CN111249299A - 大豆rna提取物在制备预防及治疗肠炎的药物中的应用 - Google Patents
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Abstract
本发明提供大豆RNA提取物在制备预防及治疗肠炎的药物中的应用,属于生物技术领域。体外实验表明大豆RNA提取物及gma‑miR159a对正常结肠上皮细胞增殖活性无影响,具有良好的安全性。体内实验表明大豆RNA能够显著改善结肠炎模型小鼠的结肠病理及炎症水平,提高了小鼠的健康水平。因此,大豆RNA提取物及gam‑miR159a能够有效预防和治疗肠炎性相关疾病。大豆RNA提取物及gam‑miR159a直接来源于大豆,具有特异性靶向,具有安全、高效、来源广泛的特点。大豆RNA提取物及gam‑miR159a提取简便,在食品、医药等领域有广阔前景,可以应用于制备预防及治疗肠炎的药物或功能性食品。
Description
技术领域
本发明属于生物技术领域,具体涉及大豆RNA提取物在制备预防及治疗肠炎的药物中的应用。
背景技术
肠炎类疾病为多发病和常见病,严重影响人们的身体健康,该类疾病顽固难愈、病程长、易反复发作。其中,结肠炎在肠炎性疾病中较多,分为急性期和慢性期两类。急性期患者,若得到恰当控制,其症状可以缓解并最终治愈,反之则炎症反复发作,最后转变成慢性,增加转变为结肠癌的风险,是胃肠道最严重的疾病之一,被世界卫生组织列为现代难治疾病之一。因此,有效的预防手段对于肠炎的预防和治疗具有重要意义。
MicroRNAs(miRNAs)是一种含有19-24个核苷酸的单链小RNAs,可通过与靶基因碱基互补配对调控靶基因的表达。研究显示超过60%的基因受miRNAs调控,参与了大多数生理过程的调控。在早期的研究中,人们已经注意到植物性饮食中含有大量的核酸类成分,然而直到近几年,人体中的植物miRNAs才被发现,miRNAs的跨物种调控现象引起了研究人员的重视。目前,植物来源的miRNAs已被发现与心血管疾病、流感、乳腺癌等多种疾病的治疗紧密相关,这些研究揭示了植物miRNAs在未来疾病治疗中的新地位。
现有技术中缺乏高效、安全的用于预防及治疗肠炎的药物。
发明内容
本发明的目的是提供大豆RNA提取物在制备预防及治疗肠炎的药物中的应用,大豆RNA提取物具有安全、高效、来源广泛的特点。
本发明的另一目的是提供大豆RNA提取物在制备具有预防和治疗肠炎性疾病的功能性食品中的应用。
本发明的又一目的是提供用于预防及治疗肠炎的药物或功能性食品。
本发明的目的采用如下技术方案实现:
本发明提供大豆RNA提取物在制备预防及治疗肠炎的药物中的应用。
在本发明中,所述大豆RNA提取物含有序列如SEQ ID NO:1所示的miRNA。
在本发明中,所述所述肠炎包括结肠炎和其他肠炎性疾病。
本发明还提供大豆RNA提取物在制备具有预防和治疗肠炎性疾病的功能性食品中的应用。
在本发明中,所述大豆RNA提取物含有序列如SEQ ID NO:1所示的miRNA。
本发明还提供用于预防及治疗肠炎的药物或功能性食品,含有大豆RNA提取物。
本发明还提供序列如SEQ ID NO:1所示的miRNA在制备预防及治疗肠炎的药物中的应用,用于预防及治疗肠炎的药物或功能性食品,其特征在于含有序列如SEQ ID NO:1所示的miRNA。
在本发明中,所述大豆RNA提取物含有序列如SEQ ID NO:1所示的miRNA。
体外实验表明大豆RNA提取物及gma-miR159a对正常结肠上皮细胞NCM460增殖活性无影响,具有良好的安全性。体内实验结果表明大豆RNA能够显著改善结肠炎模型小鼠的结肠病理及炎症水平,提高了小鼠的健康水平。因此,大豆RNA提取物及gam-miR159a能够有效预防和治疗肠炎性相关疾病。大豆RNA提取物及gam-miR159a直接来源于大豆,具有特异性靶向,具有安全、高效、来源广泛的特点。大豆RNA提取物及gam-miR159a提取纯化简便,在食品、医药等领域具有广阔的前景,因此可以应用于制备预防及治疗肠炎的药物或功能性食品。
