JP2022033444A - Germ, il10 gene expression promoting agent, and pharmaceutical preparation - Google Patents
Germ, il10 gene expression promoting agent, and pharmaceutical preparation Download PDFInfo
- Publication number
- JP2022033444A JP2022033444A JP2020137331A JP2020137331A JP2022033444A JP 2022033444 A JP2022033444 A JP 2022033444A JP 2020137331 A JP2020137331 A JP 2020137331A JP 2020137331 A JP2020137331 A JP 2020137331A JP 2022033444 A JP2022033444 A JP 2022033444A
- Authority
- JP
- Japan
- Prior art keywords
- gene expression
- lactobacillus
- preparation
- gene
- strain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 230000014509 gene expression Effects 0.000 title claims abstract description 139
- 239000003795 chemical substances by application Substances 0.000 title description 3
- 239000000825 pharmaceutical preparation Substances 0.000 title description 2
- 230000001737 promoting effect Effects 0.000 title 1
- 101150085950 IL10 gene Proteins 0.000 claims abstract description 89
- 241000218492 Lactobacillus crispatus Species 0.000 claims abstract description 13
- 238000002360 preparation method Methods 0.000 claims description 65
- 241000894006 Bacteria Species 0.000 claims description 56
- 239000012228 culture supernatant Substances 0.000 claims description 24
- 239000003623 enhancer Substances 0.000 claims description 15
- 239000004480 active ingredient Substances 0.000 claims description 11
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 7
- 241001561398 Lactobacillus jensenii Species 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 abstract description 29
- 239000007788 liquid Substances 0.000 abstract description 24
- 230000001580 bacterial effect Effects 0.000 abstract description 10
- 208000023275 Autoimmune disease Diseases 0.000 abstract description 5
- 206010028980 Neoplasm Diseases 0.000 abstract description 4
- 208000027866 inflammatory disease Diseases 0.000 abstract description 4
- 230000009385 viral infection Effects 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 58
- 239000000243 solution Substances 0.000 description 26
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 21
- 238000002474 experimental method Methods 0.000 description 20
- 238000004519 manufacturing process Methods 0.000 description 19
- 241000186660 Lactobacillus Species 0.000 description 18
- 229940039696 lactobacillus Drugs 0.000 description 18
- 108090000623 proteins and genes Proteins 0.000 description 18
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 17
- 238000003753 real-time PCR Methods 0.000 description 16
- 239000000047 product Substances 0.000 description 14
- 239000011550 stock solution Substances 0.000 description 14
- 102000003814 Interleukin-10 Human genes 0.000 description 13
- 108090000174 Interleukin-10 Proteins 0.000 description 13
- 239000002609 medium Substances 0.000 description 13
- 239000000203 mixture Substances 0.000 description 12
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 11
- 238000011529 RT qPCR Methods 0.000 description 11
- 230000000694 effects Effects 0.000 description 11
- 239000002299 complementary DNA Substances 0.000 description 10
- 239000006071 cream Substances 0.000 description 10
- 241000186605 Lactobacillus paracasei Species 0.000 description 9
- 230000001965 increasing effect Effects 0.000 description 9
- 239000003826 tablet Substances 0.000 description 9
- 240000001929 Lactobacillus brevis Species 0.000 description 8
- 235000013957 Lactobacillus brevis Nutrition 0.000 description 8
- 230000003110 anti-inflammatory effect Effects 0.000 description 8
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 241000186000 Bifidobacterium Species 0.000 description 7
- 239000002775 capsule Substances 0.000 description 7
- 230000002708 enhancing effect Effects 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 241000894007 species Species 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 102000007469 Actins Human genes 0.000 description 5
- 108010085238 Actins Proteins 0.000 description 5
- 102000004127 Cytokines Human genes 0.000 description 5
- 108090000695 Cytokines Proteins 0.000 description 5
- 206010052428 Wound Diseases 0.000 description 5
- 208000027418 Wounds and injury Diseases 0.000 description 5
- 238000004925 denaturation Methods 0.000 description 5
- 230000036425 denaturation Effects 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 235000011187 glycerol Nutrition 0.000 description 5
- 210000002540 macrophage Anatomy 0.000 description 5
- 239000002207 metabolite Substances 0.000 description 5
- 239000006872 mrs medium Substances 0.000 description 5
- 239000008188 pellet Substances 0.000 description 5
- 238000011002 quantification Methods 0.000 description 5
- 238000010839 reverse transcription Methods 0.000 description 5
- 108020004465 16S ribosomal RNA Proteins 0.000 description 4
- 206010003645 Atopy Diseases 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- 238000012300 Sequence Analysis Methods 0.000 description 4
- 229920002472 Starch Polymers 0.000 description 4
- 229940121363 anti-inflammatory agent Drugs 0.000 description 4
- 239000002260 anti-inflammatory agent Substances 0.000 description 4
- 239000012141 concentrate Substances 0.000 description 4
- 235000008504 concentrate Nutrition 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 239000003995 emulsifying agent Substances 0.000 description 4
- 235000019441 ethanol Nutrition 0.000 description 4
- 239000003205 fragrance Substances 0.000 description 4
- 230000031261 interleukin-10 production Effects 0.000 description 4
- 239000004310 lactic acid Substances 0.000 description 4
- 235000014655 lactic acid Nutrition 0.000 description 4
- 239000006210 lotion Substances 0.000 description 4
- 239000003755 preservative agent Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 208000017520 skin disease Diseases 0.000 description 4
- 239000008107 starch Substances 0.000 description 4
- 235000019698 starch Nutrition 0.000 description 4
- 210000001215 vagina Anatomy 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 208000036142 Viral infection Diseases 0.000 description 3
- 230000035606 childbirth Effects 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 230000002757 inflammatory effect Effects 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 230000002335 preservative effect Effects 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 241001608472 Bifidobacterium longum Species 0.000 description 2
- 240000006024 Lactobacillus plantarum Species 0.000 description 2
- 235000013965 Lactobacillus plantarum Nutrition 0.000 description 2
- 239000004264 Petrolatum Substances 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 235000013361 beverage Nutrition 0.000 description 2
- 229940009291 bifidobacterium longum Drugs 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 239000002537 cosmetic Substances 0.000 description 2
- 238000010195 expression analysis Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 229940076144 interleukin-10 Drugs 0.000 description 2
- 229940072205 lactobacillus plantarum Drugs 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- 238000000465 moulding Methods 0.000 description 2
- 239000004006 olive oil Substances 0.000 description 2
- 235000008390 olive oil Nutrition 0.000 description 2
- 235000019271 petrolatum Nutrition 0.000 description 2
- 229940066842 petrolatum Drugs 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000304886 Bacilli Species 0.000 description 1
- 241000186018 Bifidobacterium adolescentis Species 0.000 description 1
- 241000286268 Bifidobacterium bifidum ATCC 29521 = JCM 1255 = DSM 20456 Species 0.000 description 1
- 241000741973 Bifidobacterium breve DSM 20213 = JCM 1192 Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000700721 Hepatitis B virus Species 0.000 description 1
- 101000831567 Homo sapiens Toll-like receptor 2 Proteins 0.000 description 1
- 240000001046 Lactobacillus acidophilus Species 0.000 description 1
- 235000013956 Lactobacillus acidophilus Nutrition 0.000 description 1
- 241001104368 Lactobacillus casei DSM 20011 = JCM 1134 Species 0.000 description 1
- 241000186673 Lactobacillus delbrueckii Species 0.000 description 1
- 240000002605 Lactobacillus helveticus Species 0.000 description 1
- 235000013967 Lactobacillus helveticus Nutrition 0.000 description 1
- 241000218588 Lactobacillus rhamnosus Species 0.000 description 1
- 241001104462 Lactobacillus rhamnosus DSM 20021 = JCM 1136 = NBRC 3425 Species 0.000 description 1
- 241000186869 Lactobacillus salivarius Species 0.000 description 1
- 241000186783 Lactobacillus vaginalis Species 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 102100024333 Toll-like receptor 2 Human genes 0.000 description 1
- 208000032159 Vaginal inflammation Diseases 0.000 description 1
- 201000008100 Vaginitis Diseases 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- LFYJSSARVMHQJB-QIXNEVBVSA-N bakuchiol Chemical compound CC(C)=CCC[C@@](C)(C=C)\C=C\C1=CC=C(O)C=C1 LFYJSSARVMHQJB-QIXNEVBVSA-N 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 230000019734 interleukin-12 production Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 229940039695 lactobacillus acidophilus Drugs 0.000 description 1
- 229940054346 lactobacillus helveticus Drugs 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 241001624918 unidentified bacterium Species 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Images
Abstract
Description
本発明は、ラクトバチルス・クリスパタスに属する細菌並びにその細菌を用いたインターロイキン１０（以下「ＩＬ１０」ともいう）遺伝子発現亢進剤並びにその細菌を用いた皮膚外用剤及び経口剤等の製剤に関する。 The present invention relates to a bacterium belonging to Lactobacillus chryspatus, an interleukin 10 (hereinafter, also referred to as “IL10”) gene expression enhancer using the bacterium, and a preparation of an external skin preparation and an oral preparation using the bacterium.
妊婦による出産は自然の理であり病気ではなく、出産の際に産道が傷つくことがあっても直ぐに修復される。このように妊婦の産道には出産時に特異な機能を備えていると考えられるが、産道の疾患に関する研究及び情報はあるものの健常人、とりわけ妊婦の産道に着目した研究及び情報はなかなか見つけることができなかった。目に付くのは、産道炎症に関する商品、又は産道にも適用できる粘膜の治療薬である。例えば、膣等の粘膜へも適用可能な治療薬に関する技術は特表２０１４－５１００９０号公報（特許文献１）に記載されている。 Childbirth by a pregnant woman is a natural reason and is not a disease, and even if the birth canal is damaged during childbirth, it will be repaired immediately. In this way, it is thought that the birth canal of pregnant women has a unique function during childbirth, but although there is research and information on diseases of the birth canal, it is difficult to find research and information focusing on the birth canal of healthy people, especially pregnant women. could not. What is noticeable is a product related to vaginal inflammation or a mucosal remedy that can also be applied to the vagina. For example, a technique relating to a therapeutic agent applicable to mucous membranes such as vagina is described in Japanese Patent Application Laid-Open No. 2014-511090 (Patent Document 1).
しかしながら、上記のように妊婦の産道には傷を直ぐに修復できるような治癒力があり、これは妊婦における産道内菌叢によってもたらされるものではないかと本発明者らは推測し、産道内菌叢の研究を行った。本発明はこうした研究の成果として生まれたものである。 However, as described above, the present inventors speculate that the birth canal of a pregnant woman has a healing power that can immediately repair the wound, and that this may be caused by the intestinal flora in the pregnant woman. I did a study of. The present invention was born as a result of such research.
本発明の一態様は、ＩＬ１０遺伝子発現を亢進させるデーデルライン桿菌である細菌である。本発明の一態様が、ＩＬ１０遺伝子発現を亢進させるデーデルライン桿菌である細菌であるため、ＩＬ１０遺伝子の発現を亢進させることができるヒトの産道から取得できる細菌である。 One aspect of the present invention is a bacterium that is a Daederlein bacillus that enhances IL10 gene expression. Since one aspect of the present invention is a bacterium that is a Daederlein bacillus that enhances the expression of the IL10 gene, it is a bacterium that can be obtained from the human birth canal that can enhance the expression of the IL10 gene.
