CN103224895A - 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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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 a kind of newborn infant's of stemming from ight soil corpse or other object for laboratory examination and chemical testing, compare through 16S rDNA gene fragment order and to be accredited as Lip river moral lactobacillus bacterial classification, and compare with other Lip river moral lactobacillus reference cultures, there were significant differences to show this bacterial strain BR101 and other reference cultures, and have irritation cell and produce higher anti-inflammation hormone, reduce autoimmune inflammatory response, reach the Lip river moral lactic bacilli strains of the novelty of improving autoimmune disorders.It is deposited at China Committee for Culture Collection of Microorganisms common micro-organisms center on September 24th, 2012, address: No. 3, No. 1 institute in North Star West Road, Chaoyang District, BeiJing, China city, deposit numbering CGMCC No.6637, also be deposited at TaiWan, China Foodstuff Industrial and Development Inst., deposit numbering BCRC910512.
Background technology
Immunity system is being played the part of critical role for the existence of organism, and it helps us to resist various infective microorganisms, comprises bacterium, virus, fungi, parasite etc.When pathogenic microorganism 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 materials such as antibody exactly and tackles pathogenic bacteria.Under tight like this protection, human body can be said so strongly fortified, the city that is difficult to capture.Autoimmune disorders is exactly that oneself immunity system is out of control, attacks tissue or the organ of oneself as wild chargers.Just be called rheumatoid arthritis if attack the joint cartilage of oneself, the vertebra of attacking self is exactly an ankylosing spondylosis, attacks pancreas and can cause the first type diabetes, and attacking eyeball is exactly iritis, and the blood vessel of attacking whole body is exactly a lupus erythematosus.Though these diseases are etiology unknown at present, symptom also has nothing in common with each other, and it all is immune imbalance that morbific machine changes, so methods of treatment all is to be target with the adjusting immunity system.
Autoimmune disorders (Autoimmune disease) is the Normocellular disease of the own health of a kind of immune system attack; have antibody in the immunity system of human body and can resist external antigen; as bacterium or virus; or abnormal cell in the body; attack and remove as tumour cell etc., such behavior is a kind of physiological mechanism of protecting health.Produce Normocellular antibody in the own health body of meeting antagonism voluntarily but autoimmune disorders is an immunity system, cause abnormal inflammatory response or tissue injury, and then influence healthy disease.These antibody are called autoimmunity antibody (Autoantibody), also can cause immune imbalance by cytohormone (Cytokine) sometimes.
The modal reason of autoimmune disorders is to infect, particularly the infection of bacterium or virus.The structure of some pathogenic agent is similar to host's antigen, and therefore immunity system attacks the normal cell of oneself in order to remove these external pathogenic agent.As the molecular structure of Epstein-Barr virus that causes lupus erythematosus is similar with the intravital dna structure of body, and behind the ebv infection human body, immunity system is also attacked normal cells in vivo simultaneously in order to defend the invasion of Epstein-Barr virus, just produces the symptom of lupus erythematosus.Cause certain fragment of ankylosing spondylosis KB virus in addition, similar with the spinal joint cellular elements, under normal immune misidentification, produced the symptom of ankylosing spondylosis.The antigen molecule that these pathogenic agent expose can be thought the antigen of self by immunity system, to avoid attack.When immunity system is made a response to pathogenic agent, also can attack those cell and organs by the real autoantigen of pathogenic agent place.The disease that is widely known by the people at present comprise rheumatic fever (rheumatic fever) that multiple sclerosis that encephalitis caused that the second type adenovirus causes or streptococcal infection causes, rheumatoid arthritis (rheumatoid arthritis, RA) and some plant diabetes that viroid causes etc.
