CN1635111A - 一种获得血液中实体肿瘤相关游离蛋白的方法 - Google Patents
一种获得血液中实体肿瘤相关游离蛋白的方法 Download PDFInfo
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Abstract
本发明公开了一种获得血液中实体肿瘤相关游离蛋白的方法。本发明所提供的获得血液中实体肿瘤相关游离蛋白的方法，包括以下步骤：1)培养实体肿瘤细胞及相应正常组织获取原代细胞或将实体肿瘤及相应正常组织进行器官培养；2)培养步骤1)中获得的原代细胞或器官，制备条件培养基，进行SDS－PAGE电泳与纳升液相色谱电喷雾串联质谱仪分析，得到差异蛋白质；3)在所述实体肿瘤患者和正常人血浆中检测步骤2)中所述差异蛋白质，确定所述实体肿瘤相关游离蛋白。本发明的方法可快速、准确地获得一组实体肿瘤相关游离蛋白，利用获得的实体肿瘤相关游离蛋白可以制备其抗体，进一步制成ELISA试剂盒或蛋白芯片用于实体肿瘤病人的诊断，进而指导治疗，将在肿瘤的临床诊断和治疗中发挥重要作用。
Description
技术领域
本发明涉及一种获得血液中实体肿瘤相关游离蛋白的方法。
背景技术
肿瘤已经成为严重威胁人类生存健康的重大疾病之一，其发病率及死亡率均呈上升趋势。来医院就诊的患者大多已经处于中期或晚期，错过了治疗的最佳时机，多数患者预后不佳。因此，寻找实体肿瘤相关蛋白，研究蛋白之间的相互作用，进而做到肿瘤的早期诊断，对提高病人的诊断率和生存率，显得尤为重要。
目前日益发展并完善的大规模蛋白质组研究技术，将成为发掘肿瘤相关游离蛋白的有力手段。并且，血浆生物标志物的研究和发展促进了蛋白质组学、医学以及生物信息学等多学科的交叉协作。如果通过检测人血浆中某一组蛋白表达水平的高低，这样一种创伤较小的方法，便可得知其患肿瘤的可能性或其肿瘤的发展程度，那么将为临床医生能尽早做出正确的诊断和治疗方案大大提供帮助。然而，人类血浆中蛋白成分不仅极为复杂，并且存在高丰度的白蛋白，因此用现有手段从血浆中直接寻找肿瘤的相关游离蛋白无异于大海里捞针。
发明创造内容
本发明的目的是提供一种获得血液中实体肿瘤相关游离蛋白的方法。
本发明所提供的获得血液中实体肿瘤相关游离蛋白的方法，包括以下步骤：
1)培养实体肿瘤细胞及相应正常组织获取原代细胞或将实体肿瘤及相应正常组织进行器官培养；
2)培养步骤1)中获得的原代细胞或器官，制备条件培养基(conditionedmedium)，进行SDS-PAGE电泳与纳升液相色谱电喷雾串联质谱仪分析，得到差异蛋白质；
3)在所述实体肿瘤患者和正常人血浆中检测步骤2)中所述差异蛋白质，确定所述实体肿瘤相关游离蛋白。
所述实体肿瘤可为肺癌，食管癌，宫颈癌，膀胱癌，皮肤癌等实体肿瘤。
步骤1)中所用的培养基可为加入无血清的常规培养基，如LHC-9培养基(购自sigma公司)，培养温度可为36.5℃；所述实体肿瘤及相应正常组织的器官培养条件优选为高氧条件，如在50％O2，45％N2和5％CO2的混合气体环境中培养；所述实体肿瘤细胞是肿瘤组织经脱氧核糖核酸酶I、胶原酶Type I、透明质酸酶、EGTA和PVP消化处理得到的。步骤2)中获取的条件培养基为无垂体的LHC-9培养基；步骤3)中所述检测方法为酶联免疫吸附测定法(enzyme-linked immunosorbantassay，ELISA)和免疫组织化学方法。
上述方法中，当所实体述肿瘤为肺癌时，所述相应正常组织为正常支气管上皮组织，所述实体肿瘤相关游离蛋白为基质金属蛋白酶I(MMP1)，Fascin(Singed-likeprotein，55kDa actin bundling protein)，14-3-3 eta，Ezrin(cytovillin)，4F2cell surface antigen heavy chain(Lymphocyte actiavation antigen 4F2 largesubunit，CD98 antigen)和Annexin A4。
本发明方法得到的MMP1的浓度与肿瘤的TNM分期密切相关(p＜0.05)；Fascin(actin-bundling protein)，14-3-3 eta，在肺癌病人血浆中浓度比年龄匹配的正常人对照组的浓度低(p＜0.05)；Ezrin，CD98在肺癌病人血浆中的浓度无显著变化；Annexin A4在SCLC病人血浆中浓度比NSCLC低(p＜0.05)。
本发明的方法可快速、准确地获得一组实体肿瘤相关游离蛋白，利用获得的实体肿瘤相关游离蛋白可以制备其抗体，进一步制成ELISA试剂盒或蛋白芯片用于实体肿瘤病人的诊断，进而指导治疗，将在肿瘤的临床诊断和治疗中发挥重要作用。
