【作者】 熊和丽；

【导师】 杨鸣琦；

【作者基本信息】 西北农林科技大学 ， 细胞生物学， 2007， 硕士

【摘要】 目的:鹿茸是我国传统名贵中药,具有广泛的药理学活性。鹿茸作为一种特殊的软骨,是一个天然的细胞因子库,其中含有大量的蛋白质及蛋白多糖。近年来大量研究证明,蛋白多糖具有抗肿瘤作用,而以往对鹿茸的研究主要是醇提物,其中蛋白质和多糖类水溶性物质研究较少,对其水溶性物质进行系统的抗肿瘤研究更是鲜见,因此本研究通过缓冲液提取出水溶性蛋白质及蛋白多糖,系统的研究鹿茸水溶性提取物的抗肿瘤作用及其机理,同时研究鹿茸中蛋白多糖与化疗药物联合用药时的抗肿瘤效果并将其与鹿茸粗提物的抗肿瘤效果进行比较。方法:利用Tris缓冲液提取出鹿茸中水溶性成分,并分别用Bradford法和天青I法检测其中蛋白质及蛋白多糖含量;采用DEAE Sepharose FF阴离子交换树脂分离出水溶性提取物中的蛋白多糖,Bradford法和天青I法示踪检测洗脱液中蛋白质和蛋白多糖,根据洗脱曲线浓缩收集蛋白多糖组分,SDS聚丙酰胺凝胶电泳鉴定分离效果,冻干提取分离物;建立移植性S180肉瘤模型,随机将植瘤鼠分为7组,分别为阴性对照组(阴性对照组),阳性对照组(环磷酰胺,25mg/kg),鹿茸蛋白多糖低剂量组(40mg/kg)、中剂量组(80mg/kg)、高剂量组(160mg/kg),鹿茸粗提物组(400mg/kg),联合用药组(蛋白多糖80mg/kg+环磷酰胺25mg/kg),连续灌胃给药10d,阴性对照组灌胃生理盐水,阳性对照组隔天腹腔注射环磷酰胺,第11d脱颈椎处死小鼠,剥离肿瘤组织和脾,制备腹腔巨噬细胞悬液和脾淋巴细胞悬液,巨噬细胞吞噬中性红实验,T、B淋巴细胞转化实验,体内抑瘤活性及生命延长率,称重瘤组织和脾,计算抑瘤率和脾指数。结果:经检测鹿茸顶部蛋白质含量占鹿茸干重13.13%,蛋白多糖占鹿茸干重1.67%。离子交换层析在NaCl浓度为0.7mol/L,0.8mol/L,0.9mol/L时出现洗脱峰,经浓缩收集得到三个蛋白多糖组分Ⅰ、Ⅱ、Ⅲ,其蛋白多糖含量分别占总蛋白多糖含量的45.25%、25.15%、18.26% ,蛋白多糖提取率为88.66%。移植性肿瘤S180免疫调节试验结果表明,灌服蛋白多糖高剂量、鹿茸粗提物及联合用药荷瘤鼠各组间日增重无显著差异,脾指数,T、B淋巴细胞转化能力,巨噬细胞吞噬中性红功能均高于阴性对照组(P<0.05);蛋白多糖高剂量组荷瘤鼠T、B淋巴细胞转化能力,巨噬细胞吞噬中性红功能高于阳性对照组(P<0.05),脾指数无显著差异;鹿茸粗提物组荷瘤鼠的巨噬细胞吞噬功能和T淋巴细胞转化能力高于阳性对照组(P<0.05),B淋巴细胞转化功能和脾指数与阳性对照组间无显著差异;联合用药组荷瘤鼠脾指数,T、B淋巴细胞转化功能均高于蛋白多糖中剂量组和阳性对照组,但仅T淋巴细胞转化功能有显著差异(P<0.05)。移植性肿瘤S180抑制试验结果表明,鹿茸粗提物组抑瘤率(78.15%)与阳性对照组(39.87%)间存在极显著差异(P<0.01),联合用药组体内抑瘤率(50.16%)高于阳性对照组和蛋白多糖中剂量组(-23.07%),但与阳性对照组间不存在显著差异;蛋白多糖高剂量组抑瘤率(23.47%)低于30%;生命延长率方面蛋白多糖高剂量组(119.79%)、鹿茸粗提物组(119.79%)均高于阳性对照组(41.67%),但无显著差异;联合用药组生命延长率(212.50%)高于蛋白多糖中剂量组(103.13%)和阳性对照组,且与阳性对照组间有极显著差异(P<0.01)。结论:鹿茸顶部蛋白质含量占鹿茸干重的13.13%;蛋白多糖含量占鹿茸干重1.67%;利用DEAE Sepharose F.F阴离子交换层析能快速分离出鹿茸中蛋白多糖,此法相对快速,简便;通过移植肿瘤S180抑制试验发现,鹿茸粗提物对荷瘤小鼠的抑瘤效果最好,抑瘤率达78.15%;联合用药组荷瘤鼠的生命延长率最高,达212.50%;移植肿瘤S180免疫调节试验发现,蛋白多糖各剂量组对荷瘤鼠的免疫调节作用呈现一定剂量依赖关系,且蛋白多糖剂量达160mg/kg时,其免疫调节作用强于鹿茸粗提物和联合用药;蛋白多糖的免疫调节功能强于鹿茸粗提物;蛋白多糖与化疗药物联用可增强机体对化疗药物的耐受性。更多还原

