【作者】 李梅；

【导师】 侯凡凡； 聂静；

【作者基本信息】 南方医科大学 ， 内科学（肾脏病）， 2015， 硕士

【摘要】 研究表明,成人慢性肾脏病(chronic kidney disease,CKD)发病率高达11-13%。几乎所有的CKD都不可避免地进展至终末期肾脏病(ESRD),而且进展至ESRD后,除了肾脏替代治疗外,几乎没有好的治疗手段。肾脏间质纤维化(tubular interstitial fibrosis,TIF)是CKD进展至终末期肾病的共同途径,主要表现为细胞外基质堆积、肾单位结构损毁、肾固有细胞消失。细胞外基质(ECM)的沉积不仅仅是肾脏纤维化的重要特征,还是其主要的“恶化”因素。ECM沉积受到多种细胞因子和信号通路的调控,其中最为主要的信号通路包括转分化生长因子1(TGFβ1)及Wnt/β-catenin等信号通路。TGFβ1在促进细胞增生、分化、ECM产生及凋亡等方面均有着重要的作用。TGFβ1与TGFβ受体结合后,可以通过经典途径TGFβ1/Smads信号通路及旁路途径发挥作用。大量研究证明,TGFβ1/Smads信号通路在肾脏纤维化中发挥着极其重要的作用。TGFβ1通过结合并活化TGFβ II型受体(TβRII),后者结合并激活TGFβ Ⅰ型受体(TβRI)。活化的TβRI通过衔接蛋白(SARA)结合Smad2/3使得两者发生磷酸化。磷酸化的Smad2/3与Smad4形成复合物入核,激活下游基因包括Ⅰ型胶原、纤连蛋白及α-平滑肌肌动蛋白等表达,使得ECM增生、沉积,进而发生组织纤维化。同时,作为Smad家族的调节蛋白,Smad7可以通过泛素化作用降解磷酸化的Smad3、Smad2。另外,TGFβ1还可以通过其他信号通路引起组织纤维化,其中较为重要的几个磷酸化信号通路包括β38通路、PI3K通路及ERK通路。TGFβ1/Smads信号通路分子成为了研究抗纤维化治疗药物的靶点。TGFβ1活性的多肽抑制剂直接抑制TGFβ1活性,SB-431542、SD-208、IN-1130则抑制TGFβ1受体活性,都可以抑制TGFβ1信号通路,有效抑制细胞外基质堆积,但是由于其非选择性,同时抑制Smad3、Smad2活性,产生了一系列副作用,并未进一步成为临床治疗CKD的药物。选择性抑制剂进入了研究日程。鉴于Smad3在介导TGFβ1促纤维化作用中的重要性,有必要开发选择性抑制Smad3磷酸化的化合物。天然药物在医疗保健和药物研究中占有重要角色,资料表明约80%的抗菌药物和60%的抗癌药物直接或间接来自天然产物。自然界中的小分子化合物不仅是人类治疗药物发现的重要源泉,而且以其为化学探针,有利于揭示其生物学机制。近年来,我研究所与云南昆明植物研究所合作,从干漆即漆树科植物漆树的干燥树脂中分离纯化出小分子化合物漆酚(代号GQ-5,结构式如图所示)。该酚性化合物具有良好的抗肿瘤活性,对多种肿瘤细胞株均有细胞毒活性。进一步研究发现GQ-5对人脐静脉血管内皮细胞的增殖、迁移和成管具有明显的抑制作用。我们前期的研究已经通过体内外实验证实GQ-5能抑制肾脏间质纤维化:在UUO大鼠7天预防用药组模型中(UUO大鼠手术第1天开始给药),GQ-5减少肾脏炎症细胞浸润,减少成纤维细胞积聚,降低肾脏组织α-SMA、Fibronectin和Collagen-Ⅰ蛋白的表达,说明GQ-5能够抑制肾脏间质纤维化的形成;在大鼠肾小管上皮细胞(NRK-52E)中,我们也发现GQ-5抑制肾脏细胞TGFβ1引起的α-SMA、Fibronectin和Collagen-Ⅰ蛋白的上调。另外,我们还发现,GQ-5显著抑制了 UUO大鼠肾脏Smad3的磷酸化水平,显著抑制TGFβ1介导的肾小管上皮细胞Smad3的磷酸化水平,而对Smad2的磷酸化水平无明显的影响,同时GQ-5对TGF-β1诱导的p38、ERK与PI3K的磷酸化几乎不影响,对Smad4、Smad7也几乎没有影响。以上结果提示GO-5特异性抑制Smad3磷酸化进而抑制肾脏纤维化。那么GQ-5抑制Smad3磷酸化的具体机制又是怎样的呢?研究表明,TGFβ1通过结合并活化TGFβ Ⅱ型受体(TβRⅡ),后者结合并激活TGFβ Ⅰ型受体(TβRI)。活化的TβRⅠ通过衔接蛋白SARA结合Smad2/3使得两者发生磷酸化。SARA分为三个部分:N端、Smad结合域(SBD)及C端,其中中间部位SBD域(氨基酸665-749)是结合Smad的部位,同时需要N端(氨基酸1-664,包含一个双锌指结构域FYVE)的作用,C端(氨基酸750-1123)是结合TGFβ受体的部位。另外还有多个衔接蛋白,如ELF、Kindlin-2等。GQ-5特异抑制Smad3磷酸化,可能是抑制了 TβRI与Smad3之间的相互作用,也可能还抑制了衔接蛋白与Smad3之间的相互作用。因此,我们设计了以下实验,探讨GQ-5特异抑制Smad3磷酸化的机制。1、动物实验观察GQ-5对肾脏纤维化的治疗作用:前期实验在UUO大鼠7天预防用药组中发现GQ-5能抑制肾脏间质纤维化,且其作用机制可能是通过特异性抑制Smad3磷酸化,说明GQ-5能够预防肾脏间质纤维化的形成,那么GQ-5对已经形成的肾脏纤维化是否有治疗作用呢,我们重新制作UUO大鼠模型,在UUO手术7天后即已经形成纤维化后开始给药,观察GQ-5对药物治疗组的作用,检测 GQ-5 对 Smad3、Smad2、p38、ERK、PI3K磷酸化的作用及其对Smad4、Smad7、a-SMA、Ⅰ型胶原及纤连蛋白的影响;2、细胞实验观察GQ-5对成纤维细胞活化的影响:前期实验已经在大鼠肾小管上皮细胞(NRK-52E)中发现GQ-5对Smad3、Smad2、p38、ERK、PI3K磷酸化的作用及其对Smad4、Smad7、a-SMA、Ⅰ型胶原及纤连蛋白的影响,考虑到成纤维细胞的活化在肾脏纤维化中起着非常重要的作用,我们选取了大鼠肾脏成纤维细胞株(NRK-49F),采用TGFβ1刺激,通过观察GQ-5对以上蛋白的影响检测其对成纤维细胞活化的作用;3、探讨GQ-5特异性抑制Smad3磷酸化的机制:前面已经发现GQ-5能特异性抑制Smad3磷酸化,接下来我们进一步探讨GQ-5抑制Smad3磷酸化的机制,在NRK-52E细胞中,采用TGFβ1刺激,观察GQ-5对TβRⅠ、TβRⅡ、Smad3、Smad2之间相互作用的影响,并观察GQ-5对TβRⅠ与SARA、SARA与Smad3、Smad2之间相互作用的影响,然后在UUO大鼠模型中检测相同指标,验证在细胞实验中的结论。