【摘要】：目的： 體外應(yīng)用has-miRNA-29b mimics瞬時(shí)轉(zhuǎn)染U251人腦膠質(zhì)瘤細(xì)胞，觀察其對(duì)細(xì)胞生長(zhǎng)的影響，探討在U251人腦膠質(zhì)瘤細(xì)胞中上調(diào)miRNA-29b的表達(dá)能否抑制細(xì)胞的生長(zhǎng)，檢測(cè)細(xì)胞內(nèi)增殖及凋亡相關(guān)蛋白的表達(dá)水平，初步探索miRNA-29b抑制U251人腦膠質(zhì)瘤細(xì)胞生長(zhǎng)的機(jī)制，，為臨床上治療膠質(zhì)瘤提供理論依據(jù)。 方法： 1.用Lipofectamine2000介導(dǎo)終濃度為100nmol/l的cel-miRNA-67mimics和has-miRNA-29b mimics體外瞬時(shí)轉(zhuǎn)染的U251人腦膠質(zhì)瘤細(xì)胞分別作為對(duì)照組和實(shí)驗(yàn)組。根據(jù)實(shí)驗(yàn)需要在轉(zhuǎn)染miRNAmimics后不同時(shí)間點(diǎn)（24h，48h或72h）收取細(xì)胞，用于細(xì)胞形態(tài)學(xué)觀察、MTT、流式細(xì)胞學(xué)術(shù)或Westernbloting實(shí)驗(yàn)。 2（.1）細(xì)胞形態(tài)學(xué)觀察：在倒置相差顯微鏡下直接觀察培養(yǎng)板中24h、48h兩組細(xì)胞的生長(zhǎng)情況及細(xì)胞形態(tài)，比較兩組細(xì)胞的差異，并拍照記錄。重復(fù)3次。（2）MTT實(shí)驗(yàn)：四甲基偶氮唑藍(lán)(MTT)比色法分別檢測(cè)24h、48h、72h實(shí)驗(yàn)組和對(duì)照組細(xì)胞在波長(zhǎng)490nm和630nm處的吸光值OD490nm和OD630nm，用A值（A=OD490nm－OD630nm）評(píng)價(jià)細(xì)胞增殖情況。（3）流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期時(shí)相分布及凋亡率：PI單染法檢測(cè)兩組細(xì)胞48h的細(xì)胞周期，記錄處于不同細(xì)胞周期時(shí)相的細(xì)胞比例；Annexin V/PI雙染法測(cè)轉(zhuǎn)染miRNA mimics48h后兩組細(xì)胞凋亡情況，計(jì)算凋亡指數(shù)AI，AI（%）=凋亡細(xì)胞數(shù)/總細(xì)胞數(shù)×100%。實(shí)驗(yàn)重復(fù)5次。（4）蛋白印跡（Western blotting）實(shí)驗(yàn)：Western blotting檢測(cè)24h、48h兩組細(xì)胞周期相關(guān)蛋白CDK6、cyclinD1和凋亡相關(guān)蛋白Bcl-2、Mcl-1、Bax的表達(dá)水平，IPP6.0軟件對(duì)Western條帶進(jìn)行定量分析。實(shí)驗(yàn)重復(fù)5次。 3.統(tǒng)計(jì)學(xué)分析：所得數(shù)據(jù)資料均以均數(shù)±標(biāo)準(zhǔn)差(x±S)來表示。采用SPSS17.0統(tǒng)計(jì)學(xué)軟件對(duì)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)處理，方差齊的兩組間采用獨(dú)立樣本t檢驗(yàn)，方差不齊則采用Wilcoxon秩和檢驗(yàn)。以P0.05為差異有統(tǒng)計(jì)學(xué)意義。 結(jié)果： 1.細(xì)胞形態(tài)學(xué)觀察：對(duì)照組U251人腦膠質(zhì)瘤細(xì)胞生長(zhǎng)狀態(tài)良好，細(xì)胞貼壁生長(zhǎng)，呈多形性，輪廓清晰，細(xì)胞間界限清楚，貼壁良好，胞體透亮，顆粒較少，細(xì)胞核清晰可見；實(shí)驗(yàn)組細(xì)胞24h、48h實(shí)驗(yàn)組U251細(xì)胞生長(zhǎng)相對(duì)緩慢，貼壁現(xiàn)象減弱，腫瘤細(xì)胞失去原有多形性狀態(tài)，逐漸變圓趨勢(shì)，胞體輕度皺縮，胞漿內(nèi)顆粒增多增粗，小部分細(xì)胞脫落死亡 2. MTT實(shí)驗(yàn)結(jié)果：對(duì)照組和實(shí)驗(yàn)組細(xì)胞在24h、48h、72h的A值分別為（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），兩組間各時(shí)間點(diǎn)差異均具有統(tǒng)計(jì)學(xué)意義（P0.05），且差異在轉(zhuǎn)染后72h最為明顯。 3.流式細(xì)胞檢測(cè)結(jié)果：對(duì)照組U251細(xì)胞48h處于G1、S、G2期細(xì)胞數(shù)比值分別為（44.78±1.52）%、（50.86±1.52）%和（4.56±0.47）%；實(shí)驗(yàn)組分別為（57.27±2.27）%，（39.25±2.12）%，（3.46±0.44）%，兩組間差異有統(tǒng)計(jì)學(xué)意義（p0.05），實(shí)驗(yàn)組細(xì)胞周期阻滯在G1期。實(shí)驗(yàn)組細(xì)胞凋亡率為（14.96±1.27）%，顯著高于對(duì)照組的（4.52±1.08）%（p0.05）。 4.Western bloting結(jié)果：實(shí)驗(yàn)組24h、48h CDK6、Bcl-2、Mcl-1蛋白的表達(dá)水平較對(duì)照組減弱，Bax蛋白表達(dá)增強(qiáng),cyclinD1蛋白表達(dá)量無明顯變化。以上變化均以48h更為明顯。 結(jié)論： 1、體外上調(diào)U251人腦膠質(zhì)瘤細(xì)胞中miRNA-29b的表達(dá)可抑制腫瘤細(xì)胞的增殖并誘導(dǎo)腫瘤細(xì)胞凋亡。 2、miRNA-29b抑制U251人腦膠質(zhì)瘤細(xì)胞增殖的機(jī)制與抑制CDK6蛋白的表達(dá)相關(guān)，與cyclinD1蛋白的表達(dá)無直接相關(guān)。 3、miRNA-29b誘導(dǎo)U251人腦膠質(zhì)瘤細(xì)胞凋亡與抑制Bcl-2、Mcl-1蛋白的表達(dá)和促進(jìn)Bax蛋白的表達(dá)相關(guān)。 4、miRNA-29b可能成為靶向治療人腦膠質(zhì)瘤的靶點(diǎn)。
[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
【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】：2014
【分類號(hào)】：R739.41

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