CN103224895B - Novel lactobacillus lodelbrueckii strain and application thereof in improving autoimmune diseases - Google Patents
Novel lactobacillus lodelbrueckii strain and application thereof in improving autoimmune diseases Download PDFInfo
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- CN103224895B CN103224895B CN201210396448.0A CN201210396448A CN103224895B CN 103224895 B CN103224895 B CN 103224895B CN 201210396448 A CN201210396448 A CN 201210396448A CN 103224895 B CN103224895 B CN 103224895B
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Abstract
The invention relates to a novel separated bacterial strain BR101, which is separated from a neonatal excrement sample, is identified as a lactobacillus reuteri strain by comparing the strain characteristics with a 16S rDNA gene fragment sequence, and is subjected to colony appearance pattern difference, 16SrDNA gene fragment difference, random primer pair amplification polymorphism DNA analysis, API50CHL microbial carbohydrate biochemical reaction, screening of probiotic strains for stimulating secretion of IL-10 and function analysis thereof with other lactobacillus reuteri standard strains, thereby showing that the bacterial strain BR101 has obvious difference with other standard strains, and the separated bacterial strain can stimulate cells to generate higher anti-inflammatory hormone, reduce the inflammatory reaction of autoimmunity and be applied to improve the autoimmunity diseases.
Description
Technical field
The present invention relates to a kind of strains separation strain BR101 of novelty, particularly relate to one and stem from a Neonatal Faeces corpse or other object for laboratory examination and chemical testing, compare through 16S rDNA gene fragment order and be accredited as Lip river moral Lactobacillus species, and compare with other Lip river moral lactobacillus reference cultures, there were significant differences to show this bacterial strain BR101 and other standards bacterial strain, and there is the higher anti-inflammation hormone of irritation cell generation, reduce autoimmune inflammatory response, reach the Lip river moral lactic bacilli strains of the novelty improving autoimmune disorders.It is deposited at China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 24th, 2012, address: No. 3, No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute, deposit numbering CGMCC No.6637, also be deposited at TaiWan, China Foodstuff Industrial and Development Inst., deposit numbering BCRC910512.
Background technology
Immunity system plays critical role for the existence of organism, and it helps us to resist various infective microorganism, comprises bacterium, virus, fungi, parasite etc.When pathogenic microorganisms invasion human body, immunity system will produce a series of immune response, comprises cellular immunization and humoral immunization.Cellular immunization utilizes phagocytic cell or poisoning type T cell to kill the microorganism of invasion; Humoral immunization produces the materials such as antibody exactly and tackles pathogenic bacteria.Under protection tight like this, human body can say it is strongly fortified, is difficult to the city of capturing.Autoimmune disorders is exactly that oneself immunity system is out of control, as wild chargers attacks oneself tissue or organ.If the joint cartilage of attacking oneself is just called rheumatoid arthritis, the vertebra attacking self is exactly ankylosing spondylosis, and attack pancreas and can cause the first patients with type Ⅰ DM, attacking eyeball is exactly iritis, and the blood vessel attacking whole body is exactly lupus erythematosus.Although these diseases are etiology unknown at present, symptom is also different, and it is all immune imbalance that the machine caused a disease turns, and therefore methods for the treatment of is all for target with immunity moderation system.
Autoimmune disorders (Autoimmune disease) is the Normocellular disease of oneself health of a kind of immune system attack; there is in the immunity system of human body antibody and can resist external antigen; as bacterium or virus; or abnormal cell in body; carry out attacking as tumour cell etc. and remove, such behavior is a kind of physiological mechanism protecting health.But autoimmune disorders is immunity system to be produced voluntarily and can resist Normocellular antibody in oneself health body, causes abnormal inflammatory response or tissue injury, and then affects healthy disease.These antibody are called autoimmunity antibody (Autoantibody), sometimes also can pass through cytohormone (Cytokine) and cause immune imbalance.
The modal reason of autoimmune disorders infects, the particularly infection of bacterium or virus.The structure of some pathogenic agent is similar to the antigen of host, and immunity system, in order to remove these external pathogenic agent, therefore attacks the normal cell of oneself.As similar with the DNA structure in health in caused the molecular structure of the Epstein-Barr virus of lupus erythematosus, after ebv infection human body, immunity system, in order to defend the invasion of Epstein-Barr virus, also attacks normal cells in vivo simultaneously, just produces the symptom of lupus erythematosus.Cause certain fragment of ankylosing spondylosis KB virus in addition, similar with spinal joint cellular elements, under the misidentification of normal immune system, create the symptom of ankylosing spondylosis.The antigen molecule that these pathogenic agent expose can be thought self antigen by immunity system, to avoid attacking.When immunity system is made a response to pathogenic agent, also can be attacked by the cell at the real autoantigen place of pathogenic agent and organ those.The disease be widely known by the people at present comprise multiple sclerosis that encephalitis that Second-Type adenovirus causes causes or the rheumatic fever (rheumatic fever) that streptococcal infection causes, rheumatoid arthritis (rheumatoid arthritis, RA) and some plant the diabetes etc. that viroid causes.
