CN107698695A - 具有免疫调节活动性的均一多糖及其制备方法 - Google Patents
具有免疫调节活动性的均一多糖及其制备方法 Download PDFInfo
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Abstract
本发明涉及具有免疫调节活动性的均一多糖及其制备方法。具有免疫调节活动性的均一多糖,均一多糖为单一色谱峰,分子量为6880±5Da,旋光值为158.4±0.5°,红外光谱在847.6cm‑1有特征吸收峰,其氢谱中的氢质子化学位移分别为δ5.40brs,3.95t,J=7.2Hz,3.84m,3.61m,碳谱中的碳信号化学位移分别为δ99.6,δ76.6,δ73.3,δ71.5,δ71.1,δ60.4。该均一多糖,具有增强机体免疫功能,在免疫相关疾病的调节汇总可以起到很好的抗肿瘤、抗病毒、抗感染等作用;在治疗和预防疾病的药品保健品行业具有潜在的开发价值。
Description
技术领域
本发明涉及均一多糖的技术领域,尤其是具有免疫调节活动性的均一多糖及其制备方法。
背景技术
一些中药多糖具有激活免疫细胞、改善机体免疫功能等作用,以效果确切、毒副作用小等优点引起了诸多研究者重视。其作为一种有开发潜力的免疫调节剂,在消除疾病及增强人体抵抗力的过程中必将发挥巨大的作用。但目前多数中药多糖研究多局限于总多糖,对其具体分子组成及作用机理研究并不清楚,因此,深入系统研究中药多糖物质结构及其对免疫系统作用与机理,有望从更深层次认识中药多糖类药效物质。
猪苓均一多糖(polyperus polysaccharide)是中药猪苓水提取物中的主要活性成分,是良好的免疫调节剂,具有抗肿瘤、免疫调节、保肝、抗氧化、抗辐射、抗诱变等功效。本研究组前期研究发现猪苓总多糖对BBN诱导膀胱癌具有较好的预防作用,并对其免疫功能有明显提高作用。对猪苓总多糖的免疫调节作用及机制我们也有相关研究,但以上研究对象均为猪苓总多糖,并未涉及猪苓均一多糖中某一个具体成分及其免疫相关活性。而有关猪苓均一多糖成分研究,日本学者报道猪苓中含有水溶性多聚糖6-支链-β-1,3-葡聚糖,其单糖组成只有葡萄糖,均为β构型。也有报道从猪苓中分离到分子量约227万主含D-葡萄糖,分子量1.4万含葡萄糖和葡萄糖醛酸及分子量约8800含葡萄糖醛酸的均一多糖。
本研究采用简单有效的多糖提取工艺和方法,首次从猪苓药材中分离鉴定一个新的不同于已有文献报道均一多糖(PPS),并证实其单糖组成只有葡萄糖,其构型均为α型,其主要连接方式为1→4连接,并对其抗肿瘤免疫调节活性及作用机理进行了研究,首次发现了该均一多糖具有较强的免疫调节,治疗,预防和保健作用。
发明内容
本发明要解决的技术问题是:为了解决上述背景技术中的现有技术存在的问题,提供具有免疫调节活动性的均一多糖及其制备方法,均一多糖具有增强机体免疫功能,在免疫相关疾病的调节汇总可以起到很好的抗肿瘤、抗病毒、抗感染等作用;在治疗和预防疾病的药品保健品行业具有潜在的开发价值。
本发明解决其技术问题所采用的技术方案是:具有免疫调节活动性的均一多糖,所述均一多糖为单一色谱峰,分子量为6880±5Da,旋光值为158.4±0.5°,红外光谱在847.6cm-1有特征吸收峰,其氢谱中的氢质子化学位移分别为δ5.40brs,3.95t,J=7.2Hz,3.84m,3.61m,碳谱中的碳信号化学位移分别为δ99.6,δ76.6,δ73.3,δ71.5,δ71.1,δ60.4。
进一步具体地说,上述技术方案中,所述均一多糖中的多糖质量百分比含量为92~98%;采用Agilent1200液相色谱,示差检测器RID分析,色谱柱为TSK-GEL G4000PWxL,流动相为超纯水,流速为0.3~1ml/min,检测器温度为30~40℃,柱温为30~50℃,色谱分析为单一峰,保留时间10~30min。
