CN111533820A - 一种三七多糖及其制备方法与应用 - Google Patents
一种三七多糖及其制备方法与应用 Download PDFInfo
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- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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Abstract
本发明公开一种三七多糖及其制备方法与应用,以提取过三七总皂苷的药渣为原料,经过水提醇沉、Sevage法除蛋白、活性炭除色素、阴离子交换纤维素柱层析纯化等步骤,得到三七多糖,其主链结构为:→4)‑α‑D‑GalAp‑(1→3,4‑β‑L‑Rhap‑1→4)‑β‑D‑Galp‑(1→,支链结构为α‑L‑Araf‑1→5)‑α‑L‑Araf‑(1→;分子量为76655Da;经药理实验研究表明,本发明三七多糖能够促进小鼠骨髓源性树突状细胞成熟,增强T细胞功能,调控肿瘤相关巨噬细胞,为三七的进一步开发利用提供理论依据和实践指导。
Description
技术领域
本发明属于中药学、免疫学领域,具体涉及三七粗多糖、多糖组分和多糖结构,其制备方法及免疫调节的用途。
研究背景
植物多糖广泛存在于生物体中,是生物体重要的营养物质,具有一定的生理和生物学活性,我国对多糖的研究始于20世纪70年代,植物多糖由于它们独特的功能和低毒性,作为新药发展的方向具有广阔的应用前景。
三七Panaxnotoginsen(Burk.)F.H.Chen又名田七、参三七,为五加科人参属植物,主要用于活血、化疲、补血和止血。迄今为止,已经从三七中分离出了200多种活性化合物,包括皂苷、多糖、黄酮类、挥发油和氨基酸等。多年来三七的研究主要集中在皂苷的药理作用上,研究表明,三七总皂苷具有良好的活血化瘀作用;抗炎作用;心肌保护作用;保肝作用等。随着科学的发展,三七活性成分多糖越来越引起人们的关注,多糖的活性研究也逐渐深入。三七多糖是提取于中药三七中的一种水溶性多糖。药理学研究表明,三七多糖具有较强的免疫调节活性且属无毒级物质,是重要的活性成分。然而对于多糖结构及免疫调节机制尚未明确。
发明内容
本发明提供一种三七多糖,以提取过三七总皂苷的药渣为原料,经过水提醇沉、Sevage法除蛋白、活性炭除色素、阴离子交换纤维素柱层析纯化等步骤,得到三七多糖,其主链结构为:→4)-α-D-GalAp-(1→3,4-β-L-Rhap-1→4)-β-D-Galp-(1→,支链结构为α-L-Araf-1→5)-α-L-Araf-(1→;分子量为76655Da;单糖组成及摩尔比例为Rha:Fuc:Ara:Xly:Man:Glc:Gal=0.145:0.023:0.396:0.005:0.018:0.024:0.389,糖苷键类型及其摩尔比为Araf-(1→:→5)-Araf-(1→:→3,4)-Rhap-(1→:→3)-Galp-(1→:→4)-Galp-(1→:→6)-Galp-(1→=23:16.47:15.5:8.12:33.19:3.72。
本发明还提供所述三七多糖的制备方法,包括以下步骤:
S1、水提:将提取过三七总皂苷的药渣粉碎,过20目筛,加入蒸馏水水浴,在温度为75℃条件下水浴提取3h后纱布粗滤,循环浸提3次,合并提取液,静置一夜;
S2、脱蛋白:55℃旋转蒸发仪浓缩提取液至原体积的1/6,参照sevage法进行脱蛋白处理,收集上清液;
S3、醇沉:在上清液中加入上清液3倍体积的无水乙醇进行沉淀,静置一夜,3000rpm离心8min,取沉淀加蒸馏水复溶,沉淀与蒸馏水质量体积比为1:1g/mL,55℃下水浴除乙醇,至无乙醇味;
S4、除杂:加入活性炭脱色,40℃水浴3h,4000rpm离心10分钟,取上清,12000rpm再次离心15min,收集上清液,3500Da透析袋透析一夜,冷冻干燥,得到三七粗多糖;
