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乏氧食管鱗癌細胞放射敏感性的影響及其機制研究目的：觀察鹽酸小檗堿對乏氧食管鱗癌細胞乏氧誘導(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)外實驗研究鹽酸小檗堿對多個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乏氧食管鱗癌的敏感性機制。方法：選用2種食管鱗癌細胞株ECA109、TE13,研究梯度濃度及不同時間點下鹽酸小檗堿處理腫瘤細胞,采用四甲基偶氮唑鹽比色法(MTT法)觀察鹽酸小檗堿對食管鱗癌細胞作用；采用免疫熒光技術(shù)觀察乏氧前后ECA109細胞HIF-1α表達情況,并觀察高低濃度鹽酸小檗堿作用后HIF-1α表達；應(yīng)用蛋白免疫印跡法(Western-blot)測定高低濃度鹽酸小檗堿處理后細胞HIF-1α、VEGF表達變化；分別給予常氧細胞、乏氧細胞、乏氧細胞聯(lián)合不同濃度下鹽酸小檗堿、順鉑陽性參照組6MV-X線照射處理后,平板克隆形成實驗觀察鹽酸小檗堿對乏氧食管鱗癌放射敏感性的影響；采用流式細胞術(shù)檢測AnnexinV-PI雙染色后各組細胞凋亡率；構(gòu)建裸鼠食管鱗癌ECA109移植瘤模型,設(shè)立對照組、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,測量腫瘤體積,免疫組化法檢測腫瘤內(nèi)HIF-1α、VEGF表達情況。結(jié)果：鹽酸小檗堿抑制食管鱗癌細胞ECA109和TE13的生長,具有時間以及劑量依賴性；通過單擊多靶模型擬合曲線,可見不同濃度的鹽酸小檗堿預(yù)處理乏氧食管鱗癌細胞24小時后,與照射組相比,增加乏氧食管鱗癌細胞的輻射損傷,鹽酸小檗堿作用乏氧細胞照射組的細胞凋亡率增加,且成劑量依賴性；鹽酸小檗堿作用于乏氧食管癌細胞時,可以使乏氧相關(guān)蛋白HIF-1α、VEGF的表達降低,并且呈現(xiàn)量效關(guān)系,且減少ECA109細胞內(nèi)核內(nèi)HIF-1α表達；鹽酸小檗堿可顯著抑制放療后ECA109細胞移植瘤模型裸鼠皮下腫瘤生長,免疫組化顯示鹽酸小檗堿可降低移植瘤內(nèi)HIF-1α、VEGF表達。結(jié)論：鹽酸小檗堿降低乏氧食管鱗癌細胞HIF-1α及VEGF表達,增加乏氧食管鱗癌細胞放射敏感性；抑制食管鱗癌裸鼠移植瘤放療后腫瘤組織生長。第二部分：乏氧誘導(dǎo)因子-1α抑制劑蜂毒肽對乏氧鼻咽癌細胞CNE-2放射敏感性的影響及其機制研究目的：觀察蜂毒肽對乏氧鼻咽癌細胞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CNE-2細胞株放射敏感性的影響,探討蜂毒肽增加乏氧鼻咽癌細胞放射敏感性的機制。方法：‘選用鼻咽癌細胞CNE-2進行研究,梯度濃度及不同時間點蜂毒肽處理腫瘤細胞后,采用CCK8法觀察蜂毒肽對CNE-2細胞增殖能力的影響。應(yīng)用蛋白免疫印跡法(Western-blot)測定不同濃度蜂毒肽處理后CNE-2細胞內(nèi)HIF-1α、 VEGF表達變化；分別給予常氧細胞、乏氧細胞,乏氧細胞聯(lián)合蜂毒肽、常氧細胞聯(lián)合蜂毒肽6MV-X線照射處理后,平板克隆形成實驗觀察蜂毒肽對乏氧CNE-2細胞放射敏感性的影響；AnnexinV-PI雙染色后采用流式細胞術(shù)檢測各組細胞凋亡率；構(gòu)建裸鼠CNE-2移植瘤模型,設(shè)立對照組、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,對移植瘤拍照并測量腫瘤體積。結(jié)果：蜂毒肽抑制CNE-2細胞生長,具有時間以及劑量依賴性；通過單擊多靶模型擬合曲線,可見蜂毒肽對常氧和乏氧鼻咽癌細胞均有增敏作用,且在乏氧組增敏效果更為明顯。相比于常氧細胞照射組,可以顯著增加常氧CNE-2細胞的輻射敏感性,而蜂毒肽對乏氧CNE-2細胞照射組中的細胞凋亡率增加更為明顯。蜂毒肽作用于乏氧CNE-2細胞,誘導(dǎo)乏氧相關(guān)蛋白HIF-1α、VEGF的表達降低,呈現(xiàn)量效關(guān)系。蜂毒肽抑制放療后CNE-2細胞移植瘤模型裸鼠皮下腫瘤生長。結(jié)論：蜂毒肽抑制乏氧鼻咽癌細胞CNE-2內(nèi)HIF-1α及其VEGF的表達,增加乏氧和常氧下CNE-2細胞放射敏感性；同時抑制鼻咽癌裸鼠移植瘤放療后腫瘤組織生長。蜂毒肽對增加常氧和乏氧情況下鼻咽癌細胞的放療敏感性,且對乏氧細胞作用更為明顯,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)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R73-36

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