乏氧誘導(dǎo)因子-1α抑制劑鹽酸小檗堿和蜂毒肽對(duì)乏氧腫瘤細(xì)胞放射敏感性的影響及機(jī)制研究
發(fā)布時(shí)間:2018-04-21 09:44
本文選題:放射治療 + 鹽酸小檗堿; 參考:《南京醫(yī)科大學(xué)》2016年博士論文
【摘要】:第一部分:乏氧誘導(dǎo)因子-1α抑制劑鹽酸小檗堿對(duì)乏氧食管鱗癌細(xì)胞放射敏感性的影響及其機(jī)制研究目的:觀察鹽酸小檗堿對(duì)乏氧食管鱗癌細(xì)胞乏氧誘導(dǎo)因子-1α (hypoxia inducible factor-1α, HIF-1 α)及其下游靶基因血管內(nèi)皮生成因子(vascμ lar endothelial growth factor, VEGF)的作用,體內(nèi)外實(shí)驗(yàn)研究鹽酸小檗堿對(duì)多個(gè)乏氧食管鱗癌放射敏感性的影響,探討鹽酸小檗堿增加放射線對(duì)乏氧食管鱗癌的敏感性機(jī)制。方法:選用2種食管鱗癌細(xì)胞株ECA109、TE13,研究梯度濃度及不同時(shí)間點(diǎn)下鹽酸小檗堿處理腫瘤細(xì)胞,采用四甲基偶氮唑鹽比色法(MTT法)觀察鹽酸小檗堿對(duì)食管鱗癌細(xì)胞作用;采用免疫熒光技術(shù)觀察乏氧前后ECA109細(xì)胞HIF-1α表達(dá)情況,并觀察高低濃度鹽酸小檗堿作用后HIF-1α表達(dá);應(yīng)用蛋白免疫印跡法(Western-blot)測(cè)定高低濃度鹽酸小檗堿處理后細(xì)胞HIF-1α、VEGF表達(dá)變化;分別給予常氧細(xì)胞、乏氧細(xì)胞、乏氧細(xì)胞聯(lián)合不同濃度下鹽酸小檗堿、順鉑陽(yáng)性參照組6MV-X線照射處理后,平板克隆形成實(shí)驗(yàn)觀察鹽酸小檗堿對(duì)乏氧食管鱗癌放射敏感性的影響;采用流式細(xì)胞術(shù)檢測(cè)AnnexinV-PI雙染色后各組細(xì)胞凋亡率;構(gòu)建裸鼠食管鱗癌ECA109移植瘤模型,設(shè)立對(duì)照組、單純照射組、腹腔注射鹽酸小檗堿組、腹腔注射鹽酸小檗堿聯(lián)合照射組,予照射組皮下移植瘤8Gy單次大劑量放療,放療14天后處死裸鼠,測(cè)量腫瘤體積,免疫組化法檢測(cè)腫瘤內(nèi)HIF-1α、VEGF表達(dá)情況。結(jié)果:鹽酸小檗堿抑制食管鱗癌細(xì)胞ECA109和TE13的生長(zhǎng),具有時(shí)間以及劑量依賴性;通過(guò)單擊多靶模型擬合曲線,可見(jiàn)不同濃度的鹽酸小檗堿預(yù)處理乏氧食管鱗癌細(xì)胞24小時(shí)后,與照射組相比,增加乏氧食管鱗癌細(xì)胞的輻射損傷,鹽酸小檗堿作用乏氧細(xì)胞照射組的細(xì)胞凋亡率增加,且成劑量依賴性;鹽酸小檗堿作用于乏氧食管癌細(xì)胞時(shí),可以使乏氧相關(guān)蛋白HIF-1α、VEGF的表達(dá)降低,并且呈現(xiàn)量效關(guān)系,且減少ECA109細(xì)胞內(nèi)核內(nèi)HIF-1α表達(dá);鹽酸小檗堿可顯著抑制放療后ECA109細(xì)胞移植瘤模型裸鼠皮下腫瘤生長(zhǎng),免疫組化顯示鹽酸小檗堿可降低移植瘤內(nèi)HIF-1α、VEGF表達(dá)。結(jié)論:鹽酸小檗堿降低乏氧食管鱗癌細(xì)胞HIF-1α及VEGF表達(dá),增加乏氧食管鱗癌細(xì)胞放射敏感性;抑制食管鱗癌裸鼠移植瘤放療后腫瘤組織生長(zhǎng)。第二部分:乏氧誘導(dǎo)因子-1α抑制劑蜂毒肽對(duì)乏氧鼻咽癌細(xì)胞CNE-2放射敏感性的影響及其機(jī)制研究目的:觀察蜂毒肽對(duì)乏氧鼻咽癌細(xì)胞CNE-2乏氧誘導(dǎo)因子-1α (hypoxia inducible factor-1α, HIF-1α)及其下游靶基因血管內(nèi)皮生成因子(vascμ lar endothelial growth factor, VEGF)的抑制情況,研究蜂毒肽對(duì)CNE-2細(xì)胞株放射敏感性的影響,探討蜂毒肽增加乏氧鼻咽癌細(xì)胞放射敏感性的機(jī)制。方法:‘選用鼻咽癌細(xì)胞CNE-2進(jìn)行研究,梯度濃度及不同時(shí)間點(diǎn)蜂毒肽處理腫瘤細(xì)胞后,采用CCK8法觀察蜂毒肽對(duì)CNE-2細(xì)胞增殖能力的影響。應(yīng)用蛋白免疫印跡法(Western-blot)測(cè)定不同濃度蜂毒肽處理后CNE-2細(xì)胞內(nèi)HIF-1α、 VEGF表達(dá)變化;分別給予常氧細(xì)胞、乏氧細(xì)胞,乏氧細(xì)胞聯(lián)合蜂毒肽、常氧細(xì)胞聯(lián)合蜂毒肽6MV-X線照射處理后,平板克隆形成實(shí)驗(yàn)觀察蜂毒肽對(duì)乏氧CNE-2細(xì)胞放射敏感性的影響;AnnexinV-PI雙染色后采用流式細(xì)胞術(shù)檢測(cè)各組細(xì)胞凋亡率;構(gòu)建裸鼠CNE-2移植瘤模型,設(shè)立對(duì)照組、單純照射組、腹腔注射蜂毒肽組、腹腔注射蜂毒肽聯(lián)合照射組,予照射組皮下移植瘤6Gy單次大劑量放療,放療14天后處死裸鼠,對(duì)移植瘤拍照并測(cè)量腫瘤體積。結(jié)果:蜂毒肽抑制CNE-2細(xì)胞生長(zhǎng),具有時(shí)間以及劑量依賴性;通過(guò)單擊多靶模型擬合曲線,可見(jiàn)蜂毒肽對(duì)常氧和乏氧鼻咽癌細(xì)胞均有增敏作用,且在乏氧組增敏效果更為明顯。相比于常氧細(xì)胞照射組,可以顯著增加常氧CNE-2細(xì)胞的輻射敏感性,而蜂毒肽對(duì)乏氧CNE-2細(xì)胞照射組中的細(xì)胞凋亡率增加更為明顯。蜂毒肽作用于乏氧CNE-2細(xì)胞,誘導(dǎo)乏氧相關(guān)蛋白HIF-1α、VEGF的表達(dá)降低,呈現(xiàn)量效關(guān)系。蜂毒肽抑制放療后CNE-2細(xì)胞移植瘤模型裸鼠皮下腫瘤生長(zhǎng)。結(jié)論:蜂毒肽抑制乏氧鼻咽癌細(xì)胞CNE-2內(nèi)HIF-1α及其VEGF的表達(dá),增加乏氧和常氧下CNE-2細(xì)胞放射敏感性;同時(shí)抑制鼻咽癌裸鼠移植瘤放療后腫瘤組織生長(zhǎng)。蜂毒肽對(duì)增加常氧和乏氧情況下鼻咽癌細(xì)胞的放療敏感性,且對(duì)乏氧細(xì)胞作用更為明顯,這一現(xiàn)象可能與HIF-1 α相關(guān)。
