本文選題：HMGB1 + 食管鱗狀細(xì)胞癌��； 參考：《江蘇大學(xué)》2016年碩士論文

【摘要】：【目的】探討HMGB1表達(dá)對(duì)食管鱗狀細(xì)胞癌TE-1細(xì)胞放療敏感性的影響,并初步探討其分子調(diào)控機(jī)制�！痉椒ā�(1)應(yīng)用siRNA轉(zhuǎn)染技術(shù),通過(guò)Lipofectamine2000分別將空載體陰性對(duì)照(NCsiRNA)和HMGB1干擾載體(siHMGB1)轉(zhuǎn)染至人食管鱗狀細(xì)胞癌細(xì)胞株TE-1,按不同分組標(biāo)記為:TE-1組(空白對(duì)照組),TE-1+NCsiRNA組(空載體陰性對(duì)照組),TE-1+siHMGB1組(HMGB1沉默組);通過(guò)實(shí)時(shí)定量PCR法檢測(cè)各組TE-1細(xì)胞內(nèi)HMGB1mRNA的表達(dá)變化,Westernblot法進(jìn)一步驗(yàn)證轉(zhuǎn)染TE-1細(xì)胞內(nèi)HMGB1蛋白的表達(dá)改變;(2)對(duì)上述三組TE-1細(xì)胞在轉(zhuǎn)染后進(jìn)行不同劑量(0、2、4、6、8Gy)X射線照射培養(yǎng)一定的時(shí)間(24、48、72、96小時(shí)),通過(guò)MTT法檢測(cè)TE-1細(xì)胞的增殖率;(3)對(duì)上述三組TE-1細(xì)胞在轉(zhuǎn)染后進(jìn)行上述不同劑量X射線照射后培養(yǎng)15天,通克隆形成實(shí)驗(yàn)檢測(cè)TE-1細(xì)胞的克隆形成率;(4)上述轉(zhuǎn)染處理24小時(shí),給予4GyX射線照射后,流式細(xì)胞術(shù)檢測(cè)各組TE-1細(xì)胞的凋亡率、活性氧簇(reactiveoxygenspecies,ROS)含量和γH2AX水平;用實(shí)時(shí)定量PCR法檢測(cè)各組TE-1細(xì)胞NOX1與NOX5mRNA的表達(dá)變化;Westernblot法檢測(cè)各組TE-1細(xì)胞Caspase-3、CleavedPARP、p38、p-p38、JNK、p-JNK蛋白的表達(dá)變化�！窘Y(jié)果】(1)HMGB1沉默組TE-1細(xì)胞中HMGB1mRNA和蛋白的表達(dá)量均較空白對(duì)照組、空載體陰性對(duì)照組顯著降低(均P0.001);(2)MTT法檢測(cè)結(jié)果發(fā)現(xiàn)轉(zhuǎn)染后24、48、72、96小時(shí)HMGB1沉默組TE-1細(xì)胞增殖率低于空白對(duì)照組及空載體陰性對(duì)照組(均P0.05);轉(zhuǎn)染后24小時(shí)行4GyX射線照射24、48、72、96小時(shí)后,轉(zhuǎn)染前后三組TE-1細(xì)胞的增殖能力均受到抑制,而HMGB1沉默組TE-1細(xì)胞增殖抑制程度較其他兩組明顯(均P0.05);轉(zhuǎn)染后24小時(shí)經(jīng)不同劑量(0、2、4、6、8Gy)的X射線照射48小時(shí)后:隨著X射線照射劑量的增加,三組TE-1細(xì)胞的增殖率均降低,HMGB1沉默組細(xì)胞增殖抑制較兩對(duì)照組明顯(均P0.05);(3)Sigmaplot軟件分析結(jié)果顯示HMGB1沉默組D0、Dq、N值均低于空白對(duì)照組和空載體陰性對(duì)照組;HMGB1沉默組相對(duì)于空白對(duì)照組的放射增敏比(sensitizingenhancementratio,SER)為1.26;(4)流式細(xì)胞術(shù)檢測(cè)結(jié)果顯示:轉(zhuǎn)染后24小時(shí),HMGB1沉默組凋亡率、ROS含量和γH2AX水平高于兩對(duì)照組;實(shí)時(shí)定量PCR檢測(cè)結(jié)果顯示HMGB1沉默組NOX1和NOX5mRNA的表達(dá)高于兩對(duì)照組;Westernblot檢測(cè)結(jié)果顯示HMGB1沉默組Caspase-3、CleavedPARP、p-p38、p-JNK蛋白的表達(dá)高于兩對(duì)照組(均P0.05);轉(zhuǎn)染后24小時(shí)起給予4GyX射線照射,流式細(xì)胞術(shù)檢測(cè)結(jié)果顯示HMGB1沉默組凋亡率、ROS含量和γH2AX水平高于單純放療組;實(shí)時(shí)定量PCR檢測(cè)結(jié)果顯示HMGB1沉默組NOX1與NOX5mRNA的表達(dá)高于單純放療組;Westernblot檢測(cè)結(jié)果顯示HMGB1沉默組Caspase-3、CleavedPARP、p-p38、p-JNK蛋白的表達(dá)高于單純放療組(均P0.05)�！窘Y(jié)論】通過(guò)體外細(xì)胞實(shí)驗(yàn)研究,發(fā)現(xiàn)下調(diào)HMGB1表達(dá)能抑制人食管鱗狀細(xì)胞癌細(xì)胞株TE-1放療后的增殖率,降低其克隆形成能力,即下調(diào)HMGB1表達(dá)能提高TE-1細(xì)胞對(duì)放療的敏感性。其分子機(jī)制是下調(diào)HMGB1表達(dá)可能通過(guò)上調(diào)促細(xì)胞凋亡相關(guān)蛋白Caspase-3/CleavedPARP的表達(dá)以及上調(diào)ROS產(chǎn)生來(lái)影響DNA損傷,并通過(guò)JNK和p38通路激活,上調(diào)p-p38和p-JNK蛋白表達(dá),調(diào)控放療對(duì)TE-1細(xì)胞增殖和凋亡能力改變,進(jìn)而提高其對(duì)電離輻射的敏感性。
[Abstract]:[Objective] to investigate the effect of HMGB1 expression on the radiation sensitivity of esophageal squamous cell carcinoma TE-1 cells and to explore its molecular mechanism. [Methods] (1) siRNA transfection technique was used to transfect the empty carrier negative control (NCsiRNA) and HMGB1 interfering carrier (siHMGB1) to the human esophageal squamous cell carcinoma cell line TE, respectively. -1 was labeled as group TE-1 (blank control group), group TE-1+NCsiRNA (negative control group) and group TE-1+siHMGB1 (HMGB1 silence group); the expression of HMGB1mRNA in TE-1 cells in each group was detected by real-time quantitative PCR method, and Westernblot method was used to further verify the expression change of HMGB1 protein in the transfected TE-1 cells; (2) the three groups TE-1 were TE-1. After transfection, the cells were irradiated with different doses (0,2,4,6,8Gy) X ray for a certain time (24,48,72,96 hours), and the proliferation rate of TE-1 cells was detected by MTT method. (3) the above three groups of TE-1 cells were irradiated with different doses of X rays for 15 days after the transfection, and the cloning formation rate of TE-1 cells was detected by clonogenic formation; (4) After 24 hours of