进一步具体地说,上述技术方案中,所述均一多糖中的单糖组成为α-D-吡喃葡萄糖,构型均为α型,连接方式为1→4连接。
进一步具体地说,上述技术方案中,所述均一多糖内还添加有荧光标记产物、羧甲基化产物、羟甲基化产物、羟丙基化产物、乙二醇化产物、丙二醇化产物或聚乙二醇化产物中的一种。
具有免疫调节活动性的均一多糖的制备方法,包括如下步骤:
步骤1:水加热提取,醇沉制备总多糖;
将猪苓药材粉碎,取300g加3L去离子水,室温浸泡1小时后100℃加热回流提取2次,每次1h,过滤,合并滤液,浓缩至600ml,3000rpm离心10min,上清液调乙醇比例至大于80%,4℃静置过夜,滤过上清液,沉淀冷冻干燥得粗多糖,取粗多糖加纯水配成25~35mg·mL-1溶液;
步骤2:Sevag法除粗多糖蛋白;
准备糖液:氯仿:正丁醇按25:4:1的比例加入试剂,倾入分液漏斗中充分震摇3min后静置分层,弃去有机层及沉淀,重复除蛋白多次至无白色沉淀产生,收集上清液,用旋转蒸发仪浓缩至适量体积后转移,冷冻干燥,得除蛋白后粗多糖;
步骤3:DEAE-52纤维素脱色素;
将经去蛋白后猪苓粗多糖分别加少量水溶解后,上经预处理后的DEAE-52纤维素柱35×3cm脱色,以1mL/min超纯水、0.1~0.5%氯化钠溶液洗脱,洗脱至流出液用苯酚-浓硫酸法无糖检出,收集峰值段洗脱液用旋转蒸发仪浓缩后冷冻干燥,脱盐,即得白色疏松粉末精多糖;
步骤4:Sephadex G-100凝胶柱精制;
将精多糖适量溶于蒸馏水中,上Sephadex G-100凝胶柱,以纯水、0.1~0.5%氯化钠溶液洗脱,流速1ml/min,洗脱液每10mL收集一管,以苯酚-浓硫酸法跟踪监测;以检测管号为横坐标,吸光度为纵坐标,绘制多糖洗脱曲线,收集峰值段洗脱液,冷冻干燥,脱盐即可获得均一多糖。
进一步具体地说,上述技术方案中,所述步骤3中DEAE-52纤维素柱的预处理方法为:
步骤1:将干粉的纤维素浸泡在蒸馏水中2~5h,去除杂质后抽干;
步骤2:采用0.5mol/L的HCL溶液浸泡1~3h,用去离子水洗净置PH为中性并抽干;
步骤3:再将抽干的纤维素浸泡在0.5mol/L的NaOH溶液中1~3h,用去离子水洗净置PH为中性并抽干即可。。
本发明的有益效果是:该均一多糖具有增强机体免疫功能,在免疫相关疾病的调节汇总可以起到很好的抗肿瘤、抗病毒、抗感染等作用;在治疗和预防疾病的药品保健品行业具有潜在的开发价值。
附图说明
下面结合附图和实施例对本发明进一步说明。
图1是猪苓均一多糖PPS HPLC-RID的色谱图;
图2是猪苓均一多糖PPS的红外光谱图;
图3是猪苓均一多糖PPS 1H-NMR(400MHz,D2O)谱图;
图4是猪苓均一多糖PPS 13C-NMR(100MHz,D2O)谱图;
图5为猪苓均一多糖PPS对RAW264.7细胞存活率的影响示意图;
图6a为猪苓均一多糖PPS对RAW264.7细胞膜表面分子CD11b表达影响示意图;
图6b为猪苓均一多糖PPS对RAW264.7细胞膜表面分子CD16/32表达影响示意图;
图6c为猪苓均一多糖PPS对RAW264.7细胞膜表面分子CD40表达影响示意图;
图7A为猪苓均一多糖PPS对人移行膀胱癌T24细胞和RAW264.7巨噬细胞共培养体系NO分泌量的影响示意图;
图7B为猪苓均一多糖PPS对人移行膀胱癌T24细胞和RAW264.7巨噬细胞共培养体系炎症因子的影响示意图;
图8为猪苓均一多糖PPS对T24与RAW264.7细胞共培养体系COX2和INOS mRNA蛋白表达的影响示意图;
图9a为猪苓均一多糖PPS对CD14蛋白激酶磷酸化表达的作用示意图;
