Mfn2基因抑制血管平滑肌细胞增殖的功能序列及肽研究
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摘要
第一部分
携带Mfn2基因不同结构序列的重组腺病毒的构建、扩增和纯化
1.目的
采用DNA重组技术,构建、制备、鉴定、扩增和纯化含线粒体融合素2基因(Mitofusin 2, Mfn2)不同结构序列的重组腺病毒。
2.方法
利用聚合酶链反应(Polymerase chain reaction, PCR)扩增大鼠Mfn2不同区段的互补DNA (cDNAs),将PCR产物连接到TA克隆载体(pCR8/GW/TOPO)上,筛选出含有正确片段的质粒。利用Gateway方法将含有正确片段的重组质粒与腺病毒目的质粒(pAd/CMV/V5-DEST),在LR Clonase的帮助下进行重组反应。用PacI将所得到的重组腺病毒载体切开,再利用脂质体(Lipofectamine 2000)包装后转染到293细胞中,产生重组的腺病毒。经测序鉴定无误后进行大量扩增,利用氯化铯密度梯度离心法,超高速离心纯化病毒,分装冻存于-80℃中。用紫外分光光度仪测定病毒液光密度(Optical density, OD)值,计算病毒滴度。采用Western blot方法检测目的基因在大鼠血管平滑肌细胞(Vascular smooth muscle cells, VSMCs)中的表达。
3.结果
8种目的基因结构序列(1A、2A、3A、4A、5A、6A、7A、8A)正确,通过PCR鉴定可检测到相应条带。8种重组腺病毒的滴度分别为27.8×109 pfu/ml、24×109 pfu/ml、19×109 pfu/ml、30.8×109 pfu/ml、6.7×109 pfu/ml、12.7×109 pfu/ml、27.6×109 pfu/ml、11.8×109 pfu/ml。Western blot检测结果显示相应的Mfn2蛋白表达。
4.结论
利用DNA重组技术可成功构建携带Mfn2基因不同结构序列的重组腺病毒,并通过扩增和纯化获得大量高滴度的病毒液,为进一步研究其对VSMCs增殖和凋亡的影响奠定了基础。
第二部分
Mfn2基因不同结构序列对血管平滑肌细胞增殖和凋亡的影响
1.目的
研究大鼠线粒体融合素2基因(Mitofusin 2, Mfn2)不同结构序列对大鼠血管平滑肌细胞(Vascular smooth muscle cells, VSMCs)增殖和凋亡的影响,寻找能有效抑制VSMCs增殖和促进凋亡的最小cDNA (scDNA),并探讨其相关的信号通路。
2.方法
利用携带Mfn2基因不同结构序列的重组腺病毒感染大鼠VSMCs.采用细胞计数法、水溶性四氮唑盐(WST-8,CCK-8)法比较其对VSMCs增殖的影响；流式细胞术比较各组细胞周期的变化；流式细胞术和细胞凋亡ELISA观察各组对VSMCs凋亡的影响；Western blot法分析各组磷酸化Raf-1 (p-Raf-1)、磷酸化ERK1/2 (p-ERK1/2)、磷酸化Akt (p-Akt)蛋白表达的变化。
3.结果
细胞计数和CCK-8结果显示,与对照组相比,Mfn2基因不同结构序列对VSMCs的增殖均有不同程度的抑制作用(P<0.01),其中以1A、4A和8A的抑制效果最明显；流式细胞仪细胞周期检测结果表明,Mfn2基因不同结构序列较对照组均能不同程度使VSMCs生长停滞于G0/G1期(P<0.01),其中以1A、4A和8A最为明显,进入S期的细胞比例分别为(59.85±3.26)%、(34.73±2.65)%和(60.73±2.69)%；流式细胞仪Annexin V-FITC细胞凋亡检测结果表明,与对照组相比,1A、4A和8A均有明显的促VSMCs凋亡作用(P<0.01),且呈时间依赖性(48h-72h)；细胞凋亡ELISA结果显示,与对照组相比,1A、4A和8A均有显著的促VSMCs凋亡作用(P)<0.01); Western blot检测结果显示,1A、4A和8A的p-Raf-1、p-ERK1/2和p-Akt表达水平较对照组均有明显降低(P<0.01)。
4.结论
与Mfn2全长序列(8A, Adv-Mfn2)和去除穿膜区的Mfn2序列(4A, Adv-tMfn2)相比,Mfn2最小结构序列1A同样具有显著抑制VSMCs增殖的作用,使细胞周期停滞于G0/G1期,这一作用主要是使Ras-Raf-ERK1/2通路受到抑制,p-Raf-1和p-ERK1/2的表达下调,从而发挥抑制血管平滑肌细胞增殖的作用。此外,1A也具有促VSMCs凋亡的作用,该作用主要是通过抑制.Ras-PI3K-Akt信号通路而实现。
第三部分
Mfn2基因相关合成肽对血管平滑肌细胞增殖和凋亡的影响
1.目的
研究Mfn2基因相关合成肽(Mfn2 gene-related synthetic peptide, MRSP)对大鼠血管平滑肌细胞(Vascular smooth muscle cells, VSMCs)增殖和凋亡的影响,并探讨其相关的信号通路。
2.方法