第一部分GQ-5对UUO大鼠已形成的肾脏纤维化的作用目的通过建立大鼠单侧输尿管梗阻模型(UUO)检测GQ-5对UUO大鼠14天模型中药物治疗组肾脏纤维化的作用。方法大鼠UUO模型:雄性SD大鼠18只,体重250-280g,假手术(Sham)组6只,UUO+丙二醇组6只,UUO+GQ-5 day7(UUO大鼠手术7天后开始给药,药物GQ-5用丙二醇溶解)组6只。UUO模型制作过程:3%的戊巴比妥钠麻醉,固定大鼠,做左侧的背侧切口,在左侧输尿管近肾脏下极端结扎,然后缝合切口。Sham组做左侧的背侧切口,不做输尿管结扎,然后缝合切口;UUO+GQ-5 day7组造模第7天开始每只腹腔注射GQ-5 40mg/kg,每日一次;UUO+丙二醇组造模第7天开始腹腔注射与UUO+GQ-5 day7组等体积的5%丙二醇,每日一次,术后第14天全部处死,处死大鼠时,腹主动脉抽血,处以50ml冰PBS灌注腹主动脉至肾脏苍白色,Western Blot实验方法检测Smad2、Smad3、p38、ERK、PI3K及其磷酸化蛋白水平,同时检测Collagen-Ⅰ、α-SMA及Fibronectin蛋白的表达。结果本实验用Western Blot实验发现在UUO大鼠14天模型中,GQ-5能显著降低药物治疗组中Collagen-Ⅰ、α-SMA及Fibronectin蛋白的表达,同时也明显抑制了治疗组中Smad3的磷酸化水平而对p-Smad2 Smad4 Smad7 p-p38 p-PI3K p-ERK的表达均无明显影响。小结GQ-5对UUO大鼠已形成的肾脏纤维化具有显著的治疗作用。第二部分GQ-5对成纤维细胞活化的影响目的采用TGF-β1诱导大鼠肾脏成纤维细胞(NRK-49F),观察GQ-5对成纤维细胞活化的影响。方法NRK-49F细胞:大鼠肾脏成纤维细胞株NRK-49F用含10%胎牛血清的DMEM/F12培养基传代培养,在37℃,5%C02孵育箱培养,细胞贴壁生长,每3～4天传代一次,达50%或70%～80%融合后给予无血清培养基同步静止12 h后进行实验。Western Blot试验方法检测Smad2 Smad3及其磷酸化蛋白水平,同时检测p38 ERK PI3K及其磷酸化蛋白的表达,以及检测Smad4 Smad7、a-SMA、Collagen-Ⅰ(Ⅰ 型胶原)、Fibronectin(纤连蛋白)。结果本实验用Western Blot实验发现在NRK49F的细胞中,GQ-5抑制Collagen-Ⅰ、α-SMA及Fibronectin蛋白的表达以及选择性抑制了 Smad3的磷酸化。小结GQ-5能有效抑制TGFβ1诱导的成纤维细胞的活化。第三部分探讨GQ-5选择性抑制Smad3磷酸化的机制目的通过采用TGF-β1诱导大鼠肾小管上皮细胞(NRK-52E)活化,观察GQ-5对TβRⅠ、TβRⅡ、Smad3、Smad2之间相互作用的影响,并观察GQ-5对TβR1与SARA、SARA与Smad3、Smad2之间相互作用的影响,探讨GQ-5特异抑制Smad3磷酸化的机制;随后在大鼠单侧输尿管梗阻模型(UUO)中验证细胞实验所得。方法1;NRK-52E细胞:大鼠肾小管上皮细胞株NRK-52E用含10%胎牛血清的DMEM培养基传代培养,在37℃,5%CO2孵育箱培养,细胞贴壁生长,每2～3天传代一次,达70%～80%融合后给予无血清培养基同步静止12h后进行实验。免疫共沉淀(COIP)实验方法检测TβRI蛋白与Smad3、Smad2、TβRⅡ蛋白的结合水平,以及SARA蛋白与Smad3、Smad2蛋白的结合情况。2;UUO大鼠模型:雄性SD大鼠18只,体重250-280g,假手术(Sham)组6只,UUO+丙二醇组6只,UUO+GQ-5(药物GQ-5用丙二醇溶解)组6只,UUO模型制作过程:3%的戊巴比妥钠麻醉,固定大鼠,做左侧的背侧切口,在左侧输尿管近肾脏下极端结扎,然后缝合切口。Sham组做左侧的背侧切口,不做输尿管结扎,然后缝合切口;UUO+GQ-5组造模第1天开始每只腹腔注射GQ-5 40mg/kg,每日一次;UUO+丙二醇组造模第1天开始腹腔注射等体积的5%丙二醇,每日一次,术后第14天全部处死,处死大鼠时,腹主动脉抽血,处以50ml冰PBS灌注腹主动脉至肾脏苍白色,免疫共沉淀(COIP)实验方法检测TβRⅠ蛋白与Smad3、Smad2、TβRⅡ蛋白的结合水平,以及SARA蛋白与Smad3、Smad2蛋白的结合情况。结果本实验用免疫共沉淀(COIP)实验方法发现在大鼠肾小管上皮细胞(NRK-52E)中GQ-5明显抑制了 TGF-β1上调的TβRⅠ与Smad3的结合,而对TβRⅠ与Smad2、TβRⅡ的结合基本没有影响,还发现GQ-5显著抑制了 TGF-β1上调的SARA与Smad3的结合,而对SARA与Smad2的结合水平基本没有影响;在UUO大鼠模型中做相似实验,得到相一致结论。小结体内外实验发现GQ-5抑制Smad3磷酸化的机制是通过特异性抑制SARA与Smad3的结合从而减弱TGF-β1上调的TβRⅠ与Smad3的结合水平。结论1,GQ-5对UUO大鼠已形成的肾脏纤维化具有显著的治疗作用;2,GQ-5能有效抑制TGFβ1诱导的成纤维细胞的活化。3,GQ-5通过特异性抑制TGF-β/Smads信号通路中衔接蛋白SARA与Smad3的结合,导致Smad3与TβRⅠ的结合减弱,从而特异性抑制Smad3的磷酸化,最后有效抑制肾脏纤维化。更多还原