图9b为猪苓均一多糖PPS对CD284蛋白激酶磷酸化表达的作用示意图;
图9c为猪苓均一多糖PPS对CD282蛋白激酶磷酸化表达的作用示意图;
图9d为猪苓均一多糖PPS对P38蛋白激酶磷酸化表达的作用示意图;
图9e为猪苓均一多糖PPS对P65蛋白激酶磷酸化表达的作用示意图。
具体实施方式
现在结合附图对本发明作进一步详细的说明。这些附图均为简化的示意图,仅以示意方式说明本发明的基本结构,因此其仅显示与本发明有关的构成。
在本申请中,均一多糖可以从猪苓中提取的多糖与药用辅料组成的,其中多糖的组成单糖是α-D-吡喃葡萄糖,也可以从其它真菌、植物中分离,或采用微生物发酵、人工合成等手段获得的只要具备该分子量及理化光谱性质特征的均一多糖;产生的作用:明显促进巨噬细胞NO分泌,显著增加巨噬细胞IL-1β、IL-6、TNF-α和iNOS基因和iNOS、COX-2蛋白表达,增加巨噬细胞膜表面受体CD11b、CD16/32、CD40、CD14、CD284、CD282表达,以及P-P38、、P-P65蛋白激酶磷酸化表达的作用,具有潜在的治疗和预防疾病的药品保健品的开发价值;该均一多糖可开发制备成药剂学上任何剂型,以单一或复合用药形式,用于自身免疫相关疾病预防和治疗,包括抗衰老、抗肿瘤、抗感染、自身免疫性疾病。本申请中的均一多糖可用于所有激活氮氧合酶及NO表达,激活CD14/TRL4/P38和TRL2/NF-kB通路相关的抗肿瘤,抗病毒,抗感染以及免疫相关疾病的调节中的应用。
所述均一多糖为单一色谱峰,分子量为6880±5Da,旋光值为158.4±0.5°,红外光谱在847.6cm-1有特征吸收峰,其氢谱中的氢质子化学位移分别为δ5.40brs,3.95t,J=7.2Hz,3.84m,3.61m,碳谱中的碳信号化学位移分别为δ99.6,δ76.6,δ73.3,δ71.5,δ71.1,δ60.4。
其中,均一多糖中的多糖质量百分比含量为92~98%;采用Agilent1200液相色谱,示差检测器RID分析,色谱柱为TSK-GEL G4000PWxL,流动相为超纯水,流速为0.3~1ml/min,检测器温度为30~40℃,柱温为30~50℃,色谱分析为单一峰,保留时间10~30min。均一多糖中的单糖组成为α-D-吡喃葡萄糖,构型均为α型,连接方式为1→4连接。均一多糖内还添加有荧光标记产物、羧甲基化产物、羟甲基化产物、羟丙基化产物、乙二醇化产物、丙二醇化产物或聚乙二醇化产物中的一种。
具有免疫调节活动性的均一多糖的制备方法,包括如下步骤:步骤1:水加热提取,醇沉制备总多糖;将猪苓药材粉碎,取300g加3L去离子水,室温浸泡1小时后100℃加热回流提取2次,每次1h,过滤,合并滤液,浓缩至600ml,3000rpm离心10min,上清液调乙醇比例至大于80%,4℃静置过夜,滤过上清液,沉淀冷冻干燥得粗多糖,取粗多糖加纯水配成30mg·mL-1溶液;步骤2:Sevag法除粗多糖蛋白;准备糖液:氯仿:正丁醇按25:4:1的比例加入试剂,倾入分液漏斗中充分震摇3min后静置分层,弃去有机层及沉淀,重复除蛋白多次至无白色沉淀产生,收集上清液,用旋转蒸发仪浓缩至适量体积后转移,冷冻干燥,得除蛋白后粗多糖;步骤3:DEAE-52纤维素脱色素;将经去蛋白后猪苓粗多糖分别加少量水溶解后,上经预处理后的DEAE-52纤维素柱35×3cm脱色,以1mL/min超纯水、0.1%~0.5%氯化钠溶液洗脱,洗脱至流出液用苯酚-浓硫酸法无糖检出,收集峰值段洗脱液用旋转蒸发仪浓缩后冷冻干燥,脱盐,即得白色疏松粉末精多糖;步骤4:Sephadex G-100凝胶柱精制;将精多糖适量溶于蒸馏水中,上Sephadex G-100凝胶柱,以超纯水、0.1%-0.5%氯化钠溶液洗脱,流速1ml/min,洗脱液每10mL收集一管,以苯酚-浓硫酸法跟踪监测;以检测管号为横坐标,吸光度为纵坐标,绘制多糖洗脱曲线,收集峰值段洗脱液,冷冻干燥,脱盐即可获得均一多糖。