具体实施方式
实施例1、肺癌和正常支气管原代细胞的培养及肺癌和正常支气管的器官培养
手术室离体30分钟之内的肺癌标本，选取约0.5-1.0cm长的一段正常支气管和约2cm3的肿瘤组织，迅速放入盛有洗液(含1％青链霉素的L15培养基，sigma公司)的无菌瓶中。
1、培养正常支气管上皮获取原代细胞
将支气管切成2mm3左右的小块，放入已涂好胶原的平皿(直径60mm，costar公司)中，6块/平皿，4℃冰箱放置2-3小时。取出后，加入LHC-9培养基。36.5℃孵箱中培养1-2周。
2、肺癌细胞培养方法
(1)消化酶溶液的制备：按照表1的数据配制消化酶，将已经配制好的消化酶脱氧核糖核酸酶I、胶原酶Type I、透明质酸酶、PVP各0.5ml以及EGTA 0.25ml加入7.8ml MCDB 151工作液(购自sigma公司)，混匀，滤膜(0.22μm，MILLIPORE公司)过滤，备用。
表1 消化酶配制方法(所有试剂均购自sigma公司)
| 消化酶 | 浓度(用MCDB 151工作液配制) |
| 脱氧核糖核酸酶I | 5mg/ml |
| 胶原酶Type I | 40mg/ml |
| 透明质酸酶 | 20mg/ml |
| EGTA | 2mg/ml |
| PVP | 5％ |
(2)消化肺癌组织：从上述无菌瓶中取出肺癌组织，去除结缔组织等杂物，用洗液洗干净。另取一个小平皿，将组织移入，剪碎。将过滤好的消化酶加入组织中，混匀，放于4℃冰箱中消化4-8小时。开始每隔两小时观察一次，到有细胞消化下来后，随时观察。消化过程中可以用移液管吹吸混合液，帮助消化。到有成串的大量细胞下来，并观察到组织大部分呈空网状，终止消化。将消化后的混合液用铜网(200目)过滤，然后将铜网取出。在过滤完毕的滤液中加入几滴小牛血清，终止消化。滤液移入玻璃离心管中，1000rpm离心5min。
(3)种细胞：离心完毕后，弃上清。视细胞多少种入2-3个小平皿中，每个平皿加4ml含2％胎牛血清的LHC-9培养基。36.5℃孵箱中培养1-2周，得到肺癌的原代细胞。
3、器官培养方法
(1)将支气管或肺癌组织切成4mm3左右的小块，放入已涂好胶原的平皿中，6块/平皿，4℃冰箱放置2-3小时。
(2)取出平皿后，加入LHC-9培养基，放入培养盒中，充入含有50％ O2，45％N2和5％CO2的混合气体，封好盒子(确保不漏气)。
(3)将培养盒放在摇摆床上，摇摆频率3-4次/分钟，36.5℃孵箱中培养1-2周。
实施例2、肺癌相关游离蛋白的获得
(1)制备条件培养基(conditioned medium)
将实施例1中按步骤1和2的培养方法得到的原代细胞传代于直径100mm平皿，加入LHC-9培养基，36.5℃孵箱中培养至约106个细胞/平皿。然后用1×HBS(Hepes，NaCl，KCl，Glucose，NaH2PO4·7H2O)润洗细胞三次，加入3ml LHC-9培养基(无垂体)，放入36.5℃孵箱中培养1小时；然后HBS润洗两次，加入3ml LHC-9培养基(无垂体)，放入36.5℃孵箱中培养48小时；收集条件培养基；8000rpm离心5min，4℃；取上清，4℃保存。将实施例1中按步骤3的方法得到的培养器官，按上述方法收集培养基。
(2)SDS-PAGE电泳与质谱联用分析细胞释放的到条件培养基中的蛋白
将上一步收集到的条件培养基用双蒸水透析24小时，收集透析后培养基，冰冻干燥。干燥后样本用聚丙烯酰胺凝胶电泳(SDS-PAGE)加样缓冲液溶解，加热煮沸3分钟完全变性，用10％ SDS-PAGE分离，考马斯亮蓝染色。将SDS-PAGE胶从上至下切成对应的33个条带，进一步切成胶粒后脱色，用胰蛋白酶进行胶内多肽的酶切提取，提取后的多肽用纳升液相色谱电喷雾串联质谱仪分析，鉴定差异蛋白质。由质谱鉴定共得到近200种蛋白，如MMP1，Protein disulfide isomerase A3precursor，Annexin家族，14-3-3家族等。
(3)确定肺癌相关游离蛋白
使用ELISA试剂盒(Oncogene公司)测定血浆中MMP1的浓度。将血浆和标准蛋白加入ELISA板中，室温2小时；然后用洗液洗板5次，加入HRP标记抗体，室温1小时；用洗液洗板5次，加入显色液，室温30分钟；终止显色，于酶标仪(Bio-Rad公司)测450/570nm处吸光度；绘制标准曲线，计算浓度。用SPSS统计学软件分析实验结果。结果发现MMP1的浓度与肿瘤的TNM分期密切相关(p＜0.05)。
