广西莪术多糖生物活性与莪术醇免疫分析方法的研究
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摘要
本学位论文以广西道地药材广西莪术(Curcuma kwangsinensis S.G.Lee et C.F.Liang)为研究对象,对其中莪术多糖(CKP)调节血脂紊乱的生物活性及其机制、抗氧化作用进行了研究；同时对其另一大类成分挥发油中具有抗肿瘤活性的化合物莪术醇(Curcumol),进行单克隆抗体制备,并研究了莪术醇单克隆抗体在免疫分析方面的应用,主要结果如下。
将CKP高、中、低三个剂量组喂饲高脂血症大鼠,以常用降血脂药物洛伐他汀做阳性对照,三周后,测量给药大鼠血清中的甘油三酯(TG)、总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)、载脂蛋白AI (apoAI)、载脂蛋白B (apoB)、谷胱甘肽过氧化物酶(GSHPx)、超歧过氧化物酶(SOD)、丙二醛(MDA)的含量,结果显示CKP对高脂血症大鼠的血脂紊乱有明显的改善作用,亦有明显的抗氧化作用,作用效果与给药剂量呈正相关。
以脂滴包被蛋白(perilipin)和激素敏感酯酶(HSL)两个生脂基因为靶基因,以荧光PCR定量,分析CKP改善高脂大鼠血脂紊乱现象与生脂基因转录表达的规律与相关性。结果显示给予CKP三个剂量组的高脂大鼠脂肪组织HSL基因表达量都升高,而对于perilipin基因,其表达量均降低；CKP三个剂量组血清中的TG与HSL基因表达呈显著的负相关(P<0.05)；与perilipin的基因表达呈显著的正相关(P<0.05)。
以4-二甲基氨基吡啶(DMAP)做催化剂,莪术醇与丁二酸酐为反应物得到的莪术醇丁二酸单酯半抗原,再行碳化二亚胺(EDC)法与载体蛋白偶联合成的莪术醇完全抗原,具有很好的免疫原性,可作为免疫原应用于莪术醇单克隆抗体的制备。该合成方法反应条件温和、步骤简单、质量稳定且合成产物收率高,产率为87%。
应用杂交瘤技术制备出了能够高度分泌抗莪术醇的单克隆抗体的细胞株,经腹水制备、ProteinG琼脂糖凝胶亲和色谱柱纯化,可得到含量在90%以上的抗莪术醇的单克隆抗体。对抗体进行类型判断、效价、灵敏度、亲和常数、交叉反应率等的特征鉴定与评价,显示抗体的效价高(106以上),亲和性好(亲和常数2.45×10-8),特异性强。
初步建立了莪术醇ELISA分析方法,并进行了方法学考察,显示该方法的线性(0.16-11μng.ml-1)、重现性(9.02%)、精密度(<9%)、加样回收率(10.01%)、板间(<10%)板内(<10%)差异程度符合免疫分析要求,最低检测限为78ng.ml-1。以该法测定广西莪术、温莪术、含莪术醇的生物样本,其检测结果与气相色谱分析结果无显著差异。
研究结果对广西莪术药用价值的开发具有重要的意义；为完善广西莪术质量评价体系提供了新的思路和方法。
In this paper, Guangxi famous-region drug Curcuma kwangsinensis S.G.Lee et C.F.Liang was taken as research object, investigating the biological activity, mechanism and anti-oxidative of Curcuma Kwangsiensis Polysaccharides (CKP) on regulation of dyslipidemia; the present paper prepares the monoclonal antibodies of Curcumol acting as one essential composition and antitumor compound in Zedoary Tumetric Oil, and deepens the analysis on immune application of those antibodies. The results as follows.
Used three dose groups, CKP Ⅰ (300mg.kg-1), CKP Ⅱ (600mg.kg-1), CKPⅢ), to feed hyperlipemia rats, and lovastatin well-known was taken as one common cholesterol-lowering drug to do the positive control. After3weeks, these rats blood was collected for the content mesurement of the following items:serum triglycerides (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), apolipoprotein AI (apoAI), apolipoprotein B (apoB), glutathione peroxidase (GSHPx), super disambiguation peroxidase (SOD) and malondialdehyde (MDA). The results show that CKP has obvious improvement effect and anti-oxidative on dyslipidemia rats, as well as the effect positively correlates with dose.