附图说明
图1为大豆RNA提取物中miRNAs家族种类及水平(Top 15),其中横坐标为miRNAs家族名称,纵坐标为拷贝数。
图2为大豆RNA提取物及gma-miR159a对人正常结肠上皮细胞NCM460增殖活性的影响。
图3为大豆RNA提取物及gma-miR159a对结肠炎模型小鼠结肠长度的影响。*:与正常组相比有显著差异(p<0.05);**:与正常组相比有极显著差异(p<0.01);#:与模型+乱序RNA组相比有显著差异(p<0.05);##:与模型+乱序RNA组相比有极显著差异(p<0.01),下同。
图4为大豆RNA提取物及gma-miR159a对结肠炎模型小鼠结肠病理的影响。
图5为大豆RNA提取物及gma-miR159a对结肠炎模型小鼠结肠中CD11b蛋白表达的影响。
图6为大豆RNA提取物及gma-miR159a对结肠炎模型小鼠中TNF-α及IL-1β含量的影响。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
1.材料:Caco-2细胞株、NCM460细胞株购自上海中科院细胞库;胎牛血清(Fetal Bovine Serum,FBS)、DMEM培养基、EDTA-胰酶(0.05%)、Penicillin-Streptomycin(100×)、PBS磷酸盐缓冲液购自美国Gibco公司;miRNA mimics、Rnase-free H2O购自生工生物工程(上海)股份有限公司;Lipofectamine 3000、Opti-MEM培养基购自美国Invitrogen公司;二甲基亚砜(DMSO)购自美国Sigma公司;噻唑蓝(MTT)、1%多聚甲醛溶液购自北京索莱宝科技有限公司;C57BL/6雄性小鼠购自无锡菩禾生物医药技术有限公司;福尔马林购自美国Sigma公司;葡聚糖硫酸钠(Dextran sodium sulfate,DSS,MW:36000-50000 Da)购自美国MPbio公司;苏木素、伊红、CD11b抗体、二抗(HRP标记山羊抗兔)购自武汉赛维尔生物科技有限公司;其他试剂为国产分析纯。
2.主要仪器与设备:生物安全柜购自Thermo Fisher(中国)公司;MZE多功能酶标仪购自Molecular Devices(美国)公司;SL16R台式离心机购自Thermo Fisher(中国)公司;二氧化碳培养箱购自Thermo Fisher(中国)公司;BH-2型显微镜购自OLYMPUS(日本)公司。
实施例1 大豆RNA提取物的制备及miRNAs鉴定
采用常规方法从大豆种子中获得大豆RNA提取物。具体方法如下:将大豆种子于液氮中快速研磨,将得到的大豆粉末立刻转移至含有Trizol试剂(购自美国Invitrogen公司)的离心管中,其中大豆粉末的总体积不能超过所用Trizol体积的10%。室温下放置10 min,然后加入Trizol试剂体积1/5的氯仿,盖紧离心管,剧烈震动15 s,常温放置5 min,于4℃下12000g离心15 min。离心后液体分为三层(上层无色液体为RNA,中层白色为DNA,底层红色为蛋白质),小心吸取上层无色液体移入新的离心管中,加入等体积异丙醇,混匀后室温放置10 min,在4℃下12000g离心10 min,去除上清,留取管底沉淀。加入Trizol试剂相同体积的75%乙醇溶液,轻轻洗涤沉淀,在4℃、7500g离心5min,去除上清,再次重复75%乙醇溶液洗涤步骤。在超净台中自然静置干燥5-10 min以去除残留乙醇,加入适量体积Rnase-free H2O溶解,得到大豆RNA提取物。