S5、纯化:三七粗多糖采用阴离子交换纤维素柱层析纯化,采用蒸馏水及不同浓度的NaCl溶液进行梯度洗脱,NaCl溶液的浓度依次为0.1mol/L、0.3mol/L、0.5mol/L,选取浓度为0.3mol/L的NaCl洗脱液进一步纯化,得到三七多糖。
步骤S1药渣与蒸馏水料液比为1:20g/mL。
步骤S4活性炭脱色时活性炭的加入量为0.06g/mL。
步骤S5进一步纯化的具体工艺为:通过G-50(2.5cm×100cm)凝胶色谱柱进一步纯化,并用0.1mol/L的NaCl溶液进行洗脱,流速为0.10mL/min,采用苯酚硫酸法跟踪检测,在45℃-50℃下旋转蒸发浓缩,3500Da透析袋透析,冷冻干燥,得到纯化的三七多糖。
本发明还提供所述三七多糖的应用,三七多糖具有免疫活性,可以促进小鼠骨髓源性树突状细胞成熟,增强T细胞功能,调控肿瘤相关巨噬细胞,能够作为免疫佐剂,增强免疫活性。
本发明通过水提醇沉的方法从三七药渣中提取到三七多糖,进而分离纯化得到具有免疫活性的多糖组分,并对其进行结构及活性测定;本发明三七多糖组分能够促进小鼠骨髓源性树突状细胞成熟,增强T细胞功能,调控肿瘤相关巨噬细胞,可作为免疫佐剂,增强自身免疫系统,杀伤或抑制肿瘤细胞,可以为三七多糖开发免疫调节剂、抗肿瘤药物等方面提供坚实的实验基础。
附图说明
图1是三七多糖的DEAE-纤维素柱层析图谱及G-50凝胶色谱图;
图2是PNPS-0.3的分子量分布图谱;
图3是PNPS-0.3的红外光谱图;
图4是PNPS-0.3的单糖组成气相色谱分析图;
图5是PNPS-0.31H-NMR谱图;
图6是PNPS-0.313C-NMR谱图;
图7是PNPS-0.3Dept谱图;
图8是PNPS-0.3HSQC谱图;
图9是PNPS-0.31H-1H COSY谱图;
图10是PNPS-0.3NOESY谱图;
图11是PNPS-0.3HMBC谱图;
图12是PNPS-0.3的结构图;
图13是PNPS-0.3作用下BMDC成熟标记物的表达;
图14是PNPS-0.3作用下BMDC的形态;
图15是PNPS-0.3作用下BMDC分泌的细胞因子的表达;
图16是PNPS-0.3对BMDC介导的T细胞活力的测定;
图17是PNPS-0.3对BMDC介导的T细胞凋亡的测定;
图18是PNPS-0.3对BMDC介导的T细胞免疫应答因子的表达;
图19是PNPS-0.3对BMDC介导的T细胞分泌的细胞因子的表达。
具体实施方式
以下结合附图与实施例,对本发明进行更加详细的说明,但本发明的保护范围绝非仅局限于实施例。实施例中未注明具体条件者,按照常规条件进行,所用试剂或仪器未注明生产商者,均为可以通过市购获得的常规产品。
实施例1
三七多糖的提取分离与纯化:
(1)将提取过三七皂苷的药渣粉碎过20目筛,加入蒸馏水,药渣与蒸馏水料液比为1:20g/mL,在75℃下水浴提取3小时后纱布粗滤,循环浸提3次,合并提取液,静置一夜,55℃下旋转蒸发浓缩至原体积的1/6,参照sevage法进行脱蛋白处理,收集上清液,加入3倍体积无水乙醇析出沉淀,静置一夜,3000rpm离心8min,取沉淀加蒸馏水复溶,沉淀与蒸馏水质量体积比为1:1g/mL,55℃下水浴除乙醇,至无乙醇味;加入0.06g/mL活性炭除色素,40℃水浴3h,4000rpm离心10分钟,取上清液,12000rpm再次离心15min,收集上清液,3500Da透析袋透析一夜,冷冻干燥,得到三七粗多糖;
(2)称取步骤(1)所得的三七粗多糖300mg,将其溶于20mL去离子水中,通过阴离子交换纤维素柱DEAE-52(4cm×60cm)进行柱层析,分别用蒸馏水、浓度为0.1mol/L的NaCl溶液、浓度为0.3mol/L的NaCl溶液、浓度为0.5mol/L的NaCl溶液进行洗脱,流速为1mL/min,每个试管10mL,分离出四个组分,分别为PNPS-W、PNPS-0.1、PNPS-0.3、PNPS-0.5,如图1(a)所示,通过对四个组分进行免疫活性测定,得出PNPS-0.3为免疫活性最强的洗脱组分,并对该组分进一步纯化;
