CN116903762A - 一种具有肠道黏膜屏障修复功能的褐藻多糖及其制备方法 - Google Patents
一种具有肠道黏膜屏障修复功能的褐藻多糖及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种具有肠道黏膜屏障修复功能的褐藻多糖及其制备方法。所述褐藻多糖总多糖含量为91.80±0.54%,蛋白质含量为0.0065±0.024%,硫酸基含量26.02±3.34%,通过计算分析可知其单糖组成约为Fuc:Glc:Gal:Man:GluA:Xyl=26.56:0.58:10.78:1.32:2.07:1.00。所得的褐藻多糖可使杯状细胞的MUC2蛋白和ZO‑1蛋白表达量增加,因此判断其对黏液屏障损伤能够起到治疗作用。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种具有肠道黏膜屏障修复功能的褐藻多糖及其制备方法。
背景技术
随着现代生活节奏的加快和饮食习惯的改变,人们的肠道健康受到威胁,各种胃肠疾病发病率日益增高,严重影响了现代人们的生活和工作。健康的肠道屏障可以有效地抵御微生物和致病因子的侵袭,减少人们患肠道疾病的风险。其中杯状细胞分泌出的黏液层在肠道屏障功能中起到了非常关键的保护作用。
肠道黏膜屏障是保护肠上皮细胞的屏障,黏膜层的破坏导致屏障层通透性增加,微生物和有害代谢物对肠道上皮细胞的损伤作用增加,这是引起溃疡性结肠炎的发病机制。肠道杯状细胞分泌的黏蛋白(MUC2)和三叶因子3(TFF3)可以形成一种网状粘弹性凝胶来形成黏膜层屏障,防止大颗粒直接接触上皮细胞层。囊泡相关膜蛋白8(VAMP8)蛋白共定位于LS174T细胞中的黏蛋白囊泡,参与形成包裹黏蛋白通过胞吐作用运输出杯状细胞的过程。而粘蛋白囊泡通常被包裹在一个富含肌动蛋白的结构中,VAMP8参与囊泡运输胞吐推测是杯状细胞膜。Krüppel样因子4(KLF4)基因在促进溃疡性结肠炎的修复中起到促进杯状细胞增殖和分化的作用,并且可能与MUC2等黏蛋白有协同作用。ZO-1蛋白是紧密连接蛋白之一,调节肠上皮屏障的细胞通透性,维护细胞间紧密连接和肠道黏液层是共同维持肠道屏障功能的重要因素,因此被认为是胃肠道疾病的治疗靶点。
目前对于肠道黏膜屏障受损患者的临床治疗方案为:1.使用氨基水杨酸类(柳氮磺吡啶、5-氨基水杨酸、奥沙拉嗪、美沙拉嗪和巴柳氮等)。它们可用于治疗溃疡性结肠炎、直肠炎和克罗思氏病,但是存在局限性,如需要患者建立耐药性、存在受剂量依赖性和毒性影响的风险。2.皮质甾类(氢化波尼松、甲泼尼松、布地奈德、倍氯米松等),也具有不能用于长期的缓解和治疗,并导致药物依赖性和不可逆的并发症的弊端。3.免疫抑制剂类(硫唑嘌呤、6-巯基嘌呤、氨甲蝶呤、环孢菌素A等)会增加肾毒性和损害人体免疫系统的风险,长期使用会引起副作用。4.改善生活作息及饮食结构。
因此,临床用药具有安全性低、副作用大的局限性,而从饮食与生活习惯的调节又需花费许多时间,找到一种有效并且无副作用的天然来源药物十分重要。
发明内容
本发明提出一种具有肠道黏膜屏障修复功能的褐藻多糖,以及所述褐藻多糖的制备方法和应用领域,旨在解决上述现有背景中存在的技术问题之一。
本发明的第一面提供了一种褐藻多糖的制备方法。所述制备方法包括以下步骤:
(1)将干燥的裙带菜粉碎后,过筛得裙带菜粉;
(2)将所述裙带菜粉加入混合液,摇床振荡,离心后取沉淀,干燥,得到脱色裙带菜粉末,所述混合液包括甲醇和二氯甲烷;
