miRNA-29b抑制U251人脑胶质瘤细胞生长及其机制的初步研究

发布时间：2018-10-22 17:13

【摘要】：目的：体外应用has-miRNA-29b mimics瞬时转染U251人脑胶质瘤细胞，观察其对细胞生长的影响，探讨在U251人脑胶质瘤细胞中上调miRNA-29b的表达能否抑制细胞的生长，检测细胞内增殖及凋亡相关蛋白的表达水平，初步探索miRNA-29b抑制U251人脑胶质瘤细胞生长的机制，，为临床上治疗胶质瘤提供理论依据。方法： 1.用Lipofectamine2000介导终浓度为100nmol/l的cel-miRNA-67mimics和has-miRNA-29b mimics体外瞬时转染的U251人脑胶质瘤细胞分别作为对照组和实验组。根据实验需要在转染miRNAmimics后不同时间点（24h，48h或72h）收取细胞，用于细胞形态学观察、MTT、流式细胞学术或Westernbloting实验。 2（.1）细胞形态学观察：在倒置相差显微镜下直接观察培养板中24h、48h两组细胞的生长情况及细胞形态，比较两组细胞的差异，并拍照记录。重复3次。（2）MTT实验：四甲基偶氮唑蓝(MTT)比色法分别检测24h、48h、72h实验组和对照组细胞在波长490nm和630nm处的吸光值OD490nm和OD630nm，用A值（A=OD490nm－OD630nm）评价细胞增殖情况。（3）流式细胞术检测细胞周期时相分布及凋亡率：PI单染法检测两组细胞48h的细胞周期，记录处于不同细胞周期时相的细胞比例；Annexin V/PI双染法测转染miRNA mimics48h后两组细胞凋亡情况，计算凋亡指数AI，AI（%）=凋亡细胞数/总细胞数×100%。实验重复5次。（4）蛋白印迹（Western blotting）实验：Western blotting检测24h、48h两组细胞周期相关蛋白CDK6、cyclinD1和凋亡相关蛋白Bcl-2、Mcl-1、Bax的表达水平，IPP6.0软件对Western条带进行定量分析。实验重复5次。 3.统计学分析：所得数据资料均以均数±标准差(x±S)来表示。采用SPSS17.0统计学软件对数据进行统计处理，方差齐的两组间采用独立样本t检验，方差不齐则采用Wilcoxon秩和检验。以P0.05为差异有统计学意义。结果： 1.细胞形态学观察：对照组U251人脑胶质瘤细胞生长状态良好，细胞贴壁生长，呈多形性，轮廓清晰，细胞间界限清楚，贴壁良好，胞体透亮，颗粒较少，细胞核清晰可见；实验组细胞24h、48h实验组U251细胞生长相对缓慢，贴壁现象减弱，肿瘤细胞失去原有多形性状态，逐渐变圆趋势，胞体轻度皱缩，胞浆内颗粒增多增粗，小部分细胞脱落死亡 2. MTT实验结果：对照组和实验组细胞在24h、48h、72h的A值分别为（0.14±0.01），（0.25±0.03），（0.36±0.02）和（0.13±0.01），（0.21±0.02），（0.30±0.02），两组间各时间点差异均具有统计学意义（P0.05），且差异在转染后72h最为明显。 3.流式细胞检测结果：对照组U251细胞48h处于G1、S、G2期细胞数比值分别为（44.78±1.52）%、（50.86±1.52）%和（4.56±0.47）%；实验组分别为（57.27±2.27）%，（39.25±2.12）%，（3.46±0.44）%，两组间差异有统计学意义（p0.05），实验组细胞周期阻滞在G1期。实验组细胞凋亡率为（14.96±1.27）%，显著高于对照组的（4.52±1.08）%（p0.05）。 4.Western bloting结果：实验组24h、48h CDK6、Bcl-2、Mcl-1蛋白的表达水平较对照组减弱，Bax蛋白表达增强,cyclinD1蛋白表达量无明显变化。以上变化均以48h更为明显。结论： 1、体外上调U251人脑胶质瘤细胞中miRNA-29b的表达可抑制肿瘤细胞的增殖并诱导肿瘤细胞凋亡。 2、miRNA-29b抑制U251人脑胶质瘤细胞增殖的机制与抑制CDK6蛋白的表达相关，与cyclinD1蛋白的表达无直接相关。 3、miRNA-29b诱导U251人脑胶质瘤细胞凋亡与抑制Bcl-2、Mcl-1蛋白的表达和促进Bax蛋白的表达相关。 4、miRNA-29b可能成为靶向治疗人脑胶质瘤的靶点。
[Abstract]:Purpose: In vitro, has-miRNA-29bmitics transiently transfected U251 human glioma cells and observed its effect on cell growth. The expression of miRNA-29b up-regulated in human glioma cells of U251 could inhibit the growth of cells, detect the proliferation of cells and the expression of apoptosis-related proteins. Objective To explore the mechanism of miRNA-29b to inhibit the growth of glioma cells in U251 glioma cells, and to provide a theoretical basis for clinical treatment of glioma. Basis. Methods: 1. U251 human glioma cells were transiently transfected in vitro by Lipofectaminine 2000 with a final concentration of 100nmol/ l. Cells were collected at different time points (24h, 48h, or 72h) after transfection with miRNAs for cell morphology observation, MTT, flow cytometry or CD11b. lotting experiment. 2 (. 1) morphological observation: the growth and cell morphology of two groups of cells were observed directly under the inverted phase contrast microscope, and the two groups were compared. Differences in the cells (2) MTT assay: The absorbance values OD490nm and OD630nm at wavelengths of 490nm and 630nm were detected by MTT assay and MTT assay respectively. A value (A = OD490nm-OD630) was used in the experimental group and control group at the wavelength of 490nm and 630nm respectively. The proliferation of cells was evaluated by flow cytometry. (3) Flow