在Mfn2最小功能序列1A的C端加上TAT穿膜多肽,人工合成MRSP。采用细胞计数法、水溶性四氮唑盐(WST-8, CCK-8)法观察不同浓度的MRSP(1μM、10μM、25μM、50μM、100μM)对VSMCs增殖的影响；流式细胞术比较不同浓度MRSP对VSMCs细胞周期的影响；细胞凋亡ELISA和流式细胞术观察MRSP对VSMCs凋亡的影响；Western blot法分析各组磷酸化Raf-1 (p-Raf-1)、磷酸化ERK1/2(p-ERK1/2)、磷酸化Akt (p-Akt)蛋白表达的变化。
3.结果
人工合成MRSP为白色冻干粉末,纯度97.2%,分子量3305,水溶性良好(PBS中的溶解度为1mg/ml)。细胞计数和CCK-8结果均显示,与对照组相比,MRSP在10μM～100μM范围内可成剂量和时间依赖性的抑制VSMCs的增殖(P<0.01)。流式细胞仪细胞周期检测结果表明,不同浓度的MRSP (10μM、25μM.50μM)较对照组均能使VSMCs生长停滞于G0/G1期(P<0.01),G0/G1期的细胞比例分别为(24.90±3.46)%、(36.85±3.57)%和(62.45±2.83)%；流式细胞仪Annexin V-FITC细胞凋亡检测结果表明,与对照组相比,随着时间的延长(24h、48h、72h),25μM MRSP可有效诱导VSMCs的凋亡,作用72h时的细胞凋亡率为(30.4±2.1)%(P<0.01)；细胞凋亡ELISA结果表明,随着时间的延长,25μM MRSP与对照组相比有显著的促VSMCs凋亡作用(P<0.01)；Western blot检测结果显示,与对照组相比,MRSP可成浓度依赖性(10μM、25μM、50μM)的抑制p-Raf-1、p-ERK1/2和p-Akt的表达水平(P<0.01)。
4.结论
根据Mfn2的最小功能序列人工合成的MRSP具有显著抑制VSMCs增殖的作用,使细胞周期停滞于G0/G1期,这一作用主要是使Ras-Raf-ERK1/2通路受到抑制,p-Raf-1和p-ERK1/2的表达下调,从而发挥抑制血管平滑肌细胞增殖的作用。此外,MRSP也具有促VSMCs凋亡的作用,该作用主要是通过抑制]Ras-PI3K-Akt信号通路而实现。
Construction, amplification and purification of recombinant adenoviruses containing various sequence structures of Mfn2
1. Objective
To construct, identify and purify the recombinant adenoviral vector containing various sequence structures of Mfn2.
2. Methods
Various complementary DNA (cDNA) was first amplified and subcloned into pCR8/GW/TOPO TA cloning plasmid. Subsequently, we performed LR recombination reaction between the pCR8/GW/TOPO-tMfn2 and pAd/CMV/V5-DEST to obtain the adenoviral expression vector. After packaging the pAd/CMV/V5-DEST-Mfn2 in 293 cells, the replication-defective adenovirus was produced. CsCl density gradient centrifugation was used to measure the titer of various adenovirus. Western blot analysis was used to confirm the expression of Mfn2 at the protein level.
3. Results
The recombinant adenoviruses which carried various sequence structures of Mfn2 (1 A, 2A,3A,4A,5A,6A,7A,8A) was successfully constructed and identified by PCR. The titer of recombinant adenoviruses was 27.8×109 pfu/ml,24×109 pfu/ml,19×109 pfu/ml, 30.8×109 pfu/ml,6.7×109 pfu/ml,12.7×109 pfu/ml,27.6×109 pfu/ml and 11.8×109 pfu/ml, respectively. Western blot analysis showed the expression of Mfn2 protein in infected vascular smooth muscle cells (VSMCs).