【Abstract】 Renal fibrosis,characterized by massive interstitial myofibroblast activation and excessive matrix proteins accumulation,is the final common pathway of virtually all kinds of progressive chronic kidney disease(CKD)leading to end stage renal disease(ESRD).[1-4]Mounting evidence has established a crucial role for transforming growth factor-β(TGF-β)in mediating chronic inflammation,activation of myofibroblast,and accumulation of extracellular matrix(ECM).[4-7]TGF-β1 signals are transduced by transmembrane serine/threonine kinase receptors type I(TβRI)and type II(T(3RII)and intracellular mediators known as Smads.[8,9]Upon TGF-β1 stimulation,Smad2 and Smad3 are phosphorylated by TβRI.Phosphorylated Smads heteroligomerize with the common partner Smad4 and then translocate into the nucleus,where they control the transcription of TGF-β-responsive genes through interaction with specific cis-acting elements in the regulatory regions.[10-12]Although both Smad2 and Smad3 are strongly activated in various experimental and human fibrotic kidney diseases,it is now well recognized that Smad3 is the key mediator of TGF-β1-induced ECM production and tissue fibrosis.[13-15]Deletion of Smad3 suppresses fibrogenesis in a number of rodent models,including diabetic nephropathy,[16]obstructive nephropathy,[17,18]and drug toxicity-related nephropathy.[19]On the other hand,conditional knocking out of Smad2 from kidney tubular cells significantly enhanced renal fibrosis via up-regulation of Smad3 signaling.[20]These findings indicate that Smad3 expression and/or phosphorylation might be a potential target for the intervention of renal fibrosis.It is now well accepted that Smad2 and Smad3 are two critical downstream mediators responsible for the biological effects of TGF-β1.In the context of renal fibrosis,Smad2 and Smad3 are strongly activated in both experimental and human kidney diseases,including diabetic nephropathy obstructive kidney diseases,remnant kidney disease,hypertensive nephropathy,drug-associated nephropathy,and immunologically-mediated glomerulonephritis.Many fibrogenic genes,such as(Collal,ColIa2,ColIIIal,ColVa2,ColVIal,and ColVIa3)and tissue inhibitor of MMP-1(TIMP-1)are the downstream targets of TGF-β/Smad3 signaling,suggesting that Smad3 may be a critical mediator of TGF-β/Smad signaling in fibrosis.An essential role for Smad3 in fibrogenesis is confirmed by the findings that deletion of Smad3 from mice suppresses fibrogenesis in a number of rodent models,including diabetic nephropathy,obstructive nephropathy,and drug toxicity-related nephropathy.Furthermore,the use of a Smad3 inhibitor to inhibit endothelial-myofibroblast transition and renal fibrosis in a type-1 diabetic kidney disease demonstrates a therapeutic potential for kidney disease by targeting Smad3 signaling.In the past years,many promising targets for the treatment of renal fibrosis have been validated in various animal models,and even more new targets have been identified.Renal fibrosis,in contrast,remains a largely uncharted territory in clinical trials.The reasons for