对大豆RNA提取物中的miRNAs进行测序,由生工生物工程股份有限公司(上海,中国)完成。测序原理如下:经过3'末端衔接子连接,逆转录引物杂交和5'末端衔接子连接的处理后,小片段RNA被反转录成cDNA,并扩增17个循环。然后从PAGE凝胶中分离出约140-150 bp的片段,并使用HiSeq2500进行测序。生成数据后,执行读取的质量控制和处理以获得干净的读取。最后,与miRBase数据库22.0进行比对,将与参考miRNAs相比具有相同序列和长度的候选序列视为miRNAs匹配序列。
由测序结果可知,大豆RNA提取物中含有297个已知大豆miRNAs,分属于120个已知大豆miRNAs家族,其中含量排在前15位的miRNAs家族占大豆RNA提取物的99.9%。大豆RNA提取物中排在前15位的miRNAs家族的种类及水平如图1所示。
大豆RNA提取物中含有序列(SEQ ID NO:1)如下的miRNA:5'-UUUGGAUUGAAGGGAGCUCUA-3',该序列属于gma-miR159家族,命名为gma-miR159a,送生工生物工程股份有限公司(上海,中国)进行合成,以进行下述试验。
另外,设计乱序miRNA模拟物作为阴性对照进行下述试验,其序列(SEQ ID NO:2)如下:5'-UUCUCCGAACGUGUCACGU-3',送生工生物工程股份有限公司(上海,中国)进行合成。
实施例2大豆RNA提取物及gma-miR159a对正常结肠上皮细胞NCM460增殖活性的影响
通过MTT测定法检测实施例1制备的大豆RNA提取物及采用化学合成法制备的gma-miR159a对正常结肠上皮细胞NCM460增殖活性的影响。
将NCM460细胞培养在96孔板中,每孔中有3×103个细胞,大豆RNA组、gma-miR159a组、对照组及正常组分别设有6个孔。其中大豆RNA组的每孔中添加终浓度为40μg/mL的大豆RNA提取物,gma-miR159a组的每孔中添加终浓度为50 nM的gma-miR159a,对照组中添加终浓度为50 nM的乱序miRNA模拟物,正常组不添加任何药物。另设空白组,无细胞。转染24 h、48 h和72 h后,每孔中加入20 μL MTT试剂(5 mg/mL),并孵育4 h。 孵育后,除去培养基,并向每个孔中添加100 μL二甲基亚砜(DMSO),孵育15 min后,在570 nm下测量样品的吸光度。按照如下方法计算各组细胞增殖率:细胞增殖率=(样品OD值-空白组OD值)/(对照组OD值-空白组OD值)。
由图2实验结果可知,与正常组及对照组相比,大豆RNA提取物及gma-miR159a处理24 h、48 h及72 h,对NCM460的增殖活性无显著性影响(p > 0.05),表明大豆RNA提取物及gma-miR159a对人正常结肠上皮细胞无毒害作用,具有良好的安全性。
实施例3大豆RNA提取物及gma-miR159a对结肠炎模型小鼠的影响
(1)造模及给药
从菩禾生物医学技术有限公司(中国无锡)购买了6周龄的雄性C57BL / 6小鼠。将50只小鼠随机分为5组,每组10只,分别为正常组、模型组、模型+乱序RNA组、模型+RNA组和模型+gma-miR159a组。除正常组外,其他组采用如下方法构建结肠炎小鼠模型:实验小鼠自由饮用2 % DSS(葡聚糖硫酸钠)水溶液,持续7天;然后再饮用正常饮用水(双蒸水)14天。将上述过程重复三个循环,以构建结肠炎小鼠模型。
在构建结肠炎小鼠模型过程中,模型组小鼠无其他药物灌胃;模型+RNA组小鼠除了给予结肠炎模型药物外,每天采用大豆RNA提取物灌胃,剂量为80 μg/只;模型+gma-miR159a组小鼠,除了给予结肠炎模型药物外,每天采用gma-miR159a灌胃,剂量为0.3 nmol/只;模型+乱序RNA组小鼠,除了给予结肠炎模型药物外,每天采用乱序miRNA模拟物灌胃,剂量为0.3 nmol/只;另外,正常组小鼠正常饲养,不给予任何药物。
