自噬及PARP-1對(duì)鼻咽癌CNE-2細(xì)胞放射敏感性影響的實(shí)驗(yàn)研究
發(fā)布時(shí)間：2018-08-09 13:53
【摘要】：第一部分沉默自噬相關(guān)基因ATG5對(duì)鼻咽癌CNE-2細(xì)胞放射敏感性的影響 [目的] 本部分通過慢病毒介導(dǎo)的RNA干擾技術(shù)抑制自噬相關(guān)基因ATG5的表達(dá),觀察ATG5沉默后對(duì)鼻咽癌CNE-2細(xì)胞增殖、凋亡及放射敏感性的影響,以探討自噬與鼻咽癌CNE-2細(xì)胞放射敏感性的關(guān)系,為鼻咽癌放射增敏及基因治療提供新的靶點(diǎn)。 [方法] 針對(duì)自噬相關(guān)基因ATG5設(shè)計(jì)并合成3條干擾靶點(diǎn),篩選最佳沉默片段進(jìn)行慢病毒包裝,感染鼻咽癌CNE-2細(xì)胞并經(jīng)流式分選獲得穩(wěn)定沉默的細(xì)胞株。應(yīng)用RT-PCR及Western-blot實(shí)驗(yàn)驗(yàn)證干擾效果。采用CCK-8法、流式細(xì)胞技術(shù)和克隆形成實(shí)驗(yàn)檢測(cè)自噬相關(guān)基因ATG5沉默后鼻咽癌CNE-2細(xì)胞放射敏感性的變化。同時(shí),進(jìn)行裸鼠成瘤體內(nèi)試驗(yàn),觀察ATG5基因沉默后,射線照射對(duì)裸鼠移植瘤生長(zhǎng)的影響,進(jìn)一步驗(yàn)證自噬與鼻咽癌放射敏感性的關(guān)系。 [結(jié)果] CCK-8實(shí)驗(yàn)結(jié)果顯示,與未轉(zhuǎn)染的Control組和陰性對(duì)照NC組相比,各劑點(diǎn)ATG5沉默組的細(xì)胞存活率均顯著降低(P0.05),繪制細(xì)胞生存曲線可見下調(diào)ATG5的表達(dá)后可以增加CNE-2細(xì)胞的放射敏感性。流式細(xì)胞術(shù)結(jié)果顯示,經(jīng)6Gy射線照射后,與NC組及Control組相比,ATG5沉默組細(xì)胞的凋亡指數(shù)明顯升高(F=394.876,P0.01)�？寺⌒纬蓪�(shí)驗(yàn)結(jié)果顯示,在各照射劑量點(diǎn),ATG5組的細(xì)胞存活分?jǐn)?shù)較Control及NC組均有所下降(P0.05),二次線性模型擬合劑量存活曲線示沉默ATG5基因可增加鼻咽癌CNE-2細(xì)胞的放射敏感性。裸鼠成瘤體內(nèi)實(shí)驗(yàn)結(jié)果顯示,經(jīng)10Gy射線照射后,與Control及NC組相比,ATG5組移植瘤的生長(zhǎng)明顯減緩,ATG5組瘤體重量明顯小于其他兩組(F=4.035,P0.05)。 [結(jié)論] 沉默自噬相關(guān)基因ATG5可以增加鼻咽癌CNE-2細(xì)胞的放射敏感性,初步判斷自噬是鼻咽癌CNE-2細(xì)胞放射過程中的一種保護(hù)性機(jī)制。 第二部分沉默PARP-1基因?qū)Ρ茄拾〤NE-2細(xì)胞放射敏感性的影響 [目的] 本部分采用慢病毒介導(dǎo)的RNA干擾技術(shù)抑制基因PARP-1的表達(dá),觀察其沉默后對(duì)射線所致的自噬現(xiàn)象及鼻咽癌CNE-2細(xì)胞放射敏感性的影響,進(jìn)一步探討PARP-1與自噬及鼻咽癌放射敏感性的關(guān)系,為尋找鼻咽癌放射增敏途徑提供理論基礎(chǔ)。 [方法] 針對(duì)基因PARP-1設(shè)計(jì)并合成的3條干擾靶點(diǎn),進(jìn)行慢病毒包裝,應(yīng)用RT-PCR及Western-blot實(shí)驗(yàn)篩選出沉默效果最佳的靶點(diǎn),感染目的細(xì)胞CNE-2并流式分選獲得穩(wěn)定沉默PARP-1基因的細(xì)胞株。Western-blot實(shí)驗(yàn)分別檢測(cè)PARP-1及ATG5基因沉默的CNE-2細(xì)胞射線照射前后LC3-Ⅱ、ATG5及PARP-1蛋白的變化情況,研究PARP-1與自噬的關(guān)系。另外應(yīng)用CCK-8法、流式細(xì)胞術(shù)及克隆形成實(shí)驗(yàn)檢測(cè)]PARP-1基因沉默后鼻咽癌CNE-2細(xì)胞放射敏感性的變化。[結(jié)果] Western-blot實(shí)驗(yàn)結(jié)果顯示,經(jīng)10Gy射線照射,PARP-1基因沉默組LC3-Ⅱ的相對(duì)表達(dá)量較單純照射組明顯降低(F=34.856,P0.01),這一現(xiàn)象在ATG5沉默組中也有相似表現(xiàn)。同時(shí),沉默PARP-1基因后,PARP-1蛋白的相對(duì)表達(dá)量較單純照射組降低(F=14.853,P0.01),ATG5的蛋白表達(dá)量也相應(yīng)降低(F=10.863,P0.01),而沉默自噬相關(guān)基因ATG5后,ATG5蛋白的表達(dá)量有所降低,PARP-1的表達(dá)卻未受影響。CCK-8實(shí)驗(yàn)結(jié)果顯示,降低PARP-1的表達(dá)后各實(shí)驗(yàn)組細(xì)胞的存活率均有所降低(P0.05),繪制細(xì)胞生存曲線可見下調(diào)PARP-1的表達(dá)后可增加CNE-2細(xì)胞的放射敏感性。流式細(xì)胞術(shù)結(jié)果顯示,經(jīng)6Gy射線照射后,與NC組及Control組相比,PARP-1沉默組細(xì)胞的凋亡率明顯升高(F=501.048,P0.01)�？寺⌒纬蓪�(shí)驗(yàn)結(jié)果顯示,各劑量點(diǎn)PARP-1沉默組細(xì)胞的存活分?jǐn)?shù)均有所降低(P0.05),二次線性模型擬合劑量存活曲線示下調(diào)PARP-1的表達(dá)后可以增加鼻咽癌CNE-2細(xì)胞的放射敏感性。 [結(jié)論] 射線可以誘導(dǎo)PARP-1的激活。PARP-1參與了射線所致的鼻咽癌CNE-2細(xì)胞的自噬現(xiàn)象,且可能處于相應(yīng)信號(hào)通路的上游。PARP-1基因表達(dá)量的降低增加了CNE-2細(xì)胞對(duì)射線的敏感性,即PARP-1在射線所致鼻咽癌CNE-2細(xì)胞死亡過程中起保護(hù)性作用。
