CN107802653A - 灭活乳酸菌在防治肿瘤性疾病药物中的应用 - Google Patents
灭活乳酸菌在防治肿瘤性疾病药物中的应用 Download PDFInfo
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- CN107802653A CN107802653A CN201610809583.1A CN201610809583A CN107802653A CN 107802653 A CN107802653 A CN 107802653A CN 201610809583 A CN201610809583 A CN 201610809583A CN 107802653 A CN107802653 A CN 107802653A
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Abstract
本发明涉及一种灭活乳酸菌在制备防治肿瘤性疾病药物中的用途，属于生物医药领域。这种药物的主要成分是灭活乳酸菌，可以通过将活的乳酸菌经过常规方法灭活制备。灭活乳酸菌静脉给药可以用于人或动物的各种癌症的预防、治疗或辅助治疗，具有广阔的应用前景。
Description
技术领域
本发明属于生物医药领域，尤其涉及灭活乳酸菌在防治肿瘤性疾病中的新用途。
背景技术
肿瘤性疾病是目前对人类危害最大的疾病。能够引发肿瘤的致病因素很多，一些病毒可以致瘤，如现已发现600多种病毒中约有1/4具有致癌的特性，如人类T淋巴细胞白血病Ⅰ型病毒诱发成人T淋巴细胞白血病；乳腺肿瘤病毒诱发乳腺癌；单纯疱疹病毒Ⅱ型诱发宫颈癌；肝炎病毒导致肝癌；EB病毒与鼻咽癌有关等。一些有害物质可诱发癌症，如黄曲霉素、苯并芘和亚硝胺类是公认的三大致癌物质。不良的生活习性可能导致癌症，如吸烟、嗜酒、熬夜等。另外，环境雾霾、水源污染、手机辐射等也可能诱发癌症。当今世界癌症发病率高，而且呈上升趋势，成为了医学界的难题。
目前用于癌症治疗的药物主要有：烷化剂，如氮芥、白消安、塞替派等；天然产物类，如紫杉醇、喜树碱、长春新碱等；抗生素类，如放线菌素D、博来霉素、丝裂霉素等；抗代谢类，如氟尿嘧啶、硫鸟嘌呤、阿糖胞苷等；激素类，如他莫昔芬、福美坦、氨鲁米特等；其他类，如顺铂、曲妥珠单抗、重组人血管内皮抑素、盐酸埃克替尼等；另外，一些免疫调节类药物常用于癌症的辅助治疗，如香菇多糖、干扰素、胸腺肽、卡介苗、A群溶链球菌制剂、铜绿假单胞菌制剂等。虽然目前抗癌药物种类较多，但对于恶性肿瘤治愈率仍然很低。大多化疗药选择性差，杀灭癌细胞的同时还会损伤机体正常细胞，使机体自身的抗病能力降低，很难使肿瘤痊愈。另外，肿瘤细胞对化疗药物还会产生耐药性，如通过主动外排泵将药物排除细胞外，使药物无法发挥杀灭瘤细胞作用。
寻找新型的抗肿瘤药物一直是国际上研究的热点。本发明发现将灭活的乳酸菌进行静脉给药对肿瘤性疾病具有防治作用，目前国内外尚无相关报道，未来具有广阔的应用前景。
发明内容
本发明提供了一种全新的可以用于肿瘤性疾病防治的灭活乳酸菌混悬液，这种药物的主要成分为灭活的乳酸菌。该药物通过静脉给药可以有效防治肿瘤性疾病。