this are certainly multifactorial and may include long study durations if hard endpoints,the lack of non-invasive markers or diagnostic tools to assess kidney scarring,and thus,monitor therapy.However,the industry has noted the enormous potential market,given the possibility of developing antifibrotic therapy that might be of benefit in many different types of organ fibrosis.Furthermore,there is hope that with a large consortia search for biomarkers and advancing ultrasound,or through MR-based or molecular-imaging techniques.The potential of natural products as the candidates for drug discovery has been well recognized.[21]Resina Toxicodendrl is the dried resin secreted by Toxicodendron venicifluum and has been used as an anti-inflammatory and anti-scarring agent in traditional Chinese medicine for centuries.In the present study,we isolated and purified the major component of Resina Toxicodendri GQ5,a small molecular phenolic compound.We demonstrated that treatment with GQ5 significantly inhibited the progression of interstitial fibrosis in the unilateral ureteral obstruction(UUO)model.We also demonstrated that the anti-fibrotic effect of GQ5 might be mediated by selective inhibition of TGF-β1-induced Smad3 phosphorylation.GQ5 isolated from Resina Toxicodendri is a small molecular phenolic compound,3-[(Z)-Pentadec-8-enyl]catechol.The chemical structure was shown in the Figure as follow.In last study,we have found that GQ5 Ameliorates Renal Interstitial Fibrosis in vitro and vivo.Histology stained with HE detected that GQ-5 could make UUO rat renal interstitial inflammatory cells significantly reduced,fibroblasts was also reduced,renal tubular cell morphology had been restored.Western blot analysis showed that GQ-5 decreased alpha SMA expression,Similar findings were also demonstrated at the protein level by Immunohistochemically analysis.Western Blot and immunohistochemistry confirmed that GQ-5 decreased Fibronectin and Collagen-I expression.Western Blot and immunohistochemistry have revealed that GQ-5 significantly inhibited Smad3 phosphorylation levels,but had no obvious effect on the levels of Smad2 phosphorylation.And in NRK52E,we also have found that GQ-5 could decreased the a-SMA expression in a dose-dependent manner.GQ-5 reduced the up-regulated expression of fibronectin protein as well as α-SMA ·GQ-5 reduced phosphorylation levels of Smad3 induced by TGF-β,but not the phosphorylated levels of Smad2 and the protein expression levels of Smad4 and Smad7,GQ-5 did not affect the phosphorylation of other signaling pathways,such as MAPK/p38,ERK or phosphoinositide 3-kinase.So we supposed that GQ-5 can ameliotate renal fibrosis by inhibiting TGF-β 1-induced Smad3 phosphorylation.To confirm whether GQ-5 can treat renal fibrosis and inhibit TGF-β1-induced activation of fibroblasts and explore the mechanisms of GQ-5 inhibits TGF-β1-induced smad3 phosphorylation,we have designed experiments as follows.