Second kind of reason is that normal and antigen hiding property is released in the body, is considered as external pathogenic agent and then attack by immunity system.The human immune system is to self having the identification protection mechanism; be called immunotolerance (Tolerance); for example female intravital fetus can not repelled by normal mother's immune system, or the autoimmune disorders of locating to take place at eyes, heart and male reproductive system etc.The third reason then be have the regulatory T cells of regulating the immunity system normal operation (Regulator T cell Treg) dysfunction occurs, can't the normal regulating immunity system due to.Other reason has the influence of mhc gene, that is heredity, and for example ankylosing spondylosis is relevant with HLA-B27.The heredity of lupus erythematosus has accounted for about 10%; Or the antibody of anti-autologous tissue is made and disengaged to some lymphoma cell because of after unknown cause changes.When immunotolerance destroyed, the former antigen-exposed that should hide, immunity system is thought exotic antigen and is attacked.Modal example is face's erythema of lupus erythematosus, and when irradiation ultraviolet radiation, excessive ultraviolet ray destroys epidermic cell, and 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), for causing the most serious sacroiliitis of destruction of joint, its good sending out in joints such as hand MCP, PIP or Wrist.Cardinal symptom is a whole body arthrodynia, and usually early stage patient points stiff (Morning stiffness) above one hour with getting up morning simultaneously.Serious (the Swan-neck deformity of visible hand distortion in late period; Z-shape deformity), distortion miscellaneous is all possible, more serious will cause handicapped, lifelong wheelchair.RA also may cause abarticular symptom (because of immunity is a general), and modal symptom is xerophthalmia (dry syndrome Sjogren ' syndrome), and eyes are dry and astringent, usually need eye droppings liquid medicine; Face is done, and need usually drink water; Second common sympton is rheumatoid lung (Rheumatoid lung), refers to the fibrosis of lower lobe, the pulmonary function variation.Some patient understands concurrent vasculitis, mainly betides skin table shallow place, particularly foot.It is bad that more some patient understands the circulation of concurrent inflammatory eye or blood vessel, usually feels that trick is ice-cold; Systemic lupus erythematosis (Systemic Lupus Erythematosus), its cardinal symptom is that butterfly spot (Malarrash) appears in arthrodynia, serious hair loss, skin photaesthesia (Photosensitivity) even severe patient face, has only light face's erythema in early days, good sending out in the young woman, the woman man is than ten to one.Systemic lupus erythematous is a kind of autoimmune disorder of many organs, is excessive because of the B cellular activity, causes producing too much autoantibody.The damage of Patients with SLE organ-tissue is by due to the immune complex deposit of inflammatory reaction, and the patient has mouth to do easily, eye is done, the broken skin (Oralulcers) of repeatability face; Dermatomyositis/polymyositis (Derma tomyositis/Polymyositis), the dermatomyositis symptom is muscle weakness, myalgia, and be near-end muscle weakness (Proximal muscle weakness), some patient squats down to stand up and maybe can't sit up.Good sending out in the young woman, common sympton is the whole erythema that has on the face, even eyelid all has erythema, erythema also can appear 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 muscle weakness only, but skin does not have symptom then to be called polymyositis.Myasthenia gravis is that a kind of meeting causes muscle weakness, fatigable disease, mainly be because nerve can't effectively reach muscle to its signal, it can have influence on many different muscle, for example controls the muscle of eyeball, muscle and four limbs that control is expressed one's feelings on the face, chews, speaks, swallowed.Owing to everyone affected muscle difference, clinical symptom also just is not quite similar.Muscle will move, and will discharge a kind of chemical substance by motorius and be called acetyl choline, and acetyl choline could produce enough potential variation and cause muscular movement with after its receptor combines.Acetyl choline decomposes Enzyme and then it is decomposed, and current potential is recovered.The patient of gravis because produced a kind of antibody of acetyl choline receptor in the 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), show as face in early days or shirtfront back skin is all very tight and stiff ill, late period or even finger are all tight and stiff, be called hard finger disease (Sclerodactyly), from the near-end to the far-end, begin stiff, the fibrosis (Pulmonaryfibrosis) that some patient more can concurrent lung.The most special symptom of scleroderma is Raynaud's phenomenon (Raynaud phenomenon), because of poor circulation, finger meeting variable color is because of anoxic XianCheng white, again because of sclerosis (sclerosis) becomes blueness again, because congested (Reactive hyperemia) reddens at last again.
Comprehensively above-mentioned, under normal circumstances, oneself's immunotolerance mechanism can make individuality avoid oneself's reaction (self-reaction).Can't normal operation when this mechanism, immunity system can be impelled T cell or B cell activation, produces body fluid or cellulous reaction, resists the antigen of self, and these reactions come to harm cell and organ.Autoimmune disorders can be divided into two classes, one is organ specificity (organ-specific), the specific objective antigen of organ that the immune response directtissima is independent or body of gland (target antigen), so have only the function of a certain organ to be stimulated or blocking-up by autoantibody (autoantibodies), and pathology appears, as hemolytic anemia, myasthenia gravis, insulin type diabetes; Two is general autoimmune disorders (sys temic 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 by the reaction of cellularity that autoantibody caused or directly cause the injury of cell, as multiple sclerosis.For overactive immunity system, human body has the mechanism of self-control, that be 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 arrival coda of antagonism pathogeny microorganism, adjustment type T cell begins to carry out its task, and it plays the part of the role of brake, and immunity system is smoothed down.Its effect is mainly by discharging the cytohormone (anti-inflammatory cytokine) of anti-inflammation, white plain 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, will stop hyperplasia, suppress activation, even carve and 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 the identification autoantigen, avoids the generation of autoimmune disease.Find in the research that adjustment type T cell disappearance can cause fatal autoimmune disease, the hyperplasia of lymph corpuscle property out of control produces serious inflammatory response.Find that in the human research adjustment type T cell is played the part of important role in the pathogenic machine of autoimmune disease changes.Anti-inflammation hormone IL-10 that adjustment type T cell is secreted or TGF-β can be used as the functional parameter of assessment adjustment type T cell.Probiotic bacterium grows in enteron aisle, can stimulate human body adjustment type T proliferation of cells by the Lymphoid tissue of enteron aisle.Because the cell walls of probiotic bacterium win the poly-candy of peptide composition can with the susceptor combination in the immunocyte, make adjustment type T cell activation hyperplasia, sophisticated adjustment type T cell can be secreted IL-10 and TGF-β, cytohormone by these anti-inflammatioies, can suppress inflammation type T cell, inflammatory response is alleviated.Therefore probiotic oral has the effect of adjusting for autoimmune inflammatory response.
