CN106722973A - 针对癌细胞迁徙能力相关基因的配方食品及其制备方法 - Google Patents
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Abstract
本发明提供了一种针对癌细胞迁徙能力相关基因的配方食品,所述个体化干预配方食品通过如下步骤制成:1)检测个体的p53结合蛋白1基因、p53蛋白基因、血管内皮生长因子基因的变异;2)评估所检测的基因的变异的风险;3)选择食品基础方中的原料,根据所评估的风险选择基因方中的原料,制成所述的针对癌细胞迁徙能力相关基因的配方食品。本发明所提供的个体化干预配方食品能提高肿瘤患者的突变细胞迁徙抑制能力。
Description
技术领域
本发明涉及一种个体化干预配方食品,尤其涉及一种针对癌细胞迁徙能力相关基因的配方食品。
背景技术
根据《2012年中国肿瘤登记年报》最新数据显示,我国每年新发恶性肿瘤病例约为312万,每分钟就有6人被诊断为恶性肿瘤;每年因恶性肿瘤死亡的病例约为270万,每分钟就有5人死于恶性肿瘤。肿瘤患者因其代谢情况的特殊、肿瘤细胞需要更多的营养,手术、放化疗带来的不舒适感加重病人的厌食等原因,极易出现营养不良。营养不良会导致患者体重下降、免疫力下降、耐受性降低,会极大缩短生存时间。如果忽视了营养问题,错过了营养给予的时机,将造成1/4的肿瘤患者直接死于营养不良。全世界20%的新发恶性肿瘤患者在中国,24%的恶性肿瘤患者死亡在中国。在中国,肿瘤患者营养不良的发生率高达65%,恶性肿瘤患者营养不良的发病率约为31%-87%,其中约35%的结肠癌患者、40%的肺癌患者、45%头颈部癌患者、49%的上消化道癌症患者、80%的胰腺癌患者出现营养不良,每年约有22%的肿瘤患者直接死于营养不良,营养不良已成为我国恶性肿瘤患者常见的并发症之一。
大量的证据和临床实践表明,营养支持对于患者的治疗效果和康复速度具有十分重要和不可替代的作用。如何才能避免肿瘤患者的营养不良问题?目前,特殊医学用途配方食品是临床上实施营养支持的主要方法,根据肿瘤的共同代谢特点和对某些特定营养成分需求,人为地改变其比例,造成体内某种特定物质过剩或缺乏,使其不利于肿瘤细胞的扩增,或提高肿瘤对抗肿瘤治疗的敏感性,或减少其他治疗的毒副反应。正确的营养治疗不但可改善恶性肿瘤病人的营养状态还能起到治疗肿瘤的作用。
肿瘤本质上是基因病,各种环境的和遗传的致癌因素等以协同或序贯的方式引起DNA损害,从而激活原癌基因和(或)灭活肿瘤抑制基因,加上凋亡调节基因和(或)DNA修复基因的改变,而引起表达水平的异常,使正常细胞发生转化,最终导致肿瘤形成。
突变细胞从原发肿瘤沿着一定渠道,通过淋巴道、血液、腔道到达另一部分或多个部位,形成新的转移灶。而肿瘤转移的途径主要有四个方面:局部扩散、淋巴管渗透、血行转移、腔道种植。其中以淋巴管渗透和血行转移最多。90%的患者都是因为肿瘤发生转移、复发,导致肿瘤治疗前功尽弃的。
与突变细胞迁徙抑制能力相关的基因包括p53结合蛋白1基因(TP53BP1)、p53蛋白基因(TP53)、血管内皮生长因子基因(VEGF),其中TP53BP1基因变异导致蛋白质翻译减少引起细胞的恶变,导致肿瘤易感性增加;TP53基因的变异易导致蛋白质翻译减少引起细胞的恶变,导致肿瘤易感性增加;VEGF基因存在变异,易引起细胞生长失去控制,通过促进肿瘤新生淋巴管形成和扩张,直接和间接的促进肿瘤的淋巴扩散和淋巴转移,从而导致癌症的易感性增加。
一些食物和营养物质可以通过与突变细胞迁徙抑制能力基因的相互作用来控制肿瘤的转移,具体如下:
豌豆属于豆类植物,豌豆富含人体所需的各种营养物质,尤其是含有优质的蛋白质。而且豆类植物不含胆固醇,这一点优于动物蛋白。豌豆蛋白粉是采用先进工艺从豌豆中提取的蛋白质,豌豆蛋白含有人体所有必需的所有氨基酸,属于全价蛋白质,有利于均衡营养,提高免疫力,尤其适合术后病人食用。
壳聚糖(Chitosan)是甲壳素的脱乙酰产物,是迄今为止发现的唯一带正电荷的碱性多糖,有显著的生物活性和保健功能。由于甲壳素和壳聚糖分子质量大、晶体结构紧密、溶解性差,其应用受到限制。壳寡糖是壳聚糖降解的产物(Chitooligosaccharide,COS),一般认为由2~10个氨基葡糖通过β-l,4-糖苷键连接而成,也有学者将其扩大为2~20个单元结构的。壳寡糖的分子量小、水溶性好,更易被吸收利用,比大分子壳聚糖具有更好的生理活性,如抗肿瘤、增强免疫效应、抗氧化、调节肠道微生态、降血糖等。近年来国内外学者对壳寡糖及衍生物的抗肿瘤作用进行了较多的研究,目前认为其抗肿瘤的机制是通过直接抑制肿瘤细胞增生、影响肿瘤血管生成和迁移、诱导细胞坏死和凋亡、增强机体免疫力而实现的。何学斌等以200mg/(kg·d)水溶性壳聚糖灌胃,可显著抑制荷S180和艾氏腹水癌小鼠肿瘤的生长。壳寡糖是肿瘤血管生成的有效抑制剂,并且在对比壳寡糖与可溶性壳聚糖抑制埃利希腹水瘤细胞生长和肿瘤血管生成时发现,50μg壳寡糖比100μg可溶性壳聚糖具有更强的抑制效果。