(1)Do experients in GQ-5+UUO day7 of UU014 days rats as same as that have did in GQ-5+UUO 7days rats,and so is it in NRK49F cells.(2)Explore the deeper mechanisms of GQ-5 inhibits renal fibrosis in vitro and vivo.(3)Explore other mechanisms of GQ-5 hinders renal fibrosis,such as kindlin-2,another adapter protein.Part I Explore whether GQ-5 can treat renal fibrosisObjective:In this study,we detected whether GQ-5 can treat renal fibrosis in GQ-5+UUO day7 of UU014 days rats.Method:UUO Rats.The animals were anesthetized with an intraperitoneal(IP)injection.A midline incision was made in the abdominal wall,the left ureter was dissected out and ligated with 4.0 silk at two points along its length.The abdominal wound was approximated with the same silk suture.Three groups of rats comprising 6 animals each(total=18)were included as follows.(1)Sham group:(2)UUO+ Propylene glycol group:the rats received IP injection of Propylene glycol and underwent unilateral ureteral ligation.(3)UUO + GQ-5 day7 group:these rats in addition those in group Ⅱ received IP injection of GQ-5 7 days after surgery at a dose of 40mg/kg a day.The rats were killed on day 14 after UUO or Sham operation,and the kidneys were harvested.Western Blot were carried out by using anti-Smad2,anti-Smad3,anti-phospho-Smad2,anti-phospho-Smad3,anti-fibronectin,anti-Collagen-I,anti-Sma4,anti-a-SMA,anti-Smad7,anti-MAPK/p38,anti-ERK,anti-phosphoinositide3,kinase,anti-phospho-p38,anti-phospho-ERK.Result:Western Blot revealed that GQ-5 decreased a-SMA,Fibronectin and Collagen-1 expression.GQ-5 significantly inhibited Smad3 phosphorylation levels,but had no obvious effect on the levels of Smad2 phosphorylation,as well as Smad4,Smad7,P-MAPK/p38,P-ERK or P-phosphoinositide 3-kinase.SummaryIn this study,we proved that GQ-5 can treat renal fibrosis in GQ-5+UUO day7 of UUO 14 days rats.PartⅡ Explore whether GQ-5 can inhibit TGF-β1-induced activation of fibroblastsObjective:In this study,we detected whether GQ-5 can inhibit TGF-β1-induced activation of fibroblasts in NRK-49F cells.Method:Cell culture.NRK49F were grown in DMEM/F12containing 10%fetal bovine serum.When 60-80%cells were stick on the well,they were cultured for12h in free serum medium.Western Blot were carried out by using anti-Smad2,anti-Smad3,anti-phospho-Smad2,anti-phospho-Smad3,anti-fibronectin,anti-Collagen-I,anti-Sma4,anti-a-SMA,anti-Smad7,anti-MAPK/p38,anti-ERK or anti-phos phoino sitide 3-kinase,anti-phospho-p38,anti-phospho-ERK.Result:Western