本発明の一態様は、前記デーデルライン桿菌が、ＩＬ１０遺伝子発現を亢進させる受託番号：NITE P-03215として寄託されたラクトバチルス・クリスパタス（Lactobacillus crispatus）種に属するＤＬ７２菌株（細菌）である。 One aspect of the present invention is a DL72 strain (bacteria) belonging to the Lactobacillus crispatus species deposited under the accession number: NITE P-03215 in which the Döderlein's bacillus enhances IL10 gene expression.
本発明の一態様は、前記デーデルライン桿菌が、ＩＬ１０遺伝子発現を亢進させる受託番号：NITE P-03215として寄託されたラクトバチルス・クリスパタス（Lactobacillus crispatus）種に属するＤＬ７２菌株（細菌）であるため、ヒトの産道から取得できるラクトバチルス・クリスパタス種に属する細菌が、ＩＬ１０遺伝子の発現を亢進させることができる。 One aspect of the present invention is that the Daederlein bacillus is a DL72 strain (bacteria) belonging to the Lactobacillus crispatus species deposited under the accession number: NITE P-03215 that enhances IL10 gene expression. , Bacteria belonging to the Lactobacillus chryspatus species that can be obtained from the human birth canal can enhance the expression of the IL10 gene.
本発明の一態様は、ＩＬ１０遺伝子発現を亢進させるラクトバチルス・クリスパタス（Lactobacillus crispatus）、ラクトバチルス・ジェンセニイ(Lactobacillus jensenii)の組合せからなる細菌である。
本発明の一態様を、ＩＬ１０遺伝子発現を亢進させるラクトバチルス・クリスパタス（Lactobacillus crispatus）、ラクトバチルス・ジェンセニイ(Lactobacillus jensenii)の組合せからなる細菌としたため、ヒトの産道から取得できるラクトバチルス・クリスパタス（Lactobacillus crispatus）、ラクトバチルス・ジェンセニイ(Lactobacillus jensenii)の少なくとも何れかの種に属する菌株の組合せからなる細菌群が、ＩＬ１０遺伝子の発現を１０倍以上亢進させることができる。
One aspect of the present invention is a bacterium consisting of a combination of Lactobacillus crispatus and Lactobacillus jensenii that enhances IL10 gene expression.
Since one aspect of the present invention is a bacterium consisting of a combination of Lactobacillus crispatus and Lactobacillus jensenii that enhance IL10 gene expression, Lactobacillus can be obtained from the human birth canal. A bacterial group consisting of a combination of strains belonging to at least one of crispatus ) and Lactobacillus jensenii can enhance the expression of the IL10 gene by 10 times or more.
本発明の一態様は、上記細菌の培養上清を主成分として含有するＩＬ１０遺伝子発現亢進剤である。
本発明の一態様を、上記細菌の培養上清を主成分として含有するＩＬ１０遺伝子発現亢進剤としたため、ＩＬ１０遺伝子発現により、炎症性疾患、自己免疫疾患、ウイルス感染、腫瘍等による疾患を予防し、又は治療することができる。
One aspect of the present invention is an IL10 gene expression enhancer containing the culture supernatant of the bacterium as a main component.
Since one aspect of the present invention is an IL10 gene expression enhancer containing the culture supernatant of the above bacteria as a main component, IL10 gene expression prevents diseases caused by inflammatory diseases, autoimmune diseases, viral infections, tumors and the like. , Or can be treated.
本発明の一態様は、上記細菌を有効成分として含有するＩＬ１０遺伝子発現亢進剤である。
本発明の一態様を、上記細菌を有効成分として含有するＩＬ１０遺伝子発現亢進剤としたため、ＩＬ１０遺伝子発現により、炎症性疾患、自己免疫疾患、ウイルス感染、腫瘍等による疾患を予防し、又は治療することができる。
One aspect of the present invention is an IL10 gene expression enhancer containing the above-mentioned bacterium as an active ingredient.
Since one aspect of the present invention is an IL10 gene expression enhancer containing the above bacteria as an active ingredient, IL10 gene expression prevents or treats diseases caused by inflammatory diseases, autoimmune diseases, viral infections, tumors and the like. be able to.
本発明の一態様は、上記何れかの細菌又は上記何れかのＩＬ１０遺伝子発現亢進剤を有効成分として含有する製剤である。
本発明の一態様を、上記何れかの細菌又は上記何れかのＩＬ１０遺伝子発現亢進剤を有効成分として含有する製剤としたため、抗炎症作用剤として利用することができる。例えば医薬製剤、化粧品、食品、飲料等に利用できる。
One aspect of the present invention is a preparation containing any of the above-mentioned bacteria or any of the above-mentioned IL10 gene expression enhancer as an active ingredient.
Since one aspect of the present invention is a preparation containing any of the above bacteria or any of the above IL10 gene expression enhancer as an active ingredient, it can be used as an anti-inflammatory agent. For example, it can be used for pharmaceutical preparations, cosmetics, foods, beverages and the like.
本発明の一態様は、上記細菌又は上記ＩＬ１０遺伝子発現亢進剤を有効成分として含有する皮膚外用剤である。
本発明の一態様を、上記細菌又は上記ＩＬ１０遺伝子発現亢進剤を有効成分として含有する皮膚外用剤としたため、抗炎症作用剤として利用することができ、より具体的にはデリケートゾーンのケア製品、産後のケア製品、又はスキンケア製品等のクリーム製品、化粧液等として利用することができる。
One aspect of the present invention is a skin external preparation containing the bacterium or the IL10 gene expression enhancer as an active ingredient.
Since one aspect of the present invention is a skin external preparation containing the above-mentioned bacteria or the above-mentioned IL10 gene expression enhancer as an active ingredient, it can be used as an anti-inflammatory agent, and more specifically, a delicate zone care product. It can be used as a postpartum care product, a cream product such as a skin care product, a cosmetic solution, or the like.
本発明の一態様は、上記細菌又は上記ＩＬ１０遺伝子発現亢進剤を有効成分として含有する経口剤である。
本発明の一態様を、上記細菌又は上記ＩＬ１０遺伝子発現亢進剤を有効成分として含有する経口剤としたため、抗炎症作用剤として利用することができ、より具体的には医薬品、食品、飲料等となる錠剤、散剤、カプセル剤、シロップ剤、ドリンク剤等として利用することができる。
One aspect of the present invention is an oral preparation containing the above-mentioned bacteria or the above-mentioned IL10 gene expression enhancer as an active ingredient.
Since one aspect of the present invention is an oral preparation containing the above-mentioned bacteria or the above-mentioned IL10 gene expression enhancer as an active ingredient, it can be used as an anti-inflammatory agent, and more specifically, with pharmaceuticals, foods, beverages and the like. Can be used as tablets, powders, capsules, syrups, drinks and the like.
本発明によれば、ＩＬ１０遺伝子の発現を亢進させることができる。 According to the present invention, the expression of the IL10 gene can be enhanced.
抗炎症サイトカインであるインターロイキン１０（以下「ＩＬ１０」という）の遺伝子発現を亢進する技術について詳しく説明する。より具体的にはＩＬ１０の遺伝子発現を亢進する細菌と、この細菌から得られる上清と、この細菌を含む混合液と、これらを用いたＩＬ１０遺伝子発現亢進剤と、これらを用いた皮膚外用剤及び経口剤等の製剤である。 The technique for enhancing the gene expression of interleukin 10 (hereinafter referred to as "IL10"), which is an anti-inflammatory cytokine, will be described in detail. More specifically, a bacterium that enhances the gene expression of IL10, a supernatant obtained from this bacterium, a mixed solution containing this bacterium, an IL10 gene expression enhancer using these, and an external skin agent using these. And preparations such as oral preparations.
好適な実施形態において、ＩＬ１０遺伝子発現を亢進する細菌にはラクトバチルス・クリスパタス（Lactobacillus crispatus）種に属する細菌が挙げられ、特にＤＬ７２株がより好適に挙げられる。このＤＬ７２株は、独立行政法人製品評価技術基盤機構特許微生物寄託センター（千葉県木更津市かずさ鎌足２－５－８）に、受託番号：NITE P-03215、受託日：2020年4月30日として寄託されている。また、この菌株の１６Ｓ ｒＲＮＡ塩基配列を配列番号１として配列表に示す。 In a preferred embodiment, the bacterium that enhances IL10 gene expression includes a bacterium belonging to the Lactobacillus crispatus species, and the DL72 strain is more preferably mentioned. This DL72 strain was sent to the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture) with a consignment number: NITE P-03215 and a consignment date: April 30, 2020. Has been deposited as. The 16S rRNA base sequence of this strain is shown in the sequence listing as SEQ ID NO: 1.
ＤＬ７２株については、その１６Ｓ ｒＲＮＡ遺伝子の塩基配列をデータベース上の配列と相同性検索を行って種を同定したところ、相同値が９８％以上であったラクトバチルス・クリスパタスに属する菌株であることがわかった。なお、ＤＬ７２株の菌学的特徴は、ラクトバチルス・クリスパタスの菌学的特徴と同じである。 Regarding the DL72 strain, when the nucleotide sequence of the 16S rRNA gene was homologously searched with the sequence on the database to identify the species, it was found to be a strain belonging to Lactobacillus crispatus whose homology value was 98% or more. all right. The mycological characteristics of the DL72 strain are the same as those of Lactobacillus crispatus.
ＤＬ７２株の菌体は、通常の乳酸菌培養の手法によって培養することができ、培養物からの遠心分離等の手段によって分離、精製した菌体及び上清を利用することができる。
ＤＬ７２株の菌体、又は菌体から調製した上清は、賦形剤、崩壊剤、結合剤、安定化剤、湿潤剤等と適宜混合して、錠剤、顆粒剤、散剤、カプセル剤、溶液剤、乳剤、クリーム等として、皮膚外用剤や経口剤等の製剤として調製することができる。
The cells of the DL72 strain can be cultivated by a conventional method of culturing lactic acid bacteria, and the cells and supernatant separated and purified by means such as centrifugation from the culture can be used.
The cells of the DL72 strain or the supernatant prepared from the cells are appropriately mixed with excipients, disintegrants, binders, stabilizers, wetting agents and the like to form tablets, granules, powders, capsules and solutions. It can be prepared as a preparation such as an external preparation for skin or an oral preparation as an agent, an emulsion, a cream or the like.
そしてこれらの製剤は、抗炎症作用剤として、ＩＬ１０の産生不足による場合のみならず、そうした場合に該当しない炎症性疾患、自己免疫疾患、ウイルス感染、腫瘍等による疾患を予防し、又は治療に用いることができる。 And these preparations are used as an anti-inflammatory agent not only for the case of insufficient production of IL10 but also for the prevention or treatment of diseases caused by inflammatory diseases, autoimmune diseases, viral infections, tumors, etc. which do not correspond to such cases. be able to.
