【摘要】：背景:眾所周知,血小板源性生長因子(PDGF)可以誘導血管平滑肌細胞(VSMCs)表型轉換,即從收縮型轉換為合成型,而表型轉換又是VSMCs增殖的基礎。我們的前期研究證實了PDGF在VSMCs中可以上調Shh信號表達,同時抑制Shh信號可以阻止PDGF引起的VSMCs增殖。此外,大量研究提出KLF4作為重要的轉錄因子參與了PDGF誘導VSMCs表型轉換的過程,而我們的前期研究發(fā)現(xiàn)KLF4在Shh功能發(fā)揮中起到重要的作用。這也促使我們推測Shh可能參與了PDGF誘導的VSMCs表型轉換,而KLF4可能參與了此過程。目的:在本研究中,我們主要調查Shh信號是否參與PDGF誘導的VSMCs表型轉換。其后的機制研究中,我們進一步探討Shh信號是否通過KLF4調控PDGF誘導的VSMCs表型轉換。本研究將是我們前期實驗結果的重要補充和完善,同時為VSMCs增殖所導致的疾病提供新的治療思路。方法:(1)首先,我們利用血小板源性生長因子BB(PDGF-BB)刺激VSMCs,通過Western blot及RT-PCR檢測Shh信號通路及KLF4表達情況。然后給予伊馬替尼(Imatinib)抑制PDGF受體β(PDGFRβ),給予MEK抑制劑PD98059抑制ERK1/2信號,其后觀察Shh信號通路表達情況,評估PDGFRβ及ERK1/2信號在PDGF誘導的Shh信號表達中的作用。(2)然后,利用VSMCs分化型標志物α-actin和myocardin,VSMCs去分化型標志物去分化型標志物Tpm 4和SMemb來評估VSMCs的表型狀態(tài),觀察PDGF誘導的VSMCs表型轉換過程中Shh及KLF4信號表達情況。利用Shh-si RNA或Smoothened抑制劑cyclopamine抑制Shh信號通路,利用Shh-c DNA或重組N-Shh過表達Shh信號通路,評估Shh信號在PDGF誘導的VSMCs表型轉換中的作用,以及單獨的Shh信號是否具備促進VSMCs表型轉換的潛能。(3)最后,利用KLF4-si RNA抑制KLF4信號,并利用N-Shh或PDGF-BB誘導VSMCs去分化,觀察KLF4在PDGF或Shh誘導的VSMCs表型轉換中的作用。結果:我們發(fā)現(xiàn)PDGF通過活化PDGFRβ/ERK1/2通路促進VSMCs Shh信號通路激活。其后我們觀察到PDGF促進VSMCs表型轉換過程中伴隨Shh/Gli2信號及KLF4活化。在給予PDGF刺激同時抑制Shh信號通路,發(fā)現(xiàn)VSMCs去分化標志物Tpm 4和SMemb及KLF4信號表達下降,提示PDGF誘導VSMCs表型轉換及KLF4表達作用消失。然后在無PDGF刺激下過表達Shh信號,發(fā)現(xiàn)單獨的Shh信號可以刺激KLF4表達同時促進VSMCs去分化。最后,我們發(fā)現(xiàn)抑制KLF4后,PDGF及Shh信號誘導的VSMCs表型轉換作用均消失。結論:本研究證實了PDGF可誘導VSMCs中Shh信號通路及KLF4激活;Shh及KLF4參與PDGF誘導的VSMCs表型轉換;PDGF通過Shh信號促進VSMCs的去分化;而PDGF及Shh信號調控VSMCs表型轉換又是通過活化KLF4實現(xiàn)的。綜上所述,我們的研究提供了關于Shh在VSMCs增殖發(fā)病機制中全新的見解,也就是Shh可以通過KLF4調控PDGF誘導的VSMCs表型轉換。背景:肢體缺血再灌注(Ischemia/reperfusion,I/R)是臨床常見疾病。骨骼肌因其較高的代謝活性,在缺血血流恢復后極易發(fā)生I/R損傷。盡管嚴重缺血后血流恢復,骨骼肌損傷持續(xù)存在,后續(xù)可發(fā)生骨骼肌壞死,導致患者截肢,甚至多器官功能障礙。Shh在胚胎后時期對骨骼肌損傷及修復發(fā)揮了重要作用,同時大量研究也證實了Shh在骨骼肌缺血中發(fā)揮了保護作用。然而,目前國內尚無關于Shh在骨骼肌I/R損傷中的作用及機制研究。目的:本研究通過建立止血帶小鼠后肢I/R損傷模型,評估Shh信號通路在骨骼肌I/R損傷中發(fā)揮的作用。為尋找其中的機制,我們將研究Shh是否通過經典合成代謝通路AKT/m TOR/p70S6K發(fā)揮骨骼肌I/R損傷的保護作用。最后,我們將探討Shh信號對骨骼肌I/R損傷中相關的骨骼肌凋亡的影響。我們希望通過這些研究為骨骼肌I/R損傷提供新的治療策略。方法:(1)選取10-14周雄性C57BL/6小鼠,利用止血帶建立小鼠后肢I/R損傷模型,即單側后肢缺血3 h,其后松開止血帶恢復血流灌注1 d,3 d,5 d,7 d及14 d,并于不同時間點獲取骨骼肌標本。利用Western blot法檢測Shh信號通路相關蛋白包括Shh,Gli1及Gli2表達情況。同時,利用免疫熒光法檢測骨骼肌組織Shh信號表達。(2)為了闡明Shh信號是否參與了骨骼肌I/R損傷的調控。通過腹腔注射Smoothened直接抑制劑Cyclopamine以抑制Shh信號通路。相反,通過損傷側后肢肌肉注射質粒編碼人Shh基因(ph Shh)以過表達Shh通路。建立模型并取I/R 7 d損傷小鼠骨骼肌標本,通過Western Blot法或者免疫熒光檢測法評估抑制及過表達Shh信號效率。其后通過HE染色骨骼肌損傷評分評估骨骼肌損傷程度,通過Masson染色評估骨骼肌纖維化程度。(3)為了研究AKT/m TOR/p70S6K信號通路在骨骼肌I/R損傷中時間-表達進程,利用Western blot檢測不同灌注時間AKT,m TOR,p70S6K以及他們的磷酸化蛋白表達水平。為進一步驗證AKT/m TOR/p70S6K信號通路是否參與了Shh誘導的I/R損傷保護,利用肌肉注射ph Shh方式過表達Shh信號,并在I/R 7 d損傷時間點檢測AKT/m TOR/p70S6K信號通路表達。其后為進一步研究其中機制,利用ph Shh過表達Shh的同時,通過腹腔內注射PI3K-m TOR雙向抑制劑NVP-BEZ235抑制AKT/m TOR/p70S6K信號通路,并在I/R 7 d損傷時間點檢測AKT/m TOR/p70S6K信號通路表達、骨骼肌損傷評分及骨骼肌纖維化。(4)本研究中,我們還將評估I/R損傷所致的骨骼肌凋亡。首先,利用Western blot檢測法評估凋亡蛋白Cleaved Caspase 3及Bax,抗凋亡蛋白Bcl2的時間-表達進程。其后為進一步研究Shh在骨骼肌I/R損傷中的作用,過表達Shh信號并在I/R 7 d損傷時間點檢測凋亡蛋白Cleaved Caspase 3及Bax,抗凋亡蛋白Bcl2表達,同時還利用TUNEL染色評估骨骼肌凋亡水平。結果:(1)首先,我們發(fā)現(xiàn)I/R損傷可導致Shh,Gli1及Gli2表達升高,并在再灌注5 d達到峰值,其后表達量逐漸下降。免疫熒光檢測提示在正常組及I/R 1 d組,幾乎不能檢測到Shh信號表達。在I/R 3d,5 d組,于骨骼肌基底膜可以觀察到明顯的Shh信號表達,該信號表達靠近于骨骼肌肌核或者與骨骼肌肌核共定位。在I/R 7 d組和I/R 14d組,Shh信號表達減弱。(2)在進一步研究中發(fā)現(xiàn),Shh信號通路抑制劑Cyclopamine治療骨骼肌局部可見壞死區(qū)域,同時HE染色骨骼肌損傷評分評估增高(P0.05),提示抑制Shh信號通路后骨骼肌I/R損傷加重。