【Abstract】 Objective: Velvet antler is a kind of rarely tradional medicine,it has extensive effect on curing disease.As a special cartilage,velvet anlter is a natural cytokine‘storeroom’,and has lots of protein and proteoglycan.It is indicated that peoteoglycan has well effect of anti-tumor lately. Previous research on velvet antler is about ethanol extraction mostly, but less about protein and polysaccharide which dissolve in water,and least of them on anti-tumor research systematicly. Therefore we will extract protein and proteoglycan which is dissolvable in water using Tris buffer,and study their anti-tumor effect and mechanism systematicly .In addition,we not only study the cooperative effect of using proteoglycan and chemical medicine ,but also anti-tumor effect of crude extraction comparing to proteoglycan.Method: we extract the component of water dissolvable with Tris buffer,and determine the content of protein and proteoglycan in the extraction with Bradford method and Azure I method respectively ,isolate proteoglycan in the extraction by ion exchange chromatography,and determine the content of protein and proteoglycan in the same way,and estimate the effection of isolation to build a fast ,simple and convenient way for isolation.Extraction and isolation are freeze-dried .Build transplantional sarcoma 180 model in mice and then divide them into seven groups randomly.It’s negative control group(cacer model),positive control group(cydophosphamide,CTX,25mg/kg),low dosage of proteoglycan group (40mg/kg),middle dosage of proteoglycan group(80mg/kg),high dosage of proteoglycan group (160mg/kg),crude extraction group (400mg/kg),cooperation group (40mg/kg proteoglycan and 25mg/kg CTX).After inoculating tumor cells 24 hours ,give different treatment to every group for 10 days,negative control group are given physiological saline by gavage and positive control group are administrated by injection in traperitonealy,other group are treated by gavage .After 10 days,peel off tumor tissue and weigh,make celiac macrophage mixture and spleen lymphocyte mixture,measure macrophage function of absorbing Neutral red T、B lymphocyte transformation function ,spleen index ,tumor inhibiting index and life time prolongation index.Result: The content of protein and proteoglycan in the top of velvet antler is 13.13% and 1.67%. There were three bars of proteoglycan when eluting by DEAE Sepharose and the corresponding concentration of NaCl is 0.7mol/L,0.8mol/L,0.9mol/L, conbined and concentrate three proteoglycan fractions according as eluting bar. Each fraction was 45.25%、25.15%、18.26% of total proteoglycans respectively.The rate of proteoglycan reclaimant is 88.66%.The immunoregulating experiment of transplantion S180 sarcamo indicates that,there is no sinifigant diffence between high dosage of proteoglycan,crude extraction,cooperation group and tumor-loded mice with daily weight growth,but spleen index,T、B lymphocyte transformation function and macrophage absorbance index is higher than negative control group(P < 0.05). T、B lymphocyte transformation function and macrophage absorbance index of high dosage of proteoglycan is higher than positive control group(P<0.05) ,but no signifigant difference with spleen index. T lymphocyte transformation function and macrophage absorbance index of crude extraction is signifigant diffrent with positive control group(P<0.05),but no signifigant diffrent wiht B lymphocyte transformation function and spleen index .Spleen index,T、B lymphocyte transformation function is higher than middle dosage and CTX group ,but there is no signifigant different except T lymphocyte transformation function(P<0.05). Tumor inhibiting experiment indicates that the tumor inhibiting index of crude extraction group is 78.15%,and has signifigant diffrence with positive control group(39.87%)(P<0.01),and of cooperation group is 50.16%,higher than positive control group and middle dosage of proteoglycan,but has no signifigant difference with positive control group(39.87%).The tumor inhibiting index of high dosage of proteoglycan is 23.47%,lower than 30%. life time prolongation index of high dosage of proteoglycan group and crude extracion group are both 119.79% ,higher than positive control group but no signifigant different,and of cooperation group is higher than middle dosage(103.13%)and positive control group(P<0.01).Conclusion: It’s easy to extract water dissolvable component with Tris buffer, The content of protein and proteoglycan in the top of velvet antler is 13.13% and 1.67%. relatively. Proteoglycan can be isolated by DEAE Sepharose F.F anion exchang chromatography,also this method is fast and convenient.The effect of tumor inhibiting (78.15%)of crude extraction is best of each group, The life time prolongation (212.50%) of cooperation is highest of all.There is dosage dependment of every dosage of proteoglycan of the effect of immunregulating,and when it’s dosage is 160mg/kg,the effect is better than crude extracion group and cooperation group.immune regulation function of proteoglycan is better than crude extraction,and the cooperation of proteoglycan and chemical medicine can enhance endurance of tumor-loded mice to chemical medicine.更多还原