本文選題：葡萄糖轉(zhuǎn)運(yùn)蛋白4 + 肌動(dòng)蛋白�。� 參考：《河北醫(yī)科大學(xué)》2016年博士論文

【摘要】：目的:糖尿病患者血管成型術(shù)后再狹窄和動(dòng)脈粥樣硬化的發(fā)生率明顯高于正常人。血管平滑肌細(xì)胞(vascular smooth muscle cell,VSMC)增殖需要消耗高水平的葡萄糖,增強(qiáng)的葡萄糖轉(zhuǎn)運(yùn)、分解代謝和線粒體生物氧化是增殖的細(xì)胞能量轉(zhuǎn)換的重要特征。葡萄糖轉(zhuǎn)運(yùn)體4(GLUT4)介導(dǎo)的葡萄糖攝取是血管平滑肌細(xì)胞糖攝取的主要機(jī)制,GLUT4在損傷誘導(dǎo)的新生內(nèi)膜中表達(dá)明顯增加。PDGF可通過激活PI3K促進(jìn)GLUT4轉(zhuǎn)位進(jìn)而促進(jìn)葡萄糖攝取,而PI3K激活介導(dǎo)的皮層肌動(dòng)蛋白(actin)重構(gòu)是GLUT4轉(zhuǎn)位所必須的。平滑肌(smooth muscle,SM)22α,是一種actin細(xì)胞骨架相關(guān)蛋白,其主要功能是與actin結(jié)合,參與血管平滑肌細(xì)胞微絲聚合和骨架重構(gòu)。在血管平滑肌細(xì)胞增殖相關(guān)的疾病如動(dòng)脈粥樣硬化斑塊、腹主動(dòng)脈瘤以及腫瘤組織中表達(dá)下調(diào)。我們先前實(shí)驗(yàn)證明,高表達(dá)SM22α可通過阻斷PDGF-BB刺激所激活的Ras-ERK1/2信號(hào)通路,進(jìn)而抑制血管平滑肌細(xì)胞增殖,并且,敲低SM22α可引起類似PDGF-BB刺激所產(chǎn)生的actin細(xì)胞骨架的重構(gòu)。但是,SM22α是否參與PDGF-BB誘導(dǎo)的GLUT4轉(zhuǎn)位尚不清楚。本研究旨在探討SM22α在PDGF-BB誘導(dǎo)的GLUT4轉(zhuǎn)位中的作用及其與細(xì)胞增殖活性的關(guān)系,揭示其作用的分子機(jī)制。方法:利用免疫熒光、Western blot技術(shù)檢測(cè)PDGF-BB誘導(dǎo)的血管平滑肌細(xì)胞GLUT4轉(zhuǎn)位及與細(xì)胞骨架重構(gòu)的關(guān)系;利用熒光葡萄糖2-NBDG檢測(cè)血管平滑肌細(xì)胞及動(dòng)脈葡萄糖攝取;通過敲低SM22α,確定SM22α參與PDGF-BB誘導(dǎo)的GLUT4轉(zhuǎn)位和葡萄糖攝取;通過細(xì)胞計(jì)數(shù)和Brd U實(shí)驗(yàn)檢測(cè)GLUT4介導(dǎo)的葡萄糖攝取與血管平滑肌細(xì)胞增殖的相關(guān)性;利用Sm22α-/-小鼠建立頸總動(dòng)脈損傷模型,用高效液相色譜法檢測(cè)組織葡萄糖含量,結(jié)合膜蛋白提取和Western blot技術(shù),探討缺失SM22α在GLUT4轉(zhuǎn)位介導(dǎo)的葡萄糖攝取與損傷誘導(dǎo)的新生內(nèi)膜形成之間的關(guān)系。用免疫熒光技術(shù)檢測(cè)平滑肌細(xì)胞微管的聚合情況;通過敲低或敲除或用腺病毒過表達(dá)SM22α,觀察SM22α與微管穩(wěn)定性的關(guān)系;利用Western blot技術(shù)檢測(cè)α-Tubulin乙�；�;用JC-1法檢測(cè)線粒體膜電位。結(jié)果:1 SM22α通過調(diào)節(jié)微絲骨架重構(gòu)參與PDGF-BB誘導(dǎo)的GLUT4轉(zhuǎn)位和糖攝取1.1 PDGF-BB誘導(dǎo)血管平滑肌細(xì)胞GLUT4轉(zhuǎn)位和葡萄糖攝取PDGF-BB刺激血管平滑肌細(xì)胞0、5、10、15、20、30 min,提取膜蛋白,Western blot方法檢測(cè)膜上GLUT4的表達(dá)量。結(jié)果顯示,PDGF-BB刺激可誘導(dǎo)GLUT4向膜上轉(zhuǎn)位,膜組分中GLUT4含量于15 min達(dá)到峰值,隨后下降。熒光葡萄糖2-NBDG攝取實(shí)驗(yàn)顯示,用PDGF-BB處理的細(xì)胞其2-NBDG的攝取也在15 min達(dá)到峰值,變化趨勢(shì)與GLUT4膜轉(zhuǎn)位一致,提示細(xì)胞對(duì)葡萄糖的攝取增加。1.2肌動(dòng)蛋白重構(gòu)調(diào)節(jié)PDGF-BB誘導(dǎo)的GLUT4轉(zhuǎn)位細(xì)胞免疫熒光技術(shù)檢測(cè)細(xì)胞膜上GLUT4的表達(dá)和細(xì)胞骨架的變化。結(jié)果顯示,PDGF-BB刺激15 min,隨著細(xì)胞骨架重構(gòu),皮層F-actin聚合增加,GLUT4從胞質(zhì)轉(zhuǎn)位至細(xì)胞邊緣,與細(xì)胞皮層聚合的F-actin發(fā)生共定位。而用細(xì)胞骨架穩(wěn)定劑JPK預(yù)處理細(xì)胞后,PDGF-BB誘導(dǎo)的細(xì)胞骨架解聚和皮層F-actin聚合受抑制,同時(shí)GLUT4膜轉(zhuǎn)位明顯減少。2-NBDG攝取分析也顯示用JPK預(yù)處理后,PDGF-BB刺激引起的血管平滑肌細(xì)胞葡萄糖攝取被抑制。以上結(jié)果表明,PDGF-BB誘導(dǎo)的GLUT4膜轉(zhuǎn)位和葡萄糖攝取依賴于細(xì)胞骨架重構(gòu)。1.3 SM22α抑制GLUT4膜轉(zhuǎn)位用SM22α特異性小干擾RNA(si SM22α)敲低血管平滑肌細(xì)胞內(nèi)源性SM22α,鬼筆環(huán)肽熒光染色觀察PDGF-BB刺激前后細(xì)胞骨架的變化。結(jié)果顯示,相對(duì)于si Con組,敲低SM22α后,血管平滑肌細(xì)胞基礎(chǔ)狀態(tài)下應(yīng)力纖維密度明顯減少,而PDGF-BB刺激引起的皮層F-actin聚合則更加明顯。同時(shí),細(xì)胞免疫熒光結(jié)果顯示,敲低SM22α后PDGF-BB誘導(dǎo)的GLUT4轉(zhuǎn)位也明顯增加。Western blot方法檢測(cè)膜蛋白提取物中GLUT4的含量也得到相似結(jié)果。