(3)所述脱色裙带菜粉末加水,50~100℃水浴1~4h,分离滤液;
(4)向所述滤液中加入95%乙醇,静置于4℃冰箱,得静置液;
(5)所述静置液收集凝固沉淀,以蒸馏水溶解所述凝固沉淀,干燥,得褐藻粗多糖;
(6)将所述褐藻粗多糖采用二乙胺基乙基纤维素(DEAE)离子交换树脂进行纯化,收集组分液体,洗脱,透析,干燥,即得所述褐藻多糖。
所述制备方法的褐藻多糖得率为9.81±0.58%,且所得的褐藻多糖具有肠道年末屏障修复功能。
作为上述方案的进一步改进,步骤(1)所述裙带菜粉碎后过20~80目筛。优选地,可使用40目筛网进行筛分。适量粉碎裙带菜有助于后续的脱色、提纯。
作为上述方案的进一步改进,步骤(2)所述混合液由甲醇、二氯甲烷和水组成,质量比为(4~5):(2~3):1。优选地,所述混合液中甲醇、二氯甲烷和水的质量比为4:2:1。
作为上述方案的进一步改进,步骤(3)所述脱色裙带菜粉末和水的料液比1g:10mL。
作为上述方案的进一步改进,步骤(2)所述离心的速率为2000、2500、3500、4000、4500或5000rpm;所述离心的持续时间为4、5、6、7、8、9或10min。作为其中一种实施方式,所述离心的条件为4000rpm,持续4min。
作为上述方案的进一步改进,各步骤中所述干燥可以选用真空冷冻干燥,避免高温干燥对多糖的构型产生未知的影响。
作为上述方案的进一步改进,步骤(3)所述脱色裙带菜粉末加水的料液比为1g:40mL。
作为上述方案的进一步改进,步骤(4)所述95%乙醇的添加量为所述滤液的体积的2~5倍,优选为所述滤液的4倍体积。
本发明的第二面提供了一种褐藻多糖。所述褐藻多糖由上述制备方法制得,总多糖含量为91.80±0.54%,蛋白质含量为0.0065±0.024%,硫酸基含量26.02±3.34%,通过计算分析可知其单糖组成约为褐藻糖(Fuc):葡萄糖(Glc):半乳糖(Gal):甘露糖(Man):葡萄糖醛酸(GluA):木糖(Xyl)
=26.56:0.58:10.78:1.32:2.07:1.00。所得的褐藻多糖可使杯状细胞的MUC2蛋白和ZO-1蛋白表达量增加,因此判断其对黏液屏障损伤能够起到治疗作用。
本发明的第三面提供了所述褐藻多糖在制备预防/治疗肝脏损伤的药物或食品中的应用。
本发明的上述技术方案相对于现有技术,至少具有如下技术效果或优点:
1、所述褐藻多糖来源于食用天然海藻裙带菜,安全性好、生物相容性好、无毒副作用,有潜力作为解决当前临床药物副作用局限性的替代方案。
2、所述褐藻多糖能够促进结肠杯状细胞(LS174T)分泌黏液,在屏障损伤模型中促进mRNA水平上MUC2、TFF3、KLF4和PKCβ基因的表达,同时还促进紧密连接蛋白ZO-1表达,表现出修复肠道屏障损伤和保护肠道黏液层的活性。可为褐藻多糖在肠道屏障损伤的保护和修复提供理论依据。
附图说明
图1为实施例2中葡萄糖浓度含量标准曲线图;
图2为实施例3中牛血清蛋白标准曲线图;
图3为实施例4中检测所述褐藻多糖的硫酸根含量标准曲线图;
图4为实施例6中CCK-8法检测所述褐藻多糖处理后的细胞存活率柱状图;
图5为实施例7中AB-PAS法对LS174T细胞的黏液染色图;
图6为实施例7中激光共聚焦显微镜观察LS174T细胞分泌黏液情况图;
图7为实施例7中LS174T细胞内的mRNA水平表达柱状图。
具体实施方式