其中,步骤3中DEAE-52纤维素柱的预处理方法为:步骤11:将干粉的纤维素浸泡在蒸馏水中2~5小时,去除杂质后抽干;步骤22:采用0.5mol/L的HCL溶液浸泡1~3小时,用去离子水洗净置PH为中性并抽干;步骤33:再将抽干的纤维素浸泡在0.5mol/L的NaOH溶液中1~3小时,用去离子水洗净置PH为中性并抽干即可。
对上述方法制得的猪苓均一多糖进行检测,其多糖含量为96.73%。见图1,采用Agilent1200液相色谱,示差检测器RID分析,色谱柱为TSK-GEL G4000PWxL 10mm,7.8mmI.D.×30cm,流动相:超纯水,流速:0.5ml/min,检测器温度:35℃,柱温:40℃,色谱分析为单一峰,保留时间约20min。
猪苓均一多糖的分子量测定及结构鉴定:
分离制备的猪苓均一多糖PPS分子量和单糖组成委托第三方检测机构测定完成。见图2,采用HPGPC凝胶渗透色谱法测得其重均分子量约6.88kDa。PPS经三氟乙酸水解,醋酸酐乙酰化,GC-MS分析结果表明其单糖组成仅为D-葡萄糖。UV光谱分析190nm的末端吸收是多糖的典型吸收,而在260,280nm无吸收峰表明PPS不含核酸和蛋白质。其IR光谱显示典型多糖特征吸收峰(3304.6,2926.9,1642.9,1362.9,1148.8,1077.8,1014.2,847.6cm-1)。IR光谱中亦显示无NH吸收峰,表明该多糖不含氨基或结合蛋白,PPS的红外光谱与图2所示的红外光谱图基本一致。
1H-NMR(400MHz,D2O):δ5.40ppm归属为D-吡喃葡萄糖端基氢质子信号,其化学位移值大于5.0及宽单峰表明葡萄糖苷键的连接方式为α型,这与其红外(IR)光谱中有847.6cm-1特征吸收峰及测得旋光值为大的比旋正数(25D+158.4±0.5(c=2.5,H2O))可进一步证实。糖上其余氢化学位移区间在3.61-3.98,为糖环碳上其它质子信号,其化学位移值均小于4.0表明该多糖无其它糖残基异头氢信号。13C-NMR(100MHz,D2O)谱中显示6个主要碳信号,化学位置值分别为99.6,76.6,73.3,71.5,71.1和60.4,结合DEPT135谱显示只有δ60.4为亚甲基官能团,其它均为次甲基官能团,提示多糖的单糖组成为吡喃葡萄糖,且其6位亚甲基未被取代。进一步结合HSQC、HMBC和1H-1H-COSY相关谱分析,我们对PPS主要组成单糖单元的碳、氢信号进行了完全归属,其中δ99.6归属于葡萄糖残基的异头碳信号,δ71.5,71.1和73.3分别为葡萄糖未被取代的C-2,3,5碳信号,δ76.6是被取代的C-4共振信号,δ60.4是未取代的C-6碳信号,其单糖组成只含有α-D-吡喃葡萄糖,这与上述GC-MS和IR分析结果一致。其C-1(δ99.6)和C-4(δ76.6)化学位置向低场移动表明单糖的1,4位羟基被取代,单糖之间连接方式为α-1→4连接,这进一步在HMBC谱中H-1(δ5.40)与C-4(δ76.6)及H-4(δ3.61)与C-1(δ99.6)远程相关可进一步证实。碳谱中还可检测到化学位移值分别为99.7、76.8、72.8、72.6、71.6和69.3其它碳信号,其中δ69.3是取代的C-6信号,说明有O-6位出现支链,而δ99.7、76.8、72.8、72.6、71.6是支链1,4,6-α-D-吡喃葡萄糖糖C1-5位碳信号,但这些信号强度均较低,说明PPS的O-6位分支少。以上分析表明猪苓均一多糖PPS以1,4-为主要连接方式,并在O-6位出现部分支链或为末端糖基支链。所述多糖PPS的1H-NMR及13C-NMR谱的主要信号值与图3、图4中所示的氢、碳谱图中信号基本一致。
