CN107714685A - 甜菜碱在修复酒精对动物胚胎的损伤中的应用 - Google Patents
甜菜碱在修复酒精对动物胚胎的损伤中的应用 Download PDFInfo
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Abstract
本发明涉及甜菜碱在修复乙醇(酒精)对动物胚胎的损伤中的应用。本发明还涉及用甜菜碱修复乙醇(酒精)对动物胚胎的损伤的方法,所述方法包括用治疗有效量的作为活性成分的甜菜碱(任选地,与其他活性成分(如叶酸)组合)处理所述胚胎。
Description
技术领域
本发明涉及甜菜碱的一种新医药用途。更具体地,本发明涉及甜菜碱在修复乙醇(酒精)对动物胚胎的损伤中的应用。
背景技术
早期研究表明,高浓度的乙醇会明显影响体细胞和生殖细胞的正常生理状态((Mameli等,2008;Caires等,2012;Luo,2014),尤其是过量摄入乙醇会对如肝脏和心脏等各种组织和器官造成严重损害,并导致癌症的发生(Po¨schl等,2004;Nagy等,2016;Stickel等,2017)。但乙醇是如何影响细胞的仍很难解释清楚,这是因为乙醇会导致多种生理指标、信号传导和基因表达等一系列的变化(Liu等,2015;Ron和Barak,2016;Dorokhov等,2015)。更重要的是,较高浓度的乙醇是致畸因子,会引起一系列胚胎异常变化和缺陷,称为胎儿酒精综合症(FASD)(Astley,2004)。某些情况下,在小鼠卵母细胞研究中使用乙醇作为卵激活的方法之一(Balakier和Casper,1993;Zhang等,2005),并且使用乙醇作为胚胎冻存的保护剂(Martino等,1996)。此外,乙醇还常被用来溶解添加到胚胎培养液中的药物。
在已有研究中,乙醇对胚胎发育的毒性研究受到特别关注。过量的乙醇摄入可引起各种胎儿畸形,这将给幼儿的生命中带来长久的痛苦。同时,乙醇作为表观遗传因素,对身体健康的负面影响可以通过影响生殖细胞或胚胎发育传递到后代。来自急性剂量的实验数据表明,植入前阶段小鼠胚胎受到乙醇影响后,易导致胎儿死亡和胎儿子宫内发育迟缓(Padmanabhan和Hameed,1988)。同时,连续15天摄入10%乙醇会导致囊胚孵化率的显著降低,而且,孕前摄入乙醇会损害滋养层的扩张(Pérez-Tito等,2014)。在体外培养研究中发现,低浓度乙醇与其对胚胎发育的影响之间存在着相关性。例如,低浓度乙醇对猪胚胎发育具有毒性影响,乙醇浓度超过1%的体外成熟(IVM)体系处理卵母细胞时,会导致卵母细胞ROS水平的升高和凋亡相关基因表达的上调(Lee等,2014)。但是尚未发现关于乙醇对小鼠胚胎发育的精确剂量效应的研究。
氧化应激(Oxidative Stress,OS)是指体内氧化与抗氧化作用失衡,倾向于氧化,导致中性粒细胞炎性浸润,蛋白酶分泌增加,产生大量氧化中间产物。氧化应激是由自由基在体内产生的一种负面作用,并被认为是导致衰老和疾病的一个重要因素。乙醇产生的ROS可能破坏蛋白质的结构和性质,并使酶活性发生变化(Sid等,2013)。但这很难解释乙醇影响胚胎发育的途径和机制。在乙醇干扰的所有代谢过程中,叶酸作为甲基供体的一碳代谢受到明显影响(Trimble等,1993;Mason等,2005)。一碳单位来自各种饮食,异常的一碳代谢可能会影响DNA甲基化的过程(Ulrey等,2005)。已有的数据证明乙醇可以干扰依赖叶酸的生化反应(Po¨schl等,2004),抑制叶酸作为供体,由S-腺苷甲硫氨酸(SAM)介导的甲基化过程(Schalinske和Nieman,2005)。小鼠的实验研究表明,乙醇对植入前胚胎的暴露改变了H19印迹控制区(ICR)中DNA甲基化水平(Philip等,2009)。