Wang等以人脐静脉上皮细胞做体外实验表明COS可抑制由血管内皮生长因子引发的血管生成,1000μg/mL效果最好。刘清华、Huang、Pae、Hasegawa等研究发现,壳寡糖作用肉瘤细胞后,细胞阻滞于G0/G1期,同时细胞凋亡增加,明显提高Bax的表达,降低Bcl-2的表达,提示壳寡糖可抑制肉瘤的生长并促进其凋亡。Xu等对肝癌细胞SMMC-7221的研究中发现COS可显著介导该细胞的凋亡,并且随COS浓度升高而效率增大。用0.8mg/mL COS处理72h后,引发的凋亡率可达38%。Maeda等的研究表明,壳寡糖在抑制荷瘤小鼠肿瘤生长的同时,可激活肠上皮淋巴细胞和脾脏的NK细胞,并可加强肠上皮淋巴细胞和脾淋巴细胞对S180细胞的毒作用,认为它们主要是通过增强机体免疫功能而起抑制肿瘤的作用。朱婉萍认为壳寡糖通过提高荷瘤小鼠T淋巴细胞的转化功能,NK细胞的杀伤活性,IL-2、IFN-Y的含量和巨噬细胞的吞噬功能来发挥抗肿瘤作用。
绿豆淀粉从绿豆中提取而来,具有很高的营养价值,用途十分广泛。绿豆淀粉具有抗菌抑菌作用、降血脂作用、抗肿瘤作用、解毒作用等多种生物活性,其含有相当数量的低聚糖(戊聚糖、半乳聚糖等)。这些低聚糖是人体肠道内有益菌——双歧杆菌的增殖因子,经常食用绿豆淀粉可改善肠道菌群,减少有害物质吸收,预防某些癌症的发生。
乳清蛋白是采用先进工艺从牛奶分离提取出来的珍贵蛋白质,不但容易消化,而且还具有高生物价、高效化率、高蛋白质功效比和高利用率,是蛋白质中的精品。含有人体所需的所有必需氨基酸,其氨基酸组成模式与骨骼肌中的氨基酸组成模式几乎完全一致,极容易被人体所吸收。主要成分有β-乳球蛋白、α-乳白蛋白、免疫球蛋白、乳铁蛋白等,具有抗氧化,消灭或抑制细菌,促进正常细胞生长,提高机体免疫力的作用。其提高免疫力的作用机制为:维持谷氨酰胺水平,进而保护免疫细胞功能。谷氨酰胺是淋巴细胞和巨噬细胞在免疫反应过程的重要底物,高速利用谷氨酰胺生成嘌呤和嘧啶核苷酸有利合成更多的DNA,使免疫细胞增殖加速。乳清蛋白富含谷氨酸等谷氨酰胺前体物质,通过增加免疫细胞的增殖速度来提高机体免疫力。
小麦草是用来榨汁喝的小麦草的种子是专门培育的,从播放到生产其过程有别于普通小麦,使种子更加安全,有机,无污染环境下生长,其作用确实十分神奇。在国外及香港,台湾,十分风行的生机饮食主角之一,例如台湾的生机之母李秋凉女士长期服用小麦草,30年抗癌成功。在国内也越来越受到大家的重视。本草纲目记载小麦草“麦苗,气味辛、寒、无毒。主治消酒毒、暴热、酒疽、目黄等,捣烂绞汁日饮之,解渴,退胸隔热,利小肠,佐韭食,甚益颜色。”小麦草含有丰富的叶绿素、维生素A、维生素C、维生素E及维生素B族,和矿物质钙、镁、磷、铁、硒,以及超抗氧化剂SOD、纤维和有益酵素。酸性体质是百病之源,据日本食品分析中心所做的研究表明,小麦草每100g含量中有66.4%属于碱性的。
大米蛋白主要由清蛋白、球蛋白、醇溶性蛋白和谷蛋白等四种蛋白组成,氨基酸组成平衡合理,且氨基酸含量高,是公认的优质食品蛋白,符合WHO/FAO推荐的理想模式。大米蛋白是低抗原性蛋白,不会产生过敏反应,对应用于肿瘤人群的食品是十分有利的。大米蛋白不仅具有独特的营养功能,还有抗高血压、降胆固醇、抗癌变的作用。Molita等对大米分离蛋白(RPI)研究结果表明,饲喂大米分离蛋白的二甲基苯并蒽(DMBA)诱导雌性小白鼠肿瘤重量低于饲喂酪蛋白小白鼠,大米分离蛋白具有抗DMBA诱导癌变作用。
葛根素是从豆科植物野葛根中提取的一种单一成份黄酮苷,具有活血化瘀、改善微循环、扩张冠状动脉和脑血管、降低心肌耗氧量,抑制癌细胞增殖及耐药等作用。韩萍等研究发现,葛根粗提物和葛根素纯品浓度相关性的抑制小细胞肺癌H446细胞增殖,其机制可能与阻滞细胞周期于G0/G1期、上调Bax表达、下调Bel-2表达有关。
益生菌是指投入后通过改善宿主肠道菌群生态平衡而发挥有益作用,从而达到提高宿主健康状态的微生物。益生菌的主要来源是动物肠道正常的生理性细菌和非肠道菌。可分成三大类:乳杆菌类、双歧杆菌类和革兰阳性球菌。益生菌作为一种新型的且较安全的生物制品在肿瘤防治中逐渐成为一种趋势。乳酸菌、双歧杆菌等益生菌及其代谢产物,可诱导机体产生干扰素,促进细胞分裂素,活化免疫细胞,促进免疫球蛋白的生成,提高机体免疫功能,抑制肿瘤的发生发展。双歧杆菌具有降解N-亚硝酸,消除氧自由基、过氧化脂质等致癌因子的作用。实验证明双歧杆菌不仅可以抑制肿瘤发生,对已经形成的肿瘤仍具有抑制作用。大多数肿瘤细胞都表现出较高的端粒酶活性。而正常体细胞由于端粒酶活性低,其端粒随着细胞分裂逐渐缩短,细胞逐渐老化、死亡。在近期的研究中已经证实了经过鼠李糖乳杆菌(LGG)表面分子脂磷壁酸(LTA)处理白血病细菌株端粒酶后,端粒酶的活性明显降低,这表明益生菌的抗肿瘤机制可能与降低端粒酶的活性有关。