灭活乳酸菌在制备防治肿瘤性疾病药物中的用途，其特征在于，对所述的灭活乳酸菌进行革兰氏染色，在油镜下观察，灭活乳酸菌保持完整菌体形态，药物的给药方式为注射给药。所述的保持完整菌体形态是指与灭活前活菌菌体的轮廓和形态基本一致。所说的基本一致实质上是指乳酸菌灭活过程中菌体细胞壁可能会出现轻微变化，比如部分表面成分的流失，但这种变化微乎其微或很少发生。
灭活乳酸菌为一种或两种以上灭活乳酸菌混合物。注射时，所述的每单位剂量(mL)药物中包含灭活乳酸菌完整菌体数量为105—1012个。
用于本发明制备防治肿瘤性疾病的灭活乳酸菌混悬液的乳酸菌种类包括：(1)乳杆菌属：德氏乳杆菌(L.delbrueckii)、保加利亚乳杆菌(L.bulgaricus)、瑞士乳杆菌(L.helviticus)、嗜酸乳杆菌(L.acido phlus)、格氏乳杆菌(L.gasseri)、唾液乳杆菌(L.salivarius)、植物乳杆菌(L.plantarum)、罗伊氏乳杆菌(L.reuteri)、短乳杆菌(L.brevis)、干酪乳杆菌(L.casei)、发酵乳杆菌(L.fementi)等；(2)片球菌属：如乳酸片球菌(P.acidi1actic)、戊糖片球菌(P.pentasiaceus)、小片球菌(P.parvulus)等；(3)明串球菌属：肠膜明串球菌(L.mesenteroides)及其乳脂亚种(L.cremoris)和葡聚糖亚种(Leuc.dextranicun)、乳酸明串球菌(L.lactis)、酒明串球菌(L.oenos)等；(4)肠球菌属：屎肠球菌(E.faecium)、粪肠球菌(E.faecalis)等；(5)乳球菌属：乳酸乳球菌乳酸亚种(L.lactis subsp.lactis)、乳酸乳球菌乳脂亚种(L.lactis subsp.cremoris)、乳酸乳球菌叶蝉亚种(L.lactis subsp.hordniae)等；(6)链球菌属：乳酸链球菌(S.lactis)、丁二酮乳酸链球菌(S.diacetilactis)、乳酪链球菌(S.creamoris)、嗜热乳链球菌(S.thermophilus)等；(7)双歧杆菌属：两歧双歧杆菌(B.bifidum)、长双歧杆菌(B.longum)。
优选的，乳酸菌选自乳酸乳球菌乳酸亚种(拉丁名称：Lactococcus lactissubsp.Lactis，保藏编号：CICC 6246)、植物乳杆菌植物亚种(拉丁名称：Lactobacillusplantarum subsp.Plantarum，保藏编号：CICC 6240)、长双歧杆菌(拉丁名称：Bifidobacterium longum，保藏编号：CICC 6196)、短乳杆菌(拉丁名称：Lactobacillusbrevis，保藏编号：CICC 6239)、屎肠球菌(拉丁名称：Enterococcus faecium，保藏编号：CICC 6049)。
用于本发明的制备灭活乳酸菌的方法包括：包括高温高压灭活、紫外线灭活、化学试剂灭活、辐射灭活等。
用于本发明的制备灭活乳酸菌混悬液的方法包括：包括高温高压灭活、紫外线灭活、化学试剂灭活、辐射灭活等。
用于本发明的制备灭活乳酸菌静脉给药的剂型包括：粉针剂、混悬注射剂等各种剂型。