基于乙醇对基因组DNA甲基化的影响和对一碳代谢的干扰的研究结果,进一步的研究发现,通过使用动物模型或体外培养补充叶酸可有助于DNA甲基化的调节并降低潜在的患癌风险(Liu和Ward 2010)。尽管尚不清楚叶酸在调节细胞信号系统和多种代谢(包括核酸和蛋白质)方面起到保护作用,但补充叶酸确实可以阻断乙醇诱导的胎儿畸胎的发生,并有效减轻了实验中乙醇的有害影响,同时预防了神经管缺陷和先天性异常(Gutierrez等,2007),改变了Hoxa1和miR-10a的表达(Wang等,2009)。但叶酸对人体的使用是受到限制的,因为叶酸对细胞和人体生理功能的毒性范围尚不完全清楚。
甜菜碱(Glycine betaine,甘氨酸三甲胺内盐)是一碳代谢循环的另一个甲基供体,广泛分布在动物,植物和微生物中。甜菜碱可从天然植物的根、茎、叶及果实中提取或采用三甲胺和氯乙酸为原料化学合成。甜菜碱的已知用途包括:抗肿瘤,降血压,抗消化性溃疡及胃肠功能障碍,治疗肝脏疾病,作为饲料添加剂具有提供甲基供体功能,可节省部分蛋氨酸,调节体内渗透压,促进脂肪代谢和蛋白质合成,提高瘦肉率等功能。
尽管早期胚胎中的甜菜碱来源还不清楚,但植入前胚胎直到桑葚胚阶段都含有几乎恒定水平的内源性甜菜碱(Corbett等,2014;Anas等,2008)。卵母细胞中的内源性甜菜碱可能部分转运自减数分裂成熟期间的卵丘细胞。SIT1作为甜菜碱/脯氨酸转运蛋白之一,在受精后在小鼠卵中被激活,并且在甜菜碱被转运到卵母细胞直至2-细胞阶段胚胎中起关键作用。另一方面,甜菜碱可能起保护作用,是因为它可以帮助保细胞持正常的渗透压(Stadmiller等,2017),在培养液中加入甜菜碱可能保护胚胎免受渗透压变化所带来的影响(Dawson和Baltz,1997;Anas等,2007)。
发明内容
本发明人经过潜心研究,利用体外培养系统,首先探讨了乙醇浓度梯度对动物(小鼠)胚胎发育的影响,然后出人意料地发现,通过在培养基中加入甜菜碱,能够显著改善受乙醇(酒精)影响的胚胎的发育。此外,本发明人还研究了不同组别处理的囊胚的全基因组DNA甲基化水平的变化。我们的研究结果表明,甜菜碱补充到培养液中可有效改善乙醇处理小鼠胚胎的发育,包括囊胚形成,植入和植入后发育,可将乙醇处理后的囊胚的全基因组DNA的甲基化水平向正常状态修复。所有这些结果表明在辅助生殖治疗及其它有关胚胎损伤修复治疗中,甜菜碱具有较高的应用价值。
因此,在一方面,本发明提供了甜菜碱在预防和/或治疗乙醇(酒精)对动物的损伤中的应用,因此甜菜碱可以用于制备预防和/或治疗乙醇(酒精)对动物的损伤的药物/营养品。
在本发明的一个实施方案中,所述动物为哺乳动物,优选为胎生哺乳动物,更优选选自食肉目(如猫科动物)、啮齿目(如鼠类)、偶蹄目(如猪、牛、羊等)、奇蹄目(如马、驴等)、灵长目(如猴和猿类等)、翼手目(如蝙蝠等)、长鼻目(如象等)和鲸目(如海豚等),最优选选自猫、大鼠、小鼠、豚鼠、猪、牛、羊(山羊/绵羊)、马、驴、猴、猿、狗和人。
在本发明的一个实施方案中,所述动物为受孕动物或所述动物处于胚胎发育阶段(即为动物胚胎),例如为受精、卵裂、桑葚胚、囊胚、原肠胚或器官形成阶段的胚胎。即,所述药物/营养品可以施用于需要辅助生殖治疗及其它有关胚胎损伤修复治疗的受试者。
在本发明的一个实施方案中,所述动物摄入/被暴露于过量乙醇,和/或其中所述甜菜碱能够修复乙醇(酒精)对动物(特别是动物胚胎)的损伤,改善囊胚形成,提高胚胎植入的成功率和植入后的发育,和/或能够将受乙醇(酒精)影响的DNA的囊胚的甲基化水平正常化。
在本发明的一个实施方案中,所述药物作为活性成分仅包含甜菜碱,或还包含其他可以预防和/或治疗乙醇(酒精)导致的不利后果(如胎儿酒精综合征(FASD)、胎儿的神经管缺陷、先天性异常、畸胎等)和/或促进胚胎发育的活性成分(如叶酸)。
在另一方面,本发明提供了甜菜碱在制备改善受乙醇(酒精)损伤的动物胚胎的发育的药物/营养品中的应用。