同時Masson三色染色提示Cyclopamine治療組可以觀察到明顯增多的膠原堆積(P0.05)。并且,當利用肌肉內注射ph Shh過表達Shh信號通路時,可以觀察到相反的結果。(3)然后,Western blot結果提示骨骼肌I/R損傷后早期Shh信號表達升高同時伴隨AKT,m TOR及p70S6K信號磷酸化水平升高,其后均逐漸降低。ph Shh治療可導致p-AKT/AKT,p-m TOR/m TOR及p-p70S6K/p70S6K比值明顯升高,而這種作用又可被NVP-BEZ235治療所阻斷。(4)最后,Western blot檢測提示凋亡蛋白Cleaved Caspase 3水平及Bax/Bcl2比值在I/R損傷后逐漸升高,均在I/R 7 d達到峰值,其后開始下降。而進一步探討Shh對凋亡通路影響時發(fā)現(xiàn)過表達Shh信號可降低I/R 7 d凋亡蛋白Cleaved Caspase 3表達,及Bax/Bcl2比值(P0.05)。而TUNEL染色評估提示外源性ph Shh治療可降低I/R損傷后骨骼肌凋亡指數(shù)(P0.05)。結論:Shh信號在胚胎后時期小鼠后肢骨骼肌I/R損傷模型中可被再激活;Shh信號在小鼠后肢骨骼肌I/R損傷中通過AKT/m TOR/p70S6K信號通路發(fā)揮抗骨骼肌損傷及抗骨骼肌纖維化作用;同時,Shh信號在小鼠后肢骨骼肌I/R損傷中發(fā)揮抗凋亡作用。
[Abstract]:BACKGROUND: It is well known that platelet-derived growth factor (PDGF) can induce phenotypic transition of vascular smooth muscle cells (VSMCs), i.e. from contractile to synthetic, and phenotypic transition is the basis of VSMCs proliferation. In addition, a large number of studies have suggested that KLF4 is an important transcription factor involved in the phenotypic transition of VSMCs induced by PDGF, and our previous studies have found that KLF4 plays an important role in SH function. OBJECTIVE: In this study, we investigated whether Shh signaling is involved in the phenotypic transition of VSMCs induced by PDGF. In the subsequent mechanism study, we further investigated whether Shh signaling regulates the phenotypic transition of VSMCs induced by PDGF through KLF4. This study will be an important complement and improvement of our previous experimental results, and also be caused by the proliferation of VSMCs. Methods: (1) First, we stimulated VSMCs with platelet-derived growth factor BB (PDGF-BB), detected the expression of Shh signaling pathway and KLF4 by Western blot and RT-PCR. Then Imatinib inhibited PDGF receptor beta (PDGFR beta) and MEK inhibitor PD98059 inhibited ERK1/2 signal. Then Shh was observed. To evaluate the role of PDGFR beta and ERK1/2 in the expression of Shh signal induced by PDGF. (2) Then, the phenotypic status of VSMCs was assessed by using the differentiated markers of VSMCs, such as alpha-actin and myocardin, and the dedifferentiated markers of VSMCs, such as Tpm 4 and SMmb, to observe the phenotypic transition of VSMCs induced by PDGF. LF4 signaling. Shh-si RNA or Smoothened inhibitor cyclopamine was used to suppress Shh signaling pathway, Shh-c DNA or recombinant N-Shh was used to overexpress Shh signaling pathway to evaluate the role of Shh signaling in PDGF-induced phenotypic conversion of VSMCs, and whether single Shh signaling has the potential to promote phenotypic conversion of VSMCs. (3) Finally, KLF4-s was used. I RNA inhibited KLF4 signal and induced VSMCs to dedifferentiate by N-Shh or PDGF-BB. The role of KLF4 in phenotypic transition of VSMCs induced by PDGF or Shh was observed. Results: PDGF stimulated the activation of VSMCs Shh signaling pathway by activating PDGFR beta/ERK1/2 pathway. LF4 activation. When PDGF stimulation was given, Shh signaling pathway was inhibited. It was found that the expression of Tpm 4, SMMB and KLF4 was decreased, suggesting that the phenotypic