Summary of the invention
In view of this, the objective of the invention is to, a kind of strain isolated BR101 that produces high anti-inflammation cytohormone is provided, strain isolated BR101 of the present invention is the bacterial classification of screening from newborn infant's ight soil corpse or other object for laboratory examination and chemical testing, it is to be deposited at Foodstuff Industrial and Development Inst., deposits and is numbered BCRC910512.
The object of the invention to solve the technical problems realizes by the following technical solutions.According to a kind of separated microorganism strain isolated BR101 that the present invention proposes, it is one can produce the strain isolated of high anti-inflammation cytohormone, and it is deposited at Foodstuff Industrial and Development Inst., deposits to be 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 is to compare through 16S rDNA gene fragment order to be accredited as Lip river moral lactobacillus bacterial classification.
Aforesaid strain isolated has Lip river all characteristics of being known by mirror of moral lactobacillus bacterial classification.
Aforesaid strain isolated is to be Gram-positive bacillus through the gram stain microscopy observations.
Aforesaid strain isolated has catalase.
Aforesaid strain isolated does not have oxydase.
Aforesaid strain isolated does not have mobility.
Aforesaid strain isolated all can be grown in the 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 the anti-inflammation cytohormone.
Aforesaid strain isolated can produce the anti-inflammation cytohormone high than other probiotic bacterium kinds.
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 be between white plain 10 and group that tumour necrosis factor-β formed.
Aforesaid strain isolated, wherein this anti-inflammation cytohormone detecting is for utilizing the 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 are formed.
The object of the invention to solve the technical problems also realizes by the following technical solutions.But according to the composition that a kind of irritation cell hormone that the present invention proposes produces, it comprises: the microorganism strain isolated BR101 as previously described that a significant quantity is provided; And with filled capsules behind 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.
Aforementioned compositions, indication are autoimmune disorderss.
Aforementioned compositions, wherein this autoimmune disorders comprises the group that rheumatoid arthritis, ankylosing spondylosis and lupus erythematosus are formed.
The present invention compared with prior art has tangible advantage and beneficial effect.For achieving the above object, the invention provides a kind of strain isolated BR101 that produces high anti-inflammation cytohormone, strain isolated BR101 of the present invention is the bacterial classification of screening from newborn infant's ight soil corpse or other object for laboratory examination and chemical testing, and it is to be deposited at Foodstuff Industrial and Development Inst., deposits and is numbered BCRC910512.
The present invention utilizes gram stain microscopy to be viewed as Gram-positive bacillus the bacterial strain of separating, and its chemistry and physical property be for having catalase, do not have oxydase and mobility, all can grow under aerobic, anaerobic environment.
The present invention utilizes 16S rDNA partial sequence to compare and identifies and confirms that this strain isolated BR101 is Lip river moral lactobacillus bacterial classification, and preserve and the research centre available from Biological resources with other three strains, BCRC14625, BCRC16091, BCRC17476 reference culture carry out bacterium colony outward appearance kenel, 16S rDNA gene fragment, random primer to difference analyses such as amplification polymorphism DNA analysis, API50CHL microorganism carbohydrate biochemical reactions, there were significant differences to show this bacterial strain BR101 and other reference cultures, 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 detecting analysis.After utilizing human peripheral blood glomus cell and different probiotic bacterium kinds being carried out co-cultivation, white plain 10 analyze between carrying out with ELISPOT assay.ELI SPOT assay utilizes color reaction, on the correspondence position of emiocytosis cytohormone, manifest the spot that to distinguish, and can be directly at microscopically artificial counting spot or utilize the ELISPOT identification system that spot is counted, 1 spot is represented 1 cell, calculates the cell quantity of this cytohormone of secretion.
The present invention shows that this strain isolated BR101 is a novel Lip river moral lactic bacilli strains in comprehensive qualification result, and can produce higher anti-inflammation cytohormone, can apply to reduce autoimmune inflammatory response, reaches and improves autoimmune disorders.
In sum, the invention relates to a kind of separated novel strain BR101, this bacterial strain is to separate from newborn infant's ight soil 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 bacterial classification, 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 the 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 reference cultures, and but this strain isolated irritation cell produces higher anti-inflammation hormone, reduce autoimmune inflammatory response, can apply to improve autoimmune disorders.The present invention has obvious improvement technically, and has tangible positively effect, really is a new and innovative, progressive, practical new design.
