CN108450958A - 一种富硒组合物及制剂的应用 - Google Patents
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Abstract
本发明涉及一种富硒组合物及制剂的应用。具体是作为制备改善人体CD4 +、CD8 +的水平及提高CD4 +/CD8 +比值的药品、保健食品、功能性普通食品、特殊医学配方食品、特殊膳食食品的应用，属于大健康应用技术领域。本发明选用对小鼠腹水型肝癌细胞H22的影响、柯萨奇B4和B3M病毒重叠感染对昆明鼠T细胞亚群的影响、对FLV感染小鼠的细胞免疫功能的影响几项技术验证，发现该富硒组合物及制剂具有显著改善CD4 +、CD8 +的水平及提高CD4 +/CD8 +比值的效果。
Description
技术领域
本发明涉及一种富硒组合物及制剂的应用。具体是作为制备改善人体CD4 +、CD8 +的水平及提高CD4 +/CD8 +比值的药品、保健食品、功能性普通食品、特殊医学配方食品、特殊膳食食品的应用，属于大健康应用技术领域。
技术背景
T细胞是白血球细胞，在免疫系统中扮演很重要的角色。人体内有两种主要的T细胞，其中一种就称为CD4细胞，CD4 +T淋巴细胞是人体免疫系统中一种重要的免疫细胞---简称为CD4细胞。在T细胞的表面这些CD4细胞又称为免疫系统的辅助手，能指挥身体对抗微生物，例如病毒。还可以看到一种CD8 +T细胞，而这又是另一种T淋巴细胞，也称之为细胞毒性T细胞，其功能就像一个“杀手”或细胞毒素，可以消灭受感染的细胞。测定CD4 +、CD8 +的水平及CD4 +/CD8 +比值可在一定程度上反映机体免疫功能。由于HIV的攻击对象正是人体的CD4细胞，因此CD4记数能够直接反映人体免疫功能，是提供HIV感染患者免疫系统损害状况最明确的指标。
适量补硒不但可以提高免疫力，同时也能提高对一些病毒感染的抵抗力，并且可以使致病株的毒力下降而减轻病毒感染对心肌的损害；研究表明，饮食补硒后，人的外周血淋巴细胞或动物脾淋巴细胞对PHA、ConA或同种抗原的刺激应答显著增强，细胞内DNA合成增加，淋巴细胞增殖分化能力以及CD8 +细胞毒T细胞数量增多，杀肿瘤细胞能力增强。
申请号为201810104156.2的发明专利，公开了一种富硒组合物及制剂，其制备方法和应用。作为该专利的发明人，在实施该专利的过程中不断的探索和研究，开辟了富硒组合物及制剂的新用途。
发明内容
本发明的目的在于开辟申请号为201810104156.2，一种富硒组合物及制剂，其制备方法和应用的新用途。
本发明的目的是采用以下技术方案来实现的。
本发明选用对小鼠腹水型肝癌细胞H22的影响、柯萨奇B4和B3M病毒重叠感染对昆明鼠T细胞亚群的影响、对FLV感染小鼠的细胞免疫功能的影响几项技术验证，发现该富硒组合物及制剂具有明显改善CD4 +、CD8 +的水平及提高CD4 +/CD8 +比值的作用。
具体实施方式：
下面通过具体实施例对本发明作进一步说明，但不以任何方式对本发明限制，基于本发明教导所作的任何变化或替换，均属本发明保护范围。
实施例1：组合物料的制备
富硒酵母：直接购置保健品营养素补充剂原料，经检测硒含量(以Se计)为0.07％。
维生素C：直接购置食品级原料，经检测维生素C含量(以L-抗坏血酸计)为99.79％。
乳糖、预胶化淀粉、硬脂酸镁：直接购置食品级原料。
实施例2：富硒乳化物制备
富硒乳化物按昆明朗盛生物科技有限公司《一种富硒组合物及制剂，其制备方法和应用》(申请号：201810104156.2)专利申请制备。经检测富硒乳化物中硒含量为0.018％。
实施例3：组合物制备
取实施例2制备的富硒乳化物，按昆明朗盛生物科技有限公司《一种富硒组合物及制剂，其制备方法和应用》(申请号：201810104156.2)专利申请制备组合物，经检测组合物中硒含量为0.0062％，维生素C含量为19.96％