実験１：＜産道内に特有の細菌の取得＞
健常妊婦の産道内から採取した採取液をＢＬ寒天培地にて嫌気培養（３７℃、２日間）し、形成された代表的なコロニーを乳酸桿菌用のＭＲＳ液体培地に移してさらに嫌気培養（３７℃、２日間）した後、－８０℃下でグリセロールストックした。このようにしてストックした細菌からＤＮＡを抽出し、シーケンス解析に供した。
Experiment 1: <Acquisition of bacteria peculiar to the birth canal>
The collected liquid collected from the birth canal of a healthy pregnant woman was anaerobically cultured on BL agar medium (37 ° C. for 2 days), and the formed representative colonies were transferred to an MRS liquid medium for lactic acid bacilli and further anaerobically cultured (37 ° C.). After 2 days), glycerol was stocked at −80 ° C. DNA was extracted from the bacteria stocked in this way and used for sequence analysis.
より具体的には、ストックしていた各菌株から形成されるコロニーをピペットチップで掻き取り、ｄＨ２Ｏ ５０μＬに懸濁し、１００ｍＭのＮａＯＨを５０μＬ添加して、混合後、９５℃で１５分間処理を行なった。その後、１ＭＴｒｉｓ－ＨＣｌ（ｐＨ７．０）を１１μＬ添加し、遠心後、上清を回収してＤＮＡ液とした。得られたＤＮＡ液をテンプレートに、Ｂａｃｔｅｒｉａｌ １６Ｓ ｒＤＮＡ ＰＣＲ Ｋｉｔ（タカラバイオ社製）を用いて、メーカーのプロトコールに従って、シーケンス解析に供するＰＣＲ増幅産物を得た。 More specifically, colonies formed from each stocked strain are scraped off with a pipette tip, suspended in 50 μL of dH 2 O, 50 μL of 100 mM NaOH is added, mixed, and then treated at 95 ° C. for 15 minutes. Was done. Then, 11 μL of 1M Tris-HCl (pH 7.0) was added, and after centrifugation, the supernatant was collected and used as a DNA solution. Using the obtained DNA solution as a template, a PCR amplification product to be used for sequence analysis was obtained using a Bacteria 16S rDNA PCR Kit (manufactured by Takara Bio Inc.) according to the manufacturer's protocol.
ＰＣＲ増幅産物の一部をアガロースゲル電気泳動（１００Ｖ、３０分間）に供し、目的とする増幅産物が得られているのかを確認した後、ＱＩＡｑｕｉｃｋ ＰＣＲ Ｐｕｒｉｆｉｃａｔｉｕｏｎ Ｋｉｔ（ＱＩＡＧＥＮ社製）によってメーカーのプロトコール従って、ＰＣＲ反応液からＰＣＲ増幅産物の精製を行なった。 Part of the PCR amplification product was subjected to agarose gel electrophoresis (100V, 30 minutes) to confirm that the desired amplification product was obtained, and then the manufacturer's protocol was followed by the QIAquick PCR Purification Kit (manufactured by QIAGEN). , The PCR amplification product was purified from the PCR reaction solution.
ＰＣＲ増幅産物のシーケンス解析から得られた塩基配列を、配列編集ソフトのＡｑＥを用いて編集を行い、各コロニーの１６Ｓ ｒＲＮＡ遺伝子の塩基配列をデータベース上の配列と相同性検索を行って種を同定した。データベースとしては、ＮＣＢＩ ＢＬＡＳＴ(http://blast.ncbi.nlm.nih.gov/Blast.cgi)とＤＤＢＪ(http://blast.ddbj.nig.ac.jp/top-j.hyml)の２つのデータベースを用いた。結果として、ラクトバチルス・クリスパタス(Lactobacillus crispatus)、ラクトバチルス・ジェンセニイ(Lactobacillus jensenii)、ラクトバチルス・ヴァギナリス(Lactobacillus vaginalis)、ラクトバチルス・フェカリス(Lactobacillus faecalis)、ラクトバチルス・ガセリ(Lactobacillus gasseri)、ラクトバチルス・プランタラム (Lactobacillus plantarum)、ラクトバチルス・ラムノサス(Lactobacillus rhamnosus)、その他未同定のラクトバチルス属に属する細菌が認められた。 The nucleotide sequence obtained from the sequence analysis of the PCR amplification product is edited using the sequence editing software AqE, and the nucleotide sequence of the 16S rRNA gene of each colony is subjected to homology search with the sequence on the database to identify the species. did. There are two databases, NCBI BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and DDBJ (http://blast.ddbj.nig.ac.jp/top-j.hyml). Two databases were used. As a result, Lactobacillus crispatus , Lactobacillus jensenii , Lactobacillus vaginalis , Lactobacillus faecalis, Lactobacillus faecalis , Lactobacillus faecalis, Lactobacillus faecalis, Lactobacillus faecalis・ Plantalum ( Lactobacillus plantarum ), Lactobacillus rhamnosus , and other unidentified bacteria belonging to the genus Lactobacillus were found.
ＰＣＲによる菌株の同定や以下の実験で言及するリアルタイムＰＣＲによる遺伝子発現解析に用いたプライマーの配列情報を以下の表に示す。なお表中、参考文献ａは、Hepatitis B Virus Surface Antigen Selectively Inhibits TLR2 Ligand-Induced IL-12 Production in Monocytes/ Macrophages by Interfering with JNK Activationであり、参考文献ｂ～ｄは、ＯｒｉＧｅｎｅ社で公開している配列である。また、各プライマーの特異性はNCBI Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome)によって解析して確認した。 The table below shows the sequence information of the primers used for the identification of the strain by PCR and the gene expression analysis by real-time PCR referred to in the following experiments. In the table, reference a is Hepatitis B Virus Surface Antigen Selectively Inhibits TLR2 Ligand-Induced IL-12 Production in Monocytes / Macrophages by Interfering with JNK Activation, and references b to d are published by OriGene. It is an array. The specificity of each primer was analyzed and confirmed by NCBI Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome).
実験２：＜ＩＬ１０遺伝子発現＞
産道内に特有な細菌の代謝産物における抗炎症サイトカインＩＬ１０遺伝子の発現に対する作用を検討した。
産道内から取得した上記菌株のうち、産道内に特有の細菌であるデーデルライン桿菌（ＤＬ菌）６４菌株と、比較対象としてプロバイオティクスで有用とされるビフィドバクテリウム属５菌株、及びラクトバチルス属の１１菌株について、マクロファージ様細胞を標的にＩＬ１０遺伝子発現量への影響を調べた。なお、ヒト単球由来の細胞株であるＴＨＰ－１細胞は広く免疫分野の研究で用いられている細胞株であり、薬剤処理によってマクロファージ様に分化誘導することが知られている。ＴＨＰ－１細胞から分化誘導させたマクロファージ様細胞は、Ｍ１型とＭ２型の特徴を併せ持った形質を示すことから、ここでもＴＨＰ－１細胞を用い、ＰＭＡ濃度を２００ｎＭ、処理時間を３日間とした条件で分化誘導させたマクロファージ様細胞を用いた。
Experiment 2: <IL10 gene expression>
The effect on the expression of the anti-inflammatory cytokine IL10 gene in bacterial metabolites peculiar to the birth canal was investigated.
Among the above strains obtained from the birth canal, 64 strains of Daederlein bacillus (DL bacterium), which is a bacterium peculiar to the birth canal, 5 strains of the genus Bifidobacterium, which are useful in probiotics for comparison, and The effects of 11 strains of Lactobacillus on the expression level of the IL10 gene were investigated by targeting macrophage-like cells. The THP-1 cell, which is a cell line derived from human monocytes, is a cell line widely used in research in the field of immunity, and is known to induce macrophage-like differentiation by drug treatment. Since macrophage-like cells induced to differentiate from THP-1 cells show traits having both M1 type and M2 type characteristics, THP-1 cells are also used here, with a PMA concentration of 200 nM and a treatment time of 3 days. Macrophage-like cells induced to differentiate under the above conditions were used.
比較対象に用いたビフィドバクテリウム属の５菌株はそれぞれ、Bifidobacterium longum JCM 1217、Bifidobacterium bifidum JCM 1255、Bifidobacterium adolescentis JCM 1275、Bifidobacterium breve JCM 1192、Bifidobacterium longum subsp. infantis JCM1210である。
また、比較対象のラクトバチルス属の１１菌株は、Lactobacillus acidophilus JCM 2124、Lactobacillus casei JCM 1134、Lactobacillus delbrueckii subsp. bulgaricus JCM 20398、Lactobacillus paracasei subsp. paracasei JCM 8130、Lactobacillus delbrueckii JCM 1012、Lactobacillus gasseri JCM 5344、Lactobacillus brevis JCM 1059、Lactobacillus helveticus JCM 1120、Lactobacillus plantarum JCM 11125、Lactobacillus salivarius JCM 1040、Lactobacillus rhamnosus JCM 1136である。
The five strains of the genus Bifidobacterium used for comparison are Bifidobacterium longum JCM 1217 , Bifidobacterium bifidum JCM 1255 , Bifidobacterium adolescentis JCM 1275 , Bifidobacterium breve JCM 1192 , and Bifidobacterium longum subsp. Infantis JCM 1210, respectively.
The 11 strains of the genus Lactobacillus to be compared are Lactobacillus acidophilus JCM 2124 , Lactobacillus casei JCM 1134 , Lactobacillus delbrueckii subsp. Bulgaricus JCM 20398 , Lactobacillus paracasei subsp. Paracasei JCM 8130 , Lactobacillus . brevis JCM 1059 , Lactobacillus helveticus JCM 1120 , Lactobacillus plantarum JCM 11125 , Lactobacillus salivarius JCM 1040 , Lactobacillus rhamnosus JCM 1136 .
上記菌株について、それぞれＭＲＳ液体培地にて嫌気培養（３７℃、２日間）し、得られた培養液を遠心（１００００ｇ、５分間、室温）し、上清を回収後、０．２２μｍシリンジフィルターで菌体や交雑物の除去を行い清澄化させた。次に、清澄化した上清はＡｍｉｃｏｎ－Ｕｌｔｒａ ０．５ ３Ｋ（Ｍｉｌｌｉｐｏｒｅ社製）によって限外濾過して５倍濃縮させ、その濃縮液が２５倍希釈される濃度になる様にマクロファージ様細胞の培養液へ添加して２４時間処理した。 Each of the above strains was anaerobically cultured in MRS liquid medium (37 ° C. for 2 days), the obtained culture solution was centrifuged (10000 g, 5 minutes, room temperature), the supernatant was collected, and then a 0.22 μm syringe filter was used. The cells and hybrids were removed and clarified. Next, the clarified supernatant is ultra-filtered with Amicon-Ultra 0.5 3K (manufactured by Millipore) and concentrated 5 times, and the concentration of the macrophage-like cells is diluted 25 times. It was added to the culture broth and treated for 24 hours.