此外,本发明提供了甜菜碱在制备保护动物胚胎免受乙醇(酒精)损伤的药物/营养品中的应用。
在另一方面,本发明提供了一种用于预防和/或治疗乙醇(酒精)对动物的损伤的药物/营养品,其包含:作为活性成分的甜菜碱(任选地,与其他活性成分(如叶酸)组合);和药学/营养学上可接受的辅剂。
在另一方面,本发明提供了一种改善动物胚胎发育的药物/营养品,其包含:作为活性成分的甜菜碱(任选地,与其他活性成分(如叶酸)组合);和药学/营养学上可接受的辅剂。
本发明的药物/营养品可以口服服用。例如,以片剂、包衣片剂、糖锭剂、硬和软明胶胶囊、溶液、乳剂或混悬剂形式。然而,也可以通过直肠实现给药,例如,以栓剂的形式,或者经肠胃外给药,例如,以注射液的形式推注或滴注。
术语“药学/营养学上可接受的辅剂”是指在配制药物/营养品中使用的不具有治疗活性并且无毒的任意成分,如稀释剂、崩解剂、粘合剂、填充剂、溶剂、缓冲剂、张度剂、稳定剂、抗氧化剂、表面活性剂或润滑剂。
在还有的另一方面,本发明提供了一种改善动物胚胎发育的方法,所述动物胚胎受氧化应激(由乙醇(酒精)或糖尿病导致的)损伤,所述方法包括用治疗有效量的作为活性成分的甜菜碱(任选地,与其他活性成分(如叶酸)组合)处理所述胚胎。所述动物胚胎的发育可以是在体内或体外。在体内胚胎发育的情况下,所述胚胎通过母体摄入甜菜碱;而在体外胚胎发育的情况下,可以将甜菜碱加入到体外胚胎的培养液中。
术语“治疗有效量”意指当被给药于受试者用于治疗疾病状态时,足以实现对于疾病状态的这种治疗的药物的量。“治疗有效量”将依赖于所用药物、所治疗的疾病状态、所治疗的疾病的严重性、受试者的年龄和相对健康状况、给药的途径和形式、主治医师或兽医师的判断以及其他因素而变化。
在本发明的一个具体实施方案中,提供了一种在胚胎体外培养体系中通过补充甜菜碱来修复酒精对胚胎的损伤的方法,所述方法包括用治疗有效量的作为活性成分的甜菜碱(任选地,与其他活性成分(如叶酸)组合)处理所述胚胎。
哺乳动物胚胎由于母亲过度摄取乙醇而受到损害,乙醇会导致较高水平的氧化应激反应,并干扰一个碳单位的代谢。本发明中,利用体外培养体系发现乙醇对小鼠胚胎发育的直接作用呈现浓度依赖性。胚胎培养液中使用含有1%的乙醇可显著抑制早期胚胎的发育,并且这种影响会持续整个胚胎在子宫中的发育—包括胚胎着床和植入后发育。但在培养液中添加50μg/ml的甜菜碱能有效地促进乙醇损伤的胚胎发育过程,而添加甘氨酸没有起到类似作用。5-甲基胞嘧啶免疫检荧光测结果表明,补充甜菜碱可以降低1%乙醇引起的小鼠囊胚细胞全基因组DNA甲基化水平,但甘氨酸不能发挥同样的影响。我们的研究结果表明,甜菜碱可能通过恢复囊胚基因组DNA甲基化水平来有效地修复乙醇损伤胚胎的发育。
糖尿病中的胰岛素抵抗与氧化应激关系密切。高游离脂肪酸(FFA)刺激的后果是启动了氧化应激机制(高活性反应分子产生和抗氧化作用之间长期失衡而引起组织损伤)。这些活性分子可直接氧化和损伤DNA、蛋白质、脂类,还可作为功能性分子信号,激活细胞内多种应激敏感信号通路,这些信号通路与胰岛素抵抗和β细胞功能受损密切相关。基于各种(氧化)应激损伤的分子机理具有一定的共性特征(例如,影响DNA甲基化水平),我们推断甜菜碱应该具有较为广泛的修复各种(氧化)应激反应(ROS)造成的胚胎损伤(包括由糖尿病等产生的氧化应激反应(ROS)导致的胚胎损伤)的能力。
附图说明
图1.不同浓度的乙醇对植入前胚胎发育的影响。将精子加入含有卵母细胞的培养基中进行体外受精(IVF),约8小时后获得的原核胚胎(PN),分别在含有不同乙醇浓度的KSOM培养基中培养4天。通过计算囊胚数量与每个处理组中使用的原核胚数量的比例来分析囊胚形成率。数据显示,当浓度为0.5%以上时,乙醇囊胚形成率会明显降低。显示的数据是平均值±SEM;