cytometry was used to detect the phase distribution and apoptosis rate during cell cycle: the cell cycle of two groups of cells 48h was detected by PI single-stain method, the proportion of cells at different cell cycle was recorded; Annexin V/ PI double staining method was used to measure the apoptosis of two groups after transfection of miimics48h. In the case of apoptosis index AI, AI (%) = apoptotic cells. Number of total cells/ total number of cells The expression level of CDK6, cyclin D1 and Bcl-2, Mcl-1 and Bax were detected by Western blotting, and the expression levels of Bcl-2, Mcl-1 and Bax were detected by Western blotting. Stern strips are carried out Quantitative analysis. Repeat the experiment for 5 times. Statistical analysis: All the data obtained are The standard deviation (x, S) was expressed by means of SPSS17. 0 statistical software, and independent sample t test was used between the two groups of variance, and the variance was irregular. Wilcoxon rank sum test was used. P0. Results: 1. Cell morphology observation: The growth status of glioma cells in U251 glioma cells was good, the cells were adherent to the wall, the cells were pleomorphic, the outline was clear, the boundary of cells was clear, the adhesion was good, the cells were bright and the cells were bright. In the experimental group, the growth of U251 cells in experimental group was relatively slow, the malapposition was weakened, the tumor cells lost their original pleomorphic state, gradually rounded, and the cells were slightly wrinkled. In addition, the internal particles of the cytoplasm increased, and the small part of the cells dropped out of death 2. MT The results of T experiment: The values of A between the control group and the experimental group at 24h, 48h and 72h were (0. 14, 0. 01), (0. 25, 0. 03), (0. 36, 0. 02), (0. 13, 0. 01), (0. 21, 0. 02), and each time point difference between the two groups was statistically significant. The results of flow cytometry showed that the ratio of cells in G1, S and G2 phase of U251 cells in control group were (44. 78% 1. 52)%, (50. 86% 1. 52)% and (4.56% 0. 47)%, respectively, and the experimental group was (57. 27% 2.27)%, respectively. (39. 25, 2.12)%, (3.46. 0. 44)%, and there were differences between the two groups. In the experimental group, the cell cycle arrest was in G1 phase. The apoptosis rate of the experimental group was (14. 96% 1. 27)%. Results: The expression level of CDK6, Bcl-2 and Mcl-1 in experimental group was lower than that in control group. Increased expression of Bax protein, cyc lin There was no significant change in the expression of D1 protein. The above changes were more obvious at 48h. Conclusion: 1. U251 human glioma was up-regulated in vitro. the expression of miRNA-29b in tumor cells can inhibit the proliferation of tumor cells and induce apoptosis of tumor cells. the mechanism of colonizing is related to the inhibition of the expression of CDK6 protein and is not directly related to the expression of cyclin D1. Apoptosis and expression of Bcl-2 and Mcl-1 protein in human glioma cells
【学位授予单位】：承德医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41

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