Shh在血管平滑肌细胞表型转换和骨骼肌缺血再灌注损伤中的作用及机制研究

发布时间：2018-08-22 12:48

【摘要】：背景:众所周知,血小板源性生长因子(PDGF)可以诱导血管平滑肌细胞(VSMCs)表型转换,即从收缩型转换为合成型,而表型转换又是VSMCs增殖的基础。我们的前期研究证实了PDGF在VSMCs中可以上调Shh信号表达,同时抑制Shh信号可以阻止PDGF引起的VSMCs增殖。此外,大量研究提出KLF4作为重要的转录因子参与了PDGF诱导VSMCs表型转换的过程,而我们的前期研究发现KLF4在Shh功能发挥中起到重要的作用。这也促使我们推测Shh可能参与了PDGF诱导的VSMCs表型转换,而KLF4可能参与了此过程。目的:在本研究中,我们主要调查Shh信号是否参与PDGF诱导的VSMCs表型转换。其后的机制研究中,我们进一步探讨Shh信号是否通过KLF4调控PDGF诱导的VSMCs表型转换。本研究将是我们前期实验结果的重要补充和完善,同时为VSMCs增殖所导致的疾病提供新的治疗思路。方法:(1)首先,我们利用血小板源性生长因子BB(PDGF-BB)刺激VSMCs,通过Western blot及RT-PCR检测Shh信号通路及KLF4表达情况。然后给予伊马替尼(Imatinib)抑制PDGF受体β(PDGFRβ),给予MEK抑制剂PD98059抑制ERK1/2信号,其后观察Shh信号通路表达情况,评估PDGFRβ及ERK1/2信号在PDGF诱导的Shh信号表达中的作用。(2)然后,利用VSMCs分化型标志物α-actin和myocardin,VSMCs去分化型标志物去分化型标志物Tpm 4和SMemb来评估VSMCs的表型状态,观察PDGF诱导的VSMCs表型转换过程中Shh及KLF4信号表达情况。利用Shh-si RNA或Smoothened抑制剂cyclopamine抑制Shh信号通路,利用Shh-c DNA或重组N-Shh过表达Shh信号通路,评估Shh信号在PDGF诱导的VSMCs表型转换中的作用,以及单独的Shh信号是否具备促进VSMCs表型转换的潜能。(3)最后,利用KLF4-si RNA抑制KLF4信号,并利用N-Shh或PDGF-BB诱导VSMCs去分化,观察KLF4在PDGF或Shh诱导的VSMCs表型转换中的作用。结果:我们发现PDGF通过活化PDGFRβ/ERK1/2通路促进VSMCs Shh信号通路激活。其后我们观察到PDGF促进VSMCs表型转换过程中伴随Shh/Gli2信号及KLF4活化。在给予PDGF刺激同时抑制Shh信号通路,发现VSMCs去分化标志物Tpm 4和SMemb及KLF4信号表达下降,提示PDGF诱导VSMCs表型转换及KLF4表达作用消失。然后在无PDGF刺激下过表达Shh信号,发现单独的Shh信号可以刺激KLF4表达同时促进VSMCs去分化。最后,我们发现抑制KLF4后,PDGF及Shh信号诱导的VSMCs表型转换作用均消失。结论:本研究证实了PDGF可诱导VSMCs中Shh信号通路及KLF4激活;Shh及KLF4参与PDGF诱导的VSMCs表型转换;PDGF通过Shh信号促进VSMCs的去分化;而PDGF及Shh信号调控VSMCs表型转换又是通过活化KLF4实现的。综上所述,我们的研究提供了关于Shh在VSMCs增殖发病机制中全新的见解,也就是Shh可以通过KLF4调控PDGF诱导的VSMCs表型转换。背景:肢体缺血再灌注(Ischemia/reperfusion,I/R)是临床常见疾病。骨骼肌因其较高的代谢活性,在缺血血流恢复后极易发生I/R损伤。尽管严重缺血后血流恢复,骨骼肌损伤持续存在,后续可发生骨骼肌坏死,导致患者截肢,甚至多器官功能障碍。Shh在胚胎后时期对骨骼肌损伤及修复发挥了重要作用,同时大量研究也证实了Shh在骨骼肌缺血中发挥了保护作用。然而,目前国内尚无关于Shh在骨骼肌I/R损伤中的作用及机制研究。目的:本研究通过建立止血带小鼠后肢I/R损伤模型,评估Shh信号通路在骨骼肌I/R损伤中发挥的作用。为寻找其中的机制,我们将研究Shh是否通过经典合成代谢通路AKT/m TOR/p70S6K发挥骨骼肌I/R损伤的保护作用。最后,我们将探讨Shh信号对骨骼肌I/R损伤中相关的骨骼肌凋亡的影响。我们希望通过这些研究为骨骼肌I/R损伤提供新的治疗策略。