The second reason is normal in body and the antigen of hiding is released, and is considered as external pathogenic agent and then attack by immunity system.Human immune system is to self having identification protection mechanism; be called immunotolerance (Tolerance); such as, fetus in parent can not repel by normal mother's immune system, or at the autoimmune disorders that the place such as eyes, heart and male reproductive system occurs.The third reason is then that dysfunction appears in the regulatory T cells (Regulator T ce11, T reg) with the normal operation of immunity moderation system, cannot caused by normal regulating immunity system.Other reason has the impact of mhc gene, that is heredity, and such as ankylosing spondylosis is relevant with HLA-B27.The heredity of lupus erythematosus about account for about 10%; Or some lymphoma cell is because of after unknown cause changes, and manufactures and disengage the antibody of anti-autologous tissue.When immunotolerance is destroyed, this antigen-exposed hidden originally, immunity system is thought exotic antigen and attacks.Modal example is face's erythema of lupus erythematosus, and when irradiation ultraviolet radiation, excessive ultraviolet damage epidermic cell, the antigen-exposed that epidermic cell is hidden is disengaged, and produces face's erythema (Malarash).
Common autoimmune disorders has: systemic lupus erythematosis (SLE), rheumatoid arthritis (RA), ankylosing spondylosis (AS), chronic eczema, xerosis, scleroderma, dermatomyositis, vasculitis etc.Rheumatoid arthritis (Rheumatoid Arthritis, RA), be the sacroiliitis causing destruction of joint the most serious, it is apt to occur in the joints such as hand MCP, PIP or Wrist.Cardinal symptom is general joint pain, usually early stage patient simultaneously with finger stiffness of getting up morning (Morning stiffness) more than one hour.Serious (the Swan-neck deformity of visible hand distortion in late period; Z-shape deformity), distortion miscellaneous all likely, more seriously will cause handicapped, lifelong wheelchair.RA also may cause abarticular symptom (because immunity is general), and modal symptom is xerophthalmia (dry syndrome Sjogren ' syndrome), and eyes are dry and astringent, usually need applying eye drops in eyes; Face is done, and need usually drink water; Second common sympton is rheumatoid lung (Rheumatoid lung), refers to the fibrosis of lower lobe, and pulmonary function is deteriorated.Some patient can concurrent vasculitis, mainly betides skin table shallow place, particularly foot.More some patient can concurrent inflammatory eye or vascular circulation bad, usually feel that trick is ice-cold; Systemic lupus erythematosis (Systemic Lupus Erythematosus), its cardinal symptom is arthrodynia, serious hair loss, skin photosensitization sense (Photosensitivity) even severe patient face there is butterfly spot (Malar rash), only has light face's erythema in early days, be apt to occur in young woman, female man is than ten to one.Systemic lupus erythematous is a kind of autoimmune disorder of multiple organ, is because of B cell hyperactivity hyperkinesia, causes producing too much autoantibody.The damage of Patients with SLE organ-tissue is caused by the immune complex deposit of inflammatory reaction, and patient easily has that mouth is dry, eye dry, repeatability face broken skin (Oral ulcers); Dermatomyositis/multiple myositis (Dermatomyositis/Polymyositis), dermatomyositis symptom is muscle weakness, myalgia, and be near-end muscle weakness (Proximal muscle weakness), some patient squats down to stand up and maybe cannot sit up.Be apt to occur in young woman, common sympton is wholely have erythema on the face, and even eyelid has erythema, and erythema also appears on hand, because the inflammation of skin, the fold of hand mostly is dactylus most, so the common erythema of dactylus (Gottron ' s sign).If be only muscle weakness, but skin there is no symptom to be then called polymyositis.Myasthenia gravis is that one can cause muscle weakness, fatigable disease, mainly because nerve effectively cannot reach muscle its signal, it can have influence on much different muscle, such as, control the muscle of eyeball, controls to express one's feelings on the face, chews, speaks, the muscle swallowed and four limbs.Because everyone affected muscle is different, clinical symptom is also just not quite similar.Muscle will move, and will discharge a kind of chemical substance be called acetyl choline by motorius, after acetyl choline is combined with its receptor, could produces enough potential variation and cause muscular movement.Acetyl choline decomposes Enzyme and then it is decomposed, and current potential is recovered.The patient of gravis, because the antibody creating a kind of acetyl choline receptor in body, and then causes the destruction of receptor and the minimizing of number, makes the conduction function between nerve and muscle impaired; Scleroderma (scleroderma syndrome), be that face or shirtfront back skin are all very tight and stiff at ill Early manifestation, late period or even finger are all tight and stiff, be called and firmly refer to disease (Sclerodactyly), proximally arriving far-end starts stiff, and some patient more can the fibrosis (Pulmonary fibrosis) of Complicated with Pulmonary.The most special symptom of scleroderma is Raynaud's phenomenon (Raynaud phenomenon), because of poor circulation, finger meeting variable color, because of anoxic XianCheng white, again because sclerosis (sclerosis) becomes blue again, because congested (Reactive hyperemia) finally reddens again.