以下将结合实施例对本发明的构思及产生的技术效果进行清楚、完整地描述,以充分地理解本发明的目的、特征和效果。显然,所描述的实施例只是本发明的一部分实施例,而不是全部实施例,基于本发明的实施例,本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例,均属于本发明保护的范围。
(6)将所述褐藻粗多糖采用二乙胺基乙基纤维素离子交换树脂进行纯化,收集组分液体,洗脱,透析,干燥,即得所述褐藻多糖。
实施例1、褐藻多糖的制备
制备步骤:
(1)从农贸市场购得裙带菜,洗净后晾干;将干燥的裙带菜使用粉碎机粉碎后,过40目筛,得到裙带菜粉;
(2)称量200g所述裙带菜粉,以1:10(g/mL)料液比加入混合液(甲醇:二氯甲烷:水=4:2:1),摇床振荡过夜,离心(4000rpm,4min)后取沉淀,冷冻过夜后,用真空冻干机将沉淀冻干,得到脱色裙带菜粉末;
(3)向所述脱色裙带菜粉末加入1:40料液比的水,90℃水浴2.5h,纱布分离滤液;
(4)向所述滤液中加入4倍滤液体积的95%乙醇,静置于4℃冰箱中12小时,得静置液;
(5)所述静置液室温放置30min后,收集凝固沉淀,以蒸馏水溶解沉淀并冻干后,得褐藻粗多糖;
(6)采用DEAE离子交换树脂对所述褐藻粗多糖进行纯化,收集组分液体,洗脱,透析,冻干,得褐藻多糖,得率为15.03%。
通过对提取过程中多个的影响因素进行简要探究,得出以下结果:
方案编号 | 料液比 | 水浴温度 | 水浴时间 | 得率 |
1 | 1:30 | 95 | 2.5 | 12.86% |
2 | 1:40 | 90 | 2.5 | 15.03% |
3 | 1:50 | 85 | 2 | 12.11% |
4 | 1:60 | 80 | 1.5 | 10.03% |
经横向对比,可以发现方案编号2限定的提取参数对应的得率相对较高。
实施例2、褐藻多糖总糖含量分析
总糖含量采用苯酚-硫酸法进行测定。
配制:0.1mg/mL葡萄糖标准溶液(称取10mg烘干至恒重的葡萄糖粉末,溶解于蒸馏水,用100mL容量瓶定容)、6%苯酚溶液(60℃水浴溶解苯酚固体后,吸取600μL苯酚,加水定容至10mL)。
标准曲线的制作:分别吸取浓度为0.1mg/mL的葡萄糖标准液0、0.06、0.12、0.18、0.24、0.30mL,加蒸馏水补至0.4mL,再在每管中加入0.2mL苯酚溶液,缓慢加入1.0mL浓硫酸,混匀后静置10min,沸水浴15min。冷却至室温后,以200μL/孔加入96孔板中,每组三个平行,使用酶标仪在490nm的波长下测定吸光值,以标准葡萄糖浓度为横坐标,吸光值为纵坐标绘制标准曲线,如图1所示。得到三个平行组的吸光值分别为:0.36787、0.36601和0.3692,通过标准曲线计算得到实施例1制得的褐藻多糖样品浓度,由下式得到总多糖含量。
计算得出0.2mg/mL褐藻多糖的总多糖含量为91.80±0.54%。
实施例3、褐藻多糖中蛋白质含量分析
蛋白质含量使用BCA蛋白检测试剂盒法测定。
用蒸馏水溶解牛血清蛋白,配置浓度为0.5mg/mL的牛血清蛋白标准品溶液。按照试剂盒说明书配置BCA工作液(10.0mL BCA试剂A,0.2mL BCA试剂B)。用蒸馏水稀释标准品为0、0.025、0.5、0.1、0.2、0.3、0.4、0.5mg/mL的标准液,每个浓度设置3个平行,加20μL到96孔板标准孔中用于制作标准曲线。加待测的0.2mg/mL褐藻多糖样品溶液于96孔板的样品孔中,同样每孔加入20μL。最后各孔加入200μL BCA工作液。室温静置30min后用酶标仪测定A562 nm处的吸光度。以牛血清蛋白标准液浓度为横坐标,吸光值为纵坐标绘制标准曲线,如图2所示。测得三个平行组的吸光值分别为0.103、0.098和0.097,通过标准曲线计算得实施例1制得的褐藻多糖样品在浓度为0.2mg/mL时的蛋白质含量。