图2.乙醇和甜菜碱对囊胚形成的影响。将精子加入含有卵母细胞的培养基中进行体外受精(IVF),约8小时后获得的原核胚胎(PN),分别在补充有乙醇或(和)甜菜碱的KSOM培养基中培养4天。分析胚胎发育情况。(A)不同培养基产生的囊胚形态。在(A)中,培养基中没有乙醇或甜菜碱被称为对照组(NC),c和e指乙醇组,其培养基分别含有浓度为1%和1.5%的乙醇,b,d和f表示将甜菜碱以50μg/ml的浓度补充到含有对应于a,c和e的乙醇的培养基中。(B)乙醇和甜菜碱对囊胚形成比例的影响。数据显示,在培养基中乙醇浓度为1%或1.5%时,培养基中甜菜碱的补充可有效恢复乙醇囊泡形成的抑制作用。显示的数据是平均值±SEM。数值在统计学上具有显著差异(p<0.05,*);
图3.乙醇和甜菜碱对囊胚植入的影响。用于移植的囊胚从各个处理组中获得,包括对照组(NC),胚胎培养基中不含乙醇和甜菜碱;甜菜碱组(Bet),胚胎培养基中含有甜菜碱(50μg/ml);乙醇组(Eth),胚胎培养基中含乙醇(v/v 1%);甜菜碱+乙醇组(Eth+Bet),胚胎培养基中含有甜菜碱(50μg/ml)和乙醇(v/v 1%)。在实验中,将十枚囊胚移植到假孕鼠每个子宫的边角中。移植24和30小时后,静脉注射0.1ml 1%芝加哥蓝B,5分钟后检查植入部位,如Paria的工作(Paria BC等人,1993)所述。(A)乙醇或(和)甜菜碱补充培养基对囊胚植入的影响。通过囊胚转移到子宫边角上24小时后注射100μl的1%芝加哥蓝色染料溶液来检查植入部位的表现。(B)定量结果。括号内的数字表示,每组移植囊胚后的蓝色条带数和使用的囊胚总数的百分数。值在统计学上显著不同,p<0.05;
图4.乙醇和甜菜碱对囊胚移植8天后胚胎(胎儿)发育的影响(E11.5)。(A)对照组(NC)胎儿,在移植无任何补充剂的KOSM培养基中培养IVF囊胚4天后获得,它们具有典型的形态发育并具有一定程度的尺寸差异,并根据其大小记录在I类或II类。通过棒状标尺(10mm)来显示刻度。(B)乙醇处理组(Eth)的胎儿,在移植含有1%乙醇的KSOM培养基中培养IVF囊胚的4天后获得。它们显示不同的发育形态,甚至在个体大小上有很大差异,较小的胎儿根据其大小和形状记录在III级。(C)乙醇加甜菜碱处理组(Eth+Bet)的胎儿,在移植含有1%乙醇和50μg/ml甜菜碱的KSOM培养基中培养IVF囊胚4天后获得。这些胎儿的形态学质量与NC组相似。(D)和(E)定量分析乙醇和甜菜碱影响植入后胚胎发育。(D)结果表明,Eth+Bet组中所有胚珠中成形胎儿的百分比可以恢复比Eth组更高的水平(75%vs 61.08%,P>0.05),甚至高于NC组(75%vs 66.07%,P>0.05)。这些数据预示着甜菜碱可能是保护胚胎发育的有效试剂。同样,可以发现(E)甜菜碱可以使Eth+Bet组中I类胎儿的百分比提高到42.11%,而在Eth组中成形的胚珠中仅有20%的I型胎儿,而III类胎儿的数值尽管没有统计学差异,但也不会出现在Eth+Bet组中;
图5.不同浓度乙醇和甘氨酸对囊胚形成的影响。将精子加入含有卵母细胞的培养基中进行体外受精(IVF),约8小时后获得的原核胚胎(PN),分别在无(NC)或含有1%乙醇(Eth),1%乙醇加23μg/ml甘氨酸(Eth+Gly)的KSOM培养基中培养4天,分析胚胎发育情况。数据显示,甘氨酸不能修复乙醇损伤造成的囊胚形成率下降,甚至会明显导致囊胚形成率较低(22.2%)。显示的数据是平均值±SEM。值在统计学上显著不同(p<0.01,**);和