上清濃縮液を処理したマクロファージ様細胞から、ＩＳＯＧＥＮ ＩＩ（日本ジーン社製）によってＲＮＡを抽出し、ＡｃｃｕＲＴ ＧｅｎｏｍｉｃＤＮＡ Ｒｅｍｏｖａｌ Ｋｉｔ（ａｂｍ社製）でゲノムＤＮＡを除去した後、ＲｅｖｅｒＴｒａ Ａｃｅ ｑＰＣＲ ＲＴ Ｍａｓｔｅｒ Ｍｉｘ（ＴＯＹＯＢＯ社製）で逆転写を行い、ｃＤＮＡを合成した。これをテンプレートにＴＨＵＮＤＥＲＢＩＲＤ ＳＹＢＲ ｑＰＣＲ Ｍｉｘ（ＴＯＹＯＢＯ社製）を用いて、ＣＦＸ ＣｏｎｎｅｃｔＴＭ Ｒｅａｌ－Ｔｉｍｅ ＰＣＲ Ｄｅｔｅｃｔｉｏｎ Ｓｙｓｔｅｍ（Ｂｉｏ－Ｒａｄ社製）でリアルタイムＰＣＲを実施した。リアルタイムＰＣＲの反応条件は、９８℃・１分間で初期変性させた後、９５℃・１５秒、６０℃・３０秒を４０サイクル反応させた。遺伝子発現の定量は、リファレンス遺伝子としてｂｅｔａ－ａｃｔｉｎを用いて遺伝子発現量を補正し、ΔΔＣｔ法によってコントロールの遺伝子発現量から算出した相対値を求め、図１で示すグラフに表した。 RNA was extracted from macrophage-like cells treated with the supernatant concentrate by ISOGEN II (manufactured by Japan Gene), genomic DNA was removed by AccuRT Genomi cDNA Removal Kit (manufactured by Abm), and then ReverseTra Ace qPCR RT Master Mix (manufactured by Abm). Reverse transcription was performed with TOYOBO) to synthesize cDNA. Using this as a template, real-time PCR was performed by CFX ConnectTM Real-Time PCR Detection System (manufactured by Bio-Rad) using THUNDERBIRD SYBR qPCR Mix (manufactured by TOYOBO). The reaction conditions for real-time PCR were initial denaturation at 98 ° C. for 1 minute, followed by 40 cycles of reaction at 95 ° C. for 15 seconds and 60 ° C. for 30 seconds. For the quantification of gene expression, the gene expression level was corrected using beta-actin as a reference gene, and the relative value calculated from the control gene expression level by the ΔΔCt method was obtained and shown in the graph shown in FIG.
図１において、縦軸に各菌株の遺伝子発現量の相対値を示す。横軸については、「ＭＲＳ」が培地のみのコントロールであり、「Bifidobacterium」がビフィドバクテリウム属であり、「Lactobacillus」がラクトバチルス属であり、「ＤＬ」が産道内に特有の細菌（上記６４菌株）であって、グラフ中のドットが各菌株の遺伝子発現量の相対値を示し、また、グラフ中のバーは各種ごとの菌株の遺伝子発現量の相対値の平均値を示した。その結果、産道内から採取した上記６４菌株の産道内特有の細菌の平均値は、比較のためのビフィドバクテリウム属５菌株の平均値、及びラクトバチルス属１１菌株の平均値と比較してＩＬ１０遺伝子の発現を有意に上昇させることが見出された。また、６４菌株の中にはＩＬ１０遺伝子発現量を１０倍以上に上昇させる菌株が多く存在していることもわかった。こうしたことから、産道内に特有の細菌のいくつかは、マクロファージからのＩＬ１０産生を亢進させる。 In FIG. 1, the vertical axis shows the relative value of the gene expression level of each strain. On the horizontal axis, "MRS" is a medium-only control, "Bifidobacterium" is a genus Bifidobacterium, "Lactobacillus" is a genus Lactobacillus, and "DL" is a bacterium peculiar to the birth canal (above). 64 strains), dots in the graph indicate the relative value of the gene expression level of each strain, and bars in the graph indicate the average value of the gene expression level of each strain. As a result, the average value of the bacteria peculiar to the production canal of the above 64 strains collected from the production canal was compared with the average value of 5 strains of Bifidobacterium for comparison and the average value of 11 strains of Lactobacillus. It was found to significantly increase the expression of the IL10 gene. It was also found that among the 64 strains, there are many strains that increase the IL10 gene expression level by 10 times or more. Therefore, some of the bacteria peculiar to the birth canal enhance IL10 production from macrophages.
実験３：＜ＴＮＦａｌｐｈａ遺伝子発現＞
産道内に特有な細菌の代謝産物におけるＴＮＦａｌｐｈａ遺伝子の発現に対する作用を検討した。
ＩＬ１０遺伝子の発現に対する実験と同様に、上記産道内に特有の６４菌株と、比較対象としてプロバイオティクスで有用とされるビフィドバクテリウム属５菌株、及びラクトバチルス属の１１菌株を用い、マクロファージ様細胞を標的に炎症性サイトカインであるＴＮＦａｌｐｈａの遺伝子発現量への影響を上記と同様にして調べた。その結果を図２で示すグラフに表した。
Experiment 3: <TNFalpha gene expression>
The effect on the expression of the TNFalpha gene in the metabolites of bacteria peculiar to the birth canal was investigated.
Similar to the experiment for the expression of the IL10 gene, macrophages using 64 strains peculiar to the birth canal, 5 strains of Bifidobacterium spp. The effect of TNFalpha, an inflammatory cytokine, on the gene expression level was investigated by targeting like cells in the same manner as above. The results are shown in the graph shown in FIG.
図２の表示の仕方も図１と同様であり、グラフ中のドットは、各菌株のＴＮＦａｌｐｈａ遺伝子発現量の相対値を示す。また、各種ごとの菌株のＴＮＦａｌｐｈａ遺伝子発現量の平均値をバーで示した。
炎症性サイトカインであるＴＮＦａｌｐｈａの遺伝子発現については、ビフィドバクテリウム属５菌株、及びラクトバチルス属１１菌株と比較して、産道内特有の細菌６４種について、有意な差異は見られなかった。しかしながら、前記６４種のうちのいくつかの菌株については、コントロールと比較してＴＮＦａｌｐｈａの遺伝子発現を１０倍程度上昇させる菌株も認められたことから、ＴＮＦａｌｐｈａの遺伝子発現上昇によってパラクライン的にＩＬ１０遺伝子発現が引き起こされた可能性も考えられた。
The display method of FIG. 2 is the same as that of FIG. 1, and the dots in the graph indicate the relative value of the TNFalpha gene expression level of each strain. In addition, the average value of the TNFalpha gene expression level of each strain is shown by a bar.
Regarding the gene expression of TNFalpha, which is an inflammatory cytokine, no significant difference was observed in 64 species of bacteria peculiar to the birth canal as compared with 5 strains of the genus Bifidobacterium and 11 strains of the genus Lactobacillus. However, for some of the 64 strains, some strains increased the gene expression of TNFalpha by about 10 times as compared with the control. It was also possible that the expression was caused.
実験４：＜ＩＬ１０遺伝子発現とＴＮＦａｌｐｈａ遺伝子発現の相関＞
産道内から取得した６４菌株によるＩＬ１０遺伝子発現量とＴＮＦａｌｐｈａ遺伝子発現量との相関を調べた。縦軸にＴＮＦａｌｐｈａ遺伝子発現量を、横軸にＩＬ１０遺伝子発現量に設定して、各菌株の両遺伝子発現量をプロットして近似曲線を描写し、Ｒ２値を算出した。その結果を図３で示す。Ｒ２値は０．０１１と非常に低く、両遺伝子発現量に相関関係は認められなかった。従って上記６４菌株に関するＩＬ１０遺伝子発現亢進作用について、ＴＮＦａｌｐｈａ遺伝子発現量上昇によってパラクライン的にＩＬ１０遺伝子発現量が上昇したものではなく、上記６４菌株の上清処理によってマクロファージによるＩＬ１０遺伝子発現が亢進したものと考えられる。
Experiment 4: <Correlation between IL10 gene expression and TNFalpha gene expression>
The correlation between the IL10 gene expression level and the TNFalpha gene expression level by 64 strains obtained from the birth canal was investigated. The TNFalpha gene expression level was set on the vertical axis and the IL10 gene expression level was set on the horizontal axis, and the expression levels of both genes of each strain were plotted to draw an approximate curve, and the R2 value was calculated. The result is shown in FIG. The R2 value was very low at 0.011, and no correlation was observed between the expression levels of both genes. Therefore, regarding the IL10 gene expression-enhancing effect of the 64 strains, the IL10 gene expression was not paraclinically increased by the increase in the TNFalpha gene expression level, but the IL10 gene expression by the macrophages was enhanced by the supernatant treatment of the 64 strains. it is conceivable that.
実験５：＜ＩＬ１０遺伝子の発現を上昇させる菌株の探索＞
上記実験２において、産道内特有の６４菌株の中には、その上清処理において１０倍以上にＩＬ１０遺伝子発現を上昇させた菌株もみつかったことから、その１０倍以上にＩＬ１０遺伝子発現を上昇させた１５株を対象にして、菌個体レベルでＩＬ１０遺伝子発現を亢進させる代謝産物を菌数に依存せずにより多く産生する有用な菌株の探索を行った。
Experiment 5: <Search for strains that increase the expression of the IL10 gene>
In
各菌株をそれぞれＭＲＳ液体培地にて嫌気培養（３７℃、２日間）し、ＯＤ６００による菌数定量を行った。また、得られた培養液を遠心（１００００ｇ、５分間、室温）し、上清を回収後、０．２２μｍシリンジフィルターで菌体や交雑物の除去を行い清澄化させた。次に、清澄化した上清はＡｍｉｃｏｎ－Ｕｌｔｒａ ０．５ ３Ｋ（Ｍｉｌｌｉｐｏｒｅ社製）によって限外濾過して５倍濃縮し、その濃縮液が２５倍希釈される濃度になる様にマクロファージ様細胞の培養液へ添加して２４時間処理した。上清濃縮液を処理したマクロファージ様細胞から、ＩＳＯＧＥＮ ＩＩ（日本ジーン社製）によってＲＮＡを抽出し、ＡｃｃｕＲＴ ＧｅｎｏｍｉｃＤＮＡ Ｒｅｍｏｖａｌ Ｋｉｔ（ａｂｍ社製）でゲノムＤＮＡを除去した後、ＲｅｖｅｒＴｒａ Ａｃｅ ｑＰＣＲ ＲＴ Ｍａｓｔｅｒ Ｍｉｘ（ＴＯＹＯＢＯ社製）で逆転写を行い、ｃＤＮＡを合成した。これをテンプレートにＴＨＵＮＤＥＲＢＩＲＤ ＳＹＢＲ ｑＰＣＲ Ｍｉｘ（ＴＯＹＯＢＯ社製）を用いて、ＣＦＸ ＣｏｎｎｅｃｔＴＭ Ｒｅａｌ－Ｔｉｍｅ ＰＣＲ Ｄｅｔｅｃｔｉｏｎ Ｓｙｓｔｅｍ（Ｂｉｏ－Ｒａｄ社製）でリアルタイムＰＣＲを実施した。リアルタイムＰＣＲの反応条件は、９８℃・１分間で初期変性させた後、９５℃・１５秒、６０℃・３０秒を４０サイクル反応させた。遺伝子発現の定量は、リファレンス遺伝子としてｂｅｔａ－ａｃｔｉｎを用いて遺伝子発現量を補正し、ΔΔＣｔ法によってコントロールの遺伝子発現量から算出した相対値を求め、図４で示すグラフに表した。なお、図４の横軸において「Ｃｔｒｌ」は未処理の条件であり、「ＭＲＳ」は、ＭＲＳ培地で処理した条件でのものを示す。 Each strain was anaerobically cultured in MRS liquid medium (37 ° C. for 2 days), and the number of bacteria was quantified by OD600. Further, the obtained culture solution was centrifuged (10000 g, 5 minutes, room temperature), and after collecting the supernatant, cells and hybrids were removed with a 0.22 μm syringe filter to clarify the supernatant. Next, the clarified supernatant is ultra-filtered with Amicon-Ultra 0.5 3K (manufactured by Millipore) and concentrated 5 times, and the concentration of the macrophage-like cells is diluted 25 times. It was added to the culture broth and treated for 24 hours. RNA was extracted from macrophage-like cells treated with the supernatant concentrate by ISOGEN II (manufactured by Japan Gene), genomic DNA was removed by AccuRT Genomi cDNA Removal Kit (manufactured by Abm), and then ReverseTra Ace qPCR RT Master Mix (manufactured by Abm). Reverse transcription was performed with TOYOBO) to synthesize cDNA. Using this as a template, real-time PCR was performed by CFX ConnectTM Real-Time PCR Detection System (manufactured by Bio-Rad) using THUNDERBIRD SYBR qPCR Mix (manufactured by TOYOBO). The reaction conditions for real-time PCR were initial denaturation at 98 ° C. for 1 minute, followed by 40 cycles of reaction at 95 ° C. for 15 seconds and 60 ° C. for 30 seconds. For the quantification of gene expression, the gene expression level was corrected using beta-actin as a reference gene, and the relative value calculated from the control gene expression level by the ΔΔCt method was obtained and shown in the graph shown in FIG. In the horizontal axis of FIG. 4, "Ctrl" is an untreated condition, and "MRS" is a condition treated with an MRS medium.