益生元是指能够选择性地刺激特定肠道菌的生长、活性而有益于宿主健康的非消化性食物成分,这是1995年国际“益生元之父”Dr.Glenn Gibson对益生元进行的定义。Gibson等人在2004年又给出了最新的定义:“益生元是一种可被选择性发酵且专一性地改变肠道中对宿主健康有益菌群的组成和活性的配料(ingredient),常称“双歧因子”。益生元是食品行业中的通用名称,其实它包括多种产品,目前国际上认可较多的益生元主要包括菊粉(Inulin)、低聚乳果糖(LACT)、低聚木糖(XOS)、低聚异麦芽糖(IMO)、低聚半乳糖(GOS)、低聚果糖(FOS)、大豆低聚糖(SOS)、棉子糖、水苏糖等,有些微藻类也可作为益生元如节旋藻、螺旋藻等,天然植物中的中草药、蔬菜、野生植物等提取物也能作为益生元使用。糖类益生元与其他促益生菌生长的物质相比有着不可替代的优点,它无药物残留,细菌不会产生抗药性,无污染,耐高温,性质稳定,加工储运过程中损失少,是一种天然的食品添加剂。
多糖类(polysaccharide)是构成生命的四大基本物质之一,广泛存在于高等植物、动物、微生物、地衣和海藻等中,如植物的种子、茎和叶组织、动物粘液、昆虫及甲壳动物的壳真菌、细菌的胞内胞外等。多糖在抗肿瘤、抗炎、抗病毒、降血糖、抗衰老、抗凝血、免疫促进等方面发挥着生物活性作用。具有免疫活性的多糖其衍生物常常还具有其他活性,如硫酸化多糖具有抗HIV活性及抗凝血活性,羧甲基化多糖具有抗肿瘤活性。多糖类通过抑制VEGF基因的表达,有效调节细胞凋亡基因,抑制肿瘤细胞的分裂与增殖,并促进肿瘤细胞死亡、侵袭和转移。李娜研究发现在不同浓度灵芝多糖(0μg/ml、0.2μg/ml、0.8μg/ml、3.2μg/ml、12.8μg/ml)作用下,各组LA795肺癌细胞连续培养48h,应用ELISA技术检测显示,LA795肺癌细胞培养上清中VEGF表达水平分别为147.98±0.28、120.25±14.24、103.11±10.57、91.27±13.62、84.66±10.41,以空白对照组的VEGF的表达水平最高,随灵芝多糖浓度的不断增高,LA795肺癌细胞VEGF表达水平逐渐降低。证明灵芝多糖可下调LA795肺癌细胞分泌免疫抑制分子VEGF、TGF-β1、IL-10。
红花(Carthamus tinctorius L.)是菊科植物,为我国传统中草药,花青苷类物质是其发挥药理作用的物质基础,通过抑制血管生成相关因子VEGF、CXCR4,可抑制胃癌细胞体外生长及转移侵袭能力。同时红花中的有效成分红花多糖能够通过抑制VEGF的表达,使肿瘤生长及转移受到抑制。黄凌娜等(2016)研究表明,红花提取的花青苷能够抑制胃癌细胞SGC-7901的生长及侵袭能力,且呈浓度依赖,半数抑制浓度(IC50)=136.2mg/L。经花青苷处理48h后,SGC-7901的转移侵袭能力明显减弱,多重线性相关法分析得出侵袭能力与药物浓度间存在较强的线性相关关系,相关系数为0.913。当花青苷浓度为150mg/L时,侵袭能力抑制率达52.3%。孙伟等(2016)研究表明红花多糖显著促进人结肠癌LoVo细胞凋亡,可使结肠癌LoVo细胞增殖受到抑制,侵袭能力减弱,调节细胞周期。
三七(Panax notoginseng(Burk.)F.H.Chen),又名田七、人参三七、田三七、山漆等,为五加科(Araliaceae)人参属植物,主产于云南、广西及四川等地,是临床常用传统中药。三七味甘、微苦,性温,归肝、胃、心、小肠经,具有止血、散瘀、消肿、止痛、补虚、强壮等功效,主治咯血、衄血、外伤出血、跌打肿痛等,近年来用于治疗冠心病、糖尿病、抗心绞痛、抗血栓等。从上世纪40年代开始,诸多学者对三七进行了一系列研究,揭示了其主要成分及三七在血液系统、心血管系统、神经系统、免疫系统、物质代谢系统,以及抗炎、抗衰老、抗肿瘤等多方面的生理活性。三七具有抗肿瘤血行转移能力,通过抑制VEGF基因表达,抑制肿瘤新生血管形成,在保护心肌缺生长。邓伟等(2013)对中药三七抑制二乙基亚硝胺诱发大鼠肝癌血管生成的研究表明:三七在HCC肿瘤血管生成和进展中可能通过抑制Ang-2、Tie-2、HIF-1α及VEGF而发挥预防作用。
多酚类物质具有很强的抗氧化作用,被称为“第七类营养素”。多酚类化合物是指分子结构中有若干个酚性羟基的植物成分的总称,包括黄酮类、单宁类、酚酸类以及花色苷类等酚是在植物性食物中发现的、具有潜在促进健康作用的化合物。它存在于一些常见的植物性食物,如可可豆,茶,大豆,红酒,蔬菜和水果中。EGCG(多酚类物质)通过活化抑瘤蛋白p53信号通路,下调VEGF表达水平来抑制癌症的发生发展。田梦秋等(2015)研究茶多酚对人鼻咽癌细胞裸鼠移植瘤的生长抑制作用发现:茶多酚对人鼻咽癌HONE1细胞裸鼠移植瘤的生长具有明显抑制作用,机制可能与调节VEGF表达,从而抑制肿瘤血管生成等作用有关。金龙玉研究EGCG调节p53和EGFR信号通路抑制人类肺癌细胞的分子机制发现:EGCG(绿茶多酚)活化抑瘤蛋白p53信号通路促进其抗肺肿瘤效果。EGCG通过上调p53的磷酸化,抑制MDM2与p53的相互作用从而抑制MDM2对p53的泛素化降解促进p53的稳定与积聚。EGCG通过促进p53的胞核积聚,上调其反式激活能力,从而诱导其下游靶基因如p21等的表达上调,干扰肺癌细胞周期行进。