图6.来自含有乙醇,甜菜碱或甘氨酸的培养基的囊胚中DNA甲基化整体水平的分析。(A)不同处理组囊胚中5-MeC免疫荧光的代表性图像,分别来自对照组(NC),无任何补充剂的KOSM培养基中培养IVF原核胚4天后处理获得;乙醇处理组(Eth),在含有1%乙醇的KSOM培养基中培养IVF原核胚4天后处理获得;乙醇加甜菜碱处理组(Eth+Bet),在含有1%乙醇和50μg/ml甜菜碱的KSOM培养基中培养IVF原核胚4天后处理获得;甜菜碱处理组(Bet),在含有50μg/ml甜菜碱的KSOM培养基中培养IVF原核胚4天后处理获得;甘氨酸处理组(Gly),在含有23μg/ml甘氨酸的KSOM培养基中培养IVF原核胚4天后处理获得;乙醇加甘氨酸处理组(Eth+Gly),在含有1%乙醇和23μg/ml甘氨酸的KSOM培养基中培养IVF原核胚4天后处理获得。各组获得的囊胚用5-MeC特异性抗体(绿色)染色,并用PI(红色)复染色统一处理。(B)5-甲基胞嘧啶(5-MeC)整体水平的定量分析。通过将特异性靶标(5-MeC)的强度除以DNA染色(PI)的强度获得荧光强度的平均比例。运用单向ANOVA模型检查处理组之间差异。数值在统计学上具有显著差异,p<0.05。
具体实施方式
下文将参考实施例和附图详细描述本发明,所述实施例仅是意图举例说明本发明,而不是意图限制本发明的范围。本发明的范围由后附的权利要求具体限定。
实施例
材料和方法
除非特别说明,本研究使用的所有化学品都购自西格玛(St.Louis,MO,USA)。
1.动物:
6-8周未合笼的雄性和雌性ICR品系小鼠以及昆明白结扎鼠,均购买自安徽医科大学实验动物中心,安置在笼子里并保持12小时明暗周期,控制温度在21–23℃,自由食用标准饲料和水。尽量减少实验过程中使用的动物数量和实验动物的痛苦。
2.实验设计:
首先,我们研究了胚胎培养液(KSOM培养液,通用的胚胎培养液,成分(g/L):NaCl5.55,KCl 0.19,KH2PO4 0.05,MgSO4·7H2O 0.05,葡萄糖0.04,乳酸钠1.12,NaHCO3 2.1,酚红0.001,丙酮酸钠0.02,CaCl2·2H2O 0.25,EDTA0.004,L-谷氨酰胺0.146,牛血清白蛋白1,试剂全部购自sigma公司)中乙醇的剂量对胚胎发育的影响。将小鼠卵激活当作第0天,通过找出在第4天能完全抑制囊胚形成的乙醇剂量来定义乙醇半抑制剂量。在随后的实验中,将体外受精的胚胎分配加入半抑制剂量乙醇的培养基中培养,同时又分为加入甜菜碱(SigmaB2629-100G,分子量117.15)组和不含甜菜碱组。甘氨酸(G8790-100G)作为无甲基甜菜碱类似物用于参照研究。所有的胚胎在随后的研究都将进行处理。
3.小鼠卵母细胞的制备、体外受精、胚胎培养、胚胎移植:
如前所述,通过48小时内连续注射PMSG和HCG将雌性ICR小鼠进行超数排卵操作(Zhang等,2005)。注射hCG14–16h后从输卵管中收集MII期卵母细胞移入预热的添加4mg/ml的牛血清白蛋白(BSA级分V,Sigma)的M2培养基中。卵丘细胞在37℃含透明质酸酶(0.3mg/ml,Sigma)和BSA的M2培养基中稍作处理,脱去透明带外颗粒细胞。然后将MII卵母细胞在M2+BSA的液滴中清洗三次,转移到覆盖石蜡油的M2+BSA的液滴中等待进一步处理。雌性ICR小鼠通过连续注射超排,IVF在人类输卵管液(HTF)中进行。从成年雄性小鼠附睾尾得到的精子放在37℃环境5%CO2和95%的湿度培养箱中1–2h获能。然后,约预孵育终体积1/10的精子悬液加入卵丘复合体培养基中。精子和卵母细胞共培养3-4H,在人工授精介质中实现受精生殖。
受精卵由是否有两个原核(hCG后22–23h)确定,然后培养在优化培养条件中(KSOM培养液中没有添加氨基酸,Lawitts和Biggers,1993),评估其在体外发育效率。囊胚移植前3天,将至少8周龄的雌性ICR小鼠和昆明白雄性输精管结扎小鼠合笼交配。与结扎雄性小鼠交配的第二天早上,检查雌性小鼠阴道栓,此时计为假孕的第0.5天。根据标准程序在假孕雌数的每侧子宫中移入10枚囊胚。囊胚移植24小时后,通过静脉注射0.1毫升含1%芝加哥蓝染料溶液(Sigma,c8679)的生理盐水,可以清晰观察到着床点。