図４で示すように、ＤＬ７２とＤＬ８１として示した菌株においては、ＩＬ１０遺伝子発現に対する優れた亢進性が認められた。上記シーケンス解析及びＰＣＲによれば、相同値が９８％以上となり、ＤＬ７２は、ラクトバチルス・クリスパタスであり、ＤＬ７２と名付けたものである。 As shown in FIG. 4, in the strains shown as DL72 and DL81, excellent enhancement to IL10 gene expression was observed. According to the above sequence analysis and PCR, the homology value was 98% or more, and DL72 was Lactobacillus crispatus, which was named DL72.
また、全１５菌株のうち、ＤＬ７２とＤＬ８１以外の図４で示す１３菌株は、次のとおりである。ＤＬ６２、ＤＬ７０、ＤＬ７３、ＤＬ７４、ＤＬ１０２、ＤＬ１０４及びＤＬ１０６は、ラクトバチルス・クリスパタスであり、ＤＬ６１、ＤＬ６６、ＤＬ７６、ＤＬ８３及びＤＬ１５４は、ラクトバチルス・ジェンセニイであり、ＤＬ７７は、同定できなかった。 In addition, among all 15 strains, 13 strains shown in FIG. 4 other than DL72 and DL81 are as follows. DL62, DL70, DL73, DL74, DL102, DL104 and DL106 were Lactobacillus crispatus, DL61, DL66, DL76, DL83 and DL154 were Lactobacillus gensenii, and DL77 could not be identified.
実験６：＜ＩＬ１０遺伝子発現を亢進する産道内特有１５菌株に基づくＩＬ１０遺伝子発現と他の因子との相関＞
ＩＬ１０遺伝子発現の程度を１０倍以上に上昇させた上記１５株において、ＩＬ１０遺伝子発現上昇が細菌数に依存したものか、また他の遺伝子発現（ＴＮＦａｌｐｈａ又はＩＬ１ｂｅｔａ）に依存したものかを確認するために、これらの相関について検討した。
Experiment 6: <Correlation between IL10 gene expression and other factors based on 15 strains peculiar to the birth canal that enhance IL10 gene expression>
In order to confirm whether the increase in IL10 gene expression depends on the number of bacteria or other gene expression (TNFalpha or IL1beta) in the above 15 strains in which the degree of IL10 gene expression is increased 10 times or more. We examined these correlations.
図５には、縦軸をＩＬ１０遺伝子発現量、横軸をＯＤ６００値としたグラフを示す。図６には、縦軸をＩＬ１０遺伝子発現量、横軸をＩＬ１ｂｅｔａ遺伝子発現量としたグラフを示す。そして、図７には、縦軸をＩＬ１０遺伝子発現量、横軸をＴＮＦａｌｐｈａ遺伝子発現量としたグラフを示す。これらのグラフにおいて、各値をプロットして近似曲線を描写し、Ｒ２値を求めた。その結果、ＯＤ６００値とのＲ２値は０．３７、ＩＬ１ｂｅｔａ遺伝子発現量とのＲ２値は０．６３、ＴＮＦａｌｐｈａ遺伝子発現量とのＲ２値は０．４４であり、どの因子もＩＬ１０遺伝子発現量との相関性は低いことがわかった。なお、図６及び図７では、２遺伝子間の発現での相関性を比べているため、コントロールとＭＲＳ培地のみとした条件も加えたグラフとしている。 これらの結果から、菌数ではなく各菌株の代謝産生量の違いによってＩＬ１０遺伝子発現量への作用が異なること、そして、ＩＬ１ｂｅｔａやＴＮＦａｌｐｈａといったＩＬ１０産生を誘導する炎症性サイトカインとの相関も見られないことから、産道内特有の菌株の上清には、マクロファージからのＩＬ１０産生を亢進させる代謝産物が含まれていることがわかった。 FIG. 5 shows a graph in which the vertical axis represents the IL10 gene expression level and the horizontal axis represents the OD600 value. FIG. 6 shows a graph in which the vertical axis represents the IL10 gene expression level and the horizontal axis represents the IL1beta gene expression level. FIG. 7 shows a graph in which the vertical axis represents the IL10 gene expression level and the horizontal axis represents the TNFalpha gene expression level. In these graphs, each value was plotted to draw an approximate curve, and the R2 value was obtained. As a result, the R2 value with the OD600 value was 0.37, the R2 value with the IL1beta gene expression level was 0.63, and the R2 value with the TNFalpha gene expression level was 0.44. It was found that the correlation with the expression level was low. In addition, in FIGS. 6 and 7, since the correlation in the expression between the two genes is compared, the graph includes the condition that only the control and the MRS medium are used. From these results, there is no correlation between the effect on the IL10 gene expression level and the inflammatory cytokines that induce IL10 production, such as IL1beta and TNFalpha, depending on the difference in the metabolic production amount of each strain rather than the number of bacteria. From this, it was found that the supernatant of the strain peculiar to the birth canal contains a metabolite that enhances IL10 production from macrophages.
実験７：＜ＤＬ７２の培養時間とＩＬ１０遺伝子発現との関係＞
ＤＬ７２の培養上清処理によってマクロファージ様細胞におけるＩＬ１０遺伝子発現が亢進されることから、より亢進作用を示す上清を得るために、菌株の培養時間とＩＬ１０遺伝子の発現変動を検討した。
Experiment 7: <Relationship between DL72 culture time and IL10 gene expression>
Since the IL10 gene expression in macrophage-like cells was enhanced by the culture supernatant treatment of DL72, the culture time of the strain and the expression variation of the IL10 gene were examined in order to obtain a supernatant showing a more enhancing effect.
ＭＲＳ液体培地にて嫌気培養（３７℃）で２４、４８、７２時間培養し、得られた培養液を遠心（１００００ｇ、５分間、室温）し、上清を回収後、０．２２μｍシリンジフィルターで菌体や交雑物の除去を行い清澄化させた。次に、清澄化した上清はＡｍｉｃｏｎ－Ｕｌｔｒａ ０．５ ３Ｋ（Ｍｉｌｌｉｐｏｒｅ社製）によって限外濾過して５倍濃縮させ、その濃縮液が２５倍希釈される濃度になる様にマクロファージ様細胞の培養液へ添加して２４時間処理した。上清濃縮液を処理したマクロファージ様細胞から、ＩＳＯＧＥＮ ＩＩ（日本ジーン社製）によってＲＮＡを抽出し、ＡｃｃｕＲＴ ＧｅｎｏｍｉｃＤＮＡ Ｒｅｍｏｖａｌ Ｋｉｔ（ａｂｍ社製）でゲノムＤＮＡを除去した後、ＲｅｖｅｒＴｒａ Ａｃｅ ｑＰＣＲ ＲＴ Ｍａｓｔｅｒ Ｍｉｘ（ＴＯＹＯＢＯ社製）で逆転写を行い、ｃＤＮＡを合成した。これをテンプレートにＴＨＵＮＤＥＲＢＩＲＤ ＳＹＢＲ ｑＰＣＲ Ｍｉｘ（ＴＯＹＯＢＯ社製）を用いて、ＣＦＸ ＣｏｎｎｅｃｔＴＭ Ｒｅａｌ－Ｔｉｍｅ ＰＣＲ Ｄｅｔｅｃｔｉｏｎ Ｓｙｓｔｅｍ（Ｂｉｏ－Ｒａｄ社製）でリアルタイムＰＣＲを実施した。リアルタイムＰＣＲの反応条件は、９８℃・１分間で初期変性させた後、９５℃・１５秒、６０℃・３０秒を４０サイクル反応させた。遺伝子発現の定量は、リファレンス遺伝子としてｂｅｔａ－ａｃｔｉｎを用いて遺伝子発現量を補正し、ΔΔＣｔ法によってコントロールの遺伝子発現量から算出した相対値を求めた。そして、図８には培養時間とＩＬ１０遺伝子発現量との関係を、図９には培養時間とＴＮＦａｌｐｈａ遺伝子発現量との関係をそれぞれ示した。 Incubate in MRS liquid medium in anaerobic culture (37 ° C.) for 24, 48, 72 hours, centrifuge the obtained culture solution (10000 g, 5 minutes, room temperature), collect the supernatant, and then use a 0.22 μm syringe filter. The cells and hybrids were removed and clarified. Next, the clarified supernatant is ultra-filtered with Amicon-Ultra 0.5 3K (manufactured by Millipore) and concentrated 5 times, and the concentration of the macrophage-like cells is diluted 25 times. It was added to the culture broth and treated for 24 hours. RNA was extracted from macrophage-like cells treated with the supernatant concentrate by ISOGEN II (manufactured by Japan Gene), genomic DNA was removed by AccuRT Genomi cDNA Removal Kit (manufactured by Abm), and then ReverseTra Ace qPCR RT Master Mix (manufactured by Abm). Reverse transcription was performed with TOYOBO) to synthesize cDNA. Using this as a template, real-time PCR was performed by CFX ConnectTM Real-Time PCR Detection System (manufactured by Bio-Rad) using THUNDERBIRD SYBR qPCR Mix (manufactured by TOYOBO). The reaction conditions for real-time PCR were initial denaturation at 98 ° C. for 1 minute, followed by 40 cycles of reaction at 95 ° C. for 15 seconds and 60 ° C. for 30 seconds. For the quantification of gene expression, the gene expression level was corrected using beta-actin as a reference gene, and the relative value calculated from the control gene expression level was obtained by the ΔΔCt method. FIG. 8 shows the relationship between the culture time and the IL10 gene expression level, and FIG. 9 shows the relationship between the culture time and the TNFalpha gene expression level.