造模结束后,处死小鼠,测量小鼠结肠长度,然后收集结肠组织用于HE染色及免疫组化分析,收集血浆以检测炎症因子。
(2)小鼠结肠长度检测结果
图3实验结果表明,与正常组相比(结肠长度为6.9± 0.4 cm),模型组及模型+乱序RNA组的结肠长度显著降低(p < 0.01),分别为5.9 ± 0.7 cm及5.5 ± 0.3 cm。与模型+乱序RNA组相比,大豆RNA提取物及gma-miR159a显著恢复了肠炎小鼠的结肠长度(p < 0.05),分别为7.0 ± 0.5 cm及6.8 ± 1.0 cm。本实验结果表明,gma-miR159a及大豆RNA提取物能够显著改善结肠炎小鼠的结肠长度。
(3)HE染色结果
将各组小鼠的结肠组织进行HE染色,具体方法如下:将结肠组织采用福尔马林固定,然后用PBS清洗3次,依次放入乙醇中进行梯度脱水1 h。随后,将结肠组织取出,置于二甲苯溶液中进行透明处理2次,每次静置1 h,再将组织转入54 ℃石蜡中进行浸蜡包埋。之后于56-58 ℃进行石蜡包埋,待室温冷却后切片,然后置于温水中使其展开,用载玻片捞取并烘干。然后再次置于二甲苯溶液中进行脱蜡处理,再将切片置于100%、95%、85%、75%乙醇溶液中复水各1 min。然后,将切片用苏木素染色5 min,1%盐酸溶液分化5-10s,水洗两次,再用伊红染色2 min。再次进行乙醇脱水处理,再用二甲苯溶液分别处理 3 min、5 min。最后用中性树胶封片,于光学显微镜下根据病理学的标准进行组织学分析。
由图4可见,与正常组相比,模型组及模型+乱序RNA组中肠道粘膜遭到严重破坏,隐窝消失,而大豆RNA提取物及gma-miR159a改善了肠道粘膜的损伤,隐窝恢复,证明了大豆RNA提取物及gma-miR159a能够防止结肠炎小鼠中肠道组织的破坏。
(4)大豆RNA提取物及gma-miR159a对结肠炎模型小鼠结肠中CD11b表达的影响
检测各组小鼠结肠中CD11b表达情况,以大豆RNA提取物及gma-miR159a对结肠炎模型小鼠结肠中CD11b表达的影响。将小鼠的结肠组织进行石蜡切片,脱蜡并脱水。然后,将组织切片置于盛满EDTA抗原修复缓冲液(主要成分为乙二胺四乙酸二钠,pH 9.0)的修复盒中于微波炉内进行抗原修复,自然冷却后用PBS洗涤3次,每次5 min。放入3%双氧水溶液,室温避光孵育25 min以阻断内源性过氧化物酶,将玻片置于PBS中在脱色摇床上晃动洗涤3次,每次5min。接下来,在组化圈内滴加3% BSA溶液均匀覆盖组织,室温封闭30 min以封闭非特异结合位点。然后,轻轻甩掉封闭液,在切片上滴加PBS稀释的CD11b抗体,切片平放于湿盒内4℃孵育过夜。然后,玻片置于PBS(pH7.4)中在脱色摇床上晃动洗涤3次,每次5 min。切片稍甩干后在圈内滴加与一抗相应种属的二抗(HRP标记山羊抗兔)覆盖组织,室温孵育50 min。再次利用PBS洗涤3次,切片稍甩干后在圈内滴加新鲜配制的DAB显色液,显微镜下控制显色时间,阳性为棕黄色,自来水冲洗切片终止显色。然后,苏木素复染3 min左右,自来水洗,苏木素分化液分化数秒,自来水冲洗,苏木素返蓝液返蓝,流水冲洗。最后,将切片脱水透明,中性树胶封片。利用显微镜镜检,使用软件进行图像采集分析。苏木素染细胞核为蓝色,DAB显出的阳性表达为棕黄色。
由于CD11b参与单核细胞、巨噬细胞和粒细胞等炎症细胞的粘附相互作用,因此其含量在一定程度上反映了组织内的炎症水平。如图5所示,与正常组相比,模型组小鼠中CD11b阳性细胞的数量(深棕色区域)明显增加,但是大豆RNA提取物及gma-miR159a减少了模型小鼠中CD11b阳性细胞的数量,表明大豆RNA提取物及gma -miR159a可以有效防止免疫细胞的浸润,缓解结肠炎小鼠体内的炎症水平。