進(jìn)一步用Sm22α-/-小鼠的細(xì)胞進(jìn)行驗(yàn)證,與野生型(WT)相比較,PDGF-BB誘導(dǎo)的Sm22α-/-小鼠的細(xì)胞皮層F-actin聚合和GLUT4轉(zhuǎn)位均明顯增加。以上結(jié)果證實(shí)了我們的假設(shè),下調(diào)SM22α通過增強(qiáng)actin動(dòng)力學(xué)和皮層F-actin聚合從而促進(jìn)PDGF-BB誘導(dǎo)的GLUT4轉(zhuǎn)位。1.4下調(diào)SM22α表達(dá)可促進(jìn)PDGF-BB誘導(dǎo)的葡萄糖攝取和代謝利用分別檢測(cè)敲低SM22α的大鼠VSMC及Sm22α-/-小鼠細(xì)胞PDGF-BB誘導(dǎo)的熒光葡萄糖2-NBDG的攝取情況。結(jié)果顯示,敲低或敲除SM22α可促進(jìn)PDGF-BB誘導(dǎo)的2-NBDG攝取,與相同條件下GLUT4轉(zhuǎn)位增強(qiáng)相一致。而用GLUT4活性抑制劑Indinavir預(yù)孵育后,PDGF-BB誘導(dǎo)的2-NBDG攝取增加受到抑制,說明GLUT4是糖攝取的主要載體。相對(duì)于si Con組,用小干擾RNA敲低SM22α表達(dá),可增強(qiáng)PDGF-BB誘導(dǎo)的己糖激酶和乳酸脫氫酶活性。以上結(jié)果表明,PDGF-BB可加速細(xì)胞糖代謝,SM22α缺失能夠促進(jìn)這一過程。2敲除SM22α通過加速葡萄糖攝取而促進(jìn)血管平滑肌細(xì)胞增殖2.1 GLUT4及其介導(dǎo)的葡萄糖攝取參與PDGF-BB誘導(dǎo)的細(xì)胞增殖用C57BL/6J野生型小鼠復(fù)制左側(cè)頸總動(dòng)脈結(jié)扎模型。HE染色結(jié)果顯示,與假手術(shù)組相比,結(jié)扎的血管新生內(nèi)膜明顯增厚;組織免疫熒光染色結(jié)果也表明,新生內(nèi)膜處GLUT4表達(dá)顯著增加。體外研究顯示,PDGF-BB誘導(dǎo)的GLUT4表達(dá),呈時(shí)間依賴性;同時(shí)伴隨細(xì)胞核增殖性抗原PCNA表達(dá)的增加。在相同刺激條件下,細(xì)胞培養(yǎng)基(低糖DMEM)中葡萄糖消耗也明顯增加。細(xì)胞計(jì)數(shù)和Brd U分析結(jié)果顯示,抑制GLUT4活性的細(xì)胞其增殖活性顯著降低。以上結(jié)果表明,在PDGF-BB刺激下,GLUT4表達(dá)及其介導(dǎo)的葡萄糖攝取參與了細(xì)胞增殖。2.2 Sm22α-/-小鼠主動(dòng)脈GLUT4介導(dǎo)的2-NBDG攝取增加取野生型(WT)及Sm22α-/-小鼠頸總動(dòng)脈,加入PDGF-BB和2-NBDG共同孵育15 min,制備動(dòng)脈冰凍切片,觀察動(dòng)脈組織攝取2-NBDG的熒光強(qiáng)度。結(jié)果顯示,Sm22α-/-小鼠頸總動(dòng)脈在PDGF-BB刺激下,2-NBDG攝取顯著增加,熒光強(qiáng)度明顯高于野生型小鼠的主動(dòng)脈,這一效應(yīng)可被GLUT4特異抑制劑indinavir解除。2.3 GLUT4參與Sm22α-/-小鼠損傷后新生內(nèi)膜的形成體外研究已證實(shí)GLUT4介導(dǎo)的葡萄糖攝取參與細(xì)胞增殖,SM22α缺失可促進(jìn)這一過程。為了在體內(nèi)驗(yàn)證這一發(fā)現(xiàn),選雄性WT及Sm22α-/-小鼠復(fù)制左側(cè)頸總動(dòng)脈結(jié)扎模型。于結(jié)扎術(shù)后28天取頸總動(dòng)脈制備切片。HE染色結(jié)果表明,與WT對(duì)照組相比,Sm22α-/-小鼠在結(jié)扎的第28天血管新生內(nèi)膜明顯增厚。取WT及Sm22α-/-小鼠結(jié)扎側(cè)頸總動(dòng)脈進(jìn)行總蛋白及膜蛋白提取,檢測(cè)GLTU4表達(dá)。結(jié)果表明,相對(duì)于WT組,Sm22α-/-小鼠損傷動(dòng)脈總蛋白和膜組分中GLTU4表達(dá)均高于WT小鼠,膜組分與總蛋白GLTU4比值也有所增加。用高效液相色譜法檢測(cè)動(dòng)脈組織中葡萄糖含量,結(jié)果發(fā)現(xiàn),Sm22α-/-小鼠損傷動(dòng)脈組織葡萄糖含量也明顯高于野生型小鼠,與內(nèi)膜增生程度相一致。以上結(jié)果表明,SM22α缺失可促進(jìn)體內(nèi)GLUT4轉(zhuǎn)位和葡萄糖攝取,加重內(nèi)膜增生程度。3 SM22α增強(qiáng)血管平滑肌細(xì)胞微管穩(wěn)定性3.1 SM22a增強(qiáng)血管平滑肌細(xì)胞微管的穩(wěn)定性為檢測(cè)SM22α在微管穩(wěn)定性中的作用,用SM22α特異性小干擾RNA(si SM22α)敲低血管平滑肌細(xì)胞內(nèi)源性SM22α,α-tubulin抗體進(jìn)行免疫熒光染色,結(jié)果顯示,si Con組微管從核周向細(xì)胞邊緣呈放射狀分布;敲低SM22α后,微管解聚并向核周聚集。過表達(dá)SM22α的細(xì)胞,其微管呈束狀存在。與WT相比較,Sm22α-/-小鼠細(xì)胞微管解聚并向核周聚集;補(bǔ)救SM22α表達(dá)后,微管結(jié)構(gòu)恢復(fù)至WT狀態(tài)。結(jié)果表明,SM22α表達(dá)增強(qiáng)微管的穩(wěn)定性。3.2 SM22α增強(qiáng)a-tubulin 40位賴氨酸殘基乙酰化α-tubulin第40位賴氨酸殘基乙�；揎検枪J(rèn)的是微管穩(wěn)定性標(biāo)志。細(xì)胞免疫熒光和Westhern blot結(jié)果均表明,敲低SM22α可降低α-tubulin乙�；�,而過表達(dá)SM22α則增強(qiáng)α-tubulin的乙酰化水平,與SM22α對(duì)微管存在形式的影響相一致。