その結果、図８で示すように、培養時間に依存的にＩＬ１０遺伝子発現量は有意に増加したのに対し、図９で示すように、ＴＮＦａｌｐｈａについては培養時間に対して大きな差異は認められなかった。これらの結果から、ＩＬ１０遺伝子発現上昇をさせる代謝産物は培養時間によって依存することが示唆された。 As a result, as shown in FIG. 8, the IL10 gene expression level increased significantly depending on the culture time, whereas as shown in FIG. 9, no significant difference was observed in the culture time for TNFalpha. rice field. These results suggest that the metabolites that increase IL10 gene expression depend on the culture time.
実験８：＜ＤＬ７２の菌体からのＩＬ１０遺伝子発現＞
ＤＬ７２の培養上清処理によってマクロファージ様細胞におけるＩＬ１０遺伝子発現が亢進されることから、ＤＬ７２菌体そのものにもＩＬ１０遺伝子発現亢進作用を有するのかについて検討を行なった。ＤＬ７２が属する種は、基礎的な知見や報告が豊富にあり、詳細な性状が明確であるため、効果が確認できれば菌体自体での使用も有効であるからである。比較対象としてLactobacillus brevis JCM 1059とLactobacillus paracasei subsp. paracasei JCM 8130を用いて、ＤＬ７２の菌体によるＩＬ１０遺伝子発現亢進作用の検討を実施した。
Experiment 8: <IL10 gene expression from DL72 cells>
Since the IL10 gene expression in macrophage-like cells is enhanced by the culture supernatant treatment of DL72, it was investigated whether the DL72 cells themselves also have an IL10 gene expression enhancing effect. This is because the species to which DL72 belongs has abundant basic knowledge and reports, and its detailed properties are clear. Therefore, if the effect can be confirmed, it is also effective to use the cells themselves. Using Lactobacillus brevis JCM 1059 and Lactobacillus paracasei subsp. Paracasei JCM 8130 as comparison targets, the IL10 gene expression-enhancing effect of DL72 cells was examined.
ＭＲＳ液体培地にて７２時間、嫌気培養（３７℃）し、得られた培養液を遠心（１００００ｇ、５分間、室温）後、上清を廃棄し、ＰＢＳを加えて遠心して上清を除去することで菌体ペレットを得た。得られた菌体ペレットをＰＢＳで懸濁して菌体液を作製した。この菌体液の一部を測り取り、ＯＤ６００値が０．９～１．２の各菌体ペレットを準備し、細胞用培養液（ＲＰＭＩ １６４０、２ｍＭ Ｇｌｕｔａｍｉｎｅ、１０％ Ｆｏｅｔａｌ Ｂｏｖｉｎｅ Ｓｅｒｕｍ）で懸濁し、これを基準とした３段階の１０倍希釈系列を調製してマクロファージ様細胞に添加処理を行なった。２４時間後、マクロファージ様細胞から、ＩＳＯＧＥＮ ＩＩ（日本ジーン社製）によってＲＮＡを抽出し、ＡｃｃｕＲＴ ＧｅｎｏｍｉｃＤＮＡ Ｒｅｍｏｖａｌ Ｋｉｔ（ａｂｍ社製）でゲノムＤＮＡを除去した後、ＲｅｖｅｒＴｒａ Ａｃｅ ｑＰＣＲ ＲＴ Ｍａｓｔｅｒ Ｍｉｘ（ＴＯＹＯＢＯ社製）で逆転写を行い、ｃＤＮＡを合成した。
After anaerobic culture (37 ° C.) for 72 hours in MRS liquid medium, centrifuge the obtained culture solution (10000 g, 5 minutes, room temperature), discard the supernatant, add PBS and centrifuge to remove the supernatant. As a result, bacterial cell pellets were obtained. The obtained bacterial cell pellet was suspended in PBS to prepare a bacterial cell fluid. A part of this cell fluid is measured, each cell pellet having an OD600 value of 0.9 to 1.2 is prepared, suspended in a cell culture medium (
これをテンプレートにＴＨＵＮＤＥＲＢＩＲＤ ＳＹＢＲ ｑＰＣＲ Ｍｉｘ（ＴＯＹＯＢＯ社製）を用いて、ＣＦＸ ＣｏｎｎｅｃｔＴＭ Ｒｅａｌ－Ｔｉｍｅ ＰＣＲ Ｄｅｔｅｃｔｉｏｎ Ｓｙｓｔｅｍ（Ｂｉｏ－Ｒａｄ社製）でリアルタイムＰＣＲを実施した。リアルタイムＰＣＲの反応条件は、９８℃・１分間で初期変性させた後、９５℃・１５秒、６０℃・３０秒を４０サイクル反応させた。遺伝子発現の定量は、リファレンス遺伝子としてｂｅｔａ－ａｃｔｉｎを用いて遺伝子発現量を補正し、ΔΔＣｔ法によってコントロールの遺伝子発現量から算出した相対値を求め、その値を対数変換し比較検討を行なった。 Using this as a template, real-time PCR was performed by CFX ConnectTM Real-Time PCR Detection System (manufactured by Bio-Rad) using THUNDERBIRD SYBR qPCR Mix (manufactured by TOYOBO). The reaction conditions for real-time PCR were initial denaturation at 98 ° C. for 1 minute, followed by 40 cycles of reaction at 95 ° C. for 15 seconds and 60 ° C. for 30 seconds. For the quantification of gene expression, the gene expression level was corrected using beta-actin as a reference gene, the relative value calculated from the control gene expression level was obtained by the ΔΔCt method, and the value was logarithically converted for comparative study.
これらの結果を図１０に示す。図１０では縦軸にＩＬ１０遺伝子発現量の相対値を、横軸にはLactobacillus brevis JCM 1059 を「ＬＢ」で、Lactobacillus paracasei subsp. paracasei JCM 8130 を「ＬＰＰ」で、ＤＬ７２を「ＤＬ７２」で示している。これらの結果から、ＤＬ７２、Lactobacillus brevis JCM 1059、及びLactobacillus paracasei subsp. paracasei JCM 8130の何れの菌株もＩＬ１０遺伝子発現を強く亢進させたが、Lactobacillus brevis JCM 1059とLactobacillus paracasei subsp. paracasei JCM 8130については、１００倍に希釈した条件からＩＬ１０遺伝子発現亢進の作用がなくなるのに対し、ＤＬ７２では、１００倍希釈された条件でもＩＬ１０遺伝子発現亢進の作用を維持していることが認められた。このことから、ＤＬ７２の菌体にもＩＬ１０遺伝子発現を亢進させる作用を有しており、その作用は他の乳酸菌よりも高いことが認められた。 These results are shown in FIG. In FIG. 10, the vertical axis indicates the relative value of the IL10 gene expression level, the horizontal axis indicates Lactobacillus brevis JCM 1059 as “LB”, Lactobacillus paracasei subsp. Paracasei JCM 8130 as “LPP”, and DL72 as “DL72”. There is. From these results, all strains of DL72, Lactobacillus brevis JCM 1059 , and Lactobacillus paracasei subsp. Paracasei JCM 8130 strongly enhanced IL10 gene expression, but Lactobacillus brevis JCM 1059 and Lactobacillus paracasei subsp. Paracasei JCM 8130 It was found that the effect of enhancing IL10 gene expression disappeared from the condition of 100-fold dilution, whereas the effect of increasing IL10 gene expression was maintained in DL72 even under the condition of 100-fold dilution. From this, it was confirmed that the cells of DL72 also have an action of enhancing IL10 gene expression, and the action is higher than that of other lactic acid bacteria.
実験９：＜ＤＬ７２とラクトバチルス２株の培養上清によるＩＬ１０遺伝子発現亢進の比較＞
ＤＬ７２の培養上清処理によってマクロファージ様細胞におけるＩＬ１０遺伝子発現が亢進されたことから、抗炎症作用が知られているLactobacillus brevisとLactobacillus paracasei subsp. paracaseiを比較対象にＤＬ７２によるＩＬ１０遺伝子発現亢進の優位性を検討した。
Experiment 9: <Comparison of IL10 gene expression enhancement by culture supernatants of DL72 and
Since the IL10 gene expression in macrophage-like cells was enhanced by the culture supernatant treatment of DL72, the superiority of IL10 gene expression by DL72 was compared with Lactobacillus brevis and Lactobacillus paracasei subsp. Paracasei, which are known to have anti-inflammatory effects. It was investigated.
ＤＬ７２、Lactobacillus brevis JCM 1059（「ＬＢ」）、Lactobacillus paracasei subsp. paracasei JCM 8130（「ＬＰＰ」）のそれぞれを、ＭＲＳ液体培地にて７２時間、嫌気培養（３７℃）し、得られた培養液を遠心（１００００ｇ、５分間、室温）し、上清を回収後、０．２２μｍシリンジフィルターで菌体や交雑物の除去を行い清澄化させた。次に、清澄化した上清はＡｍｉｃｏｎ－Ｕｌｔｒａ ０．５ ３Ｋ（Ｍｉｌｌｉｐｏｒｅ社製）によって限外濾過して５倍濃縮させ、その濃縮液が２５倍希釈される濃度になる様にマクロファージ様細胞の培養液へ添加して２４時間処理した。 DL72, Lactobacillus brevis JCM 1059 (“LB”), and Lactobacillus paracasei subsp. Paracasei JCM 8130 (“LPP”) were anaerobically cultured (37 ° C.) in MRS liquid medium for 72 hours, and the obtained culture solution was used. After centrifuging (10000 g, 5 minutes, room temperature) and collecting the supernatant, cells and hybrids were removed with a 0.22 μm syringe filter for clarification. Next, the clarified supernatant is ultra-filtered with Amicon-Ultra 0.5 3K (manufactured by Millipore) and concentrated 5 times, and the concentration of the macrophage-like cells is diluted 25 times. It was added to the culture broth and treated for 24 hours.