Above-mentioned explanation only is the general introduction of technical solution of the present invention, for can clearer understanding technique means of the present invention, and can be implemented according to the content of specification sheets, and for above-mentioned and other purposes, feature and advantage of the present invention can be become apparent, below especially exemplified by preferred embodiment, and conjunction with figs., be described in detail as follows.
Description of drawings
Fig. 1 is the bacterium colony schematic appearance of BR101.
Fig. 2 is the gram stain microscopy result's of BR101 a synoptic diagram.
Fig. 3 A is the synoptic diagram of the bacterium colony outward appearance kenel of BR101.
Fig. 3 B is the make comparisons synoptic diagram of difference of the bacterium colony outward appearance of BCRC14625 and BR101.
Fig. 3 C is the make comparisons synoptic diagram of difference of the bacterium colony outward appearance of BCRC16091 and BR101
Fig. 3 D is the make comparisons synoptic diagram of difference of the bacterium colony outward appearance of BCRC17476 and BR101.
Fig. 4 is relationship evolution tree result's a synoptic diagram.
Fig. 5 is the variance analysis result schematic diagram of RAPD collection of illustrative plates.
Fig. 6 utilizes ELISPT assay to analyze the result's of each probiotic bacterium secretion IL-10 synoptic diagram.
Embodiment
Reach technique means and the effect that predetermined goal of the invention is taked for further setting forth the present invention, below in conjunction with accompanying drawing and preferred embodiment, the Lip river moral lactic bacilli strains of the novelty that foundation the present invention is proposed and be applied to improve its embodiment of purposes, structure, method, step, feature and the effect thereof of autoimmune disorders, describe in detail as after.
1. novelty microorganism BR101 strain identification
Utilize screening culture medium to separate single bacterium colony, and observe bacterium colony outward appearance kenel and remove from office blue Albert'stain Albert result, and cooperate the comparison of 16S rDNA gene fragment order, the result shows that this strain isolated BR101 is a Lip river moral lactobacillus bacterial classification (Lactobacillusreuteri).
1.1 bacterium colony outward appearance and remove from office blue Albert'stain Albert microscopy
BR101 bacterial strain of the present invention is to utilize newborn infant's ight soil corpse or other object for laboratory examination and chemical testing, with ROGOSA-agar plate (ROGOSA-AGAR plate) (Merck, Cat No.1.05413.0500) carries out strain separating as selective medium, with isolating single bacterium colony with MRS Broth (Merck, Cat No.1.10661.0500) cultivates based on cultivating 48 hours under the 37 degree environment Celsius, single bacterium colony is analyzed, selected strain isolated BR101 and carry out strain identification.Show that by test-results this strain isolated BR101 bacterium colony outward appearance is circular, white colony, smooth surface convex, result are as shown in Figure 1.Show that according to the gramstaining result be bluish voilet after the dyeing, strain isolated BR101 is a Gram-positive bacillus, the result as shown in Figure 2.Do not have mobility, have catalase, do not have oxydase, all can grow under the aerobic and anaerobic environment.
1.216S rDNA gene fragment order
Point out according to people such as 2002P.S.M.Yeung et.al. research, utilize special introduction right: Primer PAF[5 ' AGA GTT TGA TCC TGG CTC AG3 '] and Primer 536R[5 ' GTA TTA CCG CGG CTG CTG 3 '], carry out milk-acid bacteria 16S rDNA gene PCR amplification and can carry out the evaluation of genus lactubacillus.BR10116S rDNA gene fragment order (counting roughly 555bp), and nucleic acid gene storehouse sequence alignment tools (Nuclotide BLAST) comparison result shows that gene fragment order is provided through American National biotechnology resource center (NCBI) is shown in table 1-3, strain isolated BR101 is a Lip river moral lactobacillus bacterial classification (Lactobacillus reuteri), and the result shows BR10116S rDNAgene fragment and the similarity of Lip river moral lactobacillus bacterial classification (Lactobacillus reuteri) up to 99%, therefore can illustrate that BR101 of the present invention is a Lip river moral lactobacillus bacterial classification (Lactobacillusreuteri).
The species primer of table 1 Lactobacillus 16S rDNA carries out the sequence of PCR: 16S rDNA gene fragment order is identified
Table 2Sequence Blas t searches: BR-101 and Lip river moral lactobacillus have 99% similar
It is 99% similar that table 3BR-101 and Lip river moral lactobacillus have
1.3API50CHL microorganism carbohydrate biochemical reaction
Utilize API50CH microbial biochemical reaction evaluation cover group to carry out 49 kinds of carbohydrates fermentations, the result shows as shown in table 4, the BR101 of the present invention following 12 kinds of carbohydrates that can ferment are respectively D-arabinose (D-arab inose), L-arabinose (L-arab inose), methyl-β D-ratio xyloside (Methy1-β D-xylopyranoside) of muttering, D-decomposes newborn candy (D-galactose), arbutin (Arbutin), Horse Chestnut Extract glycosides candy (Esculin ferric-citrate), the two candys (D-celobiose) of D-fiber, D-maltose (D-maltose), D-lactose (D-lactose), D-pine three candys (D-melezitose) and D-lysol candy (D-lyxose).
