【摘要】：目的：神經(jīng)膠質(zhì)瘤為顱內(nèi)常見腫瘤,為預(yù)后較差的顱內(nèi)原發(fā)性惡性腫瘤,具有浸潤性生長和邊界不清等生物學(xué)特點(diǎn),手術(shù)難以徹底切除。術(shù)后放射治療是膠質(zhì)瘤綜合治療中較為重要的手段。但是惡性膠質(zhì)瘤具有放射抗性致其放射治療效果欠佳,近來多個研究證實(shí)γH2AX逐漸成為檢測DNA雙鏈斷裂的一項(xiàng)新型生物學(xué)標(biāo)志。在本研究中,我們通過體外培養(yǎng)高級別膠質(zhì)瘤細(xì)胞系(U87、U251和LN229),研究其放射敏感性以及經(jīng)X射線照射后三種細(xì)胞株γH2AX蛋白的表達(dá)變化,并分析γH2AX蛋白表達(dá)與膠質(zhì)瘤細(xì)胞放射敏感性之間的相互關(guān)系。 方法： 1.選擇高級別人腦膠質(zhì)瘤U87、U251和LN229細(xì)胞株,分成0Gy組、2Gy組、4Gy組、6Gy組、8Gy組和10Gy組,經(jīng)常規(guī)細(xì)胞培養(yǎng)、細(xì)胞計數(shù)后,行X線照射,繼續(xù)細(xì)胞培養(yǎng)2周左后,經(jīng)固定、染色后鏡下檢測細(xì)胞集落數(shù),計算細(xì)胞克隆形成率、細(xì)胞存活分?jǐn)?shù),并繪制細(xì)胞存活曲線、由生物學(xué)參數(shù)比較細(xì)胞系間放射敏感性； 2.選擇高級別人腦膠質(zhì)瘤U87、U251和LN229細(xì)胞株,按照經(jīng)X線照射后培養(yǎng)的時間分為0h組、0.5h組、1h組、2h組、6h組、12h組、24h組、36h組和48h組,常規(guī)細(xì)胞培養(yǎng)至細(xì)胞貼壁并鋪滿整個培養(yǎng)瓶約75%體積時,行2Gy照射,然后放置于細(xì)胞培養(yǎng)箱中分別培養(yǎng)0h、0.5h、1h、2h、6h、2h、24h、36h和 48h,依次分別提取制備細(xì)胞總蛋白,經(jīng)蛋白變性、聚丙烯酰胺凝膠電泳分離、蛋白質(zhì)轉(zhuǎn)移至PVDF膜、封閉、Ⅰ抗反應(yīng)、Ⅱ抗孵育后,ECL化學(xué)發(fā)光試劑盒顯影。采用Alpha Innotech凝膠成像儀檢測結(jié)果,Image J計算各電泳條帶平均光密度值,從而檢測其γH2AX蛋白表達(dá)變化。目的蛋白質(zhì)相對表達(dá)量=目的條帶灰度值／同一樣本內(nèi)參灰度值。 3.統(tǒng)計學(xué)分析γH2AX蛋白表達(dá)與膠質(zhì)瘤細(xì)胞放射敏感性之間的相關(guān)性。以Western blotting條帶中6h組與0.5h組的蛋白表達(dá)量差值代表相對衰減速度,以目的蛋白質(zhì)峰值與初始值之間差值代表目的蛋白升高程度,分別與放射增敏比行相關(guān)性分析。 結(jié)果： 1.細(xì)胞克隆形成實(shí)驗(yàn)顯示,隨著X線照射劑量的增加,膠質(zhì)瘤細(xì)胞存活分?jǐn)?shù)逐漸降低,細(xì)胞克隆形成率減少,U87、LN229、U251細(xì)胞的放射增敏比(SER)依次為1.31±0.04、1.14±0.02、1.00±0.00,并采用單因素方差分析得出：放射增敏比(SER)自高至低依次為U87、LN229、U251(均P=0.000)； 2.Western blotting法顯示,隨著經(jīng)X線照射后培養(yǎng)時間的延長,各膠質(zhì)瘤細(xì)胞系γH2AX蛋白表達(dá)呈現(xiàn)出先上升后下降的時間效應(yīng)曲線,U87、LN229和U251細(xì)胞株出現(xiàn)峰值時間依次為2h、1h和1h(P=0.000、0.000、0.015)。 3.yH2AX蛋白相對衰減速度和γH2AX升高程度均與放射增敏比呈正相關(guān)。(r=0.733,p=0.025；r=0.672,P=0.047)。 結(jié)論： 1.三種高級別人腦膠質(zhì)瘤細(xì)胞株放射敏感性自高至低依次為U87、LN229、U251; 2.電離輻射產(chǎn)生的yH2AX蛋白在放射性較抵抗的膠質(zhì)瘤細(xì)胞株中達(dá)到峰值的時間相對更早； 3.電離輻射產(chǎn)生的γH2AX蛋白在輻射較敏感的膠質(zhì)瘤細(xì)胞株中升高程度以及衰減速度均相對較高； 4.yH2AX蛋白有望成為高級別膠質(zhì)瘤的分子生物學(xué)標(biāo)志物,并作為預(yù)測膠質(zhì)瘤放射敏感性的重要指標(biāo)。
[Abstract]:Objective: The glioma is a common type of intracranial tumor. It is a kind of intracranial primary malignant tumor with poor prognosis. It has the biological characteristics of infiltrative growth and non-clear boundary, and the operation is difficult to be completely cut off. Postoperative radiation therapy is an important means in the comprehensive treatment of glioma. However, the radiation resistance of the malignant glioma is not good, and the recent researches have shown that HH2AX is a new biological marker for detecting the double-strand break of DNA. In this study, we studied the radiosensitivity of the high-level glioma cell line (U87, U251, and LN229) in vitro, and the expression of the HH2AX protein in the three cell lines after X-ray irradiation. The relationship between the expression of HH2AX and the radiosensitivity of glioma cells was analyzed. square Methods: 1. High-grade human glioma U87, U251 and LN229 cell lines were selected, divided into 0Gy group, 2Gy group, 4Gy group, 6Gy group, 8Gy group and 10Gy group. After cell count, X-ray irradiation was performed and the cell culture was continued for 2 weeks. The cell colony number, the cell clone formation rate, the cell survival score, and the cell survival curve were measured under a fixed and stained mirror, and the cell line-to-cell radiation was compared by the biological parameters. Sensitivity; 2. High-level human glioma U87, U251 and LN2 The cell line was divided into 0h group, 0.5h group, 1h group, 2h group, 6h group, 12h group, 24h group, 36h group and 48h group according to the time after the X-ray irradiation. 