上清濃縮液を処理したマクロファージ様細胞から、ＩＳＯＧＥＮ ＩＩ（日本ジーン社製）によってＲＮＡを抽出し、ＡｃｃｕＲＴ ＧｅｎｏｍｉｃＤＮＡ Ｒｅｍｏｖａｌ Ｋｉｔ（ａｂｍ社製）でゲノムＤＮＡを除去した後、ＲｅｖｅｒＴｒａ Ａｃｅ ｑＰＣＲ ＲＴ Ｍａｓｔｅｒ Ｍｉｘ（ＴＯＹＯＢＯ社製）で逆転写を行い、ｃＤＮＡを合成した。これをテンプレートにＴＨＵＮＤＥＲＢＩＲＤ ＳＹＢＲ ｑＰＣＲ Ｍｉｘ（ＴＯＹＯＢＯ社製）を用いて、ＣＦＸ ＣｏｎｎｅｃｔＴＭ Ｒｅａｌ－Ｔｉｍｅ ＰＣＲ Ｄｅｔｅｃｔｉｏｎ Ｓｙｓｔｅｍ（Ｂｉｏ－Ｒａｄ社製）でリアルタイムＰＣＲを実施した。リアルタイムＰＣＲの反応条件は、９８℃・１分間で初期変性させた後、９５℃・１５秒、６０℃・３０秒を４０サイクル反応させた。遺伝子発現の定量は、リファレンス遺伝子としてｂｅｔａ－ａｃｔｉｎを用いて遺伝子発現量を補正し、ΔΔＣｔ法によってコントロールの遺伝子発現量から算出した相対値を求め、その値を対数変換し比較検討を行なった。また、ＭＲＳ培地処理条件との有意差検定はｔ検定によって行い、ｐ値が０．０５以下の場合を有意差があるとして判定した。 RNA was extracted from macrophage-like cells treated with the supernatant concentrate by ISOGEN II (manufactured by Japan Gene), genomic DNA was removed by AccuRT Genomi cDNA Removal Kit (manufactured by Abm), and then ReverseTra Ace qPCR RT Master Mix (manufactured by Abm). Reverse transcription was performed with TOYOBO) to synthesize cDNA. Using this as a template, real-time PCR was performed by CFX ConnectTM Real-Time PCR Detection System (manufactured by Bio-Rad) using THUNDERBIRD SYBR qPCR Mix (manufactured by TOYOBO). The reaction conditions for real-time PCR were initial denaturation at 98 ° C. for 1 minute, followed by 40 cycles of reaction at 95 ° C. for 15 seconds and 60 ° C. for 30 seconds. For the quantification of gene expression, the gene expression level was corrected using beta-actin as a reference gene, the relative value calculated from the control gene expression level was obtained by the ΔΔCt method, and the value was logarithically converted for comparative study. In addition, the significance test with the MRS medium treatment conditions was performed by t-test, and when the p-value was 0.05 or less, it was determined that there was a significant difference.
その結果、Lactobacillus brevis JCM 1059及びLactobacillus paracasei subsp. paracasei JCM 8130のどちらの培養上清もＩＬ１０遺伝子発現を亢進させる傾向は見られたものの、ＭＲＳ培地処理条件との間に有意差は認められなかったのに対し、ＤＬ７２では、ＭＲＳ培地処理条件に対して有意差が認められ、ＩＬ１０遺伝子発現の亢進作用がLactobacillus brevis JCM 1059及びLactobacillus paracasei subsp. paracasei JCM 8130よりも高いことが示された（図１１）。このことから、ＤＬ７２は、他の乳酸菌よりも優位にＩＬ１０遺伝子発現を亢進させ、高い抗炎症作用を有するものと考えられる。 As a result, both the culture supernatants of Lactobacillus brevis JCM 1059 and Lactobacillus paracasei subsp. Paracasei JCM 8130 tended to enhance IL10 gene expression, but no significant difference was observed with the MRS medium treatment conditions. On the other hand, in DL72, a significant difference was observed with respect to the MRS medium treatment conditions, indicating that the effect of enhancing IL10 gene expression was higher than that of Lactobacillus brevis JCM 1059 and Lactobacillus paracasei subsp. Paracasei JCM 8130 (FIG. 11). ). From this, it is considered that DL72 has a high anti-inflammatory effect by enhancing IL10 gene expression predominantly over other lactic acid bacteria.
実験１０：＜ＤＬ７２培養上清によるタンパク質レベルでのＩＬ１０遺伝子発現亢進＞
ＤＬ７２の培養上清処理におけるＩＬ１０遺伝子発現に関して、タンパク質レベルでの評価も行った。
Experiment 10: <Enhancement of IL10 gene expression at protein level by DL72 culture supernatant>
The expression of the IL10 gene in the culture supernatant treatment of DL72 was also evaluated at the protein level.
ＤＬ７２をＭＲＳ液体培地にて嫌気培養（３７℃、７２時間）し、得られた培養液を遠心（１００００ｇ、５分間、室温）し、上清を回収後、０．２２μｍシリンジフィルターで菌体や交雑物の除去を行い清澄化した。次に、清澄化した上清はＡｍｉｃｏｎ－Ｕｌｔｒａ ０．５ ３Ｋ（Ｍｉｌｌｉｐｏｒｅ社製）によって限外濾過して５倍濃縮させ、その濃縮液が２５倍希釈される濃度になる様にマクロファージ様細胞の培養液へ添加して２４時間処理した。２４時間後、マクロファージ様細胞の培養液を回収し、培養液中に分泌されたＩＬ１０量を定量した。ＩＬ１０の定量は、Ｈｕｍａｎ ＰｒｏＱｕａｎｔｕｍ ＩＬ１０ Ｉｍｍｕｎｏａｓｓａｙ Ｋｉｔ（ｉｎｖｉｔｒｏｇｅｎ社製）によって、メーカーのプロトコールに従って定量解析を行った。未処理条件（Ｃｔｒｌ）のＩＬ１０量を基準として相対値を求め図１２に示した。その結果、ｑＰＣＲによる遺伝子発現解析による結果と同様に、ＤＬ７２の上清処理によって細胞外に分泌されたＩＬ１０量が増加していることが観察され、タンパク質レベルでもＤＬ７２によってＩＬ１０が増加していることが認められ、ＤＬ７２によるＩＬ１０発現亢進が強く支持された。 DL72 was anaerobically cultured in MRS liquid medium (37 ° C., 72 hours), the obtained culture solution was centrifuged (10000 g, 5 minutes, room temperature), and the supernatant was collected. The hybrids were removed and clarified. Next, the clarified supernatant is ultra-filtered with Amicon-Ultra 0.5 3K (manufactured by Millipore) and concentrated 5 times, and the concentration of the macrophage-like cells is diluted 25 times. It was added to the culture broth and treated for 24 hours. After 24 hours, the culture medium of macrophage-like cells was collected and the amount of IL10 secreted into the culture medium was quantified. Quantitative analysis of IL10 was performed by Human ProQuantum IL10 Immunoassay Kit (manufactured by Invitrogen) according to the manufacturer's protocol. Relative values were obtained based on the amount of IL10 under the untreated condition (Ctrl) and are shown in FIG. As a result, it was observed that the amount of IL10 secreted extracellularly was increased by the supernatant treatment of DL72, as in the result of the gene expression analysis by qPCR, and the IL10 was also increased by DL72 at the protein level. Was observed, and the enhancement of IL10 expression by DL72 was strongly supported.
実験１１：＜ＤＬ７２培養上清原液の製造＞
凍結保管したＤＬ７２菌を解凍して得た菌液を植菌重量が培地重量の０．１ｗｔ％となるようにＭＲＳ液体培地に加えて７２時間培養し、ＯＤ６００を４．５～５．０とした。そして得られた培養液を遠心し、上清を回収後、清澄化し、さらに限外濾過して５倍濃縮液を製造し、その後の各種製剤を製造するためのＤＬ７２培養上清原液とした。
Experiment 11: <Production of DL72 culture supernatant stock solution>
The bacterial solution obtained by thawing the cryopreserved DL72 bacteria was added to the MRS liquid medium so that the inoculation weight was 0.1 wt% of the medium weight and cultured for 72 hours, and the OD600 was adjusted to 4.5 to 5.0. did. Then, the obtained culture solution was centrifuged, the supernatant was collected, clarified, and further filtered to produce a 5-fold concentrated solution, which was used as the DL72 culture supernatant stock solution for producing various subsequent formulations.
実験１２：＜ＤＬ７２培養上清原液からの各種製剤の製造＞
１．液状製剤又はローション状製剤の製造
８０ｗｔ％の蒸留水、１０ｗｔ％のアルコール、数ｗｔ％のグリセリンに香料、乳化剤、防腐剤を配合した希釈液に、上記ＤＬ７２培養上清原液を加えて、その合計量のうちＤＬ７２培養上清原液が４ｗｔ％となるようにしてローション状製剤を得た。また、グリセリンの含量を蒸留水で代替して液状製剤を得た。
このローション状製剤又は液状製剤は、そのままローション又は化粧水として用いることで、肌用、デリケートゾーン周辺用のケア製剤として適用できる。
Experiment 12: <Manufacturing of various pharmaceuticals from DL72 culture supernatant stock solution>
1. 1. Production of liquid or lotion-like product Add the above DL72 culture supernatant stock solution to a diluted solution containing 80 wt% distilled water, 10 wt% alcohol, several wt% glycerin, fragrance, emulsifier, and preservative, and add up the total. A lotion-like preparation was obtained so that the DL72 culture supernatant stock solution was 4 wt% of the amount. Further, the content of glycerin was replaced with distilled water to obtain a liquid preparation.
This lotion-like preparation or liquid preparation can be applied as a care preparation for the skin and around the delicate zone by using it as it is as a lotion or a lotion.
２．クリーム製剤の製造
ワセリン、オリーブオイル、エタノール、顔料に香料、乳化剤、防腐剤を配合したクリームに、上記ＤＬ７２培養上清原液を加えて、その合計量のうちＤＬ７２培養上清原液が４ｗｔ％となるようにしてクリーム状製剤を得た。このクリーム状製剤は、そのままクリーム製品として、肌用、デリケートゾーン周辺用のケア製剤として適用できる。
2. 2. Manufacture of cream preparation The above DL72 culture supernatant stock solution is added to a cream containing petrolatum, olive oil, ethanol, and pigments with fragrances, emulsifiers, and preservatives, and the DL72 culture supernatant stock solution accounts for 4 wt% of the total amount. Thus, a creamy preparation was obtained. This cream-like preparation can be applied as a cream product as it is, as a care preparation for the skin and around the delicate zone.
３．錠剤の製造
デンプン及びデンプン糊に上記ＤＬ７２培養上清原液を加えて、その合計量のうちＤＬ７２培養上清原液が４ｗｔ％となるようにし成形し錠剤を得た。この錠剤は経口剤として、アトピー等の皮膚病に対するケア製剤、傷等に対する抗炎症製剤として適用できる。
４．カプセル剤の製造
上記液状製剤、ローション状製剤、クリーム製剤は、カプセルに詰めてカプセル化することで、膣内への挿入剤として産後の膣ケア製剤として適用できる。また、経口剤として、アトピー等の皮膚病に対するケア製剤、傷等に対する抗炎症製剤として適用できる。
3. 3. Preparation of Tablets The above DL72 culture supernatant stock solution was added to starch and starch paste, and molding was performed so that the DL72 culture supernatant stock solution accounted for 4 wt% of the total amount to obtain tablets. This tablet can be applied as an oral preparation as a care preparation for skin diseases such as atopy and as an anti-inflammatory preparation for wounds and the like.
4. Manufacture of Capsules The liquid preparations, lotion-like preparations, and cream preparations can be applied as postpartum vaginal care preparations as inserts into the vagina by encapsulating them in capsules. Further, as an oral preparation, it can be applied as a care preparation for skin diseases such as atopy and an anti-inflammatory preparation for wounds and the like.
実験１３：＜ＤＬ７２菌株含有液の製造＞
凍結保管したＤＬ７２菌を解凍して得た菌液を植菌重量が培地重量の０．１ｗｔ％となるようにＭＲＳ液体培地に加えて７２時間培養し、ＯＤ６００を４．５～５．０とした。
そして得られた培養液を遠心し、上清を廃棄し、菌体ペレットを得た。そして、この菌体ペレットをＰＢＳで希釈して、その後の各種製剤を製造するためのＤＬ７２菌体含有液とした。このＤＬ７２菌体含有液中には、ＤＬ７２の生菌が含まれる。
Experiment 13: <Manufacturing of DL72 strain-containing liquid>
The bacterial solution obtained by thawing the cryopreserved DL72 bacteria was added to the MRS liquid medium so that the inoculation weight was 0.1 wt% of the medium weight and cultured for 72 hours, and the OD600 was adjusted to 4.5 to 5.0. did.