为了更进一步对本发明技术效果进行说明，本发明选取实施例3所制备的组合物作为试验样品，编号为③。选取实施例1准备的富硒酵母、实施例2制备的富硒乳化物、依次编号为①、②开展试验，结果如下：
1.对小鼠腹水型肝癌细胞H22的影响
试验动物及器材：KM小鼠(18-22g)；小鼠腹水型肝癌细胞H22；流式细胞仪(Beck-Man)；蛋白试测定剂盒
给样剂量：根据本发明所述组合物(试验样品③)60kg成年人给样剂量确定为1g/天，据此折算每天小鼠给样剂量为150mg/kg(其中含富硒酵母13.5mg，含维生素C 30mg)；依据等剂量给样可比原则确定样品②每天给样剂量为51.3mg/kg(其中含富硒酵母13.5mg)，依据等剂量给样可比原则，结合国家药典或相关行业标准推荐富硒酵母最高日推荐剂量推算样品①给样剂量为21.5mg/kg。
造模、分组及实验：KM小鼠40只，体重18-22g,小鼠腹水型肝癌细胞H22体内传代两次，从荷腹水型肝癌细胞H22的小鼠中抽取含有H22细胞的腹水液，置离心管内，离心(1000r/min,10min),弃上清液，PBS(0.15mol/L pH7.2)漂洗3次，细胞计数，调整细胞浓度为107个/ml,分别注入小鼠腹腔，0.2ml/只。
次日，将小鼠随机分为4组，每组10只，设为模型组，富硒酵母组，富硒乳化物组，组合物组，灌胃给药1次/天，连续10天，模型组给予等剂量的生理盐水。注入腹水型肝癌细胞H22前对小鼠进行第1次体重称量，四天后每两天测1次体重，计算与第1次体重的差异，以体重差异反应腹水生成量。
于造模第10天，末次给药30min后摘除眼球取血，肝素抗凝。取血20μl加入抗凝管中，分别加入8F和4P两种试剂10μl，离心(1000r/min,5min)，弃上清液，加入1mlPBS，振荡，离心，弃清液，再加入0.5mlPBS，振荡，离心，弃清液，再加入0.5mlPBS，振荡，流式细胞仪检测CD8 +和CD4 +的含量，实验结果如表1所示。
表1：对荷瘤小鼠CD4 +和CD8 +的含量影响
| 组别 | CD4 + | CD8 + | CD4 +/CD8 + |
| 模型组 | 36.6±7.2 | 28.1±3.2 | 1.4±0.3 |
| ① | 55.4±5.9 | 23.3±4.8 | 2.2±0.4 |
| ② | 56.7±9.6 | 22.7±4.2 | 2.5±0.7 |
| ③ | 55.2±8.2 | 20.4±3.5 | 3.0±1.1 |
实验结论：样品①、②、③组与模型组相比CD8 +均降低(P<0.01)；样品①、②、③组与模型组相比CD4 +与CD4 +/CD8 +均明显升高(P<0.05)；样品③组(本发明所述组合物)与样品①、②组相比CD8 +显著降低(P<0.01)；且CD4 +与CD4 +/CD8 +显著升高(P<0.01)
2.柯萨奇B4和B3M病毒重叠感染对昆明鼠T细胞亚群的影响
试验动物及器材：KM小鼠(18-22g)；北京医科大学制备的抗小鼠T细胞单克隆抗体(CD4、CD8)；流式细胞仪。
给样剂量：根据本发明所述组合物(试验样品③)60kg成年人给样剂量确定为1g/天，据此折算每天小鼠给样剂量为150mg/kg(其中含富硒酵母13.5mg，含维生素C 30mg)；依据等剂量给样可比原则确定样品②每天给样剂量为51.3mg/kg(其中含富硒酵母13.5mg)，依据等剂量给样可比原则，结合国家药典或相关行业标准，富硒酵母最高日推荐剂量推算样品①给样剂量为21.5mg/kg。
造模、分组及实验：KM小鼠32只，体重18-22g,将雌鼠随机分成模型组，富硒酵母组,富硒乳化物组以及组合物组。
以上4组动物均自由进食,模型组每天灌胃等剂量的生理盐水,样品①、②、③每天分别灌胃相应剂量的富硒酵母，富硒乳化物和组合物。于5周后交配,取7日龄乳鼠供实验用。取7日龄鼠腹腔注入10-6TCID50CVB4 0.1ml,7天后在同一鼠再次腹腔注入102TCID50CVB3M0.1ml,于感染CVB3M 7天后处死。