Table 4BR-101 is in the carbohydrate of the positive reaction of API50CHL biochemical reaction
2.BR101 variance analysis result with other reference cultures
2.1 bacterium colony outward appearance kenel difference
Utilize BR101 and other three strains Lip river moral lactobacillus bacterial classification (Lactobacil lus reuteri) reference cultures (preserving and the research centre BCRC14625, BCRC16091, BCRC17476 available from Biological resources) to carry out variance analysis.4 strain bacterium are inoculated on the MRS substratum, place under the 37 degree environment Celsius, anaerobism was cultivated 48 hours, and the bacterium colony kenel presents shown in Fig. 3 A-D, and BR101 bacterium colony (colony) is white in color, edge (Margins) level and smooth (Entire), rat (Raised); The BCRC14625 bacterium colony is white in color, edge-smoothing has transparent ring, rat; The BCRC16091 bacterium colony is less, edge-smoothing, bacterium colony smooth (flat); The BCRC17476 bacterium colony is white in color, edge-smoothing, rat.
2.216S rDNA gene fragment variance analysis result
Utilize BR101 and other three strains Lip river moral lactobacillus bacterial classification (Lactobacillus reuteri) reference cultures (to preserve and the research centre available from Biological resources, BCRC14625, BCRC16091, BCRC17476) carry out sequence alignment with 16S rDNA gene fragment order, and with behind BR101 and other reference cultures 16SrDNA gene fragment sequencing, utilize Vec tor NTI7.0 software to compare, the sequence alignment result is as shown in table 5, and sequence alignment result 4 strain bacterium are respectively represented different Lip river moral lactobacillus bacterial classifications (Lactobacillus reuteri).Show BR101 really and other reference cultures have significant difference.
The 16S rDNA gene fragment analytical results of table 5BR-101 and other three strains reference cultures
On behalf of 4 strain bacterium sequences, the yellow base number of a tender exist together mutually, and blue base number of a tender representative at least 3 strain bacterium sequences exist together mutually, and the no base number of a tender is represented the sequence difference place
Utilize UPGMA (Unweighted Pair-Group Method Using Arithmeticaverages) operational method in addition, set up the evolution stammbaum.The result is as shown in Figure 4: BR101 is the Lip river moral lactobacillus bacterial classification (Lactobacillusreuteri) that is different from BCRC14625, BCRC16091, BCRC17476.
2.3 it is right to utilize random primer that the amplification polymorphism DNA analysis is carried out diversity ratio
The utilization random primer is sought polymorphic DNA fragment to amplification and be can be used as molecule marker.This method is RAPD (Random amplified polymorphic DNA, the polymorphic dna of random amplification).This RAPD technology builds on the round pcr basis, it is that to utilize the oligonucleotide strand (being generally 10 aggressiveness) of a series of (usually hundreds of) different random alignment base sequence be introduction, research genomic dna (this experiment is meant the milk-acid bacteria genomic dna) is carried out pcr amplification, detect the polymorphism of amplified production dna fragmentation, the polymorphism of these amplified production dna fragmentations has reflected the dna polymorphism of genome respective regions.If therefore genome just may cause these particular combination bit points distributions that corresponding variation takes place at these regional dna fragmentation insertions, disappearance or base mutations of taking place, and makes the PCR product increase, lack or take place the change of molecular weight.Therefore RAPD can carry out the polymorphism detection to whole genome DNA.Utilize introduction Primer P3[5 ' GGT GAC GCA G3 '] carry out the RAPD comparison of BR101 and BCRC14625, BCRC16091, BCRC17476 reference culture genomic dna, DNA electrophorogram result shows that as shown in Figure 5 BR101RPAD Pattern is 1050bp, 700bp, 400bp, 280bp, 190bp; 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, 2Kb-700bp.This result confirms that BR101 and BCRC14625, BCRC16091, BCRC17476 are different Lip river moral lactobacillus bacterial classifications (Lactobacillus reuteri).
2.4 utilize API50CHL to carry out the variance analysis of bacterial strain carbohydrate biochemical reaction
Utilize API50CHL to detect the situation of 4 strain bacterial strains, observe the carbohydrate fermentation difference situation of 4 strain bacterial strains in 49 kinds of carbohydrate fermentations.Wherein only BR101 for Arbtine (arbutin, also claim the Resorcinol glucoside), Esculin ferric citrate (Horse Chestnut Extract glycosides candy) and three kinds of carbohydrates of D-lyxose (D-lyxose) react positive reaction, show that BR101 is different from other Lip river moral lactobacillus bacterial classification (Lactobacillus reuteri) reference cultures, the result is as shown in table 6.