3.3敲除SM22α降低生長(zhǎng)接觸抑制,促進(jìn)線粒體分裂和膜電位增加細(xì)胞計(jì)數(shù)結(jié)果顯示,WT組的細(xì)胞在融合狀態(tài)下,繼續(xù)培養(yǎng)2,3,4天時(shí),細(xì)胞密度無明顯差異;而Sm22α-/-細(xì)胞在2,3,4天后仍保持增殖活性,數(shù)量明顯增加,在第4天時(shí),細(xì)胞密度達(dá)到WT組的1.68倍。JC-1染色結(jié)果表明,WT組細(xì)胞線粒體多呈長(zhǎng)桿狀、線狀并緊密連接的立體網(wǎng)絡(luò)的融合狀態(tài);而Sm22α-/-細(xì)胞線粒體則呈顆粒狀、點(diǎn)狀散在分布的分裂狀態(tài),而且線粒體膜電位也明顯高于野生型細(xì)胞。結(jié)論:1 PDGF-BB誘導(dǎo)的GLUT4轉(zhuǎn)位和糖攝取參與血管平滑肌細(xì)胞增殖。2缺失SM22α可誘導(dǎo)皮層細(xì)胞骨架聚合,增強(qiáng)PDGF-BB誘導(dǎo)的GLUT4膜轉(zhuǎn)位和糖攝取及代謝活性。3 SM22α是一種新的增殖相關(guān)糖代謝調(diào)節(jié)因子。4 SM22α增強(qiáng)血管平滑肌細(xì)胞微管的穩(wěn)定性,介導(dǎo)生長(zhǎng)接觸抑制效應(yīng)。
[Abstract]:Objective: the incidence of restenosis and atherosclerosis in diabetic patients after angioplasty is significantly higher than that of normal people. The proliferation of vascular smooth muscle cell (VSMC) needs to consume high levels of glucose, enhanced glucose transport, and the importance of catabolism and linear granular biological oxidation. Glucose transporter 4 (GLUT4) - mediated glucose uptake is the main mechanism of glucose uptake in vascular smooth muscle cells. The expression of GLUT4 in injured neointima increases significantly by activating PI3K to promote GLUT4 translocation by activating PI3K and promoting glucose uptake, while PI3K activated cortical actin (actin) reconfiguration is GLUT4 transposition The necessary. Smooth muscle (SM) 22 alpha is a actin cytoskeleton related protein whose main function is to combine with actin to participate in microfilament and skeleton remodeling in vascular smooth muscle cells. The expression of vascular smooth muscle cell proliferation related diseases such as atherosclerotic plaques, abdominal aortic aneurysm and tumor tissue are down regulated. Previous experiments showed that high expression of SM22 alpha could inhibit the proliferation of vascular smooth muscle cells by blocking the Ras-ERK1/2 signaling activated by PDGF-BB stimulation, and knocking low SM22 alpha could cause the remodeling of the actin cytoskeleton similar to PDGF-BB stimulation. However, it is not clear whether SM22 alpha participates in PDGF-BB induced GLUT4 transposition. The purpose of this study is to explore the role of SM22 alpha in the PDGF-BB induced GLUT4 transposition and the relationship with cell proliferation activity, and to reveal the molecular mechanism of its role. Methods: using immunofluorescence, Western blot technique to detect the GLUT4 transposition of vascular smooth muscle cells induced by PDGF-BB and the relationship with cytoskeleton reconstruction, and the use of fluorescent glucose 2-NBDG to detect blood. Tube smooth muscle cells and arterial glucose uptake; SM22 alpha involved in PDGF-BB induced GLUT4 transposition and glucose uptake by knocking low SM22 alpha; the