计算得出所述褐藻多糖中的蛋白质含量为0.065±0.025%。
实施例4、褐藻多糖中硫酸根含量检测
本实施例为褐藻多糖硫酸盐的含量测定,采用氯化钡-明胶比浊法测定硫酸根含量。较高硫酸化程度的多糖常被认为在肠炎的治疗中发挥了更好的生物学活性,例如促进肠道黏膜的修复和再生和调节肠道免疫功能,增强免疫系统的抵抗力。
所需试剂:
0.6g/L硫酸钾标准液:精准称取217.5mg干燥至恒定质量的硫酸钾固体,用浓度为1mol/L的盐酸定容至200mL;
3%(w/v)三氯乙酸:3g三氟乙酸溶解于蒸馏水中,在100mL容量瓶定容;
0.5%(w/v)明胶溶液:称取500mg明胶粉末并溶解于蒸馏水中,65℃水浴加热3h使明胶完全溶解,加蒸馏水用100mL容量瓶定容,4℃静置过夜;
0.5%(w/v)明胶-氯化钡溶液:精确称取500mg氯化钡溶解于0.5%(w/v)明胶溶液中,用50mL容量瓶定容,4℃下静置2h;
褐藻多糖样品溶液:用浓度为1mol/L的盐酸溶解实施例1制得的褐藻多糖冻干粉,得到浓度为0.2mg/mL褐藻多糖样品溶液。
绘制标准曲线及褐藻多糖硫酸根含量检测:取硫酸钾标准溶液0、0.2、0.4、0.6、0.8、1.0mL,用浓度为1mol/L的HCI盐酸溶液补全至1.0mL,加入3%三氯乙酸3.0mL和0.5%(w/v)明胶-氯化钡溶液1.0mL,摇匀,室温静置15min,于360nm波长测定吸光度A1,以0.5%(w/v)明胶溶液1.0mL代替0.5%明胶-氯化钡溶液按照上述方法操作,测吸光度为A2,每组三个平行,以硫酸根含量为横坐标,纵坐标为吸光度(A1-A2),绘制标准曲线如图3所示,线性关系良好。
取褐藻多糖样品溶液1.0mL,加入3%三氯乙酸3.0mL和0.5%(w/v)明胶-氯化钡溶液1.0mL,摇匀,室温静置15min,于360nm波长测定吸光度A1,以0.5%(w/v)明胶溶液1.0mL代替0.5%明胶-氯化钡溶液按照上述方法操作,测吸光度为A2,设置三个平行实验。三个平行组的吸光值分别为:0.062、0.058和0.052,代入标准曲线中的线性方程,得到褐藻多糖样品溶液中的硫酸根含量为26.02%±3.15%。
实施例5、单糖组成分析
采用高效液相色谱法(HPLC)分析通过1-苯基-3-甲基-5-吡唑啉酮(PMP)衍生褐藻多糖的单糖组成。
溶液配制:浓度为0.6mol/L的NaOH溶液(称量0.24g NaOH粉末,加水定容到10mL);浓度为0.5mol/L的PMP甲醇溶液(取0.87g PMP用甲醇定容到10mL);pH为6.7、浓度为0.1mol/L的磷酸盐缓冲液(称取NaCl 8g、KCl0.2g、Na2HPO4 7.1g、KH2PO4 6.8g,加蒸馏水溶解后用HCl/NaOH调节pH至6.7,最后定容到1L;抽滤除去难溶颗粒,并超声除去气泡);甘露糖(Man)、鼠李糖(Rha)、葡萄糖醛酸(GluA)、半乳糖醛酸(GalA)、葡萄糖(Glu)、半乳糖(Gal)、阿拉伯糖(Ara)和果糖(Fuc)标准品(各称取10mg溶解在1mL 50%乙醇中,4℃保存,各取100μL,再加入200μL水,即为标准品混合液);浓度为2mol/L TFA溶液(称取TFA 22.8g,加水定容至100mL)。
