Egr-1誘騙寡核苷酸下調(diào)PDGF抑制原代培養(yǎng)的血管平滑肌細(xì)胞增殖
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【摘要】： Egr-1誘騙寡核苷酸下調(diào)PDGF—A抑制原代培養(yǎng)的血管平滑肌細(xì)胞增殖 目的 通過向原代培養(yǎng)的大鼠動脈平滑肌細(xì)胞中轉(zhuǎn)染針對早期生長反應(yīng)因子-1(early growth response factor-1,Egr-1)的誘騙寡核苷酸(decoy oligonucleotide,decoy ODN),檢測血管平滑肌細(xì)胞增殖情況及早期生長反應(yīng)因子-1、血小板衍生生長因子-A(platelet derived growth factor,PDGF-A)的表達(dá)變化,探討針對早期生長反應(yīng)因子-1的誘騙寡核苷酸對體外培養(yǎng)的大鼠平滑肌細(xì)胞增殖的影響和作用機制。 方法 通過組織塊貼壁法進行原代大鼠動脈血管平滑肌細(xì)胞(Vascular smoorh muscle cells VSMC)培養(yǎng)、傳代培養(yǎng)和鑒定,設(shè)計合成Egr-1誘騙、誘騙對照寡核苷酸.轉(zhuǎn)染人工合成的針對Egr-1的decoy ODN,將培養(yǎng)的平滑肌細(xì)胞分為對照組(正常培養(yǎng)細(xì)胞)、誘騙組(轉(zhuǎn)染Egr-1的誘騙寡核苷酸)、誘騙對照組(轉(zhuǎn)染Egr-1誘騙雜碼寡核苷酸).免疫細(xì)胞化學(xué)染色法檢測轉(zhuǎn)染前后平滑肌細(xì)胞增殖情況與Egr-1及增殖細(xì)胞PDGF-A的表達(dá)變化,進行圖像分析。所有數(shù)據(jù)均采用SAS8.0統(tǒng)計軟件進行統(tǒng)計分析,計量資料以均值±標(biāo)準(zhǔn)差(x±S)表示,各組間比較應(yīng)用單因素方差分析,P0.05有統(tǒng)計學(xué)意義。 結(jié)果 成功進行原代VSMC培養(yǎng),經(jīng)細(xì)胞鑒定培養(yǎng)細(xì)胞為高純度的血管平滑肌細(xì)胞。轉(zhuǎn)染Egr-1誘騙寡核苷酸后,用免疫組織化學(xué)法分別觀察各組不同時間點的Egr-1和PDGF表達(dá)的情況發(fā)現(xiàn)：Egr-1在三個實驗組中均有表達(dá),誘騙轉(zhuǎn)染組與誘騙對照組和陰性對照組相比,均在一定程度上抑制了Egr-1和PDGF-A表達(dá),經(jīng)SAS8.0統(tǒng)計軟件進行統(tǒng)計分析,證明有統(tǒng)計學(xué)意義(P0.05)。 結(jié)論 本實驗證實Egr-1是一種重要的核轉(zhuǎn)錄因子,體外轉(zhuǎn)染針對Egr-1的誘騙寡核苷酸在一定程度上能夠抑制血管平滑肌細(xì)胞Egr-1及PDGF-A的表達(dá),從而達(dá)到抑制細(xì)胞增殖的目的,在抗動脈粥樣硬化和治療血管再狹窄方面具有重要作用,從而為基因治療再狹窄、防治動脈粥樣硬化開辟了一條新的途徑。
[Abstract]:Egr-1 decoy oligodeoxynucleotides down-regulate the proliferation of primary cultured vascular smooth muscle cells by down-regulation of PDGF-A by transfection into primary cultured rat arterial smooth muscle cells for early growth response Factor-1 (early growth response factor-1, Egr-1 was used to detect the proliferation of vascular smooth muscle cells and the expression of early growth response factor-1 and platelet-derived growth factor A (platelet derived growth factor,PDGF-A. To investigate the effect and mechanism of decoy oligonucleotides targeting early growth response factor-1 on the proliferation of rat smooth muscle cells in vitro. Methods the primary vascular smooth muscle cells (Vascular smoorh muscle cells VSMC) of rat artery were cultured, subcultured and identified by tissue block adherence method. Egr-1 decoy was designed and synthesized, and the control oligonucleotides were decoded. The cultured smooth muscle cells were divided into control group (normal cultured cells) and decoy group (decoy oligonucleotides transfected with Egr-1). Decoy control group (transfection of Egr-1 decoy code oligonucleotide). The proliferation of smooth muscle cells and the expression of Egr-1 and PDGF-A were detected by immunocytochemical staining before and after transfection. All the data were analyzed by SAS8.0 statistical software. The measurement data were expressed as mean 鹵standard deviation (x 鹵S). Results the primary VSMC was successfully cultured and the cultured cells were identified as high purity vascular smooth muscle cells. After transfection of Egr-1 to induce oligonucleotides, the expression of Egr-1 and PDGF at different time points in each group were observed by immunohistochemical method. The results showed that Egr-1 was expressed in all three experimental groups. The expression of Egr-1 and PDGF-A was inhibited to some extent in the decoy transfection group