收集洗脱浓度为0.3mol/L的NaCl溶液洗脱液,通过G-50(2.5cm×100cm)凝胶色谱柱进一步纯化,并用浓度为0.1mol/L的NaCl溶液进行洗脱,流速为0.10mL/min,采用苯酚硫酸法跟踪检测,在45℃-50℃下旋转蒸发浓缩,3500Da透析袋透析,冷冻干燥,得到纯化的三七多糖PNPS-0.3,G-50凝胶色谱图如图1(b)所示,从图中可知,苯酚硫酸法跟踪检测PNPS-0.3主要集中在14-18管样品。
实施例2
三七多糖PNPS-0.3的结构解析:
(1)分子量测定
使用高效液相色谱仪(岛津LC-10A)联同示差检测器对PNPS-0.3分子量进行检测,以不同相对分子质量的葡聚糖(Mw1152、5220、11600、23800、48600、80900、148000、273000、409800、667800)作为标准品,色谱柱为BRT105-104-102串联凝胶柱(8×300mm),样品配制成5mg/mL溶液,12000rpm离心10min,上清液用0.22μm的微孔滤膜过滤,然后将样品转置于1.8mL进样小瓶中,进样量20μL,按照上述参数,检测结果如图2所示,重复3次,得到多糖平均分子量为76655Da。
(2)近红外光谱分析
精密称取PNPS-0.3,按质量比为1:80的比例加入KBr,通过使用傅里叶变换红外分光光度计(AlphaII,Bruker)进行测定,扫描波长为500cm-1to 4000cm-1,检测结果如图3所示,在3405cm-1有强吸收峰,表示存在O-H伸缩振动;2938cm-1附近出现吸收峰表明了C-H的弯曲振动;1608cm-1有强吸收峰,表明存在-COO基团;1097cm-1和952cm-1处属于吡喃皂苷吸收峰;836cm-1和896cm-1表明存在α和β构型糖苷键。
(3)单糖组成及其摩尔比例
精密称取2mgPNPS-0.3,加入浓度为2mol/L的三氟乙酸1mL,水解90min,旋转蒸发仪蒸干,残基加入2mL双蒸水,100mg硼氢化钠还原,加入冰醋酸中和,旋蒸,110℃烘箱烘干,然后加入1mL乙酸酐乙酰化,100℃反应1h,反应结束后,减压抽干,加入3mL甲苯,减压浓缩蒸干,重复4-5次,以除去多余的醋酐,将乙酰化后的产物用3mL氯仿溶解后转移至分液漏斗,加入少量蒸馏水充分震荡后,除去上层水溶液,如此重复5次,氯仿层以适量的无水硫酸钠干燥,定容至10mL以供分析;通过气相色谱-质谱联用仪分析其组分,并与单糖标准品对照,见图4单糖标准品谱图(上)及PNPS-0.3的谱图(下);通过对比得出,乙酰化后的单糖组成为Rha:Fuc:Ara:Xly:Man:Glc:Gal=0.145:0.023:0.396:0.005:0.018:0.024:0.389,由于Fuc、Xly、Man、Glc含量极低,故不作为主要单糖成分。
将10mg三七多糖样品PNPS-0.3置于反应瓶中,加入4mL DMSO并快速加入50mgNaOH粉末,封闭,在超声作用下溶解,向反应物中加入0.5mL碘甲烷继续反应,最后加入0.5mL蒸馏水到上述混合物中终止甲基化反应,取甲基化后的多糖进行水解,1mL的浓度为2mol/L的三氟乙酸水解90min,旋转蒸发仪蒸干,残基加入2mL双蒸水,60mg硼氢化钠还原8小时,加入冰醋酸中和,旋蒸,101℃烘箱烘干,加入1mL乙酸酐乙酰化,100℃反应1h,反应结束后,减压抽干,加入3mL甲苯,减压浓缩蒸干,重复4-5次,以除去多余的醋酐,采用Shimadzu GCMS-QP 2010气相色谱-质谱联用仪测定产物样品,结果如下表1所示,根据上述结果可知,PNPS-0.3主要含有一下六种糖苷键连接,分别为Araf-(1→、→5)-Araf-(1→、→3,4)-Rhap-(1→、→3)-Galp-(1→、→4)-Galp-(1→、→6)-Galp-(1→,其中→4)-Galp-(1→所占摩尔比为33.19%,可推断其在主链结构上。
表1
保留时间 | 甲基糖苷 | 摩尔比(%) | 结构链接 | (m/z) |
