發(fā)布時(shí)間：2018-03-13 15:36

  本文選題：血管內(nèi)皮生長(zhǎng)因子　切入點(diǎn)：鼻咽癌　出處：《中南大學(xué)》2010年博士論文　論文類型：學(xué)位論文

【摘要】： 目的探討腺病毒E1A基因?qū)θ吮茄拾〤NE-2Z細(xì)胞放射治療的增敏作用及相關(guān)機(jī)制。 方法選用人鼻咽癌CNE-2Z細(xì)胞為靶細(xì)胞,分別用PBS,Ad-β-gal, Ad-E1A作用48小時(shí)后,用6MVX線分別給與0、2、4、6和8Gy劑量照射后,用MTT法和流式細(xì)胞儀檢測(cè)三組細(xì)胞成活率及細(xì)胞周期變化,并進(jìn)行克隆分析繪制細(xì)胞存活曲線,計(jì)算放射增敏比來(lái)觀察E1A基因?qū)θ吮茄拾〤NE-2Z細(xì)胞放射敏感性的影響,同時(shí)用RT-PCR法檢測(cè)三組細(xì)胞的VEGF水平的表達(dá)及野生型p53的表達(dá)。細(xì)胞免疫化學(xué)方法檢測(cè)三組細(xì)胞的VEGF蛋白的表達(dá)。 結(jié)果RT-PCR結(jié)果顯示Ad-E1A組可以檢測(cè)到E1A基因的表達(dá)。經(jīng)照射后Ad-E1A組的細(xì)胞存活率明顯低于PBS對(duì)照組和Ad-β-gal對(duì)照組。Ad-E1A組的細(xì)胞的生長(zhǎng)速度明顯慢于PBS對(duì)照組和Ad-β-gal對(duì)照組。細(xì)胞存活曲線結(jié)果顯示Ad-E1A組的CNE-2Z細(xì)胞放射增敏比分別為1.37(D0值比)、1.95(Dq值比)、1.46(SF2比)。PBS對(duì)照組和Ad-β-gal對(duì)照組的細(xì)胞照射后細(xì)胞存活曲線分析結(jié)果顯示無(wú)差異,D0、Dq、SF2值分別為1.57Gy及1.53Gy、1.82Gy及1.78Gy、0.89及0.82。RT-PCR結(jié)果和細(xì)胞免疫化學(xué)結(jié)果顯示Ad-E1A組比PBS對(duì)照組和Ad-β-gal對(duì)照組的人鼻咽癌CNE-2Z細(xì)胞的VEGF的表達(dá)明顯下降,RT-PCR結(jié)果顯示Ad-E1A組比PBS對(duì)照組和Ad-β-gal對(duì)照組的野生型p53水平明顯上升。流式細(xì)胞學(xué)實(shí)驗(yàn)顯示Ad-E1A組+放射治療組細(xì)胞的凋亡明顯高于其他組。 結(jié)論E1A基因通過(guò)下調(diào)人鼻咽癌CNE-2Z細(xì)胞VEGF的表達(dá)和上調(diào)野生型p53的表達(dá)來(lái)增加人鼻咽癌細(xì)胞對(duì)放射治療的敏感性。 目的：探討腺病毒E1A基因?qū)θ吮茄拾﹦?dòng)物模型放射增敏的實(shí)驗(yàn)研究。 方法：取對(duì)數(shù)生長(zhǎng)期的CNE-2Z細(xì)胞5×105/0.2mL,接種于4周齡左右裸鼠右前肢腋部皮下致瘤,第7天裸鼠皮下腫瘤長(zhǎng)至直徑0.6-0.8cm時(shí),隨機(jī)分為6組(每組10個(gè))：PBS組,Ad-β-gal組,放射組,Ad-β-gal+放射組,Ad-E1A組,Ad-E1A+放射組。Ad-β-gal組/Ad-E1A組在第2周開(kāi)始給予荷瘤裸鼠皮下腫瘤內(nèi)滴度為5×109PFU/50μL的Ad-E1A/Ad-β-gal注射,每周2次,連續(xù)2周,放射組在第3周給予6MV-X照射,每天2Gy,連續(xù)5天。Ad-β-gal+放射組/Ad-E1A+放射組在第2周開(kāi)始,給予荷瘤裸鼠皮下腫瘤內(nèi)滴度為5×109 PFU/50μL的Ad-E1A/Ad-β-gal注射,每周2次,連續(xù)2周,在第3周給予6MV-X照射,每天2Gy,連續(xù)5天。第1次治療后,每隔4天用卡尺測(cè)量1次腫瘤的體積,記錄其直徑,裸鼠的生存時(shí)間。當(dāng)腫瘤的直徑超過(guò)2cm時(shí),處死裸鼠,分離腫瘤組織,采用免疫組織化學(xué)方法分析VEGF和CD34表達(dá),RT-PCR分析wtp53的表達(dá),Western blot分析VEGF的表達(dá),TUNEL分析細(xì)胞凋亡。 結(jié)果：Ad-E1A+放射組較其他組能明顯延緩移植瘤生長(zhǎng)時(shí)間,Ad-E1A+放射組裸鼠的腫瘤平均體積比單獨(dú)放射組小4.7倍,比單獨(dú)Ad-E1A組的小5.3倍。Ad-E1A+放射組的裸鼠生存率顯著高于其他治療組。Ad-E1A+放射組的VEGF蛋白表達(dá)和微血管密度較其他各組明顯下降。(P0.01)。Ad-E1A組和Ad-E1A+放射組的wtp53基因表達(dá)明顯上調(diào)。TUNEL分析結(jié)果顯示Ad-E1A組和Ad-E1A+放射組及放射組的腫瘤組織內(nèi)均可明顯觀察到凋亡細(xì)胞。并且,Ad-E1A+放射組腫瘤細(xì)胞凋亡數(shù)目明顯高于Ad-E1A組或單獨(dú)放射組。 結(jié)論：E1A基因通過(guò)抑制腫瘤血管的形成和上調(diào)腫瘤組織中wtp53的表達(dá)及誘導(dǎo)腫瘤細(xì)胞的凋亡來(lái)提高人鼻咽癌細(xì)胞對(duì)放射的敏感性。
[Abstract]:Objective to investigate the sensitizing effect of adenovirus E1A gene on the radiotherapy of human nasopharyngeal carcinoma CNE-2Z cells and its related mechanism.
Methods human nasopharyngeal carcinoma CNE-2Z cells were treated with PBS, Ad- beta -gal, 48 hours after Ad-E1A, 6MV and 8Gy 0,2,4,6 were given X-ray irradiation, and flow cytometry was used to detect three groups of cell survival rate and the change of cell cycle by MTT method and clone analysis according to the cell survival curve recently, the calculation of radiosensitization effect of E1A gene on radiosensitivity of human nasopharyngeal carcinoma CNE-2Z cells, and the expression of RT-PCR was used to detect the VEGF levels of the three groups of cells and wild type p53. The detection of immunocytochemistry. The expression of the three groups of cells of VEGF protein.