4. Conclusions
The recombinant adenoviruses which carried various sequence structures of Mfn2 can be successfully constructed by Recombinant DNA technology, which provides the basis for further study on the effect of various Mfn2 sequence structures on the proliferation and apoptosis of VSMCs.
Part II
Various sequence structures of Mfn2 on proliferation and apoptosis of vascular smooth muscle cells (VSMCs)
1. Objective
To investigate the effect of various sequence structures of Mfn2 on proliferation and apoptosis of vascular smooth muscle cells (VSMCs), seek the shortest complementary DNA (scDNA) that can both effectively inhibit proliferation of VSMCs and induce apoptosis of VSMCs and explore the potential molecular mechanisms.
2. Methods
VSMCs were infected by recombinant adenoviruses containing various sequence structures of Mfn2 (1A,2A,3A,4A,5A,6A,7A,8A). The effect of various sequence structures on proliferation of VSMCs were tested by cell counting and CCK-8, the cell cycle was observed by flow cytometry; Cell death ELISA and flow cytometry were used to investigate the effect on apoptosis of VSMCs. Western blot analysis was used to detect the expression of phosphorylation of Raf (p-Raf), ERK1/2 (p-ERK1/2) and protein kinase B (p-Akt).
3. Results
Cell counting and CCK-8 both indicated that various sequence structures of Mfn2 significantly inhibited proliferation of VSMCs (P<0.01). Most of the VSMCs were blocked in the stage of G0/G1 and few entered into the S phase in all sequence structures groups (P<0.01). Cell Death ELISA assay and flow cytometry both indicated that 1A,4A and 8A promoted apoptosis of VSMCs remarkably. Western blot analysis showed that 1A, 4A and 8A significantly down-regulated expression of phosphorylation of Raf (p-Raf), ERK1/2 (p-ERK1/2) and protein kinase B (p-Akt) (P<0.01).
4. Conclusions
1A is the shortest complementary DNA (scDNA) of Mfn2 that can both effectively inhibit proliferation of VSMCs via Ras-Raf-ERK1/2 signaling pathway and induce apoptosis of VSMCs via Ras-PI3K-Akt signaling pathway.
PartⅢ
Mfn2 gene-related synthetic peptide (MRSP) on proliferation and apoptosis of vascular smooth muscle cells (VSMCs)
1. Objective
To investigate the effect of Mfn2 gene-related synthetic peptide (MRSP) on proliferation and apoptosis of vascular smooth muscle cells (VSMCs) and the potential molecular mechanisms.
2. Methods
MRSP was synthesized according to the shortest complementary DNA (scDNA) of Mfn2 (1A) and TAT-PTD (transactiviting-protein transduction domain). VSMCs were cultured in DMEM containing MRSP of various concentration (1μM,10μM,25μM,50μM, 10μM). The effect of various concentration of MRSP on proliferation of VSMCs were tested by cell counting and CCK-8, the cell cycle was observed by flow cytometry;; Cell death ELISA and flow cytometry were used to investigate the effect on apoptosis of VSMCs. Western blot analysis was used to detect the expression of phosphorylation of Raf (p-Raf), ERK1/2 (p-ERK1/2) and protein kinase B (p-Akt).
3. Results
Cell counting and CCK-8 both indicated that MRSP caused a marked dose-dependent and time-dependent proliferation inhibitory effect on VSMCs from 10μM to 100μM (P< 0.01). Flow cytometry showed that most of the VSMCs were inhibited in the stage of G0/G1 and few cells entered into the S phase in all the three MRSP groups (P<0.01). Cell Death ELISA assay and flow cytometry both indicated that MRSP promoted apoptosis of VSMCs effectively. Western blot analysis showed that MRSP significantly down-regulated expression of phosphorylation of Raf (p-Raf), ERK1/2 (p-ERK1/2) and protein kinase B (p-Akt) (P<0.01).
4. Conclusions
The MRSP synthesized according to the shortest complementary DNA (scDNA) of Mfn2 can significantly inhibit the proliferation of VSMCs via the Ras-Raf-ERK1/2 signaling pathway, it can also effectively induce the apoptosis of VSMCs via the PI3K-Akt signaling pathway.