The carbohydrate biochemical reaction difference analysis of table 6 BR-101 and other reference cultures
3. stimulate probiotic strain screening and the functional analysis thereof of secretion IL-10
Patient blood sample about 10 milliliters (ml), patient blood is contained Ficoll-Hypaque (GH Healthcare along tube wall slow the adding, Cat.No.17-1400-02) in the centrifuge tube, carry out centrifugal (per minute 1500 commentaries on classics with refrigerated centrifuge, 15 minutes) carry out the blood cell separation of whole blood, the separation surface place is peripheral blood glomus cell (PBMC).The PBMC that takes out is placed 15 milliliters of new (ml) centrifuge tubes, and add 10ml 1 * PBS to centrifuge tube, carry out centrifugal per minute 1500 with whizzer and change, (1500rpm 5min), removed supernatant liquor, stayed blood cell cell pellet in 15 minutes.Get 10 milliliters of (ml) RPMI-1640 substratum (containing 1%Penicillin-Streptomycin and 10%Calfserum) to centrifuge tube, come resorption to dash and avoid bubble to produce, make blood cell cell uniform distribution.Get the blood cell cell suspending liquid and mix, carry out cell counting with counting chamber with trypan blue (trypan blue) stain equal-volume.And to adjust blood cell cell suspending liquid concentration with the RPMI-1640 substratum be 4.0 * 10
6Cells/ml is standby.
The blood cell cell suspending liquid of getting 200 microlitres (μ l) adds aseptic 96 porose discs, and adds the probiotics bacterial liquid of 20 microlitres (μ l), places 37 degree 5%CO Celsius
2Cultivate in the environment after 48 hours, take out supernatant liquor and carry out IL-10ELISPOT assay.Its result as shown in Figure 6.By above-mentioned The selection result, the probiotic strain with irritation cell secretion IL-10 is BR101.
4. the probiotic strain that utilizes human peripheral blood glomus cell screening to stimulate secretion IL-10
In the autoimmune disorders, comparatively common with rheumatoid arthritis, so collect the blood of patient with rheumatoid arthritis, and after isolating its human peripheral blood glomus cell and different probiotic bacterium kinds being carried out co-cultivation, be fit to rheumatoid joint patient's probiotic bacterium kind with ELISPOT as say analysis and judgement.The bacterial classification that compares analysis comprises lactobacillus paraceasi (L.paracasei), Lactobacterium acidophilum (L.acidophilus), 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 six kinds of more common probiotic bacterium kinds such as (L.reuteri).
4.1BR101 and the preparation of other probiotic strains
After the probiotic bacterium of desiring to screen cultivated with MRS Broth, change with per minute 4000, centrifugal 5 minutes, remove supernatant liquor and add 1 * PBS buffer washing three times, the throw out of test tube bottom is returned melt into suspension with 1 * PBS buffer, and to adjust the bacterium number with 1 * PBS dilution be that 5.0 * 108CFU/ml (measures with OD600, OD600 is 1.0 o'clock, estimating the bacterium number is 5.0 * 108cell/ml), places 95 degree water bath heat treated Celsius after 5 minutes, places four degrees celsius standby.
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 the bright biotechnology of light company screening from an infant faeces corpse or other object for laboratory examination and chemical testing.This inoculation in the MRS liquid nutrient medium, is placed under the anaerobic environment with 37 degree Celsius and cultivates after activation in 6-8 hour amplifies, utilize the lyophilize mode to carry out freeze-drying.Freeze-dried vaccine powder powder detects the bacterium number must be greater than 1.0 * 1011CFU/g.Getting 1g bacterium powder is dissolved in 10 milliliters of (ml) sterilized waters, after the full and uniform mixing, change with per minute 4000, centrifugal 5 minutes, remove supernatant liquor and add 1 * PBS buffer washing three times, the throw out of test tube bottom is returned melt into suspension with 1 * PBS buffer, and to adjust the bacterium number with 1 * PBS dilution be that 5.0 * 108CFU/ml (measures with OD600, OD600 is 1.0 o'clock, estimating the bacterium number is 5.0 * 108cell/ml, bacterium powder mixed solution is diluted to 2.0 * 108CFU/ml, places 95 degree water bath heat treated Celsius after 5 minutes, place four degrees celsius standby.