11.827 | 2,3,4,5-Me<sub>4</sub>-Araf | 23 | Araf-(1→ | 43,71,87,101,117,129,145,161 |
17.585 | 2,3,4-Me<sub>3</sub>-Araf | 16.47 | →5)-Araf-(1→ | 43,87,99,117,129,161,207 |
17.881 | 2-Me<sub>1</sub>-Rhap | 15.5 | →3,4)-Rhap-(1→ | 43,71,87,99,113,117,129,159,173 |
24.871 | 2,4,6-Me<sub>3</sub>-Galp | 8.12 | →3)-Galp-(1→ | 43,43,71,87,99,101,117,129,159,161 |
25.194 | 2,3,6-Me<sub>3</sub>-Galp | 33.19 | →4)-Galp-(1→ | 43,87,99,101,113,117,129,131,161,173,233 |
26.268 | 2,3,4-Me<sub>3</sub>-Galp | 3.72 | →6)-Galp-(1→ | 43,71,87,99,101,117,129,161,173,189,233 |
(4)核磁共振分析
精密称取充分干燥后的PNPS-0.3样品50mg溶解于0.6mL的氘代水试剂中,转移至核磁管中,通过核磁共振仪测定其一维核磁谱1H-NMR、13C-NMR、Dept;二维核磁谱HSQC(Heteronuclear Single Quantum Correlation)、1H-1H COSY(1H-1H CorrelatedSpectroscopy)、NOESY(NuclearOverhauserEffect Spectroscopy)、HMBC(HeteronuclearMultipleBondCorrelation);并结合一维和二维谱图,归属出所有碳氢信号,如表2所示。
1H NMR谱图如图5所示,在1H NMR中,主要端基质子峰分布在4.3~5.5ppm区域内,包含5个信号峰δ4.56、δ4.57、δ5.01、δ5.03、δ5.09ppm,分别对应A~E五个残基;13C NMR(图6)和Dept谱(图7)中,在δ93~105间主要包含5个异头碳信号峰,分别对应出现在δ105.81、δ105.7、δ100.54、δ107.54、δ108.63,此外,δ176.77为糖醛酸C6的化学位移,根据碳氢归属表,归属出GalAp的化学位移;二维HSQC(图8)和1H-1H COSY(图9)图谱反应了直接相连的H、C核之间的耦合关系;NOESY(图10)和HMBC(图11)反应了C、H之间的相关峰。
在HMBC图谱(图11)中,残基C的H1与残基A的C3有相关峰,表明存在C-1→3-A;残基A的C1与残基B的H4有相关峰,表明存在A-1→4-B;此外,残基B的H1与残基C的H4有相关峰,表明存在B-1→4-C,证明残基B与下一个残基C的4号位相连;NOESY图谱(图10)中,残基D的H1与残基A的C3有相关峰,表明存在D-1→3-A;残基E的H1与D的H5a有相关峰,证明存在E-1→5-D,综上,推断出PNPS-0.3的主链连接方式为4-C-1→3-A-1→4-B-1,而支链E-1→5-D-1通过A的O-3键连接在主链上,结构单元如图12所示。
表2
实施例3
PNPS-0.3的免疫活性研究
1.实验动物
昆明小鼠(6~8周),雄性,18-25g,购于昆明医科大学,动物置于干燥、通风、安静的环境中,按自由饮水、进食普通饲料。
2.药物及试剂
三七多糖(实施例1制备得到的PNPS-0.3);1640基础培养基,1%青链霉素双抗、胎牛血清、谷氨酰胺、0.25%胰酶、ACK细胞裂解液、RBC细胞裂解液、抗体CD11c、CD40、CD80、CD86、MHC-II、CD4、CD8、CD69均购自赛默飞生物科技有限公司;抗体CCK-8试剂盒购自东仁化学科技有限公司;脂多糖LPS购自Sigma-Aldrich公司。
3.实验耗材与设备