transition of VSMCs and the expression of KLF4 disappeared. Then Shh signal was overexpressed without PDGF stimulation. It was found that single Shh signal could stimulate the expression of KLF4 and promote the depletion of VSMCs. Conclusion: PDGF can induce the activation of Shh signaling pathway and KLF4 in VSMCs; Shh and KLF4 participate in the phenotypic conversion of VSMCs induced by PDGF; PDGF promotes the dedifferentiation of VSMCs through Shh signal; PDGF and Shh signal regulate the phenotypic transformation of VSMCs. In conclusion, our study provides a new insight into the pathogenesis of Shh in VSMCs proliferation, that is, Shh can regulate the phenotypic transition of VSMCs induced by PDGF through KLF4. Although blood flow recovery after severe ischemia, skeletal muscle injury persists, followed by necrosis of the skeletal muscle, resulting in amputation, and even multiple organ dysfunction. Shh plays an important role in skeletal muscle injury and repair in the postembryonic period, and a large number of studies have also confirmed that Shh is in the skeletal muscle injury and repair. However, there is no study on the role and mechanism of Shh in I/R injury of skeletal muscle in China. Objective: To evaluate the role of Shh signaling pathway in I/R injury of skeletal muscle by establishing an I/R injury model of hindlimb in mice with tourniquet. Finally, we will explore the effect of Shh signal on skeletal muscle apoptosis associated with I/R injury. We hope that these studies will provide a new therapeutic strategy for skeletal muscle I/R injury. Methods: (1) Select male C57BL/6 mice from 10 to 14 weeks, benefit from Shh signal. I/R injury model of hindlimb in mice was established by tourniquet, i.e. unilateral hindlimb ischemia for 3 hours, then the tourniquet was loosened to restore blood perfusion for 1, 3, 5, 7 and 14 days. Skeletal muscle samples were obtained at different time points. Expression of Shh signaling pathway related proteins including Shh, Gli1 and Gli2 was detected by Western blot. To clarify whether Shh signaling is involved in the regulation of skeletal muscle I/R injury, the Shh signaling pathway is inhibited by intraperitoneal injection of Cyclopamine, a direct inhibitor of Smoothened, into the skeletal muscle. The inhibition and overexpression of Shh signal were evaluated by Western Blot or immunofluorescence assay in skeletal muscle specimens of injured mice. The degree of skeletal muscle injury was assessed by HE staining skeletal muscle injury score, and the degree of skeletal muscle fibrosis was assessed by Masson staining. (3) To study the AKT/m TOR/p70S6K signaling pathway in skeletal muscle I/R injury. In order to further verify whether AKT/m TOR/p70S6K signaling pathway is involved in the protection of Shh-induced I/R injury, Shh signal was overexpressed by intramuscular injection of pH Shh, and AKT/m was detected at the time point of I/R 7 d injury. To further study the mechanism of TOR/p70S6K signaling pathway expression, the PI3K-m TOR bidirectional inhibitor NVP-BEZ235 was injected intraperitoneally to inhibit