By the fluorescent quantitative PCR, perilipin protein and hormone sensitity lipase (HSL) as two lipogenic genes were chosen for the target genes to analyze the correlation between the improvement effects of CKP on hyperlipemia rats and transcription expression of lipogenic genes. The results indicate that the gene expression of HSL contained in adipose tissue of hyperlipemia rats in the three CKP dose groups increases, while the expression of perilipin decreases; the gene expression of TG in serum of the three CKP dose groups shows a significant negative correlation (P＜0.05) with HSL, but a significant positive correlation with perilipin (P＜0.05).
The present study curcumol and succinic anhydride was used as two reactants and4-dimethyl amino pyridine (DMAP) as one catalyst to make curcumol-succinic monoester hapten, then the hapen is conjugated to the protain by the method of EDC and finally gets curcumol antigen which shows good immunogenicity and can be applied in curcumol monoclonal antibody preparation as one immunogen. This synthesis has the following advantages:mild reaction conditions, simple procedures, stable quality and yields of synthesis high to87%.
The adoption of hybridoma technique makes us get the cell strain of monoclonal antibody possessing high-secretion ability, and gets the curcumol monoclonal antibody whose content is more than90%after ascites preparation and ProteinG agarose gel affinity chromatography column purification. The identification and evaluation on antibody from aspects of type, titer, sensitivity, affinity constant, cross reaction rate prove its high titer (above106), good affinity (affinity constant2.45X10-8) and strong specificity.
This study preliminarily established the curcumol ELISA analysis method and conducts methodology investigation, showing that the linear relationship (0.16-llμg.ml-1), reproducibility (9.02%), precision (＜9%), plus sample recovery (10.01%), the divergency of between board(＜10%) and within plate (＜10%) meet the requirements of the immune analysis, and the minimum detection limits is78ng.ml-1. The test results of Curcuma kwangsiensis, Curcuma wenyujin and organisms sample containing curcumol have no significant difference in ELISA method and gas chromatographic method.
This results has great significance to development the medicinal value of Curcuma kwangsiensis; and provide a reference for quality evaluation system of it.
将CKP高、中、低三个剂量组喂饲高脂血症大鼠,以常用降血脂药物洛伐他汀做阳性对照,三周后,测量给药大鼠血清中的甘油三酯(TG)、总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)、载脂蛋白AI (apoAI)、载脂蛋白B (apoB)、谷胱甘肽过氧化物酶(GSHPx)、超歧过氧化物酶(SOD)、丙二醛(MDA)的含量,结果显示CKP对高脂血症大鼠的血脂紊乱有明显的改善作用,亦有明显的抗氧化作用,作用效果与给药剂量呈正相关。
以脂滴包被蛋白(perilipin)和激素敏感酯酶(HSL)两个生脂基因为靶基因,以荧光PCR定量,分析CKP改善高脂大鼠血脂紊乱现象与生脂基因转录表达的规律与相关性。结果显示给予CKP三个剂量组的高脂大鼠脂肪组织HSL基因表达量都升高,而对于perilipin基因,其表达量均降低；CKP三个剂量组血清中的TG与HSL基因表达呈显著的负相关(P<0.05)；与perilipin的基因表达呈显著的正相关(P<0.05)。
以4-二甲基氨基吡啶(DMAP)做催化剂,莪术醇与丁二酸酐为反应物得到的莪术醇丁二酸单酯半抗原,再行碳化二亚胺(EDC)法与载体蛋白偶联合成的莪术醇完全抗原,具有很好的免疫原性,可作为免疫原应用于莪术醇单克隆抗体的制备。该合成方法反应条件温和、步骤简单、质量稳定且合成产物收率高,产率为87%。
应用杂交瘤技术制备出了能够高度分泌抗莪术醇的单克隆抗体的细胞株,经腹水制备、ProteinG琼脂糖凝胶亲和色谱柱纯化,可得到含量在90%以上的抗莪术醇的单克隆抗体。对抗体进行类型判断、效价、灵敏度、亲和常数、交叉反应率等的特征鉴定与评价,显示抗体的效价高(106以上),亲和性好(亲和常数2.45×10-8),特异性强。
初步建立了莪术醇ELISA分析方法,并进行了方法学考察,显示该方法的线性(0.16-11μng.ml-1)、重现性(9.02%)、精密度(<9%)、加样回收率(10.01%)、板间(<10%)板内(<10%)差异程度符合免疫分析要求,最低检测限为78ng.ml-1。以该法测定广西莪术、温莪术、含莪术醇的生物样本,其检测结果与气相色谱分析结果无显著差异。
研究结果对广西莪术药用价值的开发具有重要的意义；为完善广西莪术质量评价体系提供了新的思路和方法。