T75培养基;15mL离心管;transwell、1.5mL离心管、各量程吸头、0.22μm滤器;细胞培养箱(力康生物HF-100);低速离心机(SCIOGEX DMO-412);倒置显微镜(奥林巴斯CKX41);超净工作台(珠江再鑫EVL-53);移液器(eppendorf);4℃/-20℃冰箱;水浴锅(CFYQYB YLE-1000)。
4.实验方法
4.1细胞的收集
(1)小鼠骨髓来源树突状细胞(BMDC)分离
从BALB/c小鼠的胫骨和股骨中收集小鼠骨髓细胞,通过ACK裂解缓冲液,离心、过滤去除红细胞,加入5mL 1640培养基(1640培养基配制方法:超净工作台内取88mL 1640基础培养基于无菌、干净的瓶内,加入10mL FBS、1mL双抗、1mL谷氨酰胺、20ng/mL GM-CSF、10ng/mL IL-4,0.22μm过滤器过滤后转移至新的瓶内,盖紧瓶盖缠紧封口膜,4℃冰箱保存备用),制备单核细胞悬液;将细胞接种到T25瓶中,每瓶中有106个细胞,在显微镜下观察BMDC细胞的微观形态,在第9天,收集细胞备用。
(2)T细胞分离
在无菌条件下,从BALB/c小鼠中分离脾脏,加入PBS缓冲液,使悬浮液通过70μm细胞过滤器以除去组织残余物;1000rpm离心5分钟,在4℃下将2mL PBS裂解物添加到沉淀物中以重悬细胞(5分钟)并洗涤两次,注射器分离T细胞;用100μLPBS重悬细胞,加入5μLCD3抗体,室温避光孵育30min后用PBS将液体补至1mL后流式细胞仪下检测T细胞的纯度。
4.2多糖对BMDC细胞活化的影响
4.2.1实验分组
取实施例1所得的三七多糖(PNPS-0.3),用蒸馏水溶解制成10μg/mL、25μg/mL、50μg/mL三个不同浓度的多糖溶液,对照组药物为脂多糖LPS,具体分组如下:
空白组:BMDC细胞正常培养;
对照组:BMDC细胞+1μg/mL LPS;
实验组:1.BMDC细胞+10μg/mL多糖(PNPS-0.3L);
2.BMDC细胞+25μg/mL多糖(PNPS-0.3M);
3.BMDC细胞+50μg/mL多糖(PNPS-0.3H);
4.2.2BMDC细胞活力测定
接种BMDC细胞于96孔板,每孔接种2500个细胞,每组设置3个复孔,另接3孔BMDC细胞正常培养作为CCK-8对照组,另取3孔(未接种细胞)加入100uL 1640培养基和10μLCCK-8试剂作为空白组,于诱导第9天按照分组给予处理24h,作用完后每孔加入10μLCCK-8试剂,培养箱内孵育2h后酶标仪下检测细胞活力;
细胞活力%=(实验孔OD-空白孔OD)/(对照孔OD-空白孔OD)*100%;
4.2.3BMDC表型与细胞因子分泌测定
接种BMDC细胞于6孔板,每孔接种2×105个细胞,每组设置3个复孔,于诱导第9天按照分组给予处理24h,作用后用0.25%胰酶消化收集细胞,PBS洗三次,400μL PBS重悬,均匀分为4管,分别加入5μL CD40、CD80、CD86、MHC-II抗体,混匀,室温孵育30min后流式细胞仪下检测细胞荧光情况。
4.3多糖处理条件下BMDC对T细胞增殖的影响
4.3.1实验分组
取实施例1所得的三七多糖(PNPS-0.3),用蒸馏水溶解制成5μg/mL、10μg/mL、20μg/mL三个不同浓度的多糖溶液,具体分组如下:
空白组:T细胞正常培养;
对照组:T细胞+(BMDC细胞);
实验组:1.T细胞+(BMDC细胞+5μg/mLPNPS-0.3);(PNPS-0.3-L)
2.T细胞+(BMDC细胞+10μg/mLPNPS-0.3);(PNPS-0.3-M)
3.T细胞+(BMDC细胞+20μg/mLPNPS-0.3);(PNPS-0.3-H)
4.3.2BMDC/T共培养
接种BMDC细胞于transwell上室,每孔接种2×105个细胞,每组设置6个复孔,于诱导第9天按照分组给予不同浓度的多糖处理24h,作用完后于T细胞进行共培养,T细胞接种于transwell下室,每孔接种2×105个细胞,共培养48h;
4.3.3T细胞活力和凋亡测定