Comprehensively above-mentioned, under normal circumstances, the immunotolerance mechanism of oneself can make individuality avoid oneself's reaction (self-reaction).When this mechanism cannot normal operation, immunity system can impel T cell or B cell activation, and produce antibody mediated or cellulous reaction, resist the antigen of self, these reactions make cell and organ come to harm.Autoimmune disorders can be divided into two classes, one is organ specificity (organ-specific), the specific objective antigen (target antigen) of the organ that immune response directtissima is independent or body of gland, so only have the function of a certain organ to be stimulated by autoantibody (autoantibodies) or to block, and there is pathology, as hemolytic anemia, myasthenia gravis, insulin type diabetes; Two is general autoimmune disorders (systemic autoimmune), what act on is large-scale general target antigen, cause the tissue injury of general, it is by the accumulation of immunocomplex (immune complex) or reacts by the cellularity that autoantibody causes or directly cause the injury of cell, as multiple sclerosis.For overactive immunity system, human body has the mechanism of self-control, and that is exactly adjustment type T cell (Regulatory T cells, Tregs).Adjustment type T cell is the generation of persistence in thymus gland, and survival is in blood circulation.When the war of antagonism sex pheromone arrives coda, adjustment type T cell starts the task of performing it, and it plays the part of the role of brake, and immunity system is smoothed down.Its effect mainly through discharging the cytohormone (anti-inflammatory cytokine) of anti-inflammation, white element 10 (IL-10) and tumour necrosis factor-beta (TGF-β) between comprising.Cytohormone is exactly the language that iuntercellular is linked up, when other immunocyte similarly is dendritic cell (dendritic cell), inflammatory T cell (inflammatory T cell), scavenger cell and B cell, the signal of white element 10 and tumour necrosis factor-beta between receiving, hyperplasia will be stopped, suppress activation, even carry out carving to die, so inflammatory response will be alleviated, immunity system smooths down.According to the research of past for adjustment type T cell, one of its function is identification autoantigen, avoids the generation of autoimmune disease.Find in research that adjustment type T cell disappearance can cause fatal autoimmune disease, the hyperplasia that lymph corpuscle is runaway, produces serious inflammatory response.Find in human research, adjustment type T cell plays the part of important role in the pathogenic machine of autoimmune disease turns.Anti-inflammation hormone IL-10 secreted by adjustment type T cell or TGF-β can as the functional parameter of assessment adjustment type T cell.Probiotic bacterium grows in enteron aisle, by the Lymphoid tissue of enteron aisle, can stimulate the hyperplasia of human body adjustment type T cell.Because the composition that the cell walls victory peptide of probiotic bacterium gathers candy can combine with the susceptor in immunocyte, make adjustment type T cell activation hyperplasia, ripe adjustment type T cell can secrete IL-10 and TGF-β, by the cytohormone of these anti-inflammatioies, inflammation type T cell can be suppressed, inflammatory response is alleviated.Therefore probiotic oral has the effect of adjustment for autoimmune inflammatory response.
Summary of the invention
In view of this, the object of the invention is to, provide a kind of strain isolated BR101 producing high resistance inflammatory cells hormone, strain isolated BR101 of the present invention is the bacterial classification of screening from a Neonatal Faeces corpse or other object for laboratory examination and chemical testing, it is deposited at Foodstuff Industrial and Development Inst., deposits and be numbered BCRC910512.
The object of the invention to solve the technical problems realizes by the following technical solutions.According to the one separated microorganism strain isolated BR101 that the present invention proposes, it is a strain isolated that can produce high resistance inflammatory cells hormone, and it is deposited at Foodstuff Industrial and Development Inst., deposits and is numbered BCRC910512.
The object of the invention to solve the technical problems also can be applied to the following technical measures to achieve further.
Aforesaid strain isolated is that screening is from an infant faeces corpse or other object for laboratory examination and chemical testing.
Aforesaid strain isolated compares through 16S rDNA gene fragment order to be accredited as Lip river moral Lactobacillus species.
Aforesaid strain isolated, has Lip river moral Lactobacillus species all by the characteristic known of reflecting.
Aforesaid strain isolated, be through gram stain microscopy observations be Gram-positive bacillus.
Aforesaid strain isolated, has catalase.
Aforesaid strain isolated, does not have oxydase.
Aforesaid strain isolated, does not have mobility.
Aforesaid strain isolated, all can grow in anaerobic-aerobic environment.
Aforesaid strain isolated, the carbohydrate arbutin that can ferment, Horse Chestnut Extract glycosides Tang and D-lyxose.
Aforesaid strain isolated, has the ability that stimulating human peripheral blood glomus cell produces anti-inflammation cytohormone.
Aforesaid strain isolated, can produce the anti-inflammation cytohormone high compared with other prebiotic bacterial classifications.
Aforesaid strain isolated, the wherein group of this probiotic bacterium for forming than luxuriant and rich with fragrance De Shi dragon root fungus, lactobacillus paraceasi, Lactobacterium acidophilum, faecalis and lactobacillus rhamnosus.
Aforesaid strain isolated, wherein this anti-inflammation cytohormone between the group that forms of white element 10 and tumour necrosis factor-β.
Aforesaid strain isolated, wherein the detecting of this anti-inflammation cytohormone is for utilizing ferment immune analysis method to analyze.
Aforesaid strain isolated, wherein said ferment immune analysis method, comprises the method that ELISA assay, ELISPOT assay and other application ferment immunity principles form.
The object of the invention to solve the technical problems also realizes by the following technical solutions.According to the present invention propose a kind of can irritation cell hormone produce composition, it comprises: the strain isolated of the microorganism as elucidated before BR101 providing a significant quantity; And by filled capsules after this strain isolated interpolation excipient.
The object of the invention to solve the technical problems also can be applied to the following technical measures to achieve further.
Aforesaid composition, indication is autoimmune disorders.
Aforesaid composition, wherein this autoimmune disorders comprises the group of rheumatoid arthritis, ankylosing spondylosis and lupus erythematosus composition.
The present invention compared with prior art has obvious advantage and beneficial effect.For achieving the above object, the invention provides a kind of strain isolated BR101 producing high resistance inflammatory cells hormone, strain isolated BR101 of the present invention is the bacterial classification of screening from a Neonatal Faeces corpse or other object for laboratory examination and chemical testing, and it is deposited at Foodstuff Industrial and Development Inst., deposits and be numbered BCRC910512.