correlation between GLUT4 mediated glucose uptake and vascular smooth muscle cell proliferation through the cell count and Brd U test; the use of Sm22 A / - mice to establish a carotid artery injury model with high efficiency. The glucose content in tissue was detected by liquid chromatography, and the relationship between the glucose uptake mediated by GLUT4 transposition and the formation of the neointima induced by damage was investigated with membrane protein extraction and Western blot technology. The polymerization of microtubules in smooth muscle cells was detected by immunofluorescence technique; by knocking down or knocking down or using adenovirus. SM22 alpha was expressed and the relationship between SM22 alpha and microtubule stability was observed; Western blot technique was used to detect the level of alpha -Tubulin acetylation; the mitochondrial membrane potential was detected by JC-1. Results: 1 SM22 alpha was involved in PDGF-BB induced GLUT4 transposition and glucose uptake by 1.1 PDGF-BB to induce the GLUT4 transposition of vascular smooth muscle cells and glucose uptake by the regulation of microfilament framework remodeling. 0,5,10,15,20,30 min of vascular smooth muscle cells was stimulated by PDGF-BB, and membrane protein was extracted and Western blot was used to detect the expression of GLUT4 on the membrane. The results showed that PDGF-BB stimulation could induce the transposition of GLUT4 to the membrane. The GLUT4 content in the membrane components reached the peak of the 15 min and then decreased. The uptake of 2-NBDG was also at the peak of 15 min, the change trend was consistent with the GLUT4 membrane transposition, suggesting that the uptake of glucose increased by.1.2 actin reconfiguration and regulation of PDGF-BB induced GLUT4 transposition cell immunofluorescence technique to detect the expression of GLUT4 on the cell membrane and the change of cytoskeleton. The results showed that PDGF-BB stimulation was 15 min, with finer. Cytoskeleton reconstruction, cortical F-actin polymerization increased, GLUT4 translocated from cytoplasm to cell edge, and Co located with the F-actin of cell cortical aggregation. After the cells were pretreated with cytoskeleton stabilizer JPK, PDGF-BB induced cytoskeleton depolymerization and cortical F-actin polymerization were inhibited. GLUT4 membrane transposition significantly decreased.2-NBDG uptake analysis at the same time. The glucose uptake of vascular smooth muscle cells induced by PDGF-BB stimulation was inhibited after JPK pretreatment. The results showed that the GLUT4 membrane transposition and glucose uptake induced by PDGF-BB were dependent on the cytoskeleton remodeling of.1.3 SM22 a to