4.2 human peripheral blood glomus cell is collected and preparation
Collect patient blood sample about 10 milliliters (ml), patient blood is contained Ficoll-Hypaque (GH Healthcare along tube wall slow the adding, Cat.No.17-1400-02) in the centrifuge tube, carry out gradient centrifugation (1500rpm with refrigerated centrifuge, 15min) blood cell that carries out whole blood separates, the separation surface place is peripheral blood glomus cell (PBMC), and throw out is a red blood corpuscle.The PBMC that takes out is placed 15 milliliters of new (ml) centrifuge tubes, and add 10 milliliters of (ml) 1 * PBS to centrifuge tube, carry out centrifugal (per minute 1500 changes 5 minutes), remove supernatant liquor, stay blood cell cell pellet with whizzer.Get 10 milliliters of (ml) RPMI-1640 substratum and (contain 1%Penicillin-Streptomycin and 10%Calf
Serum) to centrifuge tube, come resorption to dash 20 times and avoid bubble to produce, make blood cell cell uniform distribution.Get the blood cell cell suspending liquid and mix, carry out cell counting with counting chamber with trypan blue stain equal-volume.And to adjust blood cell cell suspending liquid concentration with the RPMI-1640 substratum be 4.0 * 10
6Cell s/ml is standby.
4.3 the preparation of experiment control group
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); The positive regulation group be 1 mcg/ml (μ g/ml) LPS (Lipopolysaccharide, Escherichia coilserotype055:B5, Sigma).It is that a large-scale molecule is connected to form by covalent linkage by fat and polysaccharide as the positive regulation group because of it that lipopolysaccharides (Lipopolysaccharide) is used in this test.Lipopolysaccharides is the chief component of gram negative bacterium adventitia, and the integrity of bacterium with structure is provided, and the protection bacterial film is subjected to the attack of some chemical substance.Lipopolysaccharides is an intracellular toxin, can cause the strong immunization reaction.
Stimulate PBMC to produce the analysis of IL-10 4.4 utilize ELISPOT as say to carry out milk-acid bacteria
ELISPOT as say utilizes color reaction, on the correspondence position of emiocytosis cytohormone, manifest the spot that to distinguish, and can be directly at microscopically artificial counting spot or utilize the ELISPOT identification system that spot is counted, 1 spot is represented 1 cell, calculates the cell quantity of this cytohormone of secretion.It detects principle is to utilize 96 porose discs bottom PVDF material film, is used for adsorbing special selecting and the monoclonal antibody of nontoxicity (not containing sodium azide, intracellular toxin endotoxin).When a blood corpse or other object for laboratory examination and chemical testing is added on 96 porose discs through the PBMC that obtains (the human peripheral blood glomus cell) cell after separating, utilizing after the antigenic stimulation micropore dish is positioned over cultivates after 16～24 hours under the proper temperature, memory-type T cell can beginning secretory cell hormone after being subjected to antigenic stimulation a few hours, and this moment, local (around near secretory cell) oozy cytohormone can be caught by specific antibody on the PVDF film.After cell in the micropore dish is removed and cleans, captive cytohormone can further use the secondary antibodies of vitamin H (Bi o t in) mark to indicate, again with StreptAvidin in conjunction with ferment with it act on thereafter, and add ferment and be subjected to matter to make its colour generation, the cell of the effect of responding can stay dyeing speck.
Its reactions steps is as follows:
1) in the aseptic technique platform, (precoated) mAb9D7 dish (plate) of precoating is risen again to room temperature from four degrees celsius, with 4 times (200 μ l/well) of aseptic 1 * PBS washing;
2) the substratum RPMI-1640 (containing 10%FBS) that adds 200 μ l/well carried out under room temperature blacking30 minute;
3) remove substratum, with 100 μ l, 1 * PBS washing once, on thieving paper, pat;
4) (cell concn is adjusted into 1.0 * 10 to add PBMC
6-1.0 * 10
8Ce1ls/ml) and BR101 bacterium liquid (1.0 * 10
6-1.0 * 10
8CFU/ml), last volume is counted roughly 150 μ l/well, and this moment, positive control group was that mAb CD3-2 adds simultaneously, and experimental concentration is 100ng/ milliliter (ml);
5) will coil (plate) and place 37 degree 5%CO Celsius
2Reacted in the incubator 12～48 hours;
6) remove cell suspending liquid, add 200 μ l/well, 1 * PBS washing five times;
7) dilution detecting antibody 12-G8-biotin to concentration 1 μ g/ milliliter (ml) in 1 * PBS-0.5%FBS (1 * PBS contains 0.5% foetal calf serum), add the antibody of 100 μ l/well dilution, reaction is 2 hours under room temperature;
8) remove supernatant liquor, add 200 μ l/well, 1 * PBS washing five times;
9) dilution Streptavidin-ALP (1: 1000) adds 100 μ l/well and reacted under room temperature 1 hour in 1 * PBS-0.5%FBS (1 * PBS contains 0.5% foetal calf serum);
10) remove supernatant liquor, add 200 μ l/well1 * PBS and wash five times;
11) photoghraphic coupler BCIT/NBT is filtered with 0.45 μ m filter membrane and adds 100 μ l/well, as under the room temperature till spot presents;
12) fully after the colour developing, supernatant liquor is added in the corresponding dish, with the both sides of distilled water thorough washing film.Pat on the thieving paper, make the film drying.During preservation,, utilize microscope or interpreting system promptly to can read the spot number, will coil (plate) and place under the room temperature and keep in Dark Place in case plate is inverted in order to avoid residual liquid flows back on the film after the film drying.