Then, the obtained culture solution was centrifuged, the supernatant was discarded, and bacterial cell pellets were obtained. Then, the cell pellet was diluted with PBS to obtain a DL72 cell-containing solution for producing various subsequent preparations. The DL72 cell-containing liquid contains live DL72 bacteria.
実験１４：＜ＤＬ７２菌体含有液からの各種製剤の製造＞
１．液状製剤又はローション状製剤の製造
８０ｗｔ％の蒸留水、１０ｗｔ％のアルコール、数ｗｔ％のグリセリンに香料、乳化剤、防腐剤を配合した希釈液に、上記ＤＬ７２菌体含有液を加えて、その合計量１ｇのうちＤＬ７２の菌体が１×１０８～５×１０９個含まれるようにしてローション状製剤を得た。また、グリセリンの含量を蒸留水で代替して液状製剤を得た。
このローション状製剤又は液状製剤は、そのままローション又は化粧水として用いることで、肌用、デリケートゾーン周辺用のケア製剤として適用できる。
Experiment 14: <Manufacturing of various pharmaceuticals from DL72 cell-containing solution>
1. 1. Production of liquid or lotion-like product Add the above DL72 cell-containing solution to a diluted solution containing 80 wt% distilled water, 10 wt% alcohol, and several wt% glycerin with a fragrance, emulsifier, and preservative, and add up the total. A lotion-like preparation was obtained so that 1 × 10 8 to 5 × 10 9 cells of DL72 were contained in the amount of 1 g. Further, the content of glycerin was replaced with distilled water to obtain a liquid preparation.
This lotion-like preparation or liquid preparation can be applied as a care preparation for the skin and around the delicate zone by using it as it is as a lotion or a lotion.
２．クリーム製剤の製造
ワセリン、オリーブオイル、エタノール、顔料に香料、乳化剤、防腐剤を配合したクリームに、上記ＤＬ７２菌体含有液を加えて、その合計量１ｇのうちＤＬ７２の菌体が１×１０８～５×１０９個含まれるようにしてクリーム状製剤を得た。このクリーム状製剤は、そのままクリーム製品として、肌用、デリケートゾーン周辺用のケア製剤として適用できる。
2. 2. Manufacture of cream preparation The above DL72 cell-containing liquid is added to a cream containing petrolatum, olive oil, ethanol, pigment, fragrance, emulsifier, and preservative, and DL72 cells are 1 × 10 8 out of the total amount of 1 g. A creamy preparation was obtained so as to contain ~ 5 × 10 9 pieces. This cream-like preparation can be applied as a cream product as it is, as a care preparation for the skin and around the delicate zone.
３．錠剤の製造
デンプン及びデンプン糊に上記ＤＬ７２菌体含有液を加えて、その合計量１ｇのうちＤＬ７２の菌体が１×１０８～５×１０９個含まれるようにして成形し錠剤を得た。この錠剤は経口剤として、アトピー等の皮膚病に対するケア製剤、傷等に対する抗炎症製剤として適用できる。
４．カプセル剤の製造
上記液状製剤、ローション状製剤、クリーム製剤は、カプセルに詰めてカプセル化することで、膣内への挿入剤として産後の膣ケア製剤として適用できる。また、経口剤として、アトピー等の皮膚病に対するケア製剤、傷等に対する抗炎症製剤として適用できる。
3. 3. Preparation of Tablets The above DL72 cell-containing liquid was added to starch and starch paste, and molding was performed so that 1 × 10 8 to 5 × 10 9 DL72 cells were contained in the total amount of 1 g to obtain tablets. .. This tablet can be applied as an oral preparation as a care preparation for skin diseases such as atopy and as an anti-inflammatory preparation for wounds and the like.
4. Manufacture of Capsules The liquid preparations, lotion-like preparations, and cream preparations can be applied as postpartum vaginal care preparations as inserts into the vagina by encapsulating them in capsules. Further, as an oral preparation, it can be applied as a care preparation for skin diseases such as atopy and an anti-inflammatory preparation for wounds and the like.
実験１５：＜ＤＬ７２培養上清原液及びＤＬ７２菌体含有液からの各種製剤の製造＞
上記ＤＬ７２培養上清原液及び上記ＤＬ７２菌体含有液を適当量混合し、上記有効量を含むようにした以外は、上記各種製剤の製造方法と同様にして、液状製剤、ローション状製剤、錠剤、カプセル剤を製造した。より具体的には、ＤＬ７２培養上清原液と上記ＤＬ７２菌体含有液の寄与割合を決定し、その寄与割合に応じてそれぞれの有効成分量を決定して配合するように調製した。
Experiment 15: <Manufacturing of various pharmaceuticals from DL72 culture supernatant stock solution and DL72 cell-containing solution>
Liquid preparations, lotion-like preparations, tablets, etc. Manufactured a capsule. More specifically, the contribution ratios of the DL72 culture supernatant stock solution and the DL72 cell-containing solution were determined, and the amount of each active ingredient was determined and blended according to the contribution ratio.
実験１６：＜ＤＬ７２以外の菌株からの各種製剤の製造＞
上記実験５で示す１５株のうち、種が特定されなかった１株と、ＤＬ７２株を除く１３菌株についても、上記ＤＬ７２培養上清原液の製造方法や、上記ＤＬ７２菌体含有液の製造方法と同様にして、これらの何れか一株、及びＤＬ７２菌株を含めた何れかの複数株の培養上清原液や、菌体含有液を製造した。また、上記製剤の製造方法と同様にして、これらの培養上清原液や、菌体含有液を含む製剤を製造した。
Experiment 16: <Manufacturing of various formulations from strains other than DL72>
Of the 15 strains shown in
Claims (8)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2020137331A JP2022033444A (en) | 2020-08-17 | 2020-08-17 | Germ, il10 gene expression promoting agent, and pharmaceutical preparation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2020137331A JP2022033444A (en) | 2020-08-17 | 2020-08-17 | Germ, il10 gene expression promoting agent, and pharmaceutical preparation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP2022033444A true JP2022033444A (en) | 2022-03-02 |
Family
ID=80375189
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2020137331A Pending JP2022033444A (en) | 2020-08-17 | 2020-08-17 | Germ, il10 gene expression promoting agent, and pharmaceutical preparation |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2022033444A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004277381A (en) * | 2003-03-18 | 2004-10-07 | Japan Research & Development Association For New Functional Foods | Immunmodulating agent and immunomodulating food |
| JP2011004620A (en) * | 2009-06-23 | 2011-01-13 | Tokai Igaku Kensa Kenkyusho:Kk | Genus lactobacillus strain and food and medicine containing the strain |
| JP2015086214A (en) * | 2013-09-26 | 2015-05-07 | 雪印メグミルク株式会社 | Prophylactic agents for immune diseases |
-
2020
- 2020-08-17 JP JP2020137331A patent/JP2022033444A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004277381A (en) * | 2003-03-18 | 2004-10-07 | Japan Research & Development Association For New Functional Foods | Immunmodulating agent and immunomodulating food |
| JP2011004620A (en) * | 2009-06-23 | 2011-01-13 | Tokai Igaku Kensa Kenkyusho:Kk | Genus lactobacillus strain and food and medicine containing the strain |
| JP2015086214A (en) * | 2013-09-26 | 2015-05-07 | 雪印メグミルク株式会社 | Prophylactic agents for immune diseases |
Non-Patent Citations (2)
| Title |
|---|
| A. ALHOUSSEINI, ET AL., GYNECOLOGIC AND OBSTETRIC INVESTIGATION, vol. 85, JPN6022049442, 22 April 2020 (2020-04-22), pages 277 - 283, ISSN: 0005058362 * |
| A. RIZZO, ET AL., INTERNATIONAL JOURNAL OF MEDICAL MICROBIOLOGY, vol. 305, JPN6022049443, 2015, pages 815 - 827, ISSN: 0005058363 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| TWI627276B (en) | Novel lactobacillus crispatus strain | |
| JP2008518588A (en) | Probiotic Lactobacillus strains for improving vaginal health | |
| US11000558B2 (en) | Vaginal preparations for maintaining and/or restoring healthy female microbiota | |
| KR102133689B1 (en) | Manufacturing method of conjugate between lava seawater-originated minerals and dermabiotics-derived nucleotide, and functional dermabiotics cosmetic composition using the mineral-nucleotide | |
| CN113473997A (en) | Bacterial strains of the species lactobacillus paracasei for oral or topical application in the treatment of female urogenital disorders | |
| JP2008539776A (en) | Symbiotic Bifidobacterium species | |
| WO2019208151A1 (en) | Composition for type iv allergy | |
| CN116731936B (en) | Lactobacillus casei LC15 with immunoregulatory function and application, product and method thereof | |
| US11045507B2 (en) | Bifidobacterium animalis AMT30 strain and the composition containing the strain of Bifidobacterium animalis AMT30 | |
| JP7076840B1 (en) | Bacteria, IL10 gene expression enhancer, immune response control enhancer, and preparation | |
| JP2022033444A (en) | Germ, il10 gene expression promoting agent, and pharmaceutical preparation | |
| EP3897676A1 (en) | Novel dolosigranulum pigrum strains and uses therof | |
| Njeru et al. | Identification and characterisation of lactobacilli isolated from Kimere, a spontaneously fermented pearl millet dough from Mbeere, Kenya (East Africa) | |
| KR100556315B1 (en) | Lactobacillus rhamnosus strain and uses thereof | |
| JP6412865B2 (en) | Bifidobacterium breve strain specific gene | |
| EP3489350B1 (en) | Bifidobacterium animalis amt30 strain and composition containing the strain of bifidobacterium animalis amt30 | |
| CN113164531A (en) | External preparation for promoting hair growth or preventing alopecia | |
| CN117159598B (en) | Application of lactobacillus plantarum Lp18 in preparation of immunity-enhancing medicines or health-care foods and products | |
| CN115624573B (en) | Probiotic composition for improving hair health condition and application thereof | |
| CN117286045B (en) | Bifidobacterium longum subspecies longum KS2 and application thereof in preparation of anti-aging medicines | |
| CN117165497B (en) | Lactobacillus plantarum Lp18 for improving constipation, application and product thereof | |
| CN117106628B (en) | Lactobacillus acidophilus LA15 with immunoregulation capability and application, product and method thereof | |
| Raju et al. | Isolation, Characterization and Sequencing of Lactobacillus from the Oral and Fecal Samples of Healthy Dogs | |
| Hassan | Detection of probiotic microorganisms using RT-PCR, and isolation, identification, probiotic properties of lactobacillus species from kefir | |
| Montella et al. | Vaginal adhesion of Lactobacillus plantarum P17630 after probiotic food supplement oral administration: a preliminary in vivo study |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20200817 |
|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20221017 |
|
| A871 | Explanation of circumstances concerning accelerated examination |
Free format text: JAPANESE INTERMEDIATE CODE: A871 Effective date: 20221017 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20221122 |
|
| A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20230118 |
|
| A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20230516 |