The bacterial strain separated utilizes gram stain microscopy to be viewed as Gram-positive bacillus by the present invention, and its chemistry, all can grow under aerobic, anaerobic environment for having catalase, not having oxydase and mobility with physical property.
The present invention utilizes 16S rDNA partial sequence to compare and identifies and confirms that this strain isolated BR101 is Lip river moral Lactobacillus species, and preserve and research centre purchased from Biological resources with other three strains, BCRC14625, BCRC16091, BCRC17476 reference culture carries out bacterium colony outward appearance kenel, 16S rDNA gene fragment, random primer to variance analyses such as amplification polymorphism DNA analysis, API50CHL microorganism carbohydrate biochemical reactions, there were significant differences to show this bacterial strain BR101 and other standards bacterial strain, is the Lip river moral lactobacillus of a novelty.
On the other hand, the present invention and other common probiotic bacteriums carry out anti-inflammation cytohormone and detect and analyze.After utilizing human peripheral's blood cell to carry out co-cultivation from different prebiotic bacterial classification, between carrying out with ELISPOT assay, white element 10 is analyzed.ELISPOT assay utilizes color reaction, the spot that can distinguish is manifested on the correspondence position of emiocytosis cytohormone, and can direct artificial counting spot or utilize ELISPOT identification system to count spot under the microscope, 1 spot represents 1 cell, calculates the cell quantity of this cytohormone of secretion.
It is a novel Lip river moral lactic bacilli strains that the present invention shows this strain isolated BR101 in comprehensive identification result, and can produce higher anti-inflammation cytohormone, can apply to reduce autoimmune inflammatory response, reach and improve autoimmune disorders.
In sum, the invention relates to a kind of separated novel strain BR101, this bacterial strain is separated from a Neonatal Faeces corpse or other object for laboratory examination and chemical testing, utilize strain properties and 16S rDNA gene fragment order to compare and be accredited as Lip river moral Lactobacillus species, and make bacterium colony outward appearance kenel difference with other Lip river moral lactobacillus reference cultures, 16S rDNA gene fragment difference, random primer is to amplification polymorphism DNA analysis, API50CHL microorganism carbohydrate biochemical reaction, stimulate probiotic strain screening and the functional analysis thereof of secretion IL-10, there were significant differences to show this bacterial strain BR101 and other standards bacterial strain, and this strain isolated can produce higher anti-inflammation hormone by irritation cell, reduce autoimmune inflammatory response, can apply to improve autoimmune disorders.The present invention has significant progress technically, and has obvious positively effect, is really a new and innovative, progressive, practical new design.
Above-mentioned explanation is only the general introduction of technical solution of the present invention, in order to technique means of the present invention can be better understood, and can be implemented according to the content of specification sheets, and can become apparent to allow above and other object of the present invention, feature and advantage, below especially exemplified by preferred embodiment, and coordinate accompanying drawing, be described in detail as follows.
Accompanying drawing explanation
Fig. 1 is the bacterium colony schematic appearance of BR101.
Fig. 2 is the schematic diagram of the gram stain microscopy result of BR101.
Fig. 3 A is the schematic diagram of the bacterium colony outward appearance kenel of BR101.
Fig. 3 B is that the bacterium colony outward appearance of BCRC14625 and BR101 make comparisons the schematic diagram of difference.
Fig. 3 C is that the bacterium colony outward appearance of BCRC16091 and BR101 make comparisons the schematic diagram of difference
Fig. 3 D is that the bacterium colony outward appearance of BCRC17476 and BR101 make comparisons the schematic diagram of difference.
Fig. 4 is the schematic diagram of relationship evolution tree result.
Fig. 5 is the variance analysis result schematic diagram of RAPD collection of illustrative plates.
Fig. 6 is the schematic diagram utilizing ELISPT assay to analyze the result of each probiotic bacterium secretion IL-10.
Embodiment
For further setting forth the present invention for the technique means reaching predetermined goal of the invention and take and effect, below in conjunction with accompanying drawing and preferred embodiment, to according to the present invention propose novelty Lip river moral lactic bacilli strains and be applied to its embodiment of purposes, structure, method, step, feature and effect thereof of improving autoimmune disorders, be described in detail as follows.
1. novelty microorganism BR101 strain identification
Screening culture medium is utilized to be separated single bacterium colony, and observe bacterium colony outward appearance kenel and remove from office blue Albert'stain Albert result, and coordinating the comparison of 16S rDNA gene fragment order, it is Lip river moral Lactobacillus species (Lactobacillus reuteri) that result shows this strain isolated BR101.
1.1 bacterium colony outward appearances and remove from office blue Albert'stain Albert microscopy
BR101 bacterial strain of the present invention is utilize a Neonatal Faeces corpse or other object for laboratory examination and chemical testing, with ROGOSA-agar plate (ROGOSA-AGAR plate) (Merck, Cat No.1.05413.0500) carry out strain separating as selective medium, by the single bacterium colony of separation with MRS Broth (Merck, Cat No.1.10661.0500) cultivate and cultivate 48 hours based under 37 degree of environment Celsius, single bacterium colony is analyzed, selects strain isolated BR101 and carry out strain identification.Shown by test-results, this strain isolated BR101 bacterium colony outward appearance is circular, and white colony, smooth surface convex, result as shown in Figure 1.According to the display of gramstaining result, in bluish voilet after dyeing, strain isolated BR101 is Gram-positive bacillus, and result as shown in Figure 2.Not there is mobility, there is catalase, not there is oxydase, all can grow under aerobic and anaerobic environment.