用于本发明的制备灭活乳酸菌混悬液的辅料包括：辛苯氧基聚乙氧乙醇、四丁酚醛、山梨醇酐聚乙二醇单油酸酯、聚氧乙烯单硬脂酸酯、聚氧乙烯衍生物、吐温-80、聚烷基苯磺酸钠、月桂基硫酸酯钠、多糖硫酸酯碱金属盐、葡聚糖硫酸酯钠、磺基琥珀酸双辛酯、金合欢胶、阿拉伯乳胶、聚乙烯吡咯烷酮、聚硅酸乙酯、乙醇、甘油、山梨醇、蜂蜜、琼脂、淀粉、右旋糖、果糖、麦芽浸膏、可可粉、酒石酸、枸橼酸、枸橼酸钠、角叉酸、海藻酸、海藻酸钠、鞣酸、环己烷氨基磺酸、矿物油、优卡林、糖精钠、印度树胶、刺梧桐树胶、西黄蓍胶、果胶、鹿角菜胶、明胶、羧甲基纤维素、硫酸纤维素、甲基纤维素、羧甲基纤维素钠、醋酸硫酸纤维素钠、羟乙基纤维素钠、甲基聚硅、山梨酸钾、高岭土、硅藻土、膨润土、硅酸铝、氢氧化铝、胶体氢氧化铝、硅酸镁铝、蒙脱土镁、三聚硅酸镁、硅酸铝镁钠、碳酸氢钠、碳酸钠、对羟基苯甲酸甲酯、对羟基苯甲酸丙酯、乙基香草醛、柠檬油、橙皮油、香草醛、干酪素等。
用于本发明的灭活乳酸菌混悬液的给药方式包括：静脉推注和静脉滴注等。
本发明采用常规方法灭活了屎肠球菌，发现灭活的屎肠球菌仍然可以进行革兰氏染色，油镜下观察灭活的屎肠球菌与活的屎肠球菌保持一致的菌体轮廓和形态。然后对灭活屎肠球菌生理盐水溶液进行离心，弃上清留沉淀，提取DNA，采用PCR技术仍可以扩增出16S rDNA基因片段，通过测序还可以进行乳酸菌种类鉴别。进一步研究发现，将灭活屎肠球菌对小鼠静脉给药，可以减少小鼠腹水生成；还可以增强腹水瘤小鼠免疫功能以及延长腹水瘤小鼠生存时间。这说明灭活乳酸菌静脉注射给药可以用于肿瘤性疾病的防治。
本发明制备的灭活乳酸菌混悬液可以防治的肿瘤性疾病包括：鼻腔及鼻窦恶性肿瘤、鼻咽癌、口腔癌、喉癌、涎腺肿瘤、颅内肿瘤、甲状腺癌、舌癌、肺癌、食管癌、贲门癌、乳腺癌、纵膈肿瘤、胃癌、大肠癌、乙状结肠癌、直肠癌、肝癌、胰腺癌、壶腹周围癌、小肠恶性肿瘤、肾癌、前列腺癌、膀胱癌、睾丸恶性肿瘤、阴茎癌、子宫颈癌、子宫内膜癌、卵巢癌、恶性纤维组织细胞瘤、横纹肌肉瘤、滑膜肉瘤、皮肤癌、恶性黑色素瘤、骨肉瘤、尤文氏肉瘤、淋巴瘤、多发性骨髓瘤、急性白血病、慢性白血病、红细胞增多症、血小板增多症、骨髓增生异常综合症、骨髓纤维化、恶性组织细胞病等各种肿瘤性疾病。
灭活乳酸菌混悬液防治肿瘤性疾病的机制目前尚不完全清楚，推测可能的机制是：灭活乳酸菌静脉给药后激活了免疫系统，间接发挥抗肿瘤作用；也可能灭活乳酸菌静脉给药后经机体代谢产生了抗肿瘤物质；也可能灭活乳酸菌静脉给药后可与癌细胞产生相互作用抑制癌细胞增殖等。
具体实施方式
实施例1
将屎肠球菌(购于中国工业微生物菌种保藏管理中心，拉丁名称：Enterococcusfaecium，保藏编号：CICC 6049))接种于MRS培养基，于37℃温箱培养24小时，然后3000转离心5分钟，去除上层培养液保留沉淀，加入无菌生理盐水清洗沉淀，离心5分钟，重复清洗3次后，加入无菌生理盐水，与沉淀混匀。取一定量的屎肠球菌生理盐水溶液，于分光光度计690nm处测量其OD值，当用无菌生理盐水稀释的最终浓度的OD值为0.38时，将这样稀释浓度的屎肠球菌生理盐水溶液作为1倍(1×)浓度，经过THOMA细菌计数板进行细菌计数测定每mL混悬液中含有约108个屎肠球菌菌体。取少量的1×浓度的屎肠球菌生理盐水溶液，进行革兰氏染色，在油镜下观察活菌的形态。此后将配制好的1×浓度的屎肠球菌生理盐水溶液于121℃、压力0.12MPa，灭活15min，得到灭活屎肠球菌混悬液，取少量灭活屎肠球菌混悬液进行革兰氏染色，在油镜下观察灭活菌体的形态，发现灭活乳酸菌保持完整菌体形态，与其灭活前活菌菌体轮廓和形态一致，灭活前后细菌数没有明显变化。
实施例2
