平分别为(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相对表达水平分别为(0.70228±0.00695)、(0.59731±0.01057)、(0.48719±0.00457)、(0.38253±0.01126)、(0.16590±0.00932)。实验组中内参β-actin的m RNA表达水平无明显差异(P=0.93)。不同浓度RAPA与空白对照组相比较,RAPA处理后的m TOR、VEGF及HIF-1α的m RNA表达均明显降低(P0.01)。随着RAPA的浓度和作用时间增加,m TOR、VEGF和HIF-1α蛋白的m RNA表达水平明显下降,具有统计学意义(P0.05)。结论:1 RAPA具有明显抑制Raji细胞增殖,诱导其凋亡的作用。随着RAPA浓度及其作用时间的增加,Raji细胞抑制率及凋亡率逐渐升高,呈现出明显的时间、剂量效应依赖关系。2 RAPA具有抑制Raji细胞周期,使Raji细胞主要停滞G1期,但是随着RAPA浓度及其作用时间的增加Raji细胞停滞G1期细胞率变化不明显。3 RAPA处理组Raji细胞中p-m TOR、VEGF及HIF-1α蛋白表达水平明显低于空白对照组,RAPA能明显降低Raji细胞中p-m TOR、VEGF及HIF-1α蛋白表达水平。4 RAPA处理组Raji细胞中m TOR、VEGF及HIF-1α与空白对照组的m RNA相比,RAPA能明显降低Raji细胞中m TOR、VEGF及HIF-1α的m RNA表达水平,同时表达减少的HIF-1α和VEGF呈正相关性,提示降低的HIF-1α可减少VEGF的转录与翻译。5 RAPA能通过抑制Raji细胞VEGF和HIF-1α的m RNA表达,从而抑制VEGF及HIF-1α蛋白的表达,抑制Raji细胞的血管新生。
[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.
【学位授予单位】：承德医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.1
本文编号：2155651
[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.
【学位授予单位】：承德医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.1
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