4.共聚焦显微镜定量分析检测囊胚中DNA甲基化:
先前描述了5-甲基胞嘧啶免疫检测和随后观察的程序,并进行了微小的修改(Beaujean等,2004)。简言之,将胚胎在磷酸盐缓冲盐水(PBS)中洗涤,并在4%多聚甲醛中固定,然后在4℃下储存,直到不同组的胚胎可以同时开展以下步骤。准备好材料后,在进一步处理之前先在PBS中清洗胚胎,并用0.5%Triton X-100将其预处理30分钟,并在室温下用4mol/l HCl处理30分钟。然后用100mmol/l Tris-HCl(pH8.5)处理胚胎20分钟。洗涤数次后,将胚胎在含有1%牛血清白蛋白(1%PBS-BSA)的PBS中在室温下封闭1小时。使用针对5-甲基胞嘧啶的小鼠单克隆抗体(anti-5mC,NA81,Merck,Germany)将甲基化DNA可视化。与该抗体的孵育在4℃下进行过夜(1%PBS-BSA中1:75稀释),然后用1%PBS-BSA洗涤数次,然后在室温下用异硫氰酸荧光素(FITC)缀合的山羊-抗小鼠二抗孵育1小时。在1%PBS-BSA洗涤数次后,37℃下使用染色质用碘化丙啶(PI,10μg/ml)染色15分钟,然后用1%PBS-BSA洗涤数次。将胚胎固定在载玻片上。
使用装备有TCS SP5共聚焦系统(Leica Microsystems,Germany)的立式Optiphot-2显微镜(Nikon,Tokyo,Japan),Nikon Plan ApoX40油浸物镜和488和514nm的激发波长进行观察。通过每个波长的样品以1μm间隔收集串联光学部分(Z系列)。每个颜色通道的收集按顺序进行。对于每个实验,使用相同的智能增益,智能偏移,针孔和变焦参数。对于由每个细胞核发出的积分荧光的定量测量,合并图像的背景通过从整个图像中减去细胞质区域的平均强度来校正。通过手动勾画所有的核来测量核强度。使用Image Pro-Plus软件测量每个单独核发射的总荧光强度,并使用抗5mC信号与PI DNA信号的比率计算每个卵裂球的平均强度。
5.统计分析:
这里,定量数据的所有结果显示为平均值±标准偏差(SD)。所有数据通过单因素方差分析(ANOVA)进行分析,以评估不同组间的任何显着性差异。只有低于0.05的概率被认为是显著的。
结果
1.乙醇对小鼠植入前胚胎发育的影响取决于其在培养液中的浓度
为了研究乙醇在植入前阶段对胚胎发育的直接影响,我们在含有乙醇的培养液中培养IVF卵的原核(PN)胚胎,然后检查其囊胚形成的发育特征。表I中的结果表明,当培养液中乙醇浓度上升到0.5%以上时,乙醇出现了明显抑制小鼠胚胎早期发育的能力。当培养液中乙醇浓度达到2.5%时,所有胚胎被阻滞在4细胞阶段。当乙醇浓度达到1.5%时,囊胚形成的百分比会显着降低到40.3%,当乙醇浓度上升到2.0%时,几乎所有的胚胎都被抑制(图1)。所有这些数据与之前的发现具有一定程度的相似性(Leach RE等,1993)。我们的研究表明,乙醇对囊胚形成的抑制作用依赖于浓度,此外,0.5%和1%乙醇的作用似乎促进了胚胎从2细胞阶段到桑椹胚阶段的发育,尽管其囊胚形成速率明显降低。
除上述试验外,我们也将PN胚胎在含有0.1%乙醇的培养液中培养4天,发现对照组与试验组没有明显的差异,甚至实验组胚胎囊胚的形成率略高于对照组胚胎。以前的研究表明,当培养基乙醇浓度达到10%时,小鼠胚胎的正常发育在2细胞阶段就完全阻滞了。与之对应的,当本研究中乙醇浓度在2.5%时,所有胚胎在2-细胞阶段停止发育。因此,在随后的工作中,我们使用含有1%或1.5%乙醇的KSOM培养基来探究培养液中甜菜碱的补充是否有助于改善乙醇对小鼠胚胎的损伤作用并解释其潜在的机制。
2.甜菜碱可以扭转乙醇对植入前胚胎发育的影响