In this paper, Guangxi famous-region drug Curcuma kwangsinensis S.G.Lee et C.F.Liang was taken as research object, investigating the biological activity, mechanism and anti-oxidative of Curcuma Kwangsiensis Polysaccharides (CKP) on regulation of dyslipidemia; the present paper prepares the monoclonal antibodies of Curcumol acting as one essential composition and antitumor compound in Zedoary Tumetric Oil, and deepens the analysis on immune application of those antibodies. The results as follows.
Used three dose groups, CKP Ⅰ (300mg.kg-1), CKP Ⅱ (600mg.kg-1), CKPⅢ), to feed hyperlipemia rats, and lovastatin well-known was taken as one common cholesterol-lowering drug to do the positive control. After3weeks, these rats blood was collected for the content mesurement of the following items:serum triglycerides (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), apolipoprotein AI (apoAI), apolipoprotein B (apoB), glutathione peroxidase (GSHPx), super disambiguation peroxidase (SOD) and malondialdehyde (MDA). The results show that CKP has obvious improvement effect and anti-oxidative on dyslipidemia rats, as well as the effect positively correlates with dose.
By the fluorescent quantitative PCR, perilipin protein and hormone sensitity lipase (HSL) as two lipogenic genes were chosen for the target genes to analyze the correlation between the improvement effects of CKP on hyperlipemia rats and transcription expression of lipogenic genes. The results indicate that the gene expression of HSL contained in adipose tissue of hyperlipemia rats in the three CKP dose groups increases, while the expression of perilipin decreases; the gene expression of TG in serum of the three CKP dose groups shows a significant negative correlation (P＜0.05) with HSL, but a significant positive correlation with perilipin (P＜0.05).
The present study curcumol and succinic anhydride was used as two reactants and4-dimethyl amino pyridine (DMAP) as one catalyst to make curcumol-succinic monoester hapten, then the hapen is conjugated to the protain by the method of EDC and finally gets curcumol antigen which shows good immunogenicity and can be applied in curcumol monoclonal antibody preparation as one immunogen. This synthesis has the following advantages:mild reaction conditions, simple procedures, stable quality and yields of synthesis high to87%.
The adoption of hybridoma technique makes us get the cell strain of monoclonal antibody possessing high-secretion ability, and gets the curcumol monoclonal antibody whose content is more than90%after ascites preparation and ProteinG agarose gel affinity chromatography column purification. The identification and evaluation on antibody from aspects of type, titer, sensitivity, affinity constant, cross reaction rate prove its high titer (above106), good affinity (affinity constant2.45X10-8) and strong specificity.
This study preliminarily established the curcumol ELISA analysis method and conducts methodology investigation, showing that the linear relationship (0.16-llμg.ml-1), reproducibility (9.02%), precision (＜9%), plus sample recovery (10.01%), the divergency of between board(＜10%) and within plate (＜10%) meet the requirements of the immune analysis, and the minimum detection limits is78ng.ml-1. The test results of Curcuma kwangsiensis, Curcuma wenyujin and organisms sample containing curcumol have no significant difference in ELISA method and gas chromatographic method.
This results has great significance to development the medicinal value of Curcuma kwangsiensis; and provide a reference for quality evaluation system of it.
引文
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