样品处理:10mg实施例1所制得的褐藻多糖与2mL浓度为2mol/L的TFA混合,120℃油浴2h,离心后取上清用氮气吹干,重复两次加500μL甲醇吹干,用以除去TFA。吹干后加0.5mL水复溶,取200μL样品,加入200μL浓度为0.6mol/L的NaOH和400μL浓度为0.5mol/L的PMP甲醇溶液,70℃水浴100min。用二氯甲烷洗三次,即每次加入1mL二氯甲烷,剧烈摇动后打开放气,离心,吸出下层的二氯甲烷,最后加入0.5mL水稀释,用水系聚醚砜膜过滤(0.45μm)。
设置标准品对照:取200μL标准品混合液氮气吹干后,加入200μL水复溶,加入200μL浓度为0.6mol/L的NaOH和400μL浓度为0.5mol/L的PMP甲醇溶液,70℃水浴100min,用二氯甲烷洗三次,加入0.5mL水稀释,用0.45μm水系聚醚砜膜过滤。
空白对照:200μL水加200μL浓度为0.6mol/L的NaOH和400μL浓度为0.5mol/L的PMP甲醇溶液,70℃水浴100min,用二氯甲烷洗三次,加入0.5mL水稀释,用0.45μm水系聚醚砜膜过滤。
HPLC检测步骤:
柱子选择VisionHT C18HL5u,柱温30℃,上样体积20μL,流速1mL/min,A泵为pH6.7、浓度0.1mol/L的磷酸盐缓冲液,B泵为乙腈,条件为85%A、15%B等度洗脱,一个样品40min,紫外波长245nm。
使用电脑软件根据峰面积和摩尔浓度绘制单糖标准曲线。多糖样品中各单糖含量对照标准曲线得褐藻多糖的单糖组成及摩尔比为褐藻糖(Fuc):葡萄糖(Glc):半乳糖(Gal):甘露糖(Man):葡萄糖醛酸(GluA):木糖(Xyl)=26.56:0.58:10.78:1.32:2.07:1.00。
实施例6、人结肠癌细胞系(LS174T)细胞培养及细胞存活率试验
①试验试剂及分组
LS174T细胞来源于中国科学院上海细胞生物学研究所,该细胞系常被作为研究肠道杯状细胞的实验模型。LS174T细胞在完全培养基(10%胎牛血清、1%青链霉素混合液和高糖DMEM培养基(Gibco,美国)中培养,培养环境为37℃、含有5%CO2的加湿培养箱。每隔两天细胞半量换液,每4~5天传代。褐藻多糖溶解于不含胎牛血清的DMEM培养基中并用0.22μm滤膜过滤,得到褐藻多糖溶液。浓度为1mg/mL的LPS(脂多糖,Solarbio,中国)稀释于不含胎牛血清的DMEM培养基,得到不同浓度的LPS溶液。将(PKC抑制剂)用DMSO溶解后,用不含胎牛血清的DMEM培养基稀释至浓度为10μg/mL抑制剂溶液,分装保存于-20℃冰箱。
分组:
CN:空白对照组,完全培养基培养24小时后吸出,加入DMEM培养基,两天后半量换液。
L:损伤模型组,完全培养基培养24小时后吸出,加入浓度为25μg/mL的LPS溶液处理LS174T细胞48小时后换液。
LF:损伤治疗组,完全培养基培养24小时后吸出,加入浓度为25μg/mL的LPS溶液处理LS174T细胞24小时后吸出,PBS冲洗一次后加入浓度200μg/mL褐藻多糖溶液处理LS174T细胞48小时。
F:治疗组,完全培养基培养24小时后吸出,加入浓度为200μg/mL褐藻多糖溶液处理LS174T细胞48小时后换液。
CN+:完全培养基培养24小时后吸出,加入PKC抑制剂溶液,两天后半量换液。