Blot revealed that GQ-5 decreased a-SMA,Fibronectin and Collagen-I expression.GQ-5 significantly inhibited Smad3 phosphorylation levels,but had no obvious effect on the levels of Smad2 phosphorylation,as well as Smad4,Smad7,P-MAPK/p38,P-ERK or P-phosphoinositide 3-kinase.SummaryIn this study,we proved that GQ-5 can inhibit TGF-β1-induced activation of fibroblasts.Part Ⅲ Explore the mechanisms of GQ5 Selectively inhibiting TGF-β1-Induced Smad3 Phosphorylation.Objective:To research how GQ-5 Selectively inhibits Smad3 Phosphorylation,we evaluated the efficacy of GQ-5 on the interaction of TβRI with TβRII,Smad3,Smad2 and SARA with TβRI,Smad3,Smad2 in NRK52E cells and UUO 14days rats.MethodCell culture.NRK52E were grown in DMEM/F12 containing 10%fetal bovine serum.When 60-80%cells were stick on the well,they were cultured forl2h in free serum medium.Co-mmunoprecipitation were carried out by using anti-TβRI,anti-Smad3,anti-Smad2,anti-TβRII,anti-SARA.UUO Rats.The animals were anesthetized with an intraperitoneal(IP)injection.A midline incision was made in the abdominal wall,the left ureter was dissected out and ligated with 4.0 silk at two points along its length.The abdominal wound was approximated with the same silk suture.Three groups of rats comprising6 animals each(total=18)were included as follows.(1)Sham group:(2)UUO+ Propylene glycol group:the rats received IP injection of Propylene glycol and underwent unilateral ureteral ligation.(3)UUO+ GQ-5 group:these rats in addition those in group II received IP injection of GQ-5 at a dose of 40mg/kg a day.The rats were killed on day 14 after UUO or Sham operation,and the kidneys were harvested.Co-Immunoprecipitation were carried out by using anti-TβRI,anti-TβRII anti-Smad3,anti-Smad2,anti-SARA.Result:In NRK52E cells,TβRI bound with TβRII,Smad2 and Smad3 upon TGF-β1 stimulation.Treatment with GQ5 significantly blocked the interaction of Smad3 with TβRI,but did not affect the interaction of Smad2 with TβRI.SARA bound with Smad2 and Smad3 upon TGF-β1 stimulation.Treatment with GQ5 significantly blocked the interaction of Smad3 with SARA,but did not affect the interaction of Smad2 with SARA.The same experimental results in UUO 14days rats.SummaryThese data suggests that GQ5 inhibited Smad3 phosphorylation by selectively blocking the interaction of Smad3 with SARA and then blocked Smad3 bond to TβRI in vivo and vitro.In Clonclution1.GQ-5 can treat renal fibrosis in GQ-5+UUO day7 of UUO 14 days rats.2.GQ-5 can inhibit TGF-β1-induced activation of fibroblasts.3.GQ5 inhibited Smad3 phosphorylation by selectively blocking the interaction of Smad3 with SARA and then blocked Smad3 bond to TβRI in vivo and vitro.更多还原