携带Mfn2基因不同结构序列的重组腺病毒的构建、扩增和纯化
1.目的
采用DNA重组技术,构建、制备、鉴定、扩增和纯化含线粒体融合素2基因(Mitofusin 2, Mfn2)不同结构序列的重组腺病毒。
2.方法
利用聚合酶链反应(Polymerase chain reaction, PCR)扩增大鼠Mfn2不同区段的互补DNA (cDNAs),将PCR产物连接到TA克隆载体(pCR8/GW/TOPO)上,筛选出含有正确片段的质粒。利用Gateway方法将含有正确片段的重组质粒与腺病毒目的质粒(pAd/CMV/V5-DEST),在LR Clonase的帮助下进行重组反应。用PacI将所得到的重组腺病毒载体切开,再利用脂质体(Lipofectamine 2000)包装后转染到293细胞中,产生重组的腺病毒。经测序鉴定无误后进行大量扩增,利用氯化铯密度梯度离心法,超高速离心纯化病毒,分装冻存于-80℃中。用紫外分光光度仪测定病毒液光密度(Optical density, OD)值,计算病毒滴度。采用Western blot方法检测目的基因在大鼠血管平滑肌细胞(Vascular smooth muscle cells, VSMCs)中的表达。
3.结果
8种目的基因结构序列(1A、2A、3A、4A、5A、6A、7A、8A)正确,通过PCR鉴定可检测到相应条带。8种重组腺病毒的滴度分别为27.8×109 pfu/ml、24×109 pfu/ml、19×109 pfu/ml、30.8×109 pfu/ml、6.7×109 pfu/ml、12.7×109 pfu/ml、27.6×109 pfu/ml、11.8×109 pfu/ml。Western blot检测结果显示相应的Mfn2蛋白表达。
4.结论
利用DNA重组技术可成功构建携带Mfn2基因不同结构序列的重组腺病毒,并通过扩增和纯化获得大量高滴度的病毒液,为进一步研究其对VSMCs增殖和凋亡的影响奠定了基础。
第二部分
Mfn2基因不同结构序列对血管平滑肌细胞增殖和凋亡的影响
1.目的
研究大鼠线粒体融合素2基因(Mitofusin 2, Mfn2)不同结构序列对大鼠血管平滑肌细胞(Vascular smooth muscle cells, VSMCs)增殖和凋亡的影响,寻找能有效抑制VSMCs增殖和促进凋亡的最小cDNA (scDNA),并探讨其相关的信号通路。
2.方法
利用携带Mfn2基因不同结构序列的重组腺病毒感染大鼠VSMCs.采用细胞计数法、水溶性四氮唑盐(WST-8,CCK-8)法比较其对VSMCs增殖的影响；流式细胞术比较各组细胞周期的变化；流式细胞术和细胞凋亡ELISA观察各组对VSMCs凋亡的影响；Western blot法分析各组磷酸化Raf-1 (p-Raf-1)、磷酸化ERK1/2 (p-ERK1/2)、磷酸化Akt (p-Akt)蛋白表达的变化。
3.结果
细胞计数和CCK-8结果显示,与对照组相比,Mfn2基因不同结构序列对VSMCs的增殖均有不同程度的抑制作用(P<0.01),其中以1A、4A和8A的抑制效果最明显；流式细胞仪细胞周期检测结果表明,Mfn2基因不同结构序列较对照组均能不同程度使VSMCs生长停滞于G0/G1期(P<0.01),其中以1A、4A和8A最为明显,进入S期的细胞比例分别为(59.85±3.26)%、(34.73±2.65)%和(60.73±2.69)%；流式细胞仪Annexin V-FITC细胞凋亡检测结果表明,与对照组相比,1A、4A和8A均有明显的促VSMCs凋亡作用(P<0.01),且呈时间依赖性(48h-72h)；细胞凋亡ELISA结果显示,与对照组相比,1A、4A和8A均有显著的促VSMCs凋亡作用(P)<0.01); Western blot检测结果显示,1A、4A和8A的p-Raf-1、p-ERK1/2和p-Akt表达水平较对照组均有明显降低(P<0.01)。
4.结论
与Mfn2全长序列(8A, Adv-Mfn2)和去除穿膜区的Mfn2序列(4A, Adv-tMfn2)相比,Mfn2最小结构序列1A同样具有显著抑制VSMCs增殖的作用,使细胞周期停滞于G0/G1期,这一作用主要是使Ras-Raf-ERK1/2通路受到抑制,p-Raf-1和p-ERK1/2的表达下调,从而发挥抑制血管平滑肌细胞增殖的作用。此外,1A也具有促VSMCs凋亡的作用,该作用主要是通过抑制.Ras-PI3K-Akt信号通路而实现。
第三部分
Mfn2基因相关合成肽对血管平滑肌细胞增殖和凋亡的影响
1.目的
研究Mfn2基因相关合成肽(Mfn2 gene-related synthetic peptide, MRSP)对大鼠血管平滑肌细胞(Vascular smooth muscle cells, VSMCs)增殖和凋亡的影响,并探讨其相关的信号通路。
2.方法