T淋巴细胞亚群的检验：常规方法分离脾细胞,采用北京医科大学制备的抗小鼠T细胞单克隆抗体(CD4、CD8),用流式细胞检测脾脏CD4、CD8，阳性细胞数。试验结果见表2。
表2：病毒小鼠T淋巴细胞亚群检查结果
| 组别 | CD4 | CD8 | CD4/CD8 |
| 模型组 | 6.37±2.45 | 4.17±1.23 | 1.32±0.23 |
| ① | 9.27±1.51 | 6.26±1.44 | 1.48±0.19 |
| ② | 14.72±1.63 | 8.57±1.28 | 1.63±0.31 |
| ③ | 21.83±2.16 | 12.17±1.05 | 1.97±0.21 |
实验结论：样品①与模型组相比CD4、CD8及CD4/CD8有明显差异(P<0.05),样品②、样品③与模型组相比CD4、CD8及CD4/CD8均有显著差异(P<0.01)，样品③与样品②CD4、CD8及CD4/CD8有明显差异(P<0.01)，即样品③(本发明所述组合物)具有显著提高人体CD4、CD8的水平及CD4/CD8比值的作用。
3.对FLV感染小鼠的细胞免疫功能的影响
考察指标的确定：为了更进一步对本发明组合物技术效果进行说明，选取编号为①②③作为试验样品，同时设置正常对照组、染毒组和AZT组(阳性对照组)进行对照，以排除造模干扰与误差。开展对照组和实验组的小鼠外周血T淋巴细胞亚群CD4 +、CD8 +及CD4 +/CD8 +的水平的测定。
实验动物及器材：SPF级BALB/C小鼠60只，雌性，体重18～22g，由广东省实验动物中心提供。Friend型鼠白血病病毒(FriendLeukemia virus，FLV)引自美国典型培养物收藏中心(American Typical Culture Centre，ATCC)，经小鼠传代增强毒力后制成100g/L脾悬液，-70℃冻存备用。阳性对照药物齐多呋定(AZT)由厦门制药厂生产。
造模分组及实验：SPF级BALB/C小鼠60只，雌性，体重18～22g，平均分为6组，每组10只。正常组以灌胃方式给蒸馏水0.2ml/只；模型组采用FLV腹腔注射感染小鼠，以灌胃方式给蒸馏水0.2ml/只；AZT组(阳性对照组)采用FLV腹腔注射感染小鼠，并以灌胃方式给药，AZT剂量5000mg/kg；样品①组采用FLV腹腔注射感染小鼠，并以灌胃方式给药，样品①给药剂量21.5mg/kg；样品②组采用FLV腹腔注射感染小鼠，并以灌胃方式给药，样品②给药剂量51.3mg/kg(其中含富硒酵母13.5mg)；样品③组采用FLV腹腔注射感染小鼠，并以灌胃方式给药，样品③给药剂量150mg/kg(其中含富硒酵母13.5mg，含维生素C 30mg)。
在病毒攻击后1d开始灌胃给药，给药21d后剖杀小鼠,对小鼠外周血T淋巴细胞亚群CD4 +、CD8 +及CD4 +/CD8 +的水平进行测定。结果见表3：
表3：小鼠外周血T淋巴细胞亚群水平
实验结论：
从表3可以看出：与正常组和AZT组(阳性对照组)相比，模型组外周血T淋巴细胞亚群CD4 +(P<0.01)、CD8 +(P<0.05)、CD4 +/CD8 +(P<0.05)均显著降低，说明模型组小鼠FLV病毒攻击模型成功建立。
样品③组较染毒组及样品①②组CD4 +、CD8 +及CD4 +/CD8 +检测水平显著提高，说明样品③(即本发明所述组合物)能有效提升感染FLV病毒小鼠的外周血T淋巴细胞亚群CD4 +、CD8 +及CD4 +/CD8 +的水平。
Claims (1)
1.一种富硒组合物及制剂的应用，其特征在于该富硒组合物作为制备显著改善CD4 +、CD8 +水平及提高CD4 +/CD8 +比值的药品、保健食品、功能性普通食品、特殊医学配方食品、特殊膳食食品的应用。
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