1.216S rDNA gene fragment order
Point out according to people's researchs such as 2002P.S.M.Yeung et.al., utilize special introduction pair: Primer PAF [5 ' AGA GTT TGA TCC TGG CTC AG3 '] and Primer536R [5 ' GTA TTA CCG CGG CTG CTG3 '], carry out milk-acid bacteria 16S rDNA gene PCR and amplify the qualification can carrying out genus lactubacillus.BR101 16S rDNA gene fragment order (counting roughly 555bp), and gene fragment order is shown as shown in table 1-3 through nucleic acid gene storehouse sequence alignment tools (Nuclotide BLAST) comparison result that American National biotechnology resource center (NCBI) provides, strain isolated BR101 is Lip river moral Lactobacillus species (Lactobacillus reuteri), and result display BR101 16S rDNA gene fragment and Lip river moral Lactobacillus species (Lactobacillus reuteri) similarity up to 99%, therefore can illustrate that BR101 of the present invention is Lip river moral Lactobacillus species (Lactobacillus reuteri).
The species primer of table 1Lactobacillus 16S rDNA carries out the sequence of PCR: 16S rDNA gene fragment order is identified
Table 2Sequence Blast searches: BR-101 and Lip river moral lactobacillus have 99% similar
Table 3BR-101 and Lip river moral lactobacillus have 99% similar
1.3API50CHL microorganism carbohydrate biochemical reaction
Cover group carries out 49 kinds of carbohydrate fermentations to utilize API50CH microbial biochemical reaction to identify, result display is as shown in table 4, BR101 of the present invention can ferment following 12 kinds of carbohydrates, be respectively D-arabinose (D-arabinose), L-arabinose (L-arabinose), methyl-β D-ratio is muttered xyloside (Methyl-β D-xylopyranoside), D-decomposes lactose (D-galactose), arbutin (Arbutin), Horse Chestnut Extract glycosides candy (Esculin ferric-citrate), the two candy (D-celobiose) of D-fiber, D-Maltose (D-maltose), D-lactose (D-lactose), D-pine three candys (D-melezi tose) and D-lysol candy (D-lyxose).
Table 4BR-101 in API50CHL biochemical reaction be the carbohydrate of positive reaction
The variance analysis result of 2.BR101 and other standards bacterial strain
2.1 bacterium colony outward appearance kenel difference
BR101 and other three strains Lip rivers moral Lactobacillus species (Lactobacillus reuteri) reference culture (preserving and research centre purchased from Biological resources, BCRC14625, BCRC16091, BCRC17476) is utilized to carry out variance analysis.4 strain bacterium are inoculated on MRS substratum, be placed under taking the photograph the people's 37 degree of environment, Anaerobic culturel 48 hours, bacterium colony kenel presents as that illustrated in figures 3 a-d, and BR101 bacterium colony (colony) is in white, edge (Margins) level and smooth (Entire), rat (Raised); BCRC14625 bacterium colony has transparent ring, rat in white, edge-smoothing; BCRC16091 bacterium colony is less, edge-smoothing, bacterium colony smooth (flat); BCRC17476 bacterium colony is in white, edge-smoothing, rat.
2.216S rDNA gene fragment variance analysis result
BR101 and other three strains Lip river moral Lactobacillus species (Lactobac illus reuteri) reference cultures is utilized (to preserve and research centre purchased from Biological resources, BCRC14625, BCRC16091, BCRC17476) carry out sequence alignment with 16S rDNA gene fragment order, and by after BR101 and other standards bacterial strain 16SrDNA gene fragment sequencing, Vector NTI7.0 software is utilized to compare, sequence alignment result is as shown in table 5, and sequence alignment result 4 strain bacterium respectively represents different Lip rivers moral Lactobacillus species (Lactobacillus reuteri).Display BR101 really and other standards bacterial strain there is significant difference.
The 16S rDNA gene fragment analytical results of table 5BR-101 and other three strains reference cultures
The yellow base number of a tender represents 4 strain bacterium sequences and exists together mutually, and blue base number of a tender representative at least 3 strain bacterium sequences exist together mutually, and non-base price represents sequence difference place
Utilize UPGMA (Unweighted Pair-Group Method Using Arithmetic averages) operational method in addition, set up evolution stammbaum.Result is as shown in Figure 4: BR101 is the Lip river moral Lactobacillus species (Lactobacillus reuteri) being different from BCRC14625, BCRC16091, BCRC17476.
2.3 utilize random primer to carry out diversity ratio pair to amplification polymorphism DNA analysis
Use random primer to find polymorphic DNA fragment to amplification and can be used as molecule marker.This method is RAPD (Random amplified polymorphic DNA, Randomly amplified polymorphic DNA).This RAPD technology builds on round pcr basis, it is the oligonucleotide strand (being generally 10 aggressiveness) of the random alignment base sequence utilizing a series of (usual hundreds of) different is introduction, pcr amplification is carried out to studied genomic dna (this experiment refers to milk-acid bacteria genomic dna), detect the polymorphism of amplified production DNA fragmentation, the polymorphism of these amplified production DNA fragmentations reflects the DNA polymorphism of genome respective regions.If therefore genome just may cause the distribution of these particular combination bit points to occur to change accordingly at the insertion of these regions generation DNA fragmentation, disappearance or base mutation, and PCR primer is made to increase, lack or occur the change of molecular weight.Therefore RAPD can carry out polymorphic detection to whole genomic dna.Introduction Primer P3 [5 ' GGT GAC GCA G3 '] is utilized to carry out the RAPD comparison of BR101 and BCRC14625, BCRC16091, BCRC17476 reference culture genomic dna, as shown in Figure 5, BR101RPAD Pattern is 1050bp, 700bp, 400bp, 280bp, 190bp to the display of DNA electrophorogram result; BCRC14625RPAD Pattern is 1400bp, 1050bp, 700bp, 400bp, 280bp, 190bp; BCRC16091RPAD Pattern is 1050bp, 700bp, 400bp, 280bp; BCRC17476RPAD Pattern is 2Kb, 1800bp, 1400bp, 800bp, 700bp, 400bp, 190bp, 2Kb700bp.This result confirms BR101 and BCRC14625, BCRC16091, BCRC17476 are different Lip river moral Lactobacillus species (Lactobacillus reuteri).