CCK-8方法对共培养后的T细胞进行细胞活力测定;此外,将细胞与5μLAnnexinV-FITC染色液和5μLPI染色液室温避光孵育15分钟,通过流式细胞仪检测细胞凋亡情况。
4.3.4T细胞亚群和细胞因子测定
收集共培养后的T细胞,PBS洗三次,400μL PBS重悬,均匀分为4管,分别加入5μLCD4、CD8、CD69、MHC-Ⅱ抗体,混匀,室温孵育30min后流式细胞仪下检测细胞荧光情况。
结果分析:
1.三七多糖PNPS-0.3对BMDC的免疫调节作用
1.1不同浓度三七多糖PNPS-0.3对BMDC细胞活力的影响
通过CCK-8法对不同浓度多糖处理的BMDC进行活力测定,三组平行实验取平均值,结果显示,在浓度为50μg/mL时BMDC活力略微下降,如表3所示,PNPS-0.3L和PNPS-0.3M组细胞活力分别为101.779%、102.383%,而PNPS-0.3H组细胞活力为82.038%,因此,选择50μg/mL作为评估剂量-活性关系的最大浓度。
表3
实验分组 | 空白孔OD值 | 对照孔OD值 | 实验孔OD值 | 细胞活力(%) |
BMDC细胞正常培养 | 0.176 | 2.745 | 2.743 | 99.961 |
BMDC细胞+1μg/mL LPS | 0.176 | 2.745 | 2.776 | 101.215 |
BMDC细胞+10μg/L多糖 | 0.176 | 2.745 | 2.790 | 101.779 |
BMDC细胞+25μg/mL多糖 | 0.176 | 2.745 | 2.806 | 102.383 |
BMDC细胞+50μg/mL多糖 | 0.17 | 2.745 | 2.283 | 82.038 |
1.2三七多糖PNPS-0.3对BMDC表型成熟的影响
通过流式细胞仪检测与T细胞反应相关的CD40、CD80、CD86、MHC II等膜标志物的表达,进一步研究PNPS-0.3对小鼠未成熟BMDC成熟的影响,如图13所示,PNPS以剂量依赖的方式增加这四种标志物的表达,PNPS-0.3H组中,CD40、CD80、CD86和MHC II的表达比例分别为62.73%、87.19%、71.53%和74.23%,分别为空白组的1.98、2.26、2.37和2.42倍(p<0.01),此外,PNPS-0.3H组的这些标记物的表达也高于LPS处理的对照组(p<0.01)。
1.3三七多糖PNPS-0.3对BMDC形态学的影响
BMDC的形态如图14所示,未经任何处理的细胞大多呈椭圆形,树突较小;PNPS-0.3处理过的细胞形态显著改变,细胞大,呈卵圆形或不规则形状,有较多长或短树突,PNPS-0.3H组表现出最鲜明的形态特征;与其他组相比,PNPS-0.3H组有更多的树突伸出,突起与褶皱都较明显,显示出更成熟的形状,突出的突起和更多联褶皱。
1.4三七多糖PNPS-0.3对BMDC细胞因子分泌的影响
将未成熟的BMDC与PNPS-0.3作用24小时后,通过ELISA试剂盒检测IL-12和TNF-α的细胞因子分泌情况,如图15所示,PNPS-0.3以剂量依赖性方式增加两种细胞因子的分泌,PNPS-0.3H组的BMDC产生最高水平的IL-12和TNF-α,分别是空白组的5.0倍和2.5倍(p<0.01)。此外与对照组相比,PNPS-0.3H组的作用最强。
2.PNPS-0.3对BMDC介导的T细胞免疫应答的影响
2.1三七多糖PNPS-0.3对BMDC/T共培养中T细胞活力和凋亡的影响
BMDC与T细胞共培养48小时后,结果显示,PNPS-0.3-M与PNPS-0.3-H组细胞活力有略微的增加(p<0.01)(表4,图16);细胞凋亡无明显变化(表5,图17);
表4
表5
实验分组 | 早期凋亡(%) | 晚期凋亡(%) | 总凋亡(%) |
T细胞正常培养 | 2.27 | 0.43 | 2.70 |
T细胞+(BMDC细胞) | 2.40 | 0.56 | 2.96 |
T细胞+(BMDC细胞+5μg/mL多糖) | 2.18 | 0.53 | 2.73 |