[Abstract]:Part I Effect of silencing autophagy-related gene ATG5 on radiosensitivity of nasopharyngeal carcinoma CNE-2 cells
[Objective]
In this part, the expression of autophagy related gene ATG5 was suppressed by lentivirus mediated RNA interference technique, and the effects of ATG5 silence on the proliferation, apoptosis and radiosensitivity of nasopharyngeal carcinoma CNE-2 cells were observed in order to explore the relationship between autophagy and the radiosensitivity of nasopharyngeal carcinoma CNE-2 cells, and to provide new targets for the radiation sensitization and gene therapy of nasopharyngeal carcinoma.
[method]
3 interference targets were designed and synthesized for autophagy related gene ATG5, and the best silent fragment was screened for lentivirus package, CNE-2 cells of nasopharyngeal carcinoma were infected and the cell lines stable and silent by flow sorting. The interference effect was verified by RT-PCR and Western-blot experiments. The CCK-8 method, flow cytometry and clone formation test were used to detect autophagy. The changes in the radiosensitivity of the nasopharyngeal carcinoma CNE-2 cells after the ATG5 gene was silenced. At the same time, the nude mice were tested in vivo to observe the effect of radiation on the growth of xenografts in nude mice after the ATG5 gene silencing, and to further verify the relationship between autophagy and the radiosensitivity of nasopharyngeal carcinoma.
[results]
The results of CCK-8 experiment showed that compared with the untransfected Control group and the negative control NC group, the cell survival rate of each point ATG5 silencing group decreased significantly (P0.05). The cell survival curve could be shown to increase the radiosensitivity of the CNE-2 cells after the expression of the down-regulation of ATG5. The results of flow cytometry showed that after the 6Gy ray irradiation, it was associated with the NC group and Contr. Compared with the ol group, the apoptotic index of the cells in the ATG5 silencing group increased significantly (F=394.876, P0.01). The results of the clone formation experiment showed that the cell survival fraction of the ATG5 group decreased (P0.05) in the ATG5 group than that in the Control and NC groups. The two linear model fitted the dose survival curve to show that the silence of the ATG5 gene could increase the radiation of CNE-2 cells in nasopharyngeal carcinoma. The results of tumor formation in nude mice showed that after 10Gy ray irradiation, the growth of the transplanted tumor in the group ATG5 was significantly slower than that of the Control and NC groups, and the weight of the ATG5 group was significantly smaller than that of the other two groups (F=4.035, P0.05).