在Mfn2最小功能序列1A的C端加上TAT穿膜多肽,人工合成MRSP。采用细胞计数法、水溶性四氮唑盐(WST-8, CCK-8)法观察不同浓度的MRSP(1μM、10μM、25μM、50μM、100μM)对VSMCs增殖的影响；流式细胞术比较不同浓度MRSP对VSMCs细胞周期的影响；细胞凋亡ELISA和流式细胞术观察MRSP对VSMCs凋亡的影响；Western blot法分析各组磷酸化Raf-1 (p-Raf-1)、磷酸化ERK1/2(p-ERK1/2)、磷酸化Akt (p-Akt)蛋白表达的变化。
3.结果
人工合成MRSP为白色冻干粉末,纯度97.2%,分子量3305,水溶性良好(PBS中的溶解度为1mg/ml)。细胞计数和CCK-8结果均显示,与对照组相比,MRSP在10μM～100μM范围内可成剂量和时间依赖性的抑制VSMCs的增殖(P<0.01)。流式细胞仪细胞周期检测结果表明,不同浓度的MRSP (10μM、25μM.50μM)较对照组均能使VSMCs生长停滞于G0/G1期(P<0.01),G0/G1期的细胞比例分别为(24.90±3.46)%、(36.85±3.57)%和(62.45±2.83)%；流式细胞仪Annexin V-FITC细胞凋亡检测结果表明,与对照组相比,随着时间的延长(24h、48h、72h),25μM MRSP可有效诱导VSMCs的凋亡,作用72h时的细胞凋亡率为(30.4±2.1)%(P<0.01)；细胞凋亡ELISA结果表明,随着时间的延长,25μM MRSP与对照组相比有显著的促VSMCs凋亡作用(P<0.01)；Western blot检测结果显示,与对照组相比,MRSP可成浓度依赖性(10μM、25μM、50μM)的抑制p-Raf-1、p-ERK1/2和p-Akt的表达水平(P<0.01)。
4.结论
根据Mfn2的最小功能序列人工合成的MRSP具有显著抑制VSMCs增殖的作用,使细胞周期停滞于G0/G1期,这一作用主要是使Ras-Raf-ERK1/2通路受到抑制,p-Raf-1和p-ERK1/2的表达下调,从而发挥抑制血管平滑肌细胞增殖的作用。此外,MRSP也具有促VSMCs凋亡的作用,该作用主要是通过抑制]Ras-PI3K-Akt信号通路而实现。
Construction, amplification and purification of recombinant adenoviruses containing various sequence structures of Mfn2
1. Objective
To construct, identify and purify the recombinant adenoviral vector containing various sequence structures of Mfn2.
2. Methods
Various complementary DNA (cDNA) was first amplified and subcloned into pCR8/GW/TOPO TA cloning plasmid. Subsequently, we performed LR recombination reaction between the pCR8/GW/TOPO-tMfn2 and pAd/CMV/V5-DEST to obtain the adenoviral expression vector. After packaging the pAd/CMV/V5-DEST-Mfn2 in 293 cells, the replication-defective adenovirus was produced. CsCl density gradient centrifugation was used to measure the titer of various adenovirus. Western blot analysis was used to confirm the expression of Mfn2 at the protein level.
3. Results
The recombinant adenoviruses which carried various sequence structures of Mfn2 (1 A, 2A,3A,4A,5A,6A,7A,8A) was successfully constructed and identified by PCR. The titer of recombinant adenoviruses was 27.8×109 pfu/ml,24×109 pfu/ml,19×109 pfu/ml, 30.8×109 pfu/ml,6.7×109 pfu/ml,12.7×109 pfu/ml,27.6×109 pfu/ml and 11.8×109 pfu/ml, respectively. Western blot analysis showed the expression of Mfn2 protein in infected vascular smooth muscle cells (VSMCs).