叶黄素是一种含氧类胡萝卜素,是构成蔬菜、水果、花卉等植物色素的主要组分,其在甘蓝、菠菜等深绿色叶菜及金盏花等花卉中含量较高;其分子式为C40H56O2,相对分子质量为568.88,没有维生素A活性;研究表明,叶黄素游离羟基与脂肪酸酯化可增强对热和紫外线的稳定性,因此商品化叶黄素产品多为叶黄素酯型;叶黄素酯进入人体后可被高效地水解并释放出游离叶黄素,从而发挥生物学功效。叶黄素独特的化学结构及组成决定了其独特的生物学功能,在抗氧化、预防白内障、延缓动脉硬化以及抗肿瘤等方面具有重要作用。有学者发现,叶黄素对乳腺癌、胃癌、食管癌、口腔上皮癌等多种肿瘤具有明显抑制作用。叶黄素对肿瘤的抑制主要通过上调凋亡基因和P53表达来实现。王若仲等研究显示,叶黄素通过上调凋亡基因Bax和p53信使RNA的表达,从而抑制人肝癌细胞HepG2的生长。
中药丹参为唇形科多年生草本植物丹参的干燥根茎,具有活血祛瘀,通经止痛,清心除烦,凉血消痈之功效。多年来的实验研究以及临床应用表明,单味药丹参、或丹参的提取物、或丹参与其他药物组成的复方制剂均具有一定的抗肿瘤作用。其提取成分主要包括脂溶性和水溶性两大类,脂溶性成分主要为共轭醌、酮类化合物,包括丹参酮(I、ⅡA、ⅡB)、异丹参酮(I、Ⅱ)、隐丹参酮、异隐丹参酮、丹参醇(I、Ⅱ、Ⅲ)、丹参醌(A、B、C、D)和二氢丹参醌等;水溶性成分主要为酚酸类化合物,包括丹参酸(乙、丙)、丹参素、咖啡酸、原儿茶酸、原儿茶醛、紫草酸、迷迭香酸及丹参酚酸(A、B、C、D、E、F、G)。丹参提取物中对肿瘤有抑制的作用的有效成分主要为丹参酮,通过上调P53表达,阻滞其细胞周期,使细胞核裂解呈现碎片状,产生凋亡小体促进细胞凋亡。毕明慧等(2011)分别以0mg/L、10mg/L、20mg/L、30mg/L、40mg/L丹参素作用SMMC7721细胞24、48和72h后,分别观察细胞凋亡形态并检测不同浓度(0~40mg/L)丹参素作用SMMC7721细胞24h后p53mRNA表达。结果发现丹参素在一定浓度范围内可呈时间和剂量依赖性地抑制SMMC7721细胞增殖并诱导细胞凋亡,其机制可能与p53表达上调有关。
发明内容
本发明提供了一种针对癌细胞迁徙能力相关基因的配方食品,要解决的技术问题是提高肿瘤患者的突变细胞迁徙抑制能力。
为解决上述问题,本发明采取的技术方案是:一种针对癌细胞迁徙能力相关基因的配方食品,所述个体化干预配方食品通过如下步骤制成:
1)检测个体的p53结合蛋白1基因、p53蛋白基因、血管内皮生长因子基因的变异;
2)评估所检测的基因的变异的风险;
3)选择食品基础方中的原料,根据所评估的风险选择基因方中的原料,然后将所选择的原料制成粉状食品。
优选地,在所述步骤1)中,检测的p53结合蛋白1基因的位点为位点一:rs62021180;检测的p53蛋白基因的位点为位点二:rs1042522;检测的血管内皮生长因子基因的位点为位点三:rs699947。
优选地,在所述的步骤2)中,所检测的基因的变异的风险等于位点一风险度、位点二风险度和位点三风险度的乘积。
优选地,
当检测到的位点一的基因型分别为AA、AG、GG时,位点一风险度分别为0.31、0.31、4.18;
当检测到的位点二的基因型分别为CC、CG、GG时,位点二风险度分别为风险度0.22、3.71、3.71;
当检测到的位点三的基因型分别为AA、AC、CC时,位点三风险度分别为0.52、3.53、3.53。
优选地,在所述步骤3)中,基础方中的原料包括基础方必要原料,基础方必要原料按重量计包括:豌豆蛋白2~5份、壳聚糖0.5~2份、绿豆淀粉0.2~0.8份、乳清蛋白0.2~0.8份、小麦草0.2~0.8份。
优选地,在所述步骤3)中,基础方中的原料还包括基础方补充原料,基础方补充原料按重量计包括:大米蛋白1~4份、葛根粉1~4份、益生菌0.2~0.8份、益生元0.2~0.8份。
优选地,在所述步骤3)中,基因方中的原料包括基因方必要原料,基因方必要原料按重量计包括:灵芝多糖1~4份、红花0.5~2份、三七0.5~2份。
优选地,在所述步骤3)中,基因方中的原料还包括基因方补充原料,基因方补充原料按重量计还包括:茶多酚2~3份、叶黄素2~3份、丹参提取物0.5~2份。
优选地,在所述步骤3)中,
如果所检测的基因变异的风险为低风险时,选择基础方必要原料,再选择基因方必要原料;
如果所检测的基因变异的风险为中风险时,选择基础方必要原料,再选择基因方必要原料和基因方补充原料。
如果所检测的基因变异的风险为高风险时,选择基础方必要原料和基础方补充原料,再选择基因方必要原料和基因方补充原料。
4)个体化干预配方食品的制作
上述个体化干预配方食品的制作可通过自动化低温制样机装置来完成。