目前尚不清楚甜菜碱是否可以直接保护胚胎免遭酒精造成的损害。而体外胚胎培养系统能够更好的帮助我们研究甜菜碱对乙醇损伤胚胎发育的直接效应以及其潜在机制。在前期工作中,我们研究了培养液中各种甜菜碱浓度对小鼠胚胎发育的影响,并参考了甜菜碱剂量对动物细胞影响的相关工作结果。结果表明,在单独向培养液中加入50μg/ml甜菜碱的条件下,囊胚的形成速率没有明显变化。随后,我们分析了不同浓度乙醇的到培养液中补加50μg/ml甜菜碱对胚胎发育的影响。图2B中的结果显示,补充50μg/ml甜菜碱到含有1%或1.5%乙醇的培养液中,可有效地改善在其中培养的胚胎的发育状况,囊胚形成比例有着超过10%的增加(p<0.05)(表II)。早期胚胎的形态在所有试验组之间没有明显差异(图2A)。因此,以上数据让我们相信甜菜碱是保护胚胎免受乙醇损害的有效物质。
3.培养液中补充甜菜碱可以促进乙醇损伤囊胚的植入和植入后发育
通过囊胚移植方法分析胚胎植入的能力,以此来判断乙醇对囊胚植入的影响和甜菜碱对此的修复效应。在胚胎移植后24小时,通过注射芝加哥蓝染料来计数囊胚植入的数量,以此来计算囊胚的植入率。结果如图3所示,对照组(NC组)(平均值=83.33%)和甜菜碱组(Bet组)(平均值为87.33%)之间无显着性差异。同时,1%乙醇处理组(Eth组)的囊胚植入率(平均43.83%)明显低于NC组(P<0.01)。尽管Eth组和Eth+Bet组的植入比例差异不具有统计学意义(P=0.06),但后者的囊胚植入率却高达70.00%。
为了评估乙醇对胚胎长期发育的影响,并判断甜菜碱补充到含有乙醇的培养液中是否可以有效促进胚胎植入后发育。我们将包括1%乙醇组(Eth),1%乙醇+50μg/ml甜菜碱组(Eth+Bet)和对照组(NC)中不同组的囊胚移植到假孕鼠的子宫中。8天后,处死移植小鼠并收集子宫以获得胎儿。如图4D所示,在胚胎移植后第8天,收集胚珠并获得具有成形的胎儿,发现在Eth组中,所有胚珠中具有成形胎儿的胚珠比例降至61.08%,但在Eth+Bet组中上升至75%,尽管统计学分析未显示其差异有统计学意义(p>0.05)。根据获得的胎儿的形态和大小特征,三个处理组中的所有成形胎儿可以分为三种类型,分别是I级,II级和III级(图4A,B和C)。I级胎儿呈现正常大小以及形态学特征。II级胎儿呈现一定程度的稍小尺寸,但形态方面未发生明显变化。III级胎儿表现出异常的小体积并且发育明显滞后。图4E的结果表明,将补充50μg/ml甜菜碱到培养液中能够改善乙醇处理囊胚的胎儿发育。1%乙醇处理组(Eth)中I类胎儿的比例降低到20%,而其他处理组中,I型胎儿的平均比例分别是:对照组(NC)中为71.43%(P>0.05)),1%乙醇+50μg/ml甜菜碱组(Eth+Bet)中为42.11%(P>0.05)。III类胎儿只存在于Eth组(10%)。这些结果表明,培养液中补充甜菜碱可有效促进乙醇损伤胚胎的植入后发育。
4.甘氨酸不能修复被乙醇损伤胚胎的发育
甜菜碱是否可以通过调节胚胎细胞的渗透压来改善胚胎的发育。为此,设计以下实验来研究甘氨酸(没有甲基的甜菜碱类似物)是否可以起到同样的修复乙醇损伤胚胎发育的作用。
将甜菜碱替换成甘氨酸加入含有1%乙醇的培养液中培养胚胎。在已有研究中,甘氨酸常被用来探究渗透压和氧化损伤的机制(Cao等,2016;Bhagatte等,2012)。为了做出可比较的判断,采用与50μg/ml的甜菜碱相同的摩尔浓度,在培养液中以32μg/ml的补充甘氨酸。结果如图5所示,甘氨酸无法帮助恢复乙醇损伤小鼠胚胎的发育,甚至明显减少了囊胚形成的比率(p<0.01)。此外,在前期试验中,当甘氨酸浓度超过10μg/ml时,就会产生负面影响。相反,当培养基中甜菜碱的浓度达到500μg/ml时,其对胚胎发育的抑制作用才刚开始出现,这表明甜菜碱可以在较大的浓度范围内安全使用。所有这些数据表明,对胚胎细胞的渗透压保护不是修复乙醇损伤胚胎发育的主要原因。
