【摘要】：[目的]本試驗(yàn)旨在研究RNA干擾Dickkopf-2基因(DKK2)表達(dá)對小鼠子宮內(nèi)膜基質(zhì)細(xì)胞增殖和凋亡的影響。[方法]設(shè)計(jì)并合成3條針對DKK2基因的siRNA,在轉(zhuǎn)染試劑LipofectamineTM2000介導(dǎo)下轉(zhuǎn)染小鼠子宮內(nèi)膜基質(zhì)細(xì)胞,用RT-q PCR法檢測轉(zhuǎn)染前、后DKK2基因的表達(dá)量,篩選干擾效率最高的1條siRNA。用該siRNA轉(zhuǎn)染小鼠子宮內(nèi)膜基質(zhì)細(xì)胞24 h,利用Caspase3活性和Annexin-V FITC/PI雙染法檢測干擾DKK2基因?qū)?xì)胞凋亡的影響;用CCK8細(xì)胞計(jì)數(shù)法檢測該siRNA干擾DKK2基因不同時間對小鼠子宮內(nèi)膜基質(zhì)細(xì)胞增殖的影響。[結(jié)果]3條siRNA均能有效抑制DKK2基因的表達(dá),RT-q PCR結(jié)果顯示DKK2基因表達(dá)水平顯著降低(siRNA2,P0.05;siRNA1、siRNA3,P0.01);CCK8法結(jié)果顯示siRNA干擾DKK2后對小鼠子宮內(nèi)膜基質(zhì)細(xì)胞有明顯的抑制作用(24 h、48 h,P0.05);Caspase3活性檢測和Annexin-V FITC/PI雙染法結(jié)果顯示,siRNA1轉(zhuǎn)染小鼠子宮內(nèi)膜基質(zhì)細(xì)胞24 h后,Caspase3活性明顯增加(P0.01),細(xì)胞凋亡率明顯增加(P0.01),凋亡率為18.48%。[結(jié)論]利用RNA干擾技術(shù)沉默DKK2基因的表達(dá)可以明顯抑制小鼠子宮內(nèi)膜基質(zhì)細(xì)胞的增殖并促進(jìn)凋亡。
[Abstract]:[objective] to study the effect of RNA interfering Dickkopf-2 gene (DKK2) expression on the proliferation and apoptosis of mouse endometrial stromal cells. [methods] three siRNA, targeting DKK2 gene were designed and synthesized to transfect mouse endometrial stromal cells mediated by transfection reagent LipofectamineTM2000. The expression of DKK2 gene before and after transfection was detected by RT-q PCR method, and one siRNA. with the highest interfering efficiency was screened. Mouse endometrial stromal cells were transfected with siRNA for 24 h. The effect of interfering DKK2 gene on apoptosis was detected by Caspase3 activity and Annexin-V FITC/PI double staining. The effects of siRNA interfering with DKK2 gene on the proliferation of mouse endometrial stromal cells were detected by CCK8 cell count method. [results] all three siRNA could effectively inhibit the expression of DKK2 gene RT-q PCR. The results showed that the expression level of DKK2 gene decreased significantly (siRNA2,P0.05;siRNA1,siRNA3,P0.01) CCK8 method showed that siRNA interference with DKK2 could inhibit the activity of Caspase-3 in mouse endometrial stromal cells (24 h, 48 h, P0.05). The results of detection and Annexin-V FITC/PI double staining showed that the activity of caspase3 and apoptosis of mouse endometrial stromal cells were significantly increased (P0.01), and the apoptotic rate was 18.48% after transfection of siRNA1 into mouse endometrial stromal cells for 24 h (P0.01). [conclusion] silencing the expression of DKK2 gene by RNA interference can significantly inhibit the proliferation of mouse endometrial stromal cells and promote apoptosis.
【作者單位】： 南京農(nóng)業(yè)大學(xué)動物科技學(xué)院;
【分類號】：S814
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本文編號：2201196