2.4 utilize API50CHL to carry out the variance analysis of bacterial strain carbohydrate biochemical reaction
The situation utilizing API50CHL to detect 4 strain bacterial strains to ferment in 49 kinds of carbohydrates, observes the carbohydrate fermentation difference situation of 4 strain bacterial strains.Wherein only BR101 for Arbt ine (arbutin, also claim Resorcinol glucoside), Esculin ferric citrate (Horse Chestnut Extract glycosides candy) react with D-lyxose (D-lyxose) three kinds of carbohydrates is positive reaction, display BR101 is different from other Lip river moral Lactobacillus species (Lactobacillus reuteri) reference cultures, and result is as shown in table 6.
The carbohydrate biochemical reaction difference analysis of table 6BR-101 and other standards bacterial strain
3. stimulate probiotic strain screening and the functional analysis thereof of secretion IL-10
About 10 milliliters, patient blood sample (ml), patient blood is slowly added containing Ficoll-Hypaque (GH Heal thcare along tube wall, Cat.No.17-1400-02) in centrifuge tube, centrifugal (per minute 1500 turns is carried out with refrigerated centrifuge, 15 minutes) carry out whole blood blood cell be separated, separation surface place is peripheral blood glomus cell (PBMC).The PBMC of taking-up is placed in 15 milliliters of new (ml) centrifuge tubes, and adds 10ml 1 × PBS in centrifuge tube, carry out centrifugal per minute 1500 turns with whizzer, 15 minutes (1500rpm, 5min), removing supernatant liquor, leaves blood cell pellet.Get 10 milliliters of (ml) RPMI-1640 substratum (containing 1%Penicillin-Streptomycin and 10%Calf serum) in centrifuge tube, carry out resorption punching and avoid bubble to produce, blood cell is uniformly distributed.Get blood cell suspension to mix with trypan blue (trypan blue) stain equal-volume, carry out cell counting with counting chamber.And with RPMI-1640 substratum adjustment blood cell suspension concentration for 4.0 × 10
6cells/ml is for subsequent use.
The blood cell suspension getting 200 microlitres (μ l) adds aseptic 96 porose discs, and adds the probiotics bacterial liquid of 20 microlitres (μ l), is placed in 37 degree of 5%CO Celsius
2cultivate in environment after 48 hours, take out supernatant liquor and carry out IL-10ELISPOT assay.Its result as shown in Figure 6.Above the selection result, the probiotic strain with irritation cell secretion IL-10 is BR101.
4. utilize human peripheral's blood cell to screen the probiotic strain stimulating secretion IL-10
In autoimmune disorders, comparatively common with rheumatoid arthritis, therefore collect the blood of patient with rheumatoid arthritis, and isolate after its human peripheral's blood cell carries out co-cultivation from different prebiotic bacterial classification, the prebiotic bacterial classification judging to be applicable to rheumatoid joint patient is analyzed with ELISPOT assay.The bacterial classification comparing analysis comprises lactobacillus paraceasi (L.paracasei), Lactobacterium acidophilum (L.acidophilus), the prebiotic bacterial classification more common than luxuriant and rich with fragrance De Shi dragon root fungus (B.longum), faecalis (E.faecium), lactobacillus rhamnosus (L.rhamnosus), BR101 of the present invention (L.reuteri) etc. six kinds.
4.1BR101 and the preparation of other probiotic strains
After probiotic bacterium for carrying out screening is cultivated with MRS Broth, with per minute 4000 turns, centrifugal 5 minutes, removal supernatant liquor adds 1 × PBS buffer and washs three times, throw out bottom test tube is become suspension with 1 × PBS buffer back dissolving, and be that 5.0 × 108CFU/ml (measures with OD600 with 1 × PBS dilution adjustment bacterium number, when OD600 is 1.0, estimating bacterium number is 5.0 × 108cell/ml), be placed in 95 degree of water bath heat treated Celsius after 5 minutes, be placed in four degrees celsius for subsequent use.
Lip river moral lactobacillus L.reuter i BR101 is that this bacterial classification is L. reuteri through molecular biology identification by the bacterial classification of Guang Sheng biotechnology company screening from an infant faeces corpse or other object for laboratory examination and chemical testing.By this inoculation in MRS liquid nutrient medium, after cultivating activation amplification in 6-8 hour with 37 degree Celsius under being placed in anaerobic environment, lyophilize mode is utilized to carry out freeze-drying.Freeze-dried vaccine powder powder detects bacterium number must be greater than 1.0 × 1011CFU/g.Getting 1g bacterium powder is dissolved in 10 milliliters of (ml) sterilized waters, after full and uniform mixing, with per minute 4000 turns, centrifugal 5 minutes, removal supernatant liquor adds 1 × PBS buffer and washs three times, throw out bottom test tube is become suspension with 1 × PBS buffer back dissolving, and be that 5.0 × 108CFU/ml (measures with OD600 with 1 × PBS dilution adjustment bacterium number, when OD600 is 1.0, estimating bacterium number is 5.0 × 108cell/ml, bacterium powder mixed solution is diluted to 2.0 × 108CFU/ml, be placed in 95 degree of water bath heat treated Celsius after 5 minutes, be placed in four degrees celsius for subsequent use.