本申请人对猪苓均一多糖的免疫调节作用进行了多次实验,具体实验如下:
准备试剂与材料:
细胞小鼠巨噬细胞株RAW264.7,由广东省中医药科学院提供,人膀胱移行细胞癌细胞株T24ATCC;
高糖DMEM培养基购于Hyclone公司(批号:SH3024.01B);澳洲胎牛血清,FBS(Hyclone,USA,批号:SH30406.02E)双抗(GIBCO,USA);PBS缓冲溶液(Hyclone,USA,批号:SH30028.01B);IFN-γ干扰素(Peprotech,USA);Trizol(Invitrogen,USA);逆转录试剂盒Revert Aid First Strand cDNA Synthesis Kit(Thermo,USA);荧光定量PCR染料SYBRGreen试剂盒(Roche,USA)。
细胞培养
小鼠巨噬细胞株RAW264.7常规培养于含l00ml/L的胎牛血清,1%的双抗的DMEM中,置于37C,5%CO2培养箱中培养。
肿瘤细胞培养上清T24的制备
人移行膀胱癌细胞系T24细胞培养于含有10%胎牛血清、100U/ml青链霉素的DMED培养基,置于5%CO2,37℃孵箱中。将呈对数生长的T24细胞,以3.5×107cells/well的细胞数培养于培养皿,加18ml培养液,培养48h细胞生长至90%汇合度,收集培养上清备用。将收集的培养上清液经由0.22μm的滤器过滤,过滤后的上清培养液冻存于-80℃冰箱保存备用。
T24细胞与RAW264.7细胞共培养
小鼠巨噬细胞系RAW264.7细胞培养于含有10%胎牛血清、100U/ml青链霉素的DMED培养基,置于5%CO2,37℃孵箱中。将对数生长期的RAW264.7细胞培与T24上清共培养,培养液40%为T24肿瘤细胞培养上清。
测试猪苓均一多糖PPS对RAW264.7细胞存活率的影响:
RAW264.7细胞悬液以104个细胞/孔的密度接种于96孔板,培养24h后移除旧液;分别加入系列浓度为3.09625、7.8125、15.625、31.25、62.5、125、250、500、1000μg/mL的PPS的培养基。培养24h;每孔加入20μL的50g·L-1MTT溶液,继续孵育4h,弃上清液,每孔加入200μL的DMSO,室温溶解,振摇10min后于酶标仪490nm波长处测定吸光度(OD)值。每一药物浓度重复4孔,计算细胞相对存活率,公式如下:细胞相对存活率=OD490(给药)/OD490(对照),OD490(给药)和OD490(对照)分别为给药组细胞样品及对照组液的细胞样品在MTT实验中于490nm处的吸收值。PPS对RAW264.7巨噬细胞存活率的影响如图.5所示,可见在药物浓度为3.9-125mg/mL范围内时,细胞存活率与对照组相比无统计学意义,故PPS后续实验选择的梯度浓度为1、10、100mg/mL。
测试PPS对T24与RAW264.7细胞共培养体系中RAW264.7巨噬细胞膜表面分子的影响:
小鼠巨噬细胞株RAW264.7常规培养于含100ml/L的胎牛血清的DMEM培养液中。选择处于对数生长期的细胞,消化传代,计数并且调整细胞密度,按2.5×105个每孔接种于12孔细胞培养板中,每孔加入的1ml的DMEM培养基。培养24h完全贴壁。移除0.5mL的培养基,加入0.5mL的T24上清液。3h后,分别给予终浓度为100ng/mL的IFN-γ溶液,1、10、100μg/mL的PPS作用24h。消化收集细胞于流式管中,加入2mLPBS溶液洗涤3次,1000转离心去掉上清。然后依次加入相应的流式抗体CD16/32-FITC、CD40-PE、CD11b-APC各5uL,以相应的同型抗体作为阴性对照。避光4℃孵育30min,然后PBS洗涤,离心洗去游离的流式抗体。