4. Conclusions
The recombinant adenoviruses which carried various sequence structures of Mfn2 can be successfully constructed by Recombinant DNA technology, which provides the basis for further study on the effect of various Mfn2 sequence structures on the proliferation and apoptosis of VSMCs.
Part II
Various sequence structures of Mfn2 on proliferation and apoptosis of vascular smooth muscle cells (VSMCs)
1. Objective
To investigate the effect of various sequence structures of Mfn2 on proliferation and apoptosis of vascular smooth muscle cells (VSMCs), seek the shortest complementary DNA (scDNA) that can both effectively inhibit proliferation of VSMCs and induce apoptosis of VSMCs and explore the potential molecular mechanisms.
2. Methods
VSMCs were infected by recombinant adenoviruses containing various sequence structures of Mfn2 (1A,2A,3A,4A,5A,6A,7A,8A). The effect of various sequence structures on proliferation of VSMCs were tested by cell counting and CCK-8, the cell cycle was observed by flow cytometry; Cell death ELISA and flow cytometry were used to investigate the effect on apoptosis of VSMCs. Western blot analysis was used to detect the expression of phosphorylation of Raf (p-Raf), ERK1/2 (p-ERK1/2) and protein kinase B (p-Akt).
3. Results
Cell counting and CCK-8 both indicated that various sequence structures of Mfn2 significantly inhibited proliferation of VSMCs (P<0.01). Most of the VSMCs were blocked in the stage of G0/G1 and few entered into the S phase in all sequence structures groups (P<0.01). Cell Death ELISA assay and flow cytometry both indicated that 1A,4A and 8A promoted apoptosis of VSMCs remarkably. Western blot analysis showed that 1A, 4A and 8A significantly down-regulated expression of phosphorylation of Raf (p-Raf), ERK1/2 (p-ERK1/2) and protein kinase B (p-Akt) (P<0.01).
4. Conclusions
1A is the shortest complementary DNA (scDNA) of Mfn2 that can both effectively inhibit proliferation of VSMCs via Ras-Raf-ERK1/2 signaling pathway and induce apoptosis of VSMCs via Ras-PI3K-Akt signaling pathway.
PartⅢ
Mfn2 gene-related synthetic peptide (MRSP) on proliferation and apoptosis of vascular smooth muscle cells (VSMCs)
1. Objective
To investigate the effect of Mfn2 gene-related synthetic peptide (MRSP) on proliferation and apoptosis of vascular smooth muscle cells (VSMCs) and the potential molecular mechanisms.
2. Methods
MRSP was synthesized according to the shortest complementary DNA (scDNA) of Mfn2 (1A) and TAT-PTD (transactiviting-protein transduction domain). VSMCs were cultured in DMEM containing MRSP of various concentration (1μM,10μM,25μM,50μM, 10μM). The effect of various concentration of MRSP on proliferation of VSMCs were tested by cell counting and CCK-8, the cell cycle was observed by flow cytometry;; Cell death ELISA and flow cytometry were used to investigate the effect on apoptosis of VSMCs. Western blot analysis was used to detect the expression of phosphorylation of Raf (p-Raf), ERK1/2 (p-ERK1/2) and protein kinase B (p-Akt).
3. Results
Cell counting and CCK-8 both indicated that MRSP caused a marked dose-dependent and time-dependent proliferation inhibitory effect on VSMCs from 10μM to 100μM (P< 0.01). Flow cytometry showed that most of the VSMCs were inhibited in the stage of G0/G1 and few cells entered into the S phase in all the three MRSP groups (P<0.01). Cell Death ELISA assay and flow cytometry both indicated that MRSP promoted apoptosis of VSMCs effectively. Western blot analysis showed that MRSP significantly down-regulated expression of phosphorylation of Raf (p-Raf), ERK1/2 (p-ERK1/2) and protein kinase B (p-Akt) (P<0.01).
4. Conclusions
The MRSP synthesized according to the shortest complementary DNA (scDNA) of Mfn2 can significantly inhibit the proliferation of VSMCs via the Ras-Raf-ERK1/2 signaling pathway, it can also effectively induce the apoptosis of VSMCs via the PI3K-Akt signaling pathway.
引文
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