inhibit the GLUT4 membrane translocation with SM22 a specific small interference RNA (Si SM22 alpha) knocking down the endogenous SM22 alpha in the vascular smooth muscle cells. The cytoskeleton changes before and after PDGF-BB stimulation were observed before and after the stimulation of the Si Con group. The results showed that the stress density of the vascular smooth muscle cells decreased significantly compared to the Si Con group, and the F-actin polymerization caused by PDGF-BB stimulation was more obvious. PDGF-BB induced GLUT4 transposition also significantly increased the.Western blot method to detect the content of GLUT4 in the membrane protein extract. Further use Sm22 alpha / - mouse cells to verify, and compared with the wild type (WT), PDGF-BB induced Sm22 alpha / - mouse cell cortical F-actin polymerization and GLUT4 transposition were significantly increased. The result confirms our hypothesis that down regulation of SM22 alpha by enhancing actin kinetics and cortical F-actin polymerization to promote PDGF-BB induced GLUT4 transposition.1.4 down regulation of SM22 alpha expression can promote PDGF-BB induced glucose uptake and metabolic utilization to detect VSMC and Sm22 alpha / - mouse cell PDGF-BB induced fluorescence glucose induced by PDGF-BB The results of BDG uptake showed that knocking down or knocking SM22 alpha could promote the 2-NBDG uptake induced by PDGF-BB, which was consistent with the GLUT4 transposition enhancement under the same conditions. The increase of 2-NBDG uptake induced by PDGF-BB was inhibited after Indinavir preincubation with the GLUT4 active inhibitor, indicating that GLUT4 was the main carrier of sugar uptake. RNA knocks low SM22 alpha expression and enhances PDGF-BB induced hexokinase and lactate dehydrogenase activity. The above results show that PDGF-BB can accelerate cell sugar metabolism, and SM22 alpha deletion can promote the process of.2 knockout SM22 alpha by accelerating glucose uptake and promoting vascular smooth muscle cells to increase 2.1 GLUT4 and its mediated glucose uptake to participate PDG The proliferation of F-BB induced cell proliferation in the left cervical artery ligation model in C57BL/6J wild type mice showed that the neointima of ligated vascular thickening was obviously thickened compared with the sham group, and the results of tissue immunofluorescence staining showed that the expression of GLUT4 in the neointima was significantly increased. In vitro studies showed that PDGF-BB induced GLUT4 expression, It was time dependent and accompanied by an increase in the expression of nuclear proliferative antigen PCNA. The glucose consumption in the cell culture medium (low sugar DMEM) was also