RNA干擾技術(shù)是通過小的雙鏈RNA高效、特異的阻斷體內(nèi)特定基因的表達,促使mRNA降解,誘使細胞表現(xiàn)出特定基因缺失表達的技術(shù),是目前沉默某些致病基因功能的有效手段。本實驗通過構(gòu)建針對Survivin基因的siRNA真核表達載體轉(zhuǎn)入人喉癌HEP2細胞中。結(jié)果顯示:陰性對照序列表達載體轉(zhuǎn)染HEP2細胞后對Survivin表達無影響;而pRNATSurvivin轉(zhuǎn)染HEP2細胞后顯著抑制了Survivin的mRNA及蛋白表達(P<0.01),其中 pRNATSurvivin組中 Survivin mRNA表達水平比空白對照組最多下降了75.43%,而蛋白表達量比空白對照組最多下降了79.27%,提示本研究構(gòu)建的siRNA真核表達載體的確能夠在細胞內(nèi)持續(xù)地表達siRNA,高效、特異的抑制Survivin基因的表達。本實驗通過MTT法檢測轉(zhuǎn)染前后化療藥物抑制細胞生長的情況表明:穩(wěn)定轉(zhuǎn)染Survivin siRNA 真核表達載體后5FU、優(yōu)福定對細胞生長抑制作用增加。其中在轉(zhuǎn)染Survivin siRNA和5FU聯(lián)合作用下細胞生長抑制率最高達到(67.71±4.58)%,轉(zhuǎn)染Survivin siRNA和優(yōu)福定聯(lián)合作用下細胞生長抑制率最高達到(74.54±4.43)%,相比未轉(zhuǎn)染Survivin siRNA的空白對照組和陰性對照組有明顯提高(P<0.01)。以上研究結(jié)果顯示:用RNA干擾技術(shù)抑制Survivin表達造成腫瘤細胞對化療藥物的耐受性降低。
過表達Survivin可能并非激活腫瘤細胞化療藥物耐受性和抗凋亡能力所必需,但抑制Survivin表達可作為有效的調(diào)控信號,提高腫瘤細胞化療敏感性, 導(dǎo)致腫瘤細胞凋亡。本研究通過RNA干擾技術(shù)的應(yīng)用為基于調(diào)控細胞凋亡的生物治療與化療聯(lián)合治療喉癌提供了新的實驗依據(jù)。
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