500uL PBS重悬细胞上流式细胞仪检测相关膜表面分子的阳性表达率。结果如图6A所示:PPS能够增加巨噬细胞的CD11b的表达量,与对照组相比具有统计学意义(P<0.05);如图6B所示:1、10、100μg/mL的PPS均能刺激巨噬细胞,增加了CD40的表达,并有显著性统计学意义(P<0.01);如图6C所示,1、10、100μg/mL的PPS均能增强巨噬细胞CD16/32的表达,并具统计学意义,其中浓度10、100μg/mL的PPS组与对照组相比,具有显著性统计学意义(P<0.01)。CD11b,CD16/32,CD40均是巨噬细胞极化成M1的膜标志,提示了PPS具有在较低剂量1μg/mL时即能活化巨噬细胞。
测试猪苓均一多糖PPS对RAW264.7巨噬细胞NO因子分泌和IL-1β、IL-6、TNF-α和iNOS mRNA表达量的影响:
取生长状态良好的RAW264.7巨噬细胞,消化传代,计数调整细胞密度,于12孔细胞培养板当中,加入1ml的细胞悬液,每孔加RAW264.7细胞2.5×105个,置于37℃,5%CO2培养箱中培养24h之后阳性组给予终浓度100ng/mL的IFN-γ溶液,给药组分别给予3.09625、7.8125、15.625、31.25、62.5、125、250、500、1000μg/mL的PPS培养24h;共培养组则在细胞培养24h后,移除40%的上清,加入40%的T24上清夜,共培养3个小时,之后给予阳性组终浓度100ng/mL的IFN-γ溶液,各给药组分别给予3.09625、7.8125、15.625、31.25、62.5、125、250、500、1000μg/mL的PPS培养24h;收集细胞上清,Griess法检测NO细胞因子的分泌量。实验结果见图7A,结果PPS在3.9g/ml比IFN-γ(100ng/mL)刺激分泌NO更多,PPS在模拟膀胱癌肿瘤微环境(巨噬细胞和膀胱癌细胞共培养)中能明显促进巨噬细胞分泌NO,并且在较低剂量3.90625μg/mL时,NO的分泌量高于阳性对照组IFN-γ(100ng/mL)达2倍之多。
2.5×105个细胞种于12孔细胞培养板,培养24h,移除40%的上清,加入40%的T24上清夜,培养3个小时,之后给予终浓度100ng/mL的IFN-γ溶液,1、10、100μg/mL的PPS培养24h;移除细胞上清,每孔加入600μL Trizol裂液,收集细胞。采用RT-PCR法检测IL-1β、IL-6、TNF-α和INOS基因的表达量。实验结果见图.7B,在模拟膀胱癌肿瘤微环境中,PPS可显著增加巨噬细胞IL-1β、IL-6、TNF-α和INOS基因的表达量,提示了PPS在能在肿瘤微环境中活化巨噬细胞。引物序列如表1所示:
Table 1Primer sequences
表1
测试猪苓均一多糖PPS对T24与RAW264.7细胞共培养体系COX2和INOS蛋白表达的影响:
巨噬细胞株RAW264.7常规培养于含100ml/L的胎牛血清的DMEM培养液中。选择处于对数生长期的细胞接种于6孔细胞培养板中。24h完全贴壁后,移除0.8mL的培养基,加入0.8mL的T24上清液,共培养3h。给予终浓度为100ng/mL的IFN-γ溶液,1、10、100μg/mL的PPS作用24h。Western-blot数据分析得到,与对照组相比,1、10、100μg/mL的PPS均具有显著增加INOS和COX2蛋白激酶磷酸化表达的作用(图8),提示PPS在膀胱癌肿瘤微环境中活化巨噬细胞能够增加INOS和COX2蛋白的表达,发挥免疫调节作用。
研究PPS活化巨噬细胞CD14/TRL4/P38MAPK和TRL2/NF-kB相关通路机制:
小鼠巨噬细胞株RAW264.7常规培养于含100ml/L的胎牛血清的DMEM培养液中。选择处于对数生长期的细胞,消化传代,计数并且调整细胞密度,按1×106个每孔接种于6孔细胞培养板中。培养24h完全贴壁。移除0.8mL的培养基,加入0.8mL的T24上清液,培养3h。分别给予终浓度为100ng/mL的IFN-γ溶液,1、10、100μg/mL的PPS作用24h。移除上清,加入预冷的PBS缓冲液洗两次,后加入适量的RIPA裂解液(加入磷酸酶抑制剂和蛋白酶抑制剂),于冰上刮取细胞,15000×g离心15min,取上清即为细胞总蛋白溶液。加入Loading buffer和超纯水,100℃,10min。Western-blot数据分析得到:与对照组相比,1、10、100μg/mL的PPS均具有显著活化CD14、CD284(TLR4)、CD282(TLR2)受体表达以及增加P-P38、、P-P65蛋白激酶磷酸化表达的作用(图.9),提示PPS在膀胱癌肿瘤微环境中活化巨噬细胞可能是通过激活CD14/TRL4/P38MAPK和TRL2//NF-kB通路发挥免疫调节作用。
综上所述,猪苓均一多糖PPS有着重要的生物调节活性的。CD11b,CD16/32,CD40均是巨噬细胞极化成M1的膜标志蛋白。本实验猪苓均一多糖PPS干预巨噬细胞后,能显著的增加巨噬细胞RAW264.7的CD11b,CD16/32,CD40等膜分子的表达,说明PPS在肿瘤微环境中能够活化巨噬细胞。NO广泛参与免疫反应和炎症反应的机体多系统的生理和病理过程,可调节巨噬细胞、T淋巴细胞、B淋巴细胞、以及NK细胞等的免疫功能。激活的巨噬细胞产生的NO作为一种特异效应因子,可以杀灭或抑制多种病原微生物的生长,主要是杀灭细胞内病原体。本实验研究发现(纯化猪苓均一多糖)PPS能促进巨噬细胞分泌NO,并且在较低剂量3.90625μg/mL时,NO的分泌与IFN-γ(100ng/mL)接近。在膀胱癌肿瘤微环境中能促进巨噬细胞分泌NO,并且在较低剂量3.90625μg/mL时,NO的分泌量高于阳性对照组IFN-γ(100ng/mL),提示了PPS在较低剂量时无论是在共培养环境或非共培养环境中均可以增强机体的非特异性免疫反应,增强机体的免疫功能。
p38MAPK和NF-kB信号通路是两条重要的免疫炎症通路,我们的实验研究发现,在膀胱癌肿瘤微环境中,PPS作用之后,CD14、TRL4和pp38蛋白表达增高,提示了猪苓均一多糖PPS可能通过激活CD14/TRL4/P38的信号通路轴活化巨噬细胞。同时,在肿瘤微环境中,PPS作用后巨噬细胞的TRL2和PP65蛋白表达也增高,提示我们TRL2/NF-kB可能也是PPS促使巨噬细胞活化的通路之一。
干扰素(IFN-γ)是重要的细胞免疫因子,通过调节机体免疫力而起到抗肿的作用。IFN-γ能活化巨噬细胞,分泌NO,增强巨噬细胞的的抗原递呈细胞表达MHCII型分子产生的能力,增强对抗原的呈递能力。另外,IFN-γ能增强巨噬细胞及NK细胞的杀伤性,增加细胞表面抗原和受体的表达,抑制B细胞的功能,从而降低肿瘤细胞表面封闭抗体的水平。本实验研究发现(纯化猪苓均一多糖)PPS在非肿瘤微环境及膀胱癌肿瘤微环境中均能够分泌NO,提示了PPS具有活化巨噬细胞,调节免疫功能的作用。IFN-γ还是一种广谱抗病毒药物,PPS在非肿瘤微环境及膀胱癌肿瘤微环境中作用类似于IFN-γ,分泌NO抑制病毒在体内的复制。同时,IFN-γ是一种抗肿瘤药物,能够分泌TNF-a直接对肿瘤细胞进行杀伤,分泌IL-6、IL-1β调节炎症微环境,增强对肿瘤细胞的免疫监视作用。
安全性也是药物评价的重点。生物大分子抗原在临床上用于治疗肿瘤等疾病,但同时也存在较多为严重的副作用,例如抑制骨髓,降低外周血白细胞及血小板;流感样综合征;肾脏损害等。目前,还未见报道有猪苓在临床运用的不良反应,而PPS是从临床用药相对安全的中药猪苓中提取出来的大分子成分,相对于IFN-γ更为安全。因而提示PPS具有潜在的治疗和预防疾病的药品保健品的开发价值。