Results RT-PCR results showed that Ad-E1A group can detect the expression of E1A gene by Ad-E1A. The cell survival rate after irradiation group was significantly lower than that of PBS control group and Ad- control group.Ad-E1A group -gal beta cell growth was slower than the PBS control group and -gal control group. Ad- beta cell survival curves showed that CNE-2Z cells in radiation Ad-E1A group the sensitization enhancement ratio were 1.37 (D0 ratio), 1.95 (Dq ratio), 1.46 (SF2).PBS control group and -gal control group Ad- beta cells after irradiation, the cell survival curve analysis showed no difference, D0, Dq, SF2 values were 1.57Gy and 1.53Gy, 1.82Gy and 1.78Gy, 0.89 and the results of 0.82.RT-PCR and the immunocytochemistry showed that the expression of Ad-E1A in PBS group than those in control group and Ad- control group -gal beta in human nasopharyngeal carcinoma CNE-2Z cells VEGF decreased significantly, RT-PCR results showed that Ad-E1A group than in control group and PBS control group Ad- beta -gal wild type p53 level in Ming Dynasty The flow cytology test showed that the cell apoptosis in group Ad-E1A + radiation therapy group was significantly higher than that in other groups.
Conclusion E1A gene can increase the sensitivity of human nasopharyngeal carcinoma cells to radiotherapy by down regulation of the expression of VEGF in human nasopharyngeal carcinoma CNE-2Z cells and up regulation of the expression of wild type p53.
Objective: To study the experimental study of radioactivity sensitization of adenovirus E1A gene to human nasopharyngeal carcinoma (NPC) animal model.
Methods: CNE-2Z cells in log phase 5 * 105/0.2mL, about 4 week old nude mice were inoculated in the right armpit subcutaneous tumor, seventh days subcutaneous tumors grew to 0.6-0.8cm, were randomly divided into 6 groups (n = 10) group:PBS, Ad- beta -gal group, radiation group, -gal+ radiation Ad- beta group, Ad-E1A group, Ad-E1A+ radiation group.Ad- beta -gal group /Ad-E1A group was given subcutaneous tumor in nude mice was 5 * 109PFU/50 L Ad-E1A/Ad- beta -gal injection at second weeks, 2 times a week for 2 consecutive weeks, radiotherapy group received 6MV-X irradiation at third weeks 2Gy per day for 5 consecutive days of.Ad- beta -gal /Ad-E1A+ + radiation group radiation group at the beginning of the second week, tumor bearing nude mice subcutaneous tumor was 5 * 109 PFU/50 L Ad-E1A/Ad- beta -gal injection, 2 times a week for 2 consecutive weeks, given 6MV-X irradiation at week third 2Gy per day for 5 consecutive days. After the first treatment, every 4 days with caliper 1 measurements of tumor volume, record The diameter and survival time of nude mice. When the diameter of tumor was over 2cm, the nude mice were sacrificed and the tumor tissues were isolated. The expression of VEGF and CD34 was analyzed by immunohistochemistry. RT-PCR was used to analyze the expression of wtp53, Western blot was used to analyze VEGF expression, TUNEL was used to analyze apoptosis.
Results: Ad-E1A+ radiotherapy group can significantly delay tumor growth time than the other group, the average tumor volume of nude mice Ad-E1A+ radiation group than single radiation group is 4.7 times smaller than Ad-E1A group, the small 5.3 times.Ad-E1A+ radiation group the survival rate of nude mice was significantly higher than that of the other treatment groups.Ad-E1A+ radiation group VEGF protein expression and microvessel density significantly compared with the other groups decreased. (P0.01) wtp53 gene in.Ad-E1A group and radiation group Ad-E1A+ expression was significantly increased the.TUNEL analysis results showed that Ad-E1A and Ad-E1A+ can group radiation group and radiation group in tumor tissue was observed. Apoptosis and apoptosis of tumor cells, Ad-E1A+ radiation group number was significantly higher than Ad-E1A group or single radiation group.
Conclusion: E1A gene can enhance the radiosensitivity of human nasopharyngeal carcinoma cells by inhibiting tumor angiogenesis and upregulating the expression of wtp53 and inducing apoptosis of tumor cells.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R739.63

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