优选地,所述自动化低温制样机的制备单元包括加样室、微波灭菌组件、低温干燥粉碎组件、筛分装置管道、分离储存混合组件、混料组件、控制面板。将优选的基础方必要原料放入加样室的第一样品室、基础方补充原料放入加样室的第二样品室、基因方必要原料放入加样室的第三样品室、基因方补充原料放入加样室的第四样品室,各组分原料经微波灭菌组件灭菌;灭菌后各组分原料经过低温干燥粉碎组件,干燥后的细粉原料经过筛分装置管道后进入分离储存混合组件;安装在筛分装置管道的重力传感器控制不同组分原料的质量配比,形成基础方组合物、基础方补充方组合物、基因方组合物、基因方补充方组合物分别储存在第一至第四储存室中并充分混合;安装在储存分离组件的重力传感器可以根据可以需要控制不同储存室组合物的质量配比进入可移动混料组件充分混匀,上述过程均通过控制面板进行控制。收集所述组合物,真空封装,即为本发明所述针对癌细胞迁徙能力相关基因的配方食品。具体制作方法如下:
(1)精选优质原料;
(2)通过控制面板设置需要的重量配比;
(3)将各配方的组分原料加入到对应的样品室中,经微波灭菌,灭菌功率为750W,持续90s;
(4)将上步的各组分进行真空低温干燥粉碎,真空度为-0.08MPa以下,温度不超过4℃,时间约为5小时;
(5)将上步各组分原料通过60~90目筛分,即得各组分原粉;
(6)将上述原粉通过安装在筛分装置管道的重力传感器控制不同组分的质量配比,形成基础方组合物、基础方补充方组合物、基因方组合物、基因方补充方组合物分别储存在第一至第四储存室中;
(7)第一至第四储存室中的组合物在转速为60r/min~100r/min的条件下在储存室内充分混匀,时间为30~60s;
(8)上述储存在不同储存室的各配方组合物经过重力传感器控制不同配方的质量配比,分别进入到混料组件的不同产品室内,在转速为60r/min~100r/min条件下充分混匀,时间为15~20s;
(9)收集上述混料组件内对应产品室的组合物即得本发明所述针对癌细胞迁徙能力相关基因的配方食品。
本发明还提供了一种针对癌细胞迁徙能力相关基因的配方食品的制备方法,所述制备方法包括如下步骤:
1)检测个体的p53结合蛋白1基因、p53蛋白基因、血管内皮生长因子基因的变异;
2)评估所检测的基因的变异的风险;
3)选择食品基础方中的原料,根据所评估的风险选择基因方中的原料,制成所述的针对癌细胞迁徙能力相关基因的配方食品。
本发明的有益效果为:在基因检测的基础上,针对患者的基因型变异情况,给出适合患者基因型的特殊医学用途配方食品,为肿瘤患者提供合理的营养补充,预防肿瘤患者营养不良现象的发生,提高肿瘤患者的突变细胞迁徙抑制能力。
以下将结合附图对本发明的构思、具体结构及产生的技术效果作进一步说明,以充分地了解本发明的目的、特征和效果。
附图说明
图1是本发明的一个优选实施例中组合物制备系统示意图。
具体实施方式
实施例一基因变异的检测
采集个体口腔上皮细胞样本,用硅胶吸附法抽提基因组DNA,经电泳检测验证后进行荧光定量PCR反应。
将个体样本分别放入3个反应孔中,同时检测3个位点,即TP53BP1基因的rs62021180(位点一);TP53基因的rs1042522(位点二);VEGF基因的rs699947(位点三)。另外根据实验需要增设不含DNA模板的NTC空白对照孔;
在每个反应孔中加入试剂为荧光定量PCR反应体系,总体积为10μL,即浓度为20ng/μL的DNA模板2μL、10×荧光定量PCR反应缓冲液MGB1μL、25mMdNTP合成DNA的四种脱氧核苷酸底物0.1μL、25mMMgCl2溶液0.5μL、(5units/μL)TaqDNA聚合酶0.02μL、去离子水4.98μL、3个位点分别采用不同的正向引物(20μM,0.225μL)、反向引物(20μM,0.225μL)、VIC荧光探针(10μM,0.25μL)和FAM荧光探针(10μM,0.25μL);3个位点使用引物和探针如下表1所示。
表1
将反应孔和空白对照在PCR扩增仪上进行反应,先预热:50℃、2分钟,95℃、10分钟,然后进行60个循环的95℃、30秒,60℃、1分钟,反应结束后取出反应体系再放入荧光定量PCR仪上读取样品荧光量,得到三个基因的三张图。
将荧光定量PCR仪上显示的最终样本荧光信号的图与NTC空白对照比较,每个位点可能出现三种不同的信号中的一种,即纯VIC荧光信号、VIC和FAM杂合荧光信号以及纯FAM荧光信号,分别代表该位点三种不同的基因型。
实施例二评估所检测的基因的变异的风险
根据大规模中国人群中的分子流行病学相关研究成果查阅,实施例所测得的三个位点的不同基因型的风险度如下表2所示:
表2
每个与突变细胞迁徙抑制能力相关的基因多态性位点分别有三种亚型,将所选位点所有亚型出现的可能进行排列组合;然后将这些亚型的风险度相乘获得不同排列组合的风险度乘积;将获得的风险度乘积从小到大或从大到小排列,合并风险度乘积相等的组;最终按风险度乘积的大小确定各组风险度的高低。
上述方法是将各位点作为独立因素进行加权处理的,风险度乘积越大,评估风险越大;用上述方法计算得出的联合基因型遗传高风险个体可表述为其因自身突变细胞迁徙抑制能力差异而引发肿瘤的遗传风险因素较多或结果确定性较强。
进一步地,将相关多态性位点标记为a、b、c……,将不同的亚型标记为1、2、3,即携带位点1亚型1+位点2亚型1+位点3亚型1的单倍型表示为a1b1c1;将这个优选实施例检测的位点rs62021180(TP53BP1)、rs1042522(TP53)和rs699947(VEGF)分别标记为a、b、c,根据上述表格中所列的单基因风险度计算风险度乘积结果如表3所示:
表3
优选地,风险度乘积采用PHP编制程序,然后在计算机中输入各单基因风险度值后进行计算。
然后将上表3中的单倍型按风险度乘积从低到高排列并分组,如表4所示;风险等级越高,风险度乘积越大,即个体因自身突变细胞迁徙抑制能力差异而引发肿瘤的遗传风险越高。
表4
将三位点联合基因型风险等级I-IV定义为低风险,风险等级V-VII定义为中风险,风险等级VIII定义为高风险。
实施例三制备和服用个体化干预配方食品
(1)原料准备
按照如下表5~7中的数据准备相关原料,分别进行相关配方组合物的生产。其中豌豆蛋白选用烟台东方蛋白科技有限公司生产的金冠瑞豌豆蛋白质粉,壳聚糖选用广州利源食品添加剂有限公司生产的壳聚糖粉,绿豆淀粉选用衡水福桥淀粉有限公司生产的福桥牌绿豆淀粉,乳清蛋白选用石家庄春信生物科技有限公司生产的乳清蛋白,小麦草选用浙江百源食品有限公司生产的脱水小麦草,大米蛋白选用Krauterhaus Sanct Bernhard KG公司生产的大米蛋白粉,葛根粉选用兴化市嘉禾食品有限公司生产的葛根粉,益生菌选用山东向日葵生物科技有限公司生产的乳酸菌冻干粉,益生元选用上海耐今实业有限公司生产的益生元粉,灵芝多糖选用宣城市百草植物工贸有限公司生产的提取灵芝多糖粉,红花选用亳州市骏丰药业有限责任公司生产的红花干品,三七选用云南鹤明生物科技有限公司生产的三七干品,茶多酚选用湖北福润德食品原料有限公司生产的含量为99%的绿茶提取茶多酚,叶黄素选用山东西唐生物科技有限公司生产的万寿菊提取叶黄素粉,丹参提取物选用西安维特生物科技有限责任公司的产品。
表5个体化干预食品配方1
表6个体化干预食品配方2
表7个体化干预食品配方3
按照如下表8中的配比选用表5~7的相关配方组合物制备非个体化干预配方食品和个体化干预配方食品。
表8
(2)各种干预配方食品的生产
干预配方食品的制作可通过自动化低温制样机装置来完成。
如图1所示,所述自动化低温制样机的制备单元包括加样室1、微波灭菌组件2、低温干燥粉碎组件3、筛分装置管道4、分离储存混合组件5、混料组件6、控制面板7。将优选的基础方必要原料放入加样室的A样品室、基础方补充原料放入加样室的B样品室、基因方必要原料放入加样室的C样品室、基因方补充原料放入加样室的D样品室,各组分原料经微波灭菌组件灭菌;灭菌后各组分原料经过低温干燥粉碎组件,干燥后的细粉原料经过筛分装置管道后进入分离储存混合组件;安装在筛分装置管道的重力传感器控制不同组分原料的质量配比,形成基础方组合物、基础方补充方组合物、基因方组合物、基因方补充方组合物分别储存在A、B、C、D储存室中并充分混合;安装在储存分离组件的重力传感器可以根据可以需要控制不同储存室组合物的质量配比进入可移动混料组件充分混匀,上述过程均通过控制面板进行控制。收集所述组合物,真空封装,即为本发明所述针对癌细胞迁徙能力相关基因的配方食品。具体制作方法如下:
a.干预配方食品1的制备
(1)精选豌豆蛋白、壳聚糖、绿豆淀粉、乳清蛋白、小麦草、大米蛋白、葛根粉、益生菌、益生元、灵芝多糖、红花、三七、茶多酚、叶黄素、丹参提取物等优质原料;
(2)通过控制面板按照表5中各组分重量配比进行设置;
(3)将基础方必要原料放入加样室的A样品室、基础方补充原料放入加样室的B样品室、基因方必要原料放入加样室的C样品室、基因方补充原料放入加样室的D样品室,经微波灭菌,灭菌功率为750W,持续90s;
(4)将上步的各组分进行真空低温干燥粉碎,真空度为-0.08MPa以下,温度不超过4℃,时间约为5小时;
(5)将上步各组分原料通过60~90目筛分,即得各组分原粉;
(6)将上述原粉通过安装在筛分装置管道的重力传感器控制不同组分的质量配比(基础方:豌豆蛋白2份、壳聚糖0.5份、绿豆淀粉0.2份、乳清蛋白0.2份、小麦草0.2份,基础方补充方:大米蛋白1份、葛根粉1份、益生菌0.2份、益生元0.2份,基因方:灵芝多糖1份、红花0.5份、三七0.5份,基因方补充方:茶多酚2份、叶黄素2份、丹参提取物0.5份),形成基础方组合物、基础方补充方组合物、基因方组合物、基因方补充方组合物分别储存在A、B、C、D储存室中;
(7)A(基础方组合物)、B(基础方补充方组合物)、C(基因方组合物)、D(基因方补充方组合物)储存室中的组合物在转速为60r/min~100r/min的条件下在储存室内充分混匀,时间为30~60s;
(8)上述储存在不同储存室的各配方组合物经过重力传感器控制不同配方的质量配比(非个体化干预配方食品:A,低风险个体化干预配方食品:3.1份A、2份C,中风险个体化干预配方食品:3.1份A、2份C、2.5份D,高风险个体化干预配方食品:3.1份A、2.4份B、2份C、2.5份D),分别进入到混料组件的不同产品室内,在转速为60r/min~100r/min条件下充分混匀,时间为15~20s;
(9)收集上述混料组件内对应产品室的组合物,分装成20g/包真空封装,即得本发明实施例三(3)所用的四种针对突变细胞迁徙抑制肿瘤基因的干预配方食品1。