significantly increased under the same stimulation conditions. Cell count and Brd U analysis showed that the proliferation activity of cells inhibiting GLUT4 activity was significantly reduced. The above results showed that GLUT4 was stimulated by PDGF-BB, GLUT4 The expression and its mediated glucose uptake participated in the increase of 2-NBDG uptake mediated by GLUT4 in the proliferation of.2.2 Sm22 alpha - / - mouse aorta and the increasing of the common carotid artery of the wild type (WT) and Sm22 alpha / - mice. The 15 min was incubated with PDGF-BB and 2-NBDG to prepare the frozen section of the arteries and to observe the fluorescence intensity of the 2-NBDG in the arterial tissue. The results showed that Sm22 alpha - / - / - / - Under the stimulation of PDGF-BB, the uptake of 2-NBDG in the common carotid artery of mice increased significantly, and the fluorescence intensity was significantly higher than that of the aorta in the wild type mice. This effect could be relieved by.2.3 GLUT4, a specific inhibitor of GLUT4, to participate in the formation of neointima after Sm22 alpha / - mice injury, and that GLUT4 mediated glucose uptake was involved in cell proliferation, S. M22 alpha deletion could promote this process. In order to verify this discovery in the body, male WT and Sm22 alpha / - mice were selected to copy the left carotid artery ligation model. The.HE staining of the common carotid artery in the 28 day after ligation showed that the Sm22 alpha / - mice were thickened in the neointima of vascular neovascularization at the twenty-eighth day of ligation compared with the WT control group. WT and Sm were taken. The total protein and membrane protein of the 22 alpha - / - mice were ligated in 22 alpha - / - mice to detect the expression of the total protein and membrane protein. The results showed that the expression of GLTU4 in the total and membrane components of Sm22 A / - mice was higher than that of the WT group. The ratio of the membrane components to the total protein GLTU4 was also increased. The glucose content in the arterial tissue was detected by high performance liquid chromatography. The results showed that the glucose content in the injured arterial tissue of Sm22 - / - / - mice was also significantly higher than that in the wild type mice, which was consistent with the degree of intimal hyperplasia. The above results showed that the deletion of SM22 alpha could promote the translocation of GLUT4 and the uptake of glucose in the body, and increased the degree of intimal hyperplasia by.3 SM22 a to enhance the microtubule stability of the smooth muscle cells of the smooth muscle cells by 3.1 SM22a enhanced blood vessels. The stability