[Abstract]:The effects of berberine on the radiosensitivity of hypoxia inducible factor - 1偽 , HIF - 1偽 and vasc 渭lar endothelial growth factor ( VEGF ) were studied .
The expression of HIF - 1偽 in ECA109 cells before and after hypoxia was observed by immunofluorescence technique , and the expression of HIF - 1偽 was observed after berberine hydrochloride .
Western - blot was used to determine the expression of HIF - 1偽 and VEGF after treatment with berberine hydrochloride .
The effects of berberine hydrochloride on the radiosensitivity of patients with hypoxic esophageal squamous cell carcinoma were observed by using berberine hydrochloride and cisplatin positive reference group ( 6MV - X ) .
Flow cytometry was used to detect the apoptosis rate of each group after AnnexinV - PI double staining .
The growth , time and dose dependence of ECA109 and TE13 of esophageal squamous cell carcinoma were detected by using berberine hydrochloride combined with 8 Gy single - dose radiation therapy . The results showed that berberine hydrochloride could inhibit the growth of ECA109 and TE13 in esophageal squamous carcinoma cells , and had time and dose - dependent manner .
After 24 hours of pretreatment of hypoxic esophageal squamous cell carcinoma cells with berberine hydrochloride pretreatment with different concentrations of berberine hydrochloride , the apoptosis rate of cells irradiated by berberine hydrochloride was increased compared with the irradiation group , and the dose - dependent manner was observed .
When berberine hydrochloride acts on hypoxic esophageal cancer cells , the expression of HIF - 1偽 and VEGF in hypoxia - related protein can be reduced , and the expression of HIF - 1偽 in the inner core of ECA109 cells is reduced .
Conclusion : berberine hydrochloride can decrease the expression of HIF - 1偽 and VEGF and increase the radiosensitivity of hypoxic esophageal squamous carcinoma cells .
Objective : To investigate the effect of bee venom peptide on the radiosensitivity of CNE - 2 cell line CNE - 2 and its mechanism , and to investigate the effect of bee venom peptide on the radiosensitivity of CNE - 2 cell line CNE - 2 . The expression of HIF - 1偽 and VEGF in CNE - 2 cells was investigated by Western - blot .
【學(xué)位授予單位】:南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】:博士
【學(xué)位授予年份】:2016
【分類號(hào)】:R73-36
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