b.干预配方食品2的制备
(1)精选豌豆蛋白、壳聚糖、绿豆淀粉、乳清蛋白、小麦草、大米蛋白、葛根粉、益生菌、益生元、灵芝多糖、红花、三七、茶多酚、叶黄素、丹参提取物等优质原料;
(2)通过控制面板按照表5中各组分重量配比进行设置;
(3)将基础方必要原料放入加样室的A样品室、基础方补充原料放入加样室的B样品室、基因方必要原料放入加样室的C样品室、基因方补充原料放入加样室的D样品室,经微波灭菌,灭菌功率为750W,持续90s;
(4)将上步的各组分进行真空低温干燥粉碎,真空度为-0.08MPa以下,温度不超过4℃,时间约为5小时;
(5)将上步各组分原料通过60~90目筛分,即得各组分原粉;
(6)将上述原粉通过安装在筛分装置管道的重力传感器控制不同组分的质量配比(基础方:豌豆蛋白3份、壳聚糖1份、绿豆淀粉0.5份、乳清蛋白0.5份、小麦草0.5份,基础方补充方:大米蛋白2份、葛根粉2份、益生菌0.5份、益生元0.5份,基因方:灵芝多糖2份、红花1份、三七1份,基因方补充方:茶多酚2.5份、叶黄素2.5份、丹参提取物1份),形成基础方组合物、基础方补充方组合物、基因方组合物、基因方补充方组合物分别储存在A、B、C、D储存室中;
(7)A(基础方组合物)、B(基础方补充方组合物)、C(基因方组合物)、D(基因方补充方组合物)储存室中的组合物在转速为60r/min~100r/min的条件下在储存室内充分混匀,时间为30~60s;
(8)上述储存在不同储存室的各配方组合物经过重力传感器控制不同配方的质量配比(非个体化干预配方食品:A,低风险个体化干预配方食品:5.5份A、4份C,中风险个体化干预配方食品:5.5份A、4份C、6份D,高风险个体化干预配方食品:5.5份A、5份B、4份C、6份D),分别进入到混料组件的不同产品室内,在转速为60r/min~100r/min条件下充分混匀,时间为15~20s;
(9)收集上述混料组件内对应产品室的组合物,分装成20g/包真空封装,即得本发明实施例三(3)所用的四种针对突变细胞迁徙抑制肿瘤基因的干预配方食品2。
c.干预配方食品3的制备
(1)精选豌豆蛋白、壳聚糖、绿豆淀粉、乳清蛋白、小麦草、大米蛋白、葛根粉、益生菌、益生元、灵芝多糖、红花、三七、茶多酚、叶黄素、丹参提取物等优质原料;
(2)通过控制面板按照表5中各组分重量配比进行设置;
(3)将基础方必要原料放入加样室的A样品室、基础方补充原料放入加样室的B样品室、基因方必要原料放入加样室的C样品室、基因方补充原料放入加样室的D样品室,经微波灭菌,灭菌功率为750W,持续90s;
(4)将上步的各组分进行真空低温干燥粉碎,真空度为-0.08MPa以下,温度不超过4℃,时间约为5小时;
(5)将上步各组分原料通过60~90目筛分,即得各组分原粉;
(6)将上述原粉通过安装在筛分装置管道的重力传感器控制不同组分的质量配比(基础方:豌豆蛋白5份、壳聚糖2份、绿豆淀粉0.8份、乳清蛋白0.8份、小麦草0.8份,基础方补充方:大米蛋白4份、葛根粉4份、益生菌0.8份、益生元0.8份,基因方:灵芝多糖4份、红花2份、三七2份,基因方补充方:茶多酚3份、叶黄素3份、丹参提取物2份),形成基础方组合物、基础方补充方组合物、基因方组合物、基因方补充方组合物分别储存在A、B、C、D储存室中;
(7)A(基础方组合物)、B(基础方补充方组合物)、C(基因方组合物)、D(基因方补充方组合物)储存室中的组合物在转速为60r/min~100r/min的条件下在储存室内充分混匀,时间为30~60s;
(8)上述储存在不同储存室的各配方组合物经过重力传感器控制不同配方的质量配比(非个体化干预配方食品:A,低风险个体化干预配方食品:9.4份A、8份C,中风险个体化干预配方食品:9.4份A、8份C、8份D,高风险个体化干预配方食品:9.4份A、9.6份B、8份C、8份D),分别进入到混料组件的不同产品室内,在转速为60r/min~100r/min条件下充分混匀,时间为15~20s;
(9)收集上述混料组件内对应产品室的组合物,分装成20g/包真空封装,即得本发明实施例三(3)所用的四种针对突变细胞迁徙抑制肿瘤基因的干预配方食品3。
(3)个体化干预配方食品的效果验证
从肿瘤特殊项目研究中心的肿瘤患者中随机选择手术后突变细胞迁移抑制能力低风险、中风险和高风险的肺部肿瘤病人若干名,按照下表9中的数据服用以上制备的非个体化干预配方食品或个体化干预配方食品,其中,每日服用3次,早中晚各一次,每次一包,用约150ml温开水(45度)溶解后,直接饮用。服用后每月检测循环肿瘤细胞(circulatingtumor cells,CTCs)数量,共检测三次,然后取每组(30名)的CTC的平均值作为检测结果,统计受试者服用干预配方食品前后体重的差异及显效例数,结果如下表9所示。
表9
*表示P<0.05,存在显著差异。