将屎肠球菌(购于中国工业微生物菌种保藏管理中心，拉丁名称：Enterococcusfaecium，保藏编号：CICC 6049)接种于MRS培养基，于37℃温箱培养24小时，然后3000转离心5分钟，去除上层培养液保留沉淀，加入0.9％无菌生理盐清洗沉淀，离心5分钟，重复清洗3次后，加入适量0.9％无菌生理盐水，与沉淀混匀。取一定量的乳酸菌混悬液，于分光光度计690nm处测量其OD值，当用灭菌生理盐水稀释的最终浓度的OD值为0.38时，将这样稀释浓度的乳酸菌混悬液作为1倍(1×)浓度。以此为依据，对含有乳酸菌生理盐水进行不同倍数的生理盐水稀释，便可以得到5×、1×和0.2×浓度的乳酸菌混悬液。此后分别将配制好的5×、1×和0.2×浓度的屎肠球菌混悬液于121℃、压力0.12MPa，灭活15min，得到5×、1×和0.2×浓度的灭活屎肠球菌混悬液药品。检测灭活屎肠球菌静脉给药对腹水瘤小鼠体重及脾指数的影响。将体重为18-22g的ICR小鼠分为5组，即腹水瘤模型组、胸腺五肽组和高、中、低三个剂量的灭活屎肠球菌给药组，每组10只腹水瘤模型小鼠。腹水瘤模型组小鼠尾静脉注射无菌生理盐水，胸腺五肽组尾静脉注射0.2mg/kg剂量的胸腺五肽，高、中、低三个剂量的灭活屎肠球菌给药组分别尾静脉注射5×、1×和0.2×的灭活屎肠球菌混悬液，给药体积均为0.1mL/10g。制备EAC艾氏腹水瘤小鼠模型前一天给药1次，即给药后间隔24小时，小鼠腹腔接种艾氏腹水瘤细胞悬液，制备EAC艾氏腹水瘤小鼠模型。此后再连续给药7天，测定腹水瘤小鼠的体增重及脾指数，脾指数＝脾脏重量/小鼠体重。采用SPSS 11.5软件对实验数据进行显著性检验，结果见表1。由表1可知，与腹水瘤模型组相比较，高剂量灭活屎肠球菌给药组显著地降低了腹水瘤小鼠的体增重。EAC艾氏腹水瘤小鼠模型由于腹水的形成，荷瘤小鼠的体重增长迅速，而本研究发现高剂量的灭活屎肠球菌尾静脉给药可减缓荷瘤小鼠体重的增长速度，与抑制腹水生成有关。此外，高剂量和中剂量两个灭活屎肠球菌给药组的腹水瘤小鼠的脾指数比腹水瘤模型组分别极显著和显著地增加。以上说明，灭活屎肠球菌静脉给药还可以增强腹水瘤小鼠的免疫功能。
表1 灭活屎肠球菌静脉给药对腹水瘤小鼠体增重及脾指数的影响结果
注：**表示与腹水瘤模型组比较差异极显著P＜0.01，*表示与腹水瘤模型组比较差异显著P＜0.05；△△表示与胸腺五肽组比较差异极显著P＜0.01，△表示与胸腺五肽组比较差异显著P＜0.05；
实施例3
使用实施例2制备的5×、1×和0.2×浓度的灭活屎肠球菌混悬液，检测灭活屎肠球菌静脉给药对腹水瘤小鼠生存时间的影响。将体重为18-22g的ICR小鼠分为5组，即腹水瘤模型组、胸腺五肽组和高、中、低三个剂量的灭活屎肠球菌给药组，每组8只。腹水瘤模型组小鼠尾静脉注射无菌生理盐水，胸腺五肽组尾静脉注射0.2mg/kg剂量的胸腺五肽，高、中、低三个剂量的灭活屎肠球菌给药组分别尾静脉注射5×、1×和0.2×的灭活屎肠球菌混悬液，给药体积均为0.1mL/10g。制备EAC艾氏腹水瘤小鼠模型前一天给药1次，即给药后间隔24小时，小鼠腹腔接种艾氏腹水瘤细胞悬液，制备EAC艾氏腹水瘤小鼠模型。此后每隔两天给药一次，直至小鼠死亡。观察并记录小鼠生存天数，最后计算生命延长率。生命延长率＝(给药组平均生存天数-对照组平均生存天数)/对照组平均生存天数×100。采用SPSS11.5软件对实验数据进行显著性检验，结果见表2。由表2可知，与腹水瘤模型组相比较，高剂量和中剂量两个灭活屎肠球菌组分别极显著和显著地延长了小鼠的平均存活天数，其中高剂量给药组对腹水瘤小鼠的生命延长率可达34.5％。以上说明灭活乳酸菌静脉给药可以延长肿瘤动物的生存时间。