4.2 human peripheral's blood cell are collected and preparation
Collect patient blood about 10 milliliters, sample (ml), patient blood is slowly added containing Ficoll-Hypaque (GH Healthcare along tube wall, Cat.No.17-1400-02) in centrifuge tube, gradient centrifugation (1500rpm is carried out with refrigerated centrifuge, blood cell 15min) carrying out whole blood is separated, separation surface place is peripheral blood glomus cell (PBMC), and throw out is red blood corpuscle.The PBMC of taking-up is placed in 15 milliliters of new (ml) centrifuge tubes, and add 10 milliliters of (ml) 1 × PBS in centrifuge tube, carry out centrifugal (per minute 1500 turns, 5 minutes) with whizzer, removing supernatant liquor, leaves blood cell pellet.Get 10 milliliters of (ml) RPMI-1640 substratum (containing 1%Penicillin-Streptomycin and 10%Calf serum) in centrifuge tube, carry out resorption and rush 20 times and avoid bubble to produce, blood cell is uniformly distributed.Get blood cell suspension to mix with trypan blue stain equal-volume, carry out cell counting with counting chamber.And with RPMI-1640 substratum adjustment blood cell suspension concentration for 4.0 × 10
6cells/ml is for subsequent use.
The preparation of 4.3 experiment control groups
The control group of this test is normal control group (Normal control) and positive regulation group (Positive control), and normal control group is PRMI-1640 (containing 10%FBS); Positive regulation group is 1 mcg/ml (μ g/ml) LPS (Lipopolysaccharide, Escherichia coil serotype O55:B5, Sigma).This test uses lipopolysaccharides (Lipopolysaccharide) for positive regulation group because it is that a larger molecule is connected to form by covalent linkage by fat and polysaccharide.Lipopolysaccharides is the chief component of gram negative bacterium adventitia, provides bacterium with the integrity of structure, and protects bacterial film to be subject to the attack of some chemical substance.Lipopolysaccharides is intracellular toxin, and strong immunization can be caused to react.
PBMC is stimulated to produce the analysis of IL-10 4.4 utilize ELISPOT assay to carry out milk-acid bacteria
ELISPOT assay utilizes color reaction, the spot that can distinguish is manifested on the correspondence position of emiocytosis cytohormone, and can direct artificial counting spot or utilize ELISPOT identification system to count spot under the microscope, 1 spot represents 1 cell, calculates the cell quantity of this cytohormone of secretion.Its Cleaning Principle utilizes PVDF material film bottom 96 porose discs, is used for adsorbing special selecting and the monoclonal antibody of nontoxicity (not containing sodium azide, intracellular toxin endotoxin).When PBMC (human peripheral blood cell) cell that obtain of a blood corpse or other object for laboratory examination and chemical testing after being separated is added on 96 porose discs, cultivate under micropore dish being positioned over after utilizing antigenic stimulation proper temperature after 16 ~ 24 hours, memory T cells can start secretory cell hormone by after antigenic stimulation a few hours, and now local (near around secretory cell) oozy cytohormone can be caught by specific antibody in PVDF thin film.Cell in micropore dish is removed and after cleaning, the secondary antibodies that captured cytohormone can use vitamin H (Biot in) to mark further indicates, thereafter act on it with the StreptAvidin in conjunction with ferment again, and add ferment and make its colour generation by matter, the cell of the effect of responding can leave dyeing speck.
Its reactions steps is as follows:
1) in aseptic operating platform, (precoated) mAb9D7 dish (plate) of precoating is risen again to room temperature from four degrees celsius, wash 4 times (200 μ l/well) with aseptic 1 × PBS;
2) the substratum RPMI-1640 (containing 10%FBS) adding 200 μ l/well carries out blacking30 minute under room temperature;
3) remove substratum, wash once with 100 μ l1 × PBS, pat on thieving paper;
4) (cell concn is adjusted to 1.0 × 10 to add PBMC
6-1.0 × 10
8and BR101 bacterium liquid (1.0 × 10 cells/ml)
6-1.0 × 10
8cFU/ml), last volume counts roughly 150 μ l/well, and now positive control group is that mAb CD3-2 adds simultaneously, and experimental concentration is 100ng/ milliliter (ml);
5) (plate) will be coiled and be placed in 37 degree of 5%CO Celsius
2react 12 ~ 48 hours in incubator;
6) remove cell suspending liquid, add 200 μ l/well1 × PBS and wash five times;
7) dilution detecting antibody 12-G8-biotin to concentration 1 μ g/ milliliter (ml) is in 1 × PBS-0.5%FBS (1 × PBS is containing 0.5% foetal calf serum), adds the antibody that 100 μ l/well dilute, reacts 2 hours under room temperature;
8) remove supernatant liquor, add 200 μ l/well1 × PBS and wash five times;
9) dilute Streptavidin-ALP (1: 1000) in 1 × PBS-0.5%FBS (1 × PBS is containing 0.5% foetal calf serum), add 100 μ l/well and react 1 hour under room temperature;
10) remove supernatant liquor, add 200 μ l/well1 × PBS and wash five times;
11) photoghraphic coupler BCIT/NBT carried out filtering with 0.45 μm of filter membrane and add 100 μ l/well, as under room temperature until spot presents;
12), after developing the color completely, supernatant liquor is added in corresponding dish, fully wash the both sides of film with distilled water.Thieving paper is patted, makes film dry.During preservation, plate is inverted in order to avoid residual liquid to flow back on film once after film drying, utilizes microscope or interpreting system namely to can read spot number, will coil under (plate) is placed in room temperature and keep in Dark Place.