L+:完全培养基培养24小时后吸出,加入浓度为25μg/mL且含PKC抑制剂溶液的LPS溶液处理LS174T细胞48小时后换液。
LF+:完全培养基培养24小时后吸出,加入浓度为25μg/mL且含PKC抑制剂溶液的LPS溶液处理LS174T细胞24小时后吸出,PBS冲洗一次后加入浓度为200μg/mL且含PKC抑制剂溶液的褐藻多糖溶液处理LS174T细胞48小时。
F+:完全培养基培养24小时后吸出,加入浓度为200μg/mL且含PKC抑制剂溶液的褐藻多糖溶液处理LS174T细胞48小时后换液。
②CCK-8法检测褐藻多糖和脂多糖分别处理LS174T细胞的细胞存活率
细胞复苏后取对数生长期细胞均匀铺于96孔板,105个/孔,每个浓度设置4个复孔,孵育24小时后吸出培养液,加入不同浓度(0、20、200、500、1000、2500μg/mL)的褐藻多糖溶液处理LS174T细胞48小时,按照CCK-8试剂盒(MCE,USA)说明书要求处理后,用酶标仪检测450nm处的吸光度,计算细胞存活率。
细胞复苏后取对数生长期细胞均匀铺于96孔板,10000个/孔,每个浓度设置4个复孔,孵育24小时后吸出培养液,加入不同浓度(0、5、10、25、50、100μg/mL)的LPS溶液处理LS174T细胞72小时,按照CCK-8试剂盒(MCE,USA)说明书要求处理后,用酶标仪检测450nm处的吸光度,计算细胞存活率。
图4为CCK-8法检测褐藻多糖作用LS174T细胞对其细胞活性的影响图,图4A中横坐标表示褐藻多糖的浓度,图4B中横坐标表示脂多糖浓度,纵坐标cell viability表示细胞活性。
由图4A可知,当褐藻多糖浓度为20~500μg/mL时,LS174T细胞活力大于80%。另外,与空白对照组相比,当褐藻多糖浓度高于1000μg/mL时对LS174T细胞的增殖活性具有抑制作用。
由图4B可知,脂多糖浓度为5~100μg/mL时,LS174T细胞活力均大于80%,细胞活力大于80%表明脂多糖对细胞增殖没有抑制作用。
实施例7、褐藻多糖通过促进结肠杯状细胞分泌黏液修复肠道黏膜屏障
①AB-PAS法检测LS174T细胞分泌黏蛋白
AB-PAS染色是一种将糖类物质结构中相邻碳的羟基氧化为醛基,再将醛基染色为红色或紫红色的方法,以观察细胞表面糖类和中性黏液物质分泌情况。取对数生长期细胞以每孔2×105个每孔均匀铺于六孔板,完全培养基培养24小时后吸出,按照如前的实验分组:CN组、L组、LF组和F组。各组分别加入对应溶液培养,72小时后吸出六孔板内液体,按照AB-PAS染色试剂盒对细胞染色。洗净染色液后每孔加入1mL纯水,光学显微镜下拍照观察。实验重复三次。
图5为倒置显微镜拍摄AB-PAS染色的LS174T细胞,图5A表示空白对照组(CN),图5B表示损伤模型组(L),图5C表示损伤治疗组(LF),图5D表示治疗组(F)。图5结果显示,L组紫红色明显弱于空白对照组,这表明LPS刺激会使杯状细胞分泌黏液减少,褐藻多糖处理后紫红色增多,表明褐藻多糖对LPS引起的黏液分泌减少有恢复作用。另外,褐藻多糖单独处理后细胞也显示出强于空白对照组的紫红色,说明在褐藻多糖可以促进杯状细胞分泌黏液。
②激光共聚焦显微镜观察MUC2和ZO-1蛋白表达
