目的研究ghrelin是否能夠抑制結(jié)腸上皮細胞凋亡,觀察一些凋亡相關(guān)蛋白表達水平的變化,并進一步探討ghrelin是否通過PI3K/Akt和ERK1/2信號通路抑制結(jié)腸上皮細胞凋亡。方法1.通過RT-PCR檢測HT-29結(jié)腸癌細胞株上是否有GHS-R1a的表達。2.將細胞分為對照組和處理組,處理組細胞由5-Fu（80μM）單獨或與ghrelin(10^-9M-10^-6M)共同處理24h-48h。3.通過MTT法對各組細胞進行生長活性的檢測,計算細胞生存率。4.通過DAPI染色后在熒光顯微鏡下觀察各組細胞核的形態(tài)。5.通過Annexin V/PI雙染色后行流式細胞術(shù),測定并比較各組細胞的凋亡率。6.通過Western-blot觀察凋亡相關(guān)蛋白caspase3、bcl-2、bax及信號傳導(dǎo)蛋白Akt、P-Akt、ERK1/2、P-ERK1/2的表達水平在各組中的變化。結(jié)果1.RT-PCR檢測到HT-29細胞上有GHS-R1a的表達。2.MTT法顯示ghrelin呈劑量依賴性地降低了5-Fu引起的HT-29細胞生長抑制,提高了細胞生存率（P＜0.05... 

【文章來源】：復(fù)旦大學(xué)上海市 211工程院校 985工程院校 教育部直屬院校

【文章頁數(shù)】：52 頁

【學(xué)位級別】：碩士

【部分圖文】：

凋亡調(diào)節(jié)通路

選用卜aetion，引物序列為(「):5一e丁ee奸eeAee仃eAee6仃ee一3，引物擴增長度為26sbp。擴增以94oC305，50℃305，72oCZmin為一循環(huán)，10min。圖1為PcR產(chǎn)物的凝膠電泳掃描圖像，第道是內(nèi)參p一action，其位置在250bp偏上;第三oobp之間有一明顯條帶，與GHs一Rla大小相符有GHS一Rla的表達。

圖2ghrelin對H下29細胞生長的影響注:與對照組比較，*p<0.05;與s一「u組比較，△P<0，05。OAPI染色典型的凋亡細胞核呈現(xiàn)染色質(zhì)的濃縮、邊集、核固縮、核碎裂，染色3中A、B、e依次是對照組、5一「uso協(xié)M處理組，s一「u+Gio一6M處理組，時間為24h。A圖中細胞核基本都是正常形態(tài);B圖中可見核固縮、

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