[Conclusion]
The silencing of autophagy related gene ATG5 can increase the radiosensitivity of CNE-2 cells in nasopharyngeal carcinoma and preliminarily determine that autophagy is a protective mechanism in the radiation process of nasopharyngeal carcinoma CNE-2 cells.
Part 2 Effect of PARP-1 gene silencing on radiosensitivity of nasopharyngeal carcinoma CNE-2 cells
[Objective]
This part uses the slow virus mediated RNA interference technique to inhibit the expression of gene PARP-1, and observe the effect of its silence on the autophagy and the radiosensitivity of CNE-2 cells in nasopharyngeal carcinoma, and further explore the relationship between PARP-1 and the radiosensitivity of the autophagy and nasopharyngeal carcinoma, and provide a theoretical basis for the search for the radiosensitivity pathway of nasopharyngeal carcinoma.
[method]
3 interfering targets designed and synthesized by gene PARP-1 were designed and packed in lentivirus. RT-PCR and Western-blot experiments were used to screen the best target of silence effect. The.Western-blot experiment of cells with stable and silent PARP-1 gene from infected target cells was selected to detect the CNE-2 cell shoot of PARP-1 and ATG5 gene silencing by.Western-blot. The changes of LC3- II, ATG5 and PARP-1 protein before and after line irradiation were used to study the relationship between PARP-1 and autophagy. In addition, the changes of radiosensitivity of CNE-2 cells in nasopharyngeal carcinoma after]PARP-1 gene silencing were detected by CCK-8, flow cytometry and cloning formation. [results]
The results of Western-blot experiment showed that the relative expression of LC3- II in the PARP-1 gene silencing group was significantly lower than that in the simple irradiation group (F=34.856, P0.01), and the phenomenon was also similar in the ATG5 silence group. At the same time, the relative expression of the PARP-1 protein decreased (F=14.853, P0.01) after the silence of PARP-1 gene (F=14.853, P0.01). The expression of protein was also reduced (F=10.863, P0.01), while the expression of ATG5 protein was reduced after the silence of autophagy related gene ATG5, but the expression of PARP-1 was not affected by the.CCK-8 experimental results. The survival rate of the cells in the experimental groups decreased (P0.05) after the reduction of the expression of PARP-1, and the survival curve of the cells was reduced to PARP-1. The expression of CNE-2 cells increased the radiosensitivity of the cells. The results of flow cytometry showed that after the 6Gy ray irradiation, the apoptosis rate of the cells in the PARP-1 silence group was significantly increased (F=501.048, P0.01) compared with the NC group and the Control group (F=501.048, P0.01). The results of the clone formation experiment showed that the survival fraction of the cells of each dose point PARP-1 sink group decreased (P0.05), two times. Linear model fitting dose survival curve showed that down-regulation of PARP-1 expression could increase radiosensitivity of nasopharyngeal carcinoma CNE-2 cells.
[Conclusion]
Radiation can induce PARP-1 activation.PARP-1 to participate in the autophagy of nasopharyngeal carcinoma CNE-2 cells induced by ray, and the decrease of.PARP-1 gene expression may be in the corresponding signal pathway to increase the sensitivity of CNE-2 cells to radiation, that is to say, PARP-1 plays a protective role in the death of nasopharyngeal carcinoma CNE-2 cells caused by radiation.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.63
本文編號(hào)：2174275
[Abstract]:Part I Effect of silencing autophagy-related gene ATG5 on radiosensitivity of nasopharyngeal carcinoma CNE-2 cells
[Objective]
In this part, the expression of autophagy related gene ATG5 was suppressed by lentivirus mediated RNA interference technique, and the effects of ATG5 silence on the proliferation, apoptosis and radiosensitivity of nasopharyngeal carcinoma CNE-2 cells were observed in order to explore the relationship between autophagy and the radiosensitivity of nasopharyngeal carcinoma CNE-2 cells, and to provide new targets for the radiation sensitization and gene therapy of nasopharyngeal carcinoma.
[method]
3 interference targets were designed and synthesized for autophagy related gene ATG5, and the best silent fragment was screened for lentivirus package, CNE-2 cells of nasopharyngeal carcinoma were infected and the cell lines stable and silent by flow sorting. The interference effect was verified by RT-PCR and Western-blot experiments. The CCK-8 method, flow cytometry and clone formation test were used to detect autophagy. The changes in the radiosensitivity of the nasopharyngeal carcinoma CNE-2 cells after the ATG5 gene was silenced. At the same time, the nude mice were tested in vivo to observe the effect of radiation on the growth of xenografts in nude mice after the ATG5 gene silencing, and to further verify the relationship between autophagy and the radiosensitivity of nasopharyngeal carcinoma.