表2 灭活屎肠球菌静脉给药对腹水瘤小鼠生命延长率的影响结果
注：**表示与腹水瘤模型组比较差异极显著P＜0.01，*表示与腹水瘤模型组比较差异显著P＜0.05；△△表示与胸腺五肽组比较差异极显著P＜0.01，△表示与胸腺五肽组比较差异显著P＜0.05。
Claims (10)
1.灭活乳酸菌在制备防治肿瘤性疾病药物中的用途，其特征在于，对所述的灭活乳酸菌进行革兰氏染色，在油镜下观察，灭活乳酸菌保持完整菌体形态，药物为混悬剂或粉针剂，给药方式为注射给药。
2.根据权利要求1的用途，其特征在于，所述的乳酸菌选自乳杆菌属、肠球菌属、乳球菌属、双歧杆菌属、明串球菌属、链球菌属。
3.根据权利要求1所述的用途，其特征在于，所述乳酸菌选自乳酸乳球菌乳酸亚种(拉丁名称：Lactococcus lactis subsp.Lactis，保藏编号：CICC 6246)、植物乳杆菌植物亚种(拉丁名称：Lactobacillus plantarum subsp.Plantarum，保藏编号：CICC 6240)、长双歧杆菌(拉丁名称：Bifidobacterium longum，保藏编号：CICC 6196)、短乳杆菌(拉丁名称：Lactobacillus brevis，保藏编号：CICC 6239)、屎肠球菌(拉丁名称：Enterococcusfaecium，保藏编号：CICC 6049)。
4.如权利要求1所述的用途，其特征在于，灭活乳酸菌为一种或两种以上灭活乳酸菌混合物。
5.根据权利要求1所述的用途，其特征在于，利用高温高压灭活、紫外线灭活、化学试剂灭活或辐射灭活中的任一种灭活方法得到所述灭活乳酸菌。
6.根据权利要求1-5的用途，其特征在于，所述的注射给药为静脉注射给药。
7.根据权利要求6所述的用途，其特征在于，注射时，每ml剂量药物中包含灭活乳酸菌完整菌体数量为105—1012个。
8.根据权利要求7任一项所述的用途，其特征在于，所述防治肿瘤性疾病药物可以制成粉针剂或混悬注射剂。
9.根据权利要求8所述的注射剂，其特征在于，所述的注射剂由如下方法制备而成：将乳酸菌进行常规液体培养基培养12-36小时后，3000-5000转离心保留沉淀，再将沉淀充分清洗后，配制成所需浓度混悬液，于120-122℃、压力0.1-0.2MPa，灭活15-30min得到灭活乳酸菌注射剂。
10.根据权利要求1-9任一项所述的用途，其特征在于，所述肿瘤性疾病包括：鼻腔及鼻窦恶性肿瘤、鼻咽癌、口腔癌、喉癌、涎腺肿瘤、颅内肿瘤、甲状腺癌、舌癌、肺癌、食管癌、贲门癌、乳腺癌、纵膈肿瘤、胃癌、大肠癌、乙状结肠癌、直肠癌、肝癌、胰腺癌、壶腹周围癌、小肠恶性肿瘤、肾癌、前列腺癌、膀胱癌、睾丸恶性肿瘤、阴茎癌、子宫颈癌、子宫内膜癌、卵巢癌、恶性纤维组织细胞瘤、横纹肌肉瘤、滑膜肉瘤、皮肤癌、恶性黑色素瘤、骨肉瘤、尤文氏肉瘤、淋巴瘤、多发性骨髓瘤、急性白血病、慢性白血病、红细胞增多症、血小板增多症、骨髓增生异常综合症、骨髓纤维化疾病。