方法:(1)选取10-14周雄性C57BL/6小鼠,利用止血带建立小鼠后肢I/R损伤模型,即单侧后肢缺血3 h,其后松开止血带恢复血流灌注1 d,3 d,5 d,7 d及14 d,并于不同时间点获取骨骼肌标本。利用Western blot法检测Shh信号通路相关蛋白包括Shh,Gli1及Gli2表达情况。同时,利用免疫荧光法检测骨骼肌组织Shh信号表达。(2)为了阐明Shh信号是否参与了骨骼肌I/R损伤的调控。通过腹腔注射Smoothened直接抑制剂Cyclopamine以抑制Shh信号通路。相反,通过损伤侧后肢肌肉注射质粒编码人Shh基因(ph Shh)以过表达Shh通路。建立模型并取I/R 7 d损伤小鼠骨骼肌标本,通过Western Blot法或者免疫荧光检测法评估抑制及过表达Shh信号效率。其后通过HE染色骨骼肌损伤评分评估骨骼肌损伤程度,通过Masson染色评估骨骼肌纤维化程度。(3)为了研究AKT/m TOR/p70S6K信号通路在骨骼肌I/R损伤中时间-表达进程,利用Western blot检测不同灌注时间AKT,m TOR,p70S6K以及他们的磷酸化蛋白表达水平。为进一步验证AKT/m TOR/p70S6K信号通路是否参与了Shh诱导的I/R损伤保护,利用肌肉注射ph Shh方式过表达Shh信号,并在I/R 7 d损伤时间点检测AKT/m TOR/p70S6K信号通路表达。其后为进一步研究其中机制,利用ph Shh过表达Shh的同时,通过腹腔内注射PI3K-m TOR双向抑制剂NVP-BEZ235抑制AKT/m TOR/p70S6K信号通路,并在I/R 7 d损伤时间点检测AKT/m TOR/p70S6K信号通路表达、骨骼肌损伤评分及骨骼肌纤维化。(4)本研究中,我们还将评估I/R损伤所致的骨骼肌凋亡。首先,利用Western blot检测法评估凋亡蛋白Cleaved Caspase 3及Bax,抗凋亡蛋白Bcl2的时间-表达进程。其后为进一步研究Shh在骨骼肌I/R损伤中的作用,过表达Shh信号并在I/R 7 d损伤时间点检测凋亡蛋白Cleaved Caspase 3及Bax,抗凋亡蛋白Bcl2表达,同时还利用TUNEL染色评估骨骼肌凋亡水平。结果:(1)首先,我们发现I/R损伤可导致Shh,Gli1及Gli2表达升高,并在再灌注5 d达到峰值,其后表达量逐渐下降。免疫荧光检测提示在正常组及I/R 1 d组,几乎不能检测到Shh信号表达。在I/R 3d,5 d组,于骨骼肌基底膜可以观察到明显的Shh信号表达,该信号表达靠近于骨骼肌肌核或者与骨骼肌肌核共定位。在I/R 7 d组和I/R 14d组,Shh信号表达减弱。(2)在进一步研究中发现,Shh信号通路抑制剂Cyclopamine治疗骨骼肌局部可见坏死区域,同时HE染色骨骼肌损伤评分评估增高(P0.05),提示抑制Shh信号通路后骨骼肌I/R损伤加重。同时Masson三色染色提示Cyclopamine治疗组可以观察到明显增多的胶原堆积(P0.05)。并且,当利用肌肉内注射ph Shh过表达Shh信号通路时,可以观察到相反的结果。(3)然后,Western blot结果提示骨骼肌I/R损伤后早期Shh信号表达升高同时伴随AKT,m TOR及p70S6K信号磷酸化水平升高,其后均逐渐降低。ph Shh治疗可导致p-AKT/AKT,p-m TOR/m TOR及p-p70S6K/p70S6K比值明显升高,而这种作用又可被NVP-BEZ235治疗所阻断。(4)最后,Western blot检测提示凋亡蛋白Cleaved Caspase 3水平及Bax/Bcl2比值在I/R损伤后逐渐升高,均在I/R 7 d达到峰值,其后开始下降。而进一步探讨Shh对凋亡通路影响时发现过表达Shh信号可降低I/R 7 d凋亡蛋白Cleaved Caspase 3表达,及Bax/Bcl2比值(P0.05)。而TUNEL染色评估提示外源性ph Shh治疗可降低I/R损伤后骨骼肌凋亡指数(P0.05)。结论:Shh信号在胚胎后时期小鼠后肢骨骼肌I/R损伤模型中可被再激活;Shh信号在小鼠后肢骨骼肌I/R损伤中通过AKT/m TOR/p70S6K信号通路发挥抗骨骼肌损伤及抗骨骼肌纤维化作用;同时,Shh信号在小鼠后肢骨骼肌I/R损伤中发挥抗凋亡作用。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R68

【相似文献】