T细胞+(BMDC细胞+10μg/mL多糖) | 1.88 | 0.46 | 2.34 |
T细胞+(BMDC细胞+20μg/mL多糖) | 1.89 | 0.88 | 2.77 |
2.2三七多糖PNPS-0.3对BMDC/T共培养中T细胞免疫应答的影响
通过检测T细胞亚群,进一步验证PNS-0.3能够激活T细胞免疫应答,如图18所示,与未处理的BMDC/T的对照组相比,PNPS-0.3处理的BMDC/T在剂量依赖方式下可以显著增加CD4+或CD8+T细胞的增殖,在PNPS-0.3-H组中,CD4+(88.23%)和CD8+(98.14%)T细胞分别是对照组(BMDC/T共培养组)的8.27倍和7.13倍;此外,PNPS-0.3-H活化BMDC刺激CD69细胞(95.97%)和MHC II细胞(97.34%)时,其细胞增殖明显强于未处理的BMDC/T(CD69为16.08%,MHC II为11.11%),此外,如图19所示,经PNPS-0.3处理过的T细胞分泌了更多INF-γ细胞因子,并且INF-γ在PNPS-0.3-L、PNPS-0.3-M、PNPS-0.3-H水平下的表达量分别为对照组的12.93、23.10和36.52倍。
综上,充分证明PNPS-0.3能够促进小鼠骨髓来源树突状细胞成熟,增强T细胞功能,调控肿瘤相关巨噬细胞;能够作为免疫佐剂,增强自身免疫系统,杀伤或抑制肿瘤细胞。
Claims (7)
1.一种三七多糖,其特征在于,其主链结构为→4)-α-D-GalAp-(1→3,4-β-L-Rhap-1→4)-β-D-Galp-(1→,支链结构为α-L-Araf-1→5)-α-L-Araf-(1→;分子量为76655Da;单糖组成及摩尔比为Rha:Fuc:Ara:Xly:Man:Glc:Gal=0.145:0.023:0.396:0.005:0.018:0.024:0.389。
2.根据权利要求1所述三七多糖,其特征在于,其糖苷键类型及摩尔比为Araf-(1→:→5)-Araf-(1→:→3,4)-Rhap-(1→:→3)-Galp-(1→:→4)-Galp-(1→:→6)-Galp-(1→=23:16.47:15.5:8.12:33.19:3.72。
3.权利要求1所述三七多糖的制备方法,其特征在于,包括以下步骤:
S1、将提取过三七总皂苷的药渣粉碎,过20目筛,加入蒸馏水,在75℃水浴提取3h后纱布粗滤,循环浸提3次,合并提取液,静置一夜;
S2、55℃旋转蒸发浓缩提取液至原体积的1/6,参照sevage法进行脱蛋白处理,收集上清液;
S3、在上清液中加入上清液3倍体积的无水乙醇进行沉淀,静置一夜,3000rpm离心8min,取沉淀加蒸馏水复溶,55℃下水浴除乙醇至无乙醇味;
S4、加入活性炭脱色,40℃水浴3h,4000rpm离心10分钟,取上清,12000rpm再次离心15min,收集上清液,3500Da透析袋透析一夜,冷冻干燥得到三七粗多糖;
S5、三七粗多糖采用阴离子交换纤维素柱层析纯化,通过蒸馏水及不同浓度的NaCl溶液进行梯度洗脱,NaCl溶液的浓度依次为0.1mol/L、0.3mol/L、0.5mol/L,选取浓度为0.3mol/L的NaCl溶液洗脱液进一步纯化,得到三七多糖。
4.根据权利要求3所述三七多糖的制备方法,其特征在于,步骤S1药渣与蒸馏水料液比为为1:20g/mL。
5.根据权利要求3所述三七多糖的制备方法,其特征在于,步骤S4活性炭的加入量为0.06g/mL。
6.根据权利要求3所述三七多糖的制备方法,其特征在于,步骤S5进一步纯化的具体工艺为:通过G-50凝胶色谱柱进一步纯化,并用0.1mol/L的NaCl溶液进行洗脱,流速为0.10mL/min,采用苯酚硫酸法跟踪检测,在45℃-50℃下旋转蒸发浓缩,3500Da透析袋透析,冷冻干燥,得到纯化的三七多糖。
7.一种如权利要求1所述三七多糖在作为免疫佐剂上的应用。
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