取对数生长期细胞以每孔1×105个每孔均匀铺于20mm激光共聚焦培养皿,为了观察LS174T细胞中的黏蛋白分泌量和紧密连接蛋白分泌量,采用免疫荧光法检测MUC2和ZO-1的表达。首先用免疫染色固定液每皿加1mL,室温下固定细胞10min,加入1mL免疫染色洗涤液静置洗涤8分钟,然后用免疫染色封闭液4℃静置封闭过夜。用一抗稀释液分别按照比例1:200、1:500稀释MUC2和ZO-1蛋白的一抗。4℃静置孵育过夜后回收一抗。加入免疫染色洗涤液1mL,浸泡8分钟,重复洗涤5次,然后用1:1000稀释的FITC标记山羊抗兔IgG抗体在摇床上避光室温孵育1小时。回收二抗,加入1mL免疫染色洗涤液,每次洗涤5分钟,共洗涤3次。取少量DAPI(4',6-二脒基-2-苯基吲哚)染液,在37℃培养箱孵育半小时。免疫染色洗涤液洗1次,5分钟。最后,使用激光共聚焦显微镜观察染色细胞并拍照。实验重复三次,并设置不加一抗的阴性对照组,以避免假阳性结果。
图6显示,相比于空白对照组,LPS处理杯状细胞后使MUC2蛋白和ZO-1蛋白减少,表明黏液层黏液减少、细胞间紧密连接的连接作用减弱。加入褐藻多糖后,MUC2蛋白和ZO-1蛋白表达量增加,LPS引起的损伤被逆转,表明褐藻多糖对黏液屏障损伤有治疗作用。另外,单独添加褐藻多糖处理杯状细胞同样显示出MUC2蛋白和ZO-1蛋白表达得到促进。
③qRT-PCR法检测MUC2(A)、TFF3(B)、KLF4(C)、VAMP8(D)和PKCβ(E)在LS174T细胞内的mRNA水平表达。
取对数生长期细胞以每孔2×105个每孔均匀铺于6孔板,完全培养基培养24小时后吸出,按照如前的实验分组:CN组、L组、LF组、F组、CN+组、L+组、LF+组和F+组。各组分别加入对应溶液培养,72小时后吸出六孔板内液体。使用Trizol进行总RNA分离。按照试剂盒说明进行逆转录,扩增。用MUC2、TFF3、KLF4、VAMP8、PKCβ和甘油醛-3-磷酸脱氢酶基因(GAPDH)特异性引物进行扩增。以GAPDH的产物作为加载对照。所用引物列表见表1。
表1.扩增引物列表
Primer名称 | 序列(5'to 3') | SEQ ID No |
MUC2-F | TCCACCAACCACCACTTCC | 1 |
MUC2-R | AGAATCCAGCCAGCCAGTC | 2 |
TFF3-F | GGACAGTTCTTCGTGCCTGAGA | 3 |
TFF3-R | GTGGGTGCCAGTCTGGATTCAA | 4 |
KLF4-F | TCGGACCACCTCGCCTTACA | 5 |
KLF4-R | TCCTGATTATCCACTCACAAGATGACT | 6 |
VAMP8-F | AATGATCGTGTGCGGAACCT | 7 |
VAMP8-R | TGAAGTGCTCAGATGTGGCT | 8 |
PKCβ-F | CGCCATCCACCAGTCCTAACA | 9 |
PKCβ-R | GTCACCACAATAGCCGTTGAGC | 10 |
GAPDH-F | TATGACAACAGCCTCAAGAT | 11 |
GAPDH-R | AGTCCTTCCACGATACCA | 12 |
图7为qRT-PCR法检测MUC2(A)、TFF3(B)、KLF4(C)、VAMP8(D)和PKCβ(E)在LS174T细胞内的mRNA水平表达结果。横坐标表示实验分组,纵坐标表示在mRNA水平上各基因相对GAPDH内参基因的相对表达量。图7A显示褐藻多糖显著上调LPS引起的MUC2的mRNA表达减少(P<0.001),LF组的MUC2的mRNA相对表达量约为空白对照组的13倍,单独褐藻多糖处理后也显著上调MUC2的mRNA上调(P<0.05),F组MUC2的mRNA相对表达量增加接近8倍,表明褐藻多糖处理可以有效上调MUC2的mRNA,有显著促进杯状细胞分泌黏蛋白的作用。