compared with the decoy control group and the negative control group. The statistical analysis by SAS8.0 software proved that there was statistical significance (P0.05). Conclusion Egr-1 is an important nuclear transcription factor. In vitro transfection of decoy oligonucleotides against Egr-1 can inhibit the expression of Egr-1 and PDGF-A in vascular smooth muscle cells to some extent. Thus, it can inhibit cell proliferation and play an important role in anti-atherosclerosis and vascular restenosis, thus opening a new way for gene therapy and prevention and treatment of atherosclerosis.
【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R363
本文編號：2347018
[Abstract]:Egr-1 decoy oligodeoxynucleotides down-regulate the proliferation of primary cultured vascular smooth muscle cells by down-regulation of PDGF-A by transfection into primary cultured rat arterial smooth muscle cells for early growth response Factor-1 (early growth response factor-1, Egr-1 was used to detect the proliferation of vascular smooth muscle cells and the expression of early growth response factor-1 and platelet-derived growth factor A (platelet derived growth factor,PDGF-A. To investigate the effect and mechanism of decoy oligonucleotides targeting early growth response factor-1 on the proliferation of rat smooth muscle cells in vitro. Methods the primary vascular smooth muscle cells (Vascular smoorh muscle cells VSMC) of rat artery were cultured, subcultured and identified by tissue block adherence method. Egr-1 decoy was designed and synthesized, and the control oligonucleotides were decoded. The cultured smooth muscle cells were divided into control group (normal cultured cells) and decoy group (decoy oligonucleotides transfected with Egr-1). Decoy control group (transfection of Egr-1 decoy code oligonucleotide). The proliferation of smooth muscle cells and the expression of Egr-1 and PDGF-A were detected by immunocytochemical staining before and after transfection. All the data were analyzed by SAS8.0 statistical software. The measurement data were expressed as mean 鹵standard deviation (x 鹵S). Results the primary VSMC was successfully cultured and the cultured cells were identified as high purity vascular smooth muscle cells. After transfection of Egr-1 to induce oligonucleotides, the expression of Egr-1 and PDGF at different time points in each group were observed by immunohistochemical method. The results showed that Egr-1 was expressed in all three experimental groups. The expression of Egr-1 and PDGF-A was inhibited to some extent in the decoy transfection group compared with the decoy control group and the negative control group. The statistical analysis by SAS8.0 software proved that there was statistical significance (P0.05). Conclusion Egr-1 is an important nuclear transcription factor. In vitro transfection of decoy oligonucleotides against Egr-1 can inhibit the expression of Egr-1 and PDGF-A in vascular smooth muscle cells to some extent. Thus, it can inhibit cell proliferation and play an important role in anti-atherosclerosis and vascular restenosis, thus opening a new way for gene therapy and prevention and treatment of atherosclerosis.
【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R363
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