用下述方法检测血浆中无标准ELISA试剂盒蛋白(Fascin，14-3-3 eta，Ezrin，CD98和Annexin A4)：抗体(Santa Cruz公司)以2μg/ml浓度包被ELISA板(Costar公司)4℃过夜；加入1％BSA封闭液，37℃，4小时；1×PBST(0.5％Tween 20)洗板3次，加入待测血浆，室温1小时；1×PBST洗板7次，加入与包被抗体不同种属的抗体(Santa Cruz公司)，300-600ng/ml，室温30分钟；1×PBST洗板7次，加入抗上一步相同种属的HRP标记二抗(Jackson或DAKO公司)，室温30分钟；1×PBST洗板7次，加入0.1mg/mlTMB显色，室温30分钟；2M H2SO4终止反应；于酶标仪(Bio-Rad公司)测450/570nm处吸光度。用SPSS统计学软件分析实验结果。Fascin，14-3-3 eta，在肺癌病人血浆中浓度比年龄匹配的正常人对照组的浓度低(p＜0.05)；Ezrin，CD98在肺癌病人血浆中的浓度无显著变化；Annexin A4在SCLC病人血浆中浓度比NSCLC低(p＜0.05)。
实施例3、设计肺癌组织微阵列，进行免疫组织化学分析
一张免疫组化片子上设计100个病例。将石蜡切片脱蜡至水，3％H2O2处理10分钟，1×PBS洗3次；血清封闭10分钟；加一抗(Santa Cruz公司)，4℃过夜；1×PBS洗3次，加生物素标记的二抗(中山公司免疫组化试剂盒)，37℃，10-30分钟；1×PBS洗3次，加辣根过氧化物酶标记的链霉卵白素，37℃，10分钟；DAB显色，苏木素复染；切片经二甲苯脱水，中性树脂胶封片。显微镜下观察。MMP1在转移性鳞癌(175例)表达的阳性率显著高于非转移鳞癌(141例)(p＜0.05)。
Claims (9)
1、一种获得血液中实体肿瘤相关游离蛋白的方法，包括以下步骤：
1)培养实体肿瘤细胞及相应正常组织获取原代细胞或将实体肿瘤及相应正常组织进行器官培养；
2)培养步骤1)中获得的原代细胞或器官，制备条件培养基(conditionedmedium)，进行SDS-PAGE电泳与纳升液相色谱电喷雾串联质谱仪分析，得到差异蛋白质；
3)在所述实体肿瘤患者和正常人血浆中检测步骤2)中所述差异蛋白质，确定所述实体肿瘤相关游离蛋白。
2、根据权利要求1所述的方法，其特征在于：步骤1)中所用的培养基为加入无血清的常规培养基。
3、根据权利要求2所述的方法，其特征在于：所述培养基为LHC-9培养基。
4、根据权利要求1所述的方法，其特征在于：步骤1)中所实体述肿瘤及相应正常组织的器官在50％O2，45％N2和5％CO2的环境中培养。
5、根据权利要求1所述的方法，其特征在于：步骤1)中所述实体肿瘤细胞是实体肿瘤组织经脱氧核糖核酸酶I、胶原酶TypeI、透明质酸酶、EGTA和PVP消化处理得到的。
6、根据权利要求1所述的方法，其特征在于：步骤2)中获取的条件培养基为无垂体的LHC-9培养基
7、根据权利要求1所述的方法，其特征在于：步骤3)中所述检测方法为ELISA和免疫组化方法。
8、根据权利要求1-7任一权利要求所述的方法，其特征在于：所述实体肿瘤为肺癌；所述相应正常组织为支气管上皮组织。
9、根据权利要求8所述的方法，其特征在于：所述实体肿瘤相关游离蛋白为MMP1，Fascin，14-3-3 eta， Ezrin，CD98和Annexin A4。
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| CN111705051A (zh) * | 2020-07-28 | 2020-09-25 | 金紫晶(南京)生物医药技术有限公司 | 一种复合消化酶及复合消化酶冻干粉 |
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| CN102735840A (zh) * | 2011-04-13 | 2012-10-17 | 中国医学科学院肿瘤研究所 | 用于诊断或筛查卵巢癌的试剂 |
| CN102735840B (zh) * | 2011-04-13 | 2014-06-04 | 中国医学科学院肿瘤研究所 | 用于诊断或筛查卵巢癌的试剂 |
| CN111705051A (zh) * | 2020-07-28 | 2020-09-25 | 金紫晶(南京)生物医药技术有限公司 | 一种复合消化酶及复合消化酶冻干粉 |
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