transfection, the apoptosis rate of TE-1 cells, the content of reactiveoxygenspecies (reactiveoxygenspecies, ROS) and the level of gamma H2AX were detected by flow cytometry, and the expression of NOX1 and NOX5mRNA in TE-1 cells in each group was detected by real-time quantitative PCR; Westernblot method was used to detect TE-1 cell Caspase-3. The expression of p38, JNK and p-JNK protein was changed. [results] the expression of HMGB1mRNA and protein in TE-1 cells of HMGB1 silencing group were all more than that in the blank control group, and the negative control group decreased significantly (P0.001). (2) the MTT assay results showed that the proliferation rate of TE-1 cells in the 24,48,72,96 hour HMGB1 silence group was lower than that of the blank control group and the empty body negative group. In the sex control group (all P0.05), the proliferation ability of the three groups of TE-1 cells after transfection for 24,48,72,96 hours after 24 hours of transfection was inhibited, while the proliferation inhibition of TE-1 cells in the HMGB1 silent group was more obvious than that in the other two groups (P0.05), and 24 hours after the transfection, the X ray of the same dose (0,2,4,6,8Gy) was irradiated for 48 hours after the transfection: with X. The proliferation rate of the three groups of TE-1 cells decreased, and the proliferation inhibition in the HMGB1 silencing group was significantly lower than that in the two control group (P0.05). (3) the Sigmaplot software analysis showed that the HMGB1 silenced group D0, Dq, N values were lower than the blank control group and the empty carrier negative control group; the HMGB1 silencing group was compared with the blank control group with the radiosensitivity ratio (SEN). Sitizingenhancementratio, SER) was 1.26. (4) flow cytometry results showed that the apoptosis rate of HMGB1 silencing group, ROS content and gamma H2AX level were higher than that of two control groups at 24 hours after transfection; real-time quantitative PCR detection showed that the expression of NOX1 and NOX5mRNA in HMGB1 silencing group was higher than that of the two control group; Westernblot test results showed HMGB1 silence group Caspase. The expression of -3, CleavedPARP, p-p38, p-JNK protein was higher than that of the two control group (all P0.05); 4GyX ray irradiation was given after the transfection. The results of flow cytometry showed the apoptosis rate of HMGB1 silencing group. The content of ROS and the level of gamma H2AX were higher than those in the simple radiotherapy group. Real time quantitative PCR detection showed that the expression of NOX1 in HMGB1 silence group was higher than that of simple group. The results of Westernblot test showed that the expression of Caspase-3, CleavedPARP, p-p38 and p-JNK protein in the HMGB1 silencing group was higher than that in the radiotherapy group (P0.05). [Conclusion] the expression of down regulated HMGB1 could inhibit the proliferation rate of human esophageal squamous cell carcinoma cell line after TE-1 radiotherapy and reduce the clone formation ability by in vitro cell test. Down regulation of HMGB1 expression can increase the sensitivity of TE-1 cells to radiotherapy. Its molecular mechanism is that the expression of HMGB1 may be regulated by up regulation of the expression of apoptosis related protein Caspase-3/CleavedPARP and up regulation of ROS production to affect DNA damage. The expression of p-p38 and p-JNK proteins is up regulated by JNK and p38 pathway, and the regulation of radiotherapy to TE-1 cells is regulated. The sensitivity of colony and apoptosis to ionizing radiation is improved.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.1

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