以上述依据本发明的理想实施例为启示,通过上述的说明内容,相关工作人员完全可以在不偏离本项发明技术思想的范围内,进行多样的变更以及修改。本项发明的技术性范围并不局限于说明书上的内容,必须要如权利要求范围来确定其技术性范围。
Claims (6)
1.具有免疫调节活动性的均一多糖,其特征是:所述均一多糖为单一色谱峰,分子量为6880±5Da,旋光值为158.4±0.5°,红外光谱在847.6cm-1有特征吸收峰,其氢谱中的氢质子化学位移分别为δ5.40brs,3.95t,J=7.2Hz,3.84m,3.61m,碳谱中的碳信号化学位移分别为δ99.6,δ76.6,δ73.3,δ71.5,δ71.1,δ60.4。
2.如权利要求1所述的具有免疫调节活动性的均一多糖,其特征是:所述均一多糖中的多糖质量百分比含量为92~98%;采用Agilent1200液相色谱,示差检测器RID分析,色谱柱为TSK-GEL G4000 PWxL,流动相为超纯水,流速为0.3~1ml/min,检测器温度为30~40℃,柱温为30~50℃,色谱分析为单一峰,保留时间10~30min。
3.如权利要求1所述的具有免疫调节活动性的均一多糖,其特征是:所述均一多糖中的单糖组成为α-D-吡喃葡萄糖,构型均为α型,连接方式为1→4连接。
4.如权利要求1所述的具有免疫调节活动性的均一多糖,其特征是:所述均一多糖内还添加有荧光标记产物、羧甲基化产物、羟甲基化产物、羟丙基化产物、乙二醇化产物、丙二醇化产物或聚乙二醇化产物中的一种。
5.具有免疫调节活动性的均一多糖的制备方法,其特征是,包括如下步骤:
步骤1:水加热提取,醇沉制备总多糖;
将猪苓药材粉碎,取300g加3L去离子水,室温浸泡1小时后100℃加热回流提取2次,每次1h,过滤,合并滤液,浓缩至600ml,3000rpm离心10min,上清液调乙醇比例至大于80%,4℃静置过夜,滤过上清液,沉淀冷冻干燥得粗多糖,取粗多糖加纯水配成25~35mg·mL-1溶液;
步骤2:Sevag法除粗多糖蛋白;
准备糖液:氯仿:正丁醇按25:4:1的比例加入试剂,倾入分液漏斗中充分震摇3min后静置分层,弃去有机层及沉淀,重复除蛋白多次至无白色沉淀产生,收集上清液,用旋转蒸发仪浓缩至适量体积后转移,冷冻干燥,得除蛋白后粗多糖;
步骤3:DEAE-52纤维素脱色素;
将经去蛋白后猪苓粗多糖分别加少量水溶解后,上经预处理后的DEAE-52纤维素柱35×3cm脱色,以1mL/min超纯水、0.1~0.5%氯化钠溶液洗脱,洗脱至流出液用苯酚-浓硫酸法无糖检出,收集峰值段洗脱液用旋转蒸发仪浓缩后冷冻干燥,脱盐,即得白色疏松粉末精多糖;
步骤4:Sephadex G-100凝胶柱精制;
将精多糖适量溶于蒸馏水中,上Sephadex G-100凝胶柱,以纯水、0.1~0.5%氯化钠溶液洗脱,流速1ml/min,洗脱液每10mL收集一管,以苯酚-浓硫酸法跟踪监测;以检测管号为横坐标,吸光度为纵坐标,绘制多糖洗脱曲线,收集峰值段洗脱液,冷冻干燥,脱盐即可获得均一多糖。
6.如权利要求5所述的具有免疫调节活动性的均一多糖的制备方法,其特征是:所述步骤3中DEAE-52纤维素柱的预处理方法为:
步骤1:将干粉的纤维素浸泡在蒸馏水中2~5h,去除杂质后抽干;
步骤2:采用0.5mol/L的HCL溶液浸泡1~3h,用去离子水洗净置PH为中性并抽干;
步骤3:再将抽干的纤维素浸泡在0.5mol/L的NaOH溶液中1~3h,用去离子水洗净置PH为中性并抽干即可。
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