of smooth muscle cell microtubules was used to detect the role of SM22 alpha in microtubule stability. SM22 alpha specific small interference RNA (Si SM22 alpha) knocks endogenous SM22 A and alpha -tubulin antibody for immunofluorescence staining. The results showed that the microtubules of Si Con group were distributed radially from the periphery to the edge of the cell; after knocking down SM22 alpha, microtubules were knocked down. The microtubules in the cells expressed SM22 alpha were in a fascicular structure. Compared with WT, the microtubules of Sm22 alpha / - mouse cells were depolymerization and aggregated to the pericarp. After remedial SM22 alpha expression, the microtubule structure was restored to WT state. The results showed that SM22 alpha expression enhanced the stability of microtubules by.3.2 SM22 A and enhanced the a-tubulin 40 lysine residue acetylation The acetylation modification of alpha -tubulin fortieth lysine residues is recognized as a marker of microtubule stability. Both cell immunofluorescence and Westhern blot results show that knocking low SM22 alpha can reduce the level of alpha -tubulin acetylation, while overexpression of SM22 a enhances the level of acetylation of alpha -tubulin, and is consistent with the existence of SM22 a in the presence of microtubules in.3.3 knockout SM2. 2 alpha decreased growth contact inhibition, promoting mitochondrial division and increasing cell count by membrane potential showed that cells in WT group had no significant difference in cell density when they continued to cultivate 2,3,4 days in fusion state, while Sm22 alpha / - cells still maintained proliferation activity after 2,3,4 days, and the cell density reached 1.68 times.J in WT group at the time of fourth days. The results of C-1 staining showed that the mitochondria in the WT group were mostly rod like, linear and tightly connected stereoscopic network, while Sm22 alpha / - cell mitochondria were granular, punctate scattered in the distribution of split state, and the mitochondrial membrane potential was obviously higher than that of wild type cells. Conclusion: 1 PDGF-BB induced GLUT4 transposition and sugar uptake participate in blood Vascular smooth muscle cell proliferation,.2 deletion, SM22 a can induce cortical cytoskeleton polymerization, enhanced PDGF-BB induced GLUT4 membrane transposition, and glucose uptake and metabolic activity.3 SM22 alpha, a new proliferation related glycometabolic regulator.4 SM22 a to enhance the stability of vascular smooth muscle cells microtubules and mediate growth contact inhibition.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R587.2;R54

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