循环肿瘤细胞是指因自发或诊疗操作由原发灶和转移灶脱落进入外周血的肿瘤细胞,其存在于多种实体瘤中,少量能够长期生存的循环肿瘤细胞是肿瘤转移的重要原因,CTCs受到突变细胞迁移抑制能力的影响,其数量能够反应机体突变细胞迁移抑制能力的强弱;体重的差异反应肿瘤病人的营养状态,用以评估该个体化干预配方食品的营养价值。由表9的数据可以看出,不同类型的干预配方食品对相应的病人均有的效果,个体化干预配方食品的效果更好(CTC数量的差异具有显著性且显效人数更多),均能保证肿瘤病人的营养需求(服用前后体重没有显著变化),且风险等级越高的病人对个体化干预配方食品的效果越明显。
以上详细描述了本发明的较佳具体实施例。应当理解,本领域的普通技术人员无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此,凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案,皆应在由权利要求书所确定的保护范围内。
Claims (10)
1.一种针对癌细胞迁徙能力相关基因的配方食品,其特征在于,所述个体化干预配方食品通过如下步骤制成:
1)检测个体的p53结合蛋白1基因、p53蛋白基因、血管内皮生长因子基因的变异;
2)评估所检测的基因的变异的风险;
3)选择食品基础方中的原料,根据所评估的风险选择基因方中的原料,制成所述的针对癌细胞迁徙能力相关基因的配方食品。
2.如权利要求1所述的针对癌细胞迁徙能力相关基因的配方食品,其特征在于,在所述步骤1)中,检测的p53结合蛋白1基因的位点为位点一:rs62021180;检测的p53蛋白基因的位点为位点二:rs1042522;检测的血管内皮生长因子基因的位点为位点三:rs699947。
3.如权利要求2所述的针对癌细胞迁徙能力相关基因的配方食品,其特征在于,在所述的步骤2)中,所检测的基因的变异的风险等于位点一风险度、位点二风险度和位点三风险度的乘积;
当检测到的位点一的基因型分别为AA、AG、GG时,位点一风险度分别为0.31、0.31、4.18;
当检测到的位点二的基因型分别为CC、CG、GG时,位点二风险度分别为风险度0.22、3.71、3.71;
当检测到的位点三的基因型分别为AA、AC、CC时,位点三风险度分别为0.52、3.53、3.53。
4.如权利要求1所述的针对癌细胞迁徙能力相关基因的配方食品,其特征在于,在所述步骤3)中,基础方中的原料包括基础方必要原料,基础方必要原料按重量计包括:豌豆蛋白2~5份、壳聚糖0.5~2份、绿豆淀粉0.2~0.8份、乳清蛋白0.2~0.8份、小麦草0.2~0.8份。
5.如权利要求4所述的针对癌细胞迁徙能力相关基因的配方食品,其特征在于,在所述步骤3)中,基础方中的原料还包括基础方补充原料,基础方补充原料按重量计包括:大米蛋白1~4份、葛根粉1~4份、益生菌0.2~0.8份、益生元0.2~0.8份。
6.如权利要求5所述的针对癌细胞迁徙能力相关基因的配方食品,其特征在于,在所述步骤3)中,基因方中的原料包括基因方必要原料,基因方必要原料按重量计包括:灵芝多糖1~4份、红花0.5~2份、三七0.5~2份。
7.如权利要求6所述的针对癌细胞迁徙能力相关基因的配方食品,其特征在于,在所述步骤3)中,基因方中的原料还包括基因方补充原料,基因方补充原料按重量计还包括:茶多酚2~3份、叶黄素2~3份、丹参提取物0.5~2份。
8.如权利要求7所述的针对癌细胞迁徙能力相关基因的配方食品,其特征在于,在所述步骤3)中,
如果所检测的基因变异的风险为低风险时,选择基础方必要原料,再选择基因方必要原料;
如果所检测的基因变异的风险为中风险时,选择基础方必要原料,再选择基因方必要原料和基因方补充原料。
如果所检测的基因变异的风险为高风险时,选择基础方必要原料和基础方补充原料,再选择基因方必要原料和基因方补充原料。
9.如权利要求8所述的针对癌细胞迁徙能力相关基因的配方食品,其特征在于,采用自动化低温制样机制成所述的针对癌细胞迁徙能力相关基因的配方食品,所述自动化低温制样机的制备单元包括加样室、微波灭菌组件、低温干燥粉碎组件、筛分装置管道、分离储存混合组件、混料组件、控制面板,将基础方必要原料放入加样室的第一样品室、基础方补充原料放入加样室的第二样品室、基因方必要原料放入加样室的第三样品室、基因方补充原料放入加样室的第四样品室,各组分原料经微波灭菌组件灭菌;灭菌后各组分原料经过低温干燥粉碎组件,干燥后的细粉原料经过筛分装置管道后进入分离储存混合组件;安装在筛分装置管道的重力传感器控制不同组分原料的质量配比,形成基础方组合物、基础方补充方组合物、基因方组合物、基因方补充方组合物分别储存在第一至第四储存室中并充分混合;收集所述组合物,真空封装,即为所述针对癌细胞迁徙能力相关基因的配方食品。
10.一种针对癌细胞迁徙能力相关基因的配方食品的制备方法,其特征在于,所述制备方法包括如下步骤:
1)检测个体的p53结合蛋白1基因、p53蛋白基因、血管内皮生长因子基因的变异;
2)评估所检测的基因的变异的风险;
3)选择食品基础方中的原料,根据所评估的风险选择基因方中的原料,制成所述的针对癌细胞迁徙能力相关基因的配方食品。
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