After utilizing ELISPOT assay to analyze six probiotics and human peripheral's blood cell co-cultivation, carry out the analysis stimulating secretion IL-10, calculate the cell quantity of this cytohormone of secretion according to mottle number, result as shown in Figure 6.It is 324 that its spot number result is sequentially BR101; B.longum is 306; E.faecium is 304; L.rhamnosus is 298; L.acidophilus is 294; L.paracasei is 240.Therefore the probiotic bacterium filtering out stimulation secretory volume the highest is BR101 Lip river moral Lactobacillus species (Lactobacillus reuteri).
5. utilize BR101 to carry out unofficial human trial
Experimenter is preferential regularly can follow the trail of curer in outpatient service, and need meet following condition: 1) 19 to 75 years old ages; 2) in nearest 1 year, once blood letting found that Rheumatoid factors, polyclonal was positive; 3) phase of falling ill is 6 weeks ~ 1 year; 4) fixing drug administration person please regularly follows the trail of; 5) steroid medicine dosage > every day 7.5 milligrams; 6) or in nearly 4 weeks intra-articular injection person is accepted.
Other are got rid of and participate in trier standard and comprise: being diagnosed as the rheumatoid arthritis phase is greater than more than 1 year person, hepatic and renal function sufferer, chronic or frequent infection (as chronic bronchitis, recurrent chronic sinusitis) or other catch, other administration factor, as removal of home, economic factors or compatibility test etc. then be unwilling to get rid of test.Complete trier and amount to 14, test period is 1 month.
Get Lip river moral Lactobacillus species (Lactobacillus reuteri) BR101 to carry out after activation amplifies, carrying out powder processing with freeze-drying method, bacterium powder being added capsule charge after excipient, makes every capsule bacterium number be greater than 5 × 10
9cFU.This experiment sieving phase is 4 weeks, confirm and obtain after patient agrees to test in outpatient service, and fill in first time Symptoms index, eat 6 capsules every day afterwards, experimenter please fill in second time Symptoms index again after 1 month, and analyze according to the Symptoms index that experimenter fills in.This Symptoms index is amendment DAS (Di sease Act ivity Score) and HAQ (Health Assessment Questionnaire), content comprises five major joint (elbow joint, wrist joint, finger-joint, knee joint and podarthral symptom) in the occurrence frequency of pain, stiff, the symptom such as swelling and heating and severity, comprise the assessment of daily life in addition, as shown in table 7.
Table 7 " rheumatoid arthritis " paresthesia inquiry schedule
After Data acquisition, data analysis is carried out with statistic software SPSS 17.0 editions, add up with nonparametric test, add up with Wilcoxon Signed Rank Test, after analyzing, P value < 0.05 has statistically significant difference meaning, main assessment foundation is the mark on symptoms of rheumatoid arthritis questionnaire, and all data represent with mean number (Mean) ± S.D..
Statistics is as shown in table 8, and experimenter ate capsule containing BR101 bacterium powder after 30 days, and before and after experimenter's self-assessment is edible, whether rheumatoid arthritis related symptoms has difference situation.After statistical analysis, with regard to the severity that symptom occurs, always grade in finger-joint, daily life and all there is statistically significant difference (Table6., P < 0.05); And on symptom occurrence frequency, all there were significant differences in wrist joint, finger-joint and daily life for experimenter.Display experimenter ate the capsule containing BR101 bacterium powder after 30 days, improve significantly (P value < 0.05) on severity of symptom and occurrence frequency, represent that edible BR101 can make patient with rheumatoid arthritis really except reducing severity of symptom, also there is the ability of quality of making the life better.All experimenters all complete the experiment of 30 days, do not have seriously bad reaction to produce in experimental session.
The unofficial human test results of table 8 analyzes (N=14)
The above, it is only preferred embodiment of the present invention, not any pro forma restriction is done to the present invention, although the present invention discloses as above with preferred embodiment, but and be not used to limit the present invention, any those skilled in the art, do not departing within the scope of technical solution of the present invention, when the method and technology contents that can utilize above-mentioned announcement are made a little change or be modified to the Equivalent embodiments of equivalent variations, in every case be the content not departing from technical solution of the present invention, according to any simple modification that technical spirit of the present invention is done above embodiment, equivalent variations and modification, all still belong in the scope of technical solution of the present invention.
Claims (4)
1. separated Lip river moral lactobacillus (Lactobacillus reuteri) strain isolated BR101, it is characterized in that: described strain isolated is a strain isolated that can produce high resistance inflammatory cells hormone, it is deposited at China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposits and is numbered CGMCC No.6637.
2. can irritation cell hormone produce a composition, it is characterized in that: described composition comprises:
The microorganism strain isolated BR101 as claimed in claim 1 of one significant quantity is provided; And
By filled capsules after this strain isolated interpolation excipient.
3. composition according to claim 2, is characterized in that: the indication of described composition is autoimmune disorders.
4. composition according to claim 3, is characterized in that: this autoimmune disorders comprises the group of rheumatoid arthritis, ankylosing spondylosis and lupus erythematosus composition.
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