[results]
The results of CCK-8 experiment showed that compared with the untransfected Control group and the negative control NC group, the cell survival rate of each point ATG5 silencing group decreased significantly (P0.05). The cell survival curve could be shown to increase the radiosensitivity of the CNE-2 cells after the expression of the down-regulation of ATG5. The results of flow cytometry showed that after the 6Gy ray irradiation, it was associated with the NC group and Contr. Compared with the ol group, the apoptotic index of the cells in the ATG5 silencing group increased significantly (F=394.876, P0.01). The results of the clone formation experiment showed that the cell survival fraction of the ATG5 group decreased (P0.05) in the ATG5 group than that in the Control and NC groups. The two linear model fitted the dose survival curve to show that the silence of the ATG5 gene could increase the radiation of CNE-2 cells in nasopharyngeal carcinoma. The results of tumor formation in nude mice showed that after 10Gy ray irradiation, the growth of the transplanted tumor in the group ATG5 was significantly slower than that of the Control and NC groups, and the weight of the ATG5 group was significantly smaller than that of the other two groups (F=4.035, P0.05).
[Conclusion]
The silencing of autophagy related gene ATG5 can increase the radiosensitivity of CNE-2 cells in nasopharyngeal carcinoma and preliminarily determine that autophagy is a protective mechanism in the radiation process of nasopharyngeal carcinoma CNE-2 cells.
Part 2 Effect of PARP-1 gene silencing on radiosensitivity of nasopharyngeal carcinoma CNE-2 cells
[Objective]
This part uses the slow virus mediated RNA interference technique to inhibit the expression of gene PARP-1, and observe the effect of its silence on the autophagy and the radiosensitivity of CNE-2 cells in nasopharyngeal carcinoma, and further explore the relationship between PARP-1 and the radiosensitivity of the autophagy and nasopharyngeal carcinoma, and provide a theoretical basis for the search for the radiosensitivity pathway of nasopharyngeal carcinoma.
[method]
3 interfering targets designed and synthesized by gene PARP-1 were designed and packed in lentivirus. RT-PCR and Western-blot experiments were used to screen the best target of silence effect. The.Western-blot experiment of cells with stable and silent PARP-1 gene from infected target cells was selected to detect the CNE-2 cell shoot of PARP-1 and ATG5 gene silencing by.Western-blot. The changes of LC3- II, ATG5 and PARP-1 protein before and after line irradiation were used to study the relationship between PARP-1 and autophagy. In addition, the changes of radiosensitivity of CNE-2 cells in nasopharyngeal carcinoma after]PARP-1 gene silencing were detected by CCK-8, flow cytometry and cloning formation. [results]
The results of Western-blot experiment showed that the relative expression of LC3- II in the PARP-1 gene silencing group was significantly lower than that in the simple irradiation group (F=34.856, P0.01), and the phenomenon was also similar in the ATG5 silence group. At the same time, the relative expression of the PARP-1 protein decreased (F=14.853, P0.01) after the silence of PARP-1 gene (F=14.853, P0.01). The expression of protein was also reduced (F=10.863, P0.01), while the expression of ATG5 protein was reduced after the silence of autophagy related gene ATG5, but the expression of PARP-1 was not affected by the.CCK-8 experimental results. The survival rate of the cells in the experimental groups decreased (P0.05) after the reduction of the expression of PARP-1, and the survival curve of the cells was reduced to PARP-1. The expression of CNE-2 cells increased the radiosensitivity of the cells. The results of flow cytometry showed that after the 6Gy ray irradiation, the apoptosis rate of the cells in the PARP-1 silence group was significantly increased (F=501.048, P0.01) compared with the NC group and the Control group (F=501.048, P0.01). The results of the clone formation experiment showed that the survival fraction of the cells of each dose point PARP-1 sink group decreased (P0.05), two times. Linear model fitting dose survival curve showed that down-regulation of PARP-1 expression could increase radiosensitivity of nasopharyngeal carcinoma CNE-2 cells.
[Conclusion]
Radiation can induce PARP-1 activation.PARP-1 to participate in the autophagy of nasopharyngeal carcinoma CNE-2 cells induced by ray, and the decrease of.PARP-1 gene expression may be in the corresponding signal pathway to increase the sensitivity of CNE-2 cells to radiation, that is to say, PARP-1 plays a protective role in the death of nasopharyngeal carcinoma CNE-2 cells caused by radiation.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.63
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