(5)大豆RNA提取物及gma-miR159a对结肠炎模型小鼠中IL-1β及TNF-α含量的影响
根据制造商的说明书,使用小鼠IL-1β和TNF-αELISA试剂盒测定小鼠血浆中IL-1β和TNF-α的含量。
从图6可见,正常组IL-1β、TNF-α的含量分别为15.33 ± 2.00 pg/mL及115.70 ± 17.95 pg/mL;模型组中IL-1β、TNF-α的含量分别为35.33 ± 5.95 pg/mL及282.74 ± 67.78 pg/mL;模型+乱序RNA组IL-1β、TNF-α的含量分别为38.74 ± 4.17 pg/mL及281.07 ± 24.04 pg/mL;模型+RNA组小鼠,IL-1β含量20.88± 4.88 pg/mL,TNF-α的含量为178.80 ± 51.22 pg/mL;模型+gma-miR159a组小鼠,IL-1β含量31.08 ± 3.07 pg/mL,TNF-α的含量为215.64 ± 12.01 pg/mL。
与正常组相比,模型组及模型+乱序RNA组中IL-1β和TNF-α的含量显著增加(p < 0.01),但是大豆RNA提取物及gma-miR159a显著改善了结肠炎小鼠体内的炎症水平(p < 0.01),IL-1β含量分别降低为20.88± 4.88 pg/mL、31.08 ± 3.07 pg/mL,TNF-α的含量分别降低为178.80 ± 51.22 pg/mL、215.64 ± 12.01 pg/mL,表明大豆RNA提取物及gma-miR159a在预防和改善结肠炎中的重要作用。
SEQUENCE LISTING
<110> 南京财经大学
<120> 大豆RNA提取物在制备预防及治疗肠炎的药物中的应用
<130> 20200331
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> RNA
<213> 大豆
<400> 1
uuuggauuga agggagcucu a 21
Claims (9)
1.大豆RNA提取物在制备预防及治疗肠炎的药物中的应用。
2.根据权利要求1所述应用,其特征在于:所述大豆RNA提取物含有序列如SEQ ID NO:1所示的miRNA。
3.根据权利要求1或2所述的应用,其特征在于:所述肠炎包括结肠炎和其他肠炎性疾病。
4.大豆RNA提取物在制备具有预防和治疗肠炎性疾病的功能性食品中的应用。
5.根据权利要求4所述应用,其特征在于所述大豆RNA提取物含有序列如SEQ ID NO:1所示的miRNA。
6.用于预防及治疗肠炎的药物或功能性食品,其特征在于含有大豆RNA提取物。
7.根据权利要求6用于预防及治疗肠炎的药物或功能性食品,其特征在于所述大豆RNA提取物含有序列如SEQ ID NO:1所示的miRNA。
8.序列如SEQ ID NO:1所示的miRNA在制备预防及治疗肠炎的药物中的应用。
9.用于预防及治疗肠炎的药物或功能性食品,其特征在于含有序列如SEQ ID NO:1所示的miRNA。
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CN114606236A (zh) * | 2022-04-21 | 2022-06-10 | 南通大学 | 一种翘芪复方中药汤剂来源的微小核糖核酸及其制备方法和应用 |
WO2023201836A1 (zh) * | 2022-04-21 | 2023-10-26 | 南通大学 | 一种翘芪复方中药汤剂来源的微小核糖核酸及其制备方法和应用 |
CN114606236B (zh) * | 2022-04-21 | 2024-01-05 | 南通大学 | 一种翘芪复方中药汤剂来源的微小核糖核酸及其制备方法和应用 |
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