图7B显示褐藻多糖显著上调LPS引起的TFF3的mRNA减少(P<0.01),单独褐藻多糖处理后也上调TFF3的mRNA上调。表明褐藻多糖处理可以上调TFF3的mRNA表达,有利于杯状细胞分泌黏液。图7C显示褐藻多糖显著上调LPS引起的KLF4的mRNA抑制(P<0.01),单独褐藻多糖处理后也显著上调KLF4的mRNA水平(P<0.05),表明褐藻多糖处理可以有效增加KLF4表达,褐藻多糖处理具有促进杯状细胞分化和增殖的潜在作用。图7D显示褐藻多糖处理LPS损伤组后上调了VAMP8的mRNA,表明褐藻多糖可能具有促进炎症环境中肠杯状细胞分泌黏液的作用。而单独褐藻多糖处理组显示出VAMP8的mRNA水平无明显变化,表明褐藻多糖在正常环境中不会显著上调或下调囊泡分泌蛋白,从而使黏液分泌保持在平衡的水平。图7E显示LPS处理杯状细胞后会显著抑制PKCβ的mRNA表达(P<0.05),褐藻多糖处理后显著上调LPS引起的PKCβ的mRNA表达量减少(P<0.001),单独褐藻多糖处理后也显著上调PKCβ的mRNA上调(P<0.05),表明褐藻多糖处理可以有效增加PKCβ的mRNA表达,通过PKC信号通路对杯状细胞增殖和分泌黏液起促进作用。
上面结合附图对本发明实施例作了详细说明,但是本发明不限于上述实施例,在所属技术领域普通技术人员所具备的知识范围内,还可以在不脱离本发明宗旨的前提下作出各种变化。此外,在不冲突的情况下,本发明的实施例及实施例中的特征可以相互组合。
Claims (10)
1.一种褐藻多糖的制备方法,其特征在于,包括步骤:
(1)将干燥的裙带菜粉碎后,过筛得裙带菜粉;
(2)将所述裙带菜粉加入混合液,摇床振荡,离心后取沉淀,干燥,得到脱色裙带菜粉末,所述混合液包括甲醇和二氯甲烷;
(3)所述脱色裙带菜粉末加水,50~100℃水浴1~4h,纱布分离滤液;
(4)向所述滤液中加入95%乙醇,静置于4℃冰箱,得静置液;
(5)所述静置液收集凝固沉淀,以蒸馏水溶解所述凝固沉淀,干燥,得褐藻粗多糖;
(6)将所述褐藻粗多糖采用二乙胺基乙基纤维素离子交换树脂进行纯化,收集组分液体,洗脱,透析,干燥,即得所述褐藻多糖。
2.根据权利要求1所述制备方法,其特征在于,步骤(1)所述裙带菜粉碎后过20~80目筛。
3.根据权利要求1所述制备方法,其特征在于,步骤(2)所述混合液由甲醇、二氯甲烷和水组成,质量比为(4~5):(2~3):1。
4.根据权利要求1所述制备方法,其特征在于,步骤(3)所述脱色裙带菜粉末和水的料液比1g:10mL。
5.根据权利要求1所述制备方法,其特征在于,步骤(2)所述离心的条件为2000~5000rpm,持续4~10min。
6.根据权利要求1所述制备方法,其特征在于,步骤(2)和/或步骤(5)和/或步骤(6)所述干燥为真空冷冻干燥。
7.根据权利要求1所述制备方法,其特征在于,步骤(3)所述脱色裙带菜粉末加水的料液比为1g:40mL。
8.根据权利要求1所述制备方法,其特征在于,步骤(4)所述95%乙醇的添加量为所述滤液的体积的2~5倍。
9.一种褐藻多糖,其特征在于,由权利要求1至8任一项所述制备方法制得。
10.权利要求9所述褐藻多糖在制备预防/治疗肝脏损伤的药物或食品中的应用。
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