1h,2h,6h,2h,24 h, 36h and 48h, respectively, respectively extracting the total protein of the preparation cell, the protein denaturation, the polytetramine amine gel electrophoresis separation, the protein transfer to the PVDF membrane, the sealing, the first anti-reaction, the second anti-incubation, the ECL chemistry, Development of the light-emitting kit. As a result of the detection using the Alpha Innotech Gel imager, the image J calculates the average optical density values for each of the electrophoretic bands, thereby detecting its BHC2A X-protein expression change. Target protein relative expression quantity = target band gray value/ same Statistical analysis of the expression of HH2AX and the radiation of glioma cells The correlation between the sensitivity and sensitivity was expressed by the difference of the protein expression between the 6 h group and the 0. 5 h group in the Western blotting band. The difference between the peak value of the target protein and the initial value represents the degree of increase of the target protein, and the difference between the peak value of the target protein and the initial value represents the degree of increase of the target protein, respectively. increase-to-sensitivity ratio The results were as follows: 1. The cell clone formation experiment showed that, with the increase of X-ray irradiation dose, the survival score of glioma cells decreased gradually, the formation rate of cell clone decreased, and the radiosensitivity ratio (SER) of U87, LN229 and U251 cells was 1.31, 0.04, 1.14, and 0.. 02, 1. 00-0. 00, and using a single-factor analysis of variance, the radiosensitivity ratio (SER) is from high to low in order of U87, LN229, U, 251 (P = 0.000); With the extension of culture time after X-ray irradiation, the expression of HH2AX protein in glioma cell lines showed a time-effect curve which decreased first, and the peak time of U87, LN229 and U251 cell lines was 2h, 1h and 1h (P = 0. 0). 00, 0. 000, 0. 015). 3. yH2AX protein relative decay rate and FH2 The degree of increase of AX was positively correlated with the radiosensitivity ratio. (r = 0.733, p = 0.025 錛況= Conclusion: 1. The radiosensitivity of three high-grade human glioma cell lines from high to low, U87, LN229, U251; 2. The yH2AX protein produced by ionizing radiation is in radiation. the time to reach the peak in the resistance-resistant glioma cell line is relatively early; 3. the h2ax protein produced by the ionizing radiation is relatively high in radiation, The degree of increase and the rate of attenuation in the sensitive glioma cell line are relatively high; the 4. yH2AX protein is expected to be a high-grade glioma
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R739.41

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