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| CN111004757A (zh) * | 2019-12-31 | 2020-04-14 | 深圳市沁帆科技有限公司 | 一种罗伊氏乳杆菌、微生物菌剂及食物制品 |
| CN111849805A (zh) * | 2020-06-17 | 2020-10-30 | 天津科技大学 | 一种具有抗宫颈癌作用的乳酸片球菌及应用 |
| CN114470008A (zh) * | 2022-01-26 | 2022-05-13 | 佛山市第一人民医院 | 短乳杆菌或罗伊氏乳杆菌在制备治疗卵巢早衰的产品中的应用 |
| CN115806901A (zh) * | 2022-09-01 | 2023-03-17 | 昆明医科大学 | 一株短乳杆菌及其抗宫颈癌应用 |
| CN117535175A (zh) * | 2023-10-12 | 2024-02-09 | 善恩康生物科技（苏州）有限公司 | 一种复合益生菌及其在制备预防或辅助治疗结直肠癌的产品中的应用 |
| CN118001305A (zh) * | 2024-04-09 | 2024-05-10 | 山东中科嘉亿生物工程有限公司 | 长双歧杆菌jblc-141在制备改善副肿瘤性血小板增多症药物中的应用 |
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| CN111004757A (zh) * | 2019-12-31 | 2020-04-14 | 深圳市沁帆科技有限公司 | 一种罗伊氏乳杆菌、微生物菌剂及食物制品 |
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| CN111849805A (zh) * | 2020-06-17 | 2020-10-30 | 天津科技大学 | 一种具有抗宫颈癌作用的乳酸片球菌及应用 |
| CN114470008A (zh) * | 2022-01-26 | 2022-05-13 | 佛山市第一人民医院 | 短乳杆菌或罗伊氏乳杆菌在制备治疗卵巢早衰的产品中的应用 |
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| CN117535175A (zh) * | 2023-10-12 | 2024-02-09 | 善恩康生物科技（苏州）有限公司 | 一种复合益生菌及其在制备预防或辅助治疗结直肠癌的产品中的应用 |
| CN117535175B (zh) * | 2023-10-12 | 2024-05-31 | 善恩康生物科技（苏州）有限公司 | 一种复合益生菌及其在制备预防或辅助治疗结直肠癌的产品中的应用 |
| CN118001305A (zh) * | 2024-04-09 | 2024-05-10 | 山东中科嘉亿生物工程有限公司 | 长双歧杆菌jblc-141在制备改善副肿瘤性血小板增多症药物中的应用 |
| CN118001305B (zh) * | 2024-04-09 | 2024-06-11 | 山东中科嘉亿生物工程有限公司 | 长双歧杆菌jblc-141在制备改善副肿瘤性血小板增多症药物中的应用 |
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