5.甜菜碱降低乙醇导致的囊胚全基因组DNA的5-MeC水平的升高
囊胚期胚胎的发育状态是确定胚胎植入后发育相关基因表达的关键,它是通过基因组DNA和组蛋白建立表观遗传修饰来进行的,即建立正确的基因组印记和基因表达的再程序化。现在已经知道,DNA甲基化在胚胎发育过程中的基因表达调控中起关键作用,称为基因组印记(Bird A,2002)。胚胎发育过程中囊胚期的全基因组DNA甲基化的重建是十分关键的阶段,同时DNA甲基化水平差异分别呈现于内细胞团(ICM)和滋养外胚层(TE)。
为了确定甜菜碱是否能够帮助修复被乙醇破坏的基因组DNA甲基化修饰,我们用抗5-甲基胞嘧啶(5-MeC)抗体通过免疫荧光方法检测了不同处理组囊胚的基因组DNA甲基化状态。图6中的结果表明,单独补充乙醇(1%)或甜菜碱(50μg/ml)可能导致DNA甲基化整体水平在囊胚的ICM和TE中显著升高,甜菜碱组甚至表现出更高的水平。然而,在Eth+Bet组中,可以发现ICM和TE中的DNA甲基化水平明显下降,这与两组的囊胚形成率类似(平均值=0.88,0.47,P<0.05)。不同的是,使用与本研究中50μg/ml甜菜碱(0.427mM)相同摩尔浓度的甘氨酸加入到无乙醇的培养夜中,也提高了DNA甲基化水平(甘氨酸,32μg/ml等于0.427mM),但不能降低乙醇处理后囊胚中基因组DNA的甲基化水平(P>0.05)。
a数据展示为平均值±标准差
a数据展示为平均值±标准差
以上所述仅为本发明的优选实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
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Claims (10)
1.甜菜碱在制备预防和/或治疗乙醇(酒精)对动物的损伤的药物/营养品中的应用。
2.根据权利要求1所述的应用,其中所述动物为哺乳动物,优选为胎生哺乳动物,更优选选自食肉目(如猫科动物)、啮齿目(如鼠类)、偶蹄目(如猪、牛、羊等)、奇蹄目(如马、驴等)、灵长目(如猴和猿类等)、翼手目(如蝙蝠等)、长鼻目(如象等)和鲸目(如海豚等),最优选选自猫、大鼠、小鼠、豚鼠、猪、牛、羊(山羊/绵羊)、马、驴、猴、猿、狗和人。
3.根据权利要求1或2所述的应用,其中所述动物为受孕动物或所述动物处于胚胎发育阶段(即为动物胚胎),例如为受精、卵裂、桑葚胚、囊胚、原肠胚或器官形成阶段的胚胎。
4.根据权利要求1-3中任一项所述的应用,其中所述动物摄入/被暴露于过量乙醇,和/或其中所述甜菜碱能够修复乙醇(酒精)对动物(特别是动物胚胎)的损伤,和/或能够将受乙醇(酒精)影响的DNA的甲基化水平正常化。
5.根据权利要求1-4中任一项所述的应用,其中所述药物作为活性成分仅包含甜菜碱,或还包含叶酸作为活性成分。
6.甜菜碱在制备改善受乙醇(酒精)损伤的动物胚胎的发育的药物/营养品中的应用。
7.甜菜碱在制备保护动物胚胎免受乙醇(酒精)损伤的药物/营养品中的应用。
8.一种用于预防和/或治疗乙醇(酒精)对动物的损伤的药物/营养品,其包含:作为活性成分的甜菜碱(任选地,与其他活性成分(如叶酸)组合);和药学/营养学上可接受的辅剂。
9.一种改善动物胚胎发育的药物/营养品,其包含:作为活性成分的甜菜碱(任选地,与其他活性成分(如叶酸)组合);和药学/营养学上可接受的辅剂。
10.一种改善动物胚胎发育的方法,所述动物胚胎受(例如由乙醇(酒精)或糖尿病导致的)氧化应激损伤,所述方法包括用治疗有效量的作为活性成分的甜菜碱(任选地,与其他活性成分(如叶酸)组合)处理所述胚胎。
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