AKT/m TOR/p70S6K signaling pathway, and the expression of AKT/m TOR/p70S6K signaling pathway, skeletal muscle injury score and skeletal muscle fiber were detected at the time point of I/R 7 d injury. (4) In this study, we will also evaluate skeletal muscle apoptosis induced by I/R injury. First, the time-expression process of apoptotic proteins Cleaved Caspase-3 and Bax, and anti-apoptotic proteins Bcl-2 were evaluated by Western blot assay. Then, to further investigate the role of Shh in skeletal muscle I/R injury, Shh signaling was overexpressed and at the time point of I/R 7 d injury. The expression of apoptotic proteins Cleaved Caspase 3 and Bax, anti-apoptotic protein Bcl 2 were detected, and the level of skeletal muscle apoptosis was assessed by TUNEL staining. Results: (1) First, we found that I/R injury could induce increased expression of Shh, Gli 1 and Gli 2, and reached a peak at 5 days after reperfusion, and then decreased gradually. The expression of Shh signal was almost undetectable in the 1st day group. In the 3rd and 5th day groups, the expression of Shh signal was observed in the basement membrane of skeletal muscle, which was close to or co-localized with the nucleus of skeletal muscle. Cyclopamine treatment of skeletal muscle necrosis in the local area, and HE staining skeletal muscle injury score increased (P 0.05), suggesting that inhibition of Shh signaling pathway skeletal muscle I/R injury aggravated. Masson trichrome staining suggesting that Cyclopamine treatment group can observe a significant increase in collagen accumulation (P 0.05). Over-expression of Shh signaling pathway, the opposite results were observed. (3) Western blot results suggested that the early expression of Shh signaling increased after skeletal muscle I/R injury accompanied by increased levels of AKT, m TOR and p70S6K signaling phosphorylation, and then gradually decreased. Ph Shh treatment can lead to p-AKT/AKT, P-M TOR/m TOR and p-p70S6K/p70S6K ratio increased significantly. (4) Western blot analysis showed that the level of apoptotic protein Cleaved Caspase 3 and the ratio of Bax to Bcl 2 increased gradually after I/R injury, both peaked at the 7th day of I/R, and then began to decline. Further investigation of the effect of Shh on apoptotic pathway showed that the expression of Shh signal decreased I/R 7 d. Expression of apoptotic protein Cleaved Caspase 3 and Bax/Bcl 2 ratio (P 0.05). TUNEL staining showed that exogenous pH Shh treatment could reduce the apoptotic index of skeletal muscle after I/R injury (P 0.05). Conclusion: Shh signal could be reactivated in the I/R injury model of hindlimb skeletal muscle in mice at postembryonic stage; Shh signal could be activated through A in I/R injury of hindlimb skeletal muscle in mice. KT/m TOR/p70S6K signaling pathway plays an anti-skeletal muscle injury and anti-skeletal muscle fibrosis role; at the same time, Shh signaling plays an anti-apoptosis role in the hind limb skeletal muscle I/R injury of mice.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R68

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