After utilizing ELISPOT assay to analyze six probiotics and human peripheral blood glomus cell co-cultivation, stimulate the analysis of secretion IL-10, calculate the cell quantity of secreting this cytohormone according to the mottle number, the result is as shown in table 7.It is 324 for BR101 in regular turn that its spot is counted the result; B.longum is 306; E.faecium is 304; L.rhamnosus is 298; L.acidophilus is 294; L.paracasei is 240.So filtering out the highest probiotic bacterium of stimulation secretory volume is BR101 Lip river moral lactobacillus bacterial classification (Lactobacillus reuteri).
5. utilize BR101 to carry out unofficial human trial
The experimenter serves as preferential regularly following the trail of the curer in outpatient service, and need meet following condition: 1) age 19 was to 75 years old; 2) the positive person of Rheumatoid factors, polyclonal is found in blood drawing check once in nearest 1 year; 3) phase of falling ill is 6 week～1 year; 4) person please regularly follows the trail of fixedly to take the medicine; 5) steroid medicine dosage every day＞7.5 milligram; 6) or in nearly 4 weeks accept intra-articular injection person.
Other get rid of to participate in trier's standard and comprise: be diagnosed as the rheumatoid arthritis phase greater than person, hepatic and renal function sufferer, chronic or frequent infection more than 1 year (as chronic bronchitis, the recurrent sinusitis paranasal sinusitis) or other catch etc., other administrative factors are as removal of home, economic factors or be unwilling to compatibility test etc. then get rid of test.Finish the trier and amount to 14, test period is 1 month.
After getting Lip river moral lactobacillus bacterial classification (Lactobacillus reuteri) BR101 and activating amplification, carry out powder processing, the bacterium powder is added capsule charge behind the excipient, make every capsule bacterium number greater than 5 * 10 with freeze-drying method
9CFU.This experiment sieving phase was 4 weeks, after outpatient service is confirmed and obtained patient's agreement, test, and fill in symptom score table for the first time, eat 6 capsules afterwards every day, after 1 month, please the experimenter fill in symptom score table for the second time again, and analyze according to the symptom score table that the experimenter filled in.This symptom score table is to revise DAS (Di sease Act ivity Score) and HAQ (Health Assessment Questionnaire), content comprises occurrence frequency and the severity of five main joints (elbow joint, wrist joint, finger-joint, knee joint and podarthral symptom) in symptoms such as pain, stiff, swelling and heatings, comprise the assessment of daily life in addition, shown in table 7.
Table 7 " rheumatoid arthritis " paresthesia inquiry schedule
Behind the Data acquisition,, carry out data analysis with the SPSS17.0 of statistical software version, add up with nonpa rametrictest, add up with Wilcoxon Signed Rank Test, analyze back P value＜0.05 and have upward significant difference meaning of statistics, main assessment foundation is the mark on the symptoms of rheumatoid arthritis questionnaire, and all data are represented with mean number (Mean) ± S.D..
Statistics is as shown in table 8, and the edible capsule that contains BR101 bacterium powder of experimenter is after 30 days, and whether the rheumatoid arthritis related symptoms had the difference situation before and after experimenter's self-assessment was edible.After analyzing by statistics, the severity that takes place with regard to symptom, always grading in finger-joint, daily life all has significant difference on the statistics (Table6., P＜0.05); And on the symptom occurrence frequency, all there were significant differences in wrist joint, finger-joint and daily life for the experimenter.The demonstration experimenter is contained the capsule of BR101 bacterium powder after 30 days edible, (P value＜0.05) improves significantly on severity of symptom and occurrence frequency, the edible BR101 of expression can make patient with rheumatoid arthritis really except reducing severity of symptom, also has the ability of the quality of making the life better.All experimenters all finish 30 days experiment, do not have serious bad reaction to produce in experimental session.
The unofficial human trial interpretation of result (N=14) of table 8
The above, it only is preferred embodiment of the present invention, be not that the present invention is done any pro forma restriction, though the present invention discloses as above with preferred embodiment, yet be not in order to limit the present invention, any those skilled in the art, in not breaking away from the technical solution of the present invention scope, when the method that can utilize above-mentioned announcement and technology contents are made a little change or be modified to the equivalent embodiment of equivalent variations, in every case be the content that does not break away from technical solution of the present invention, according to technical spirit of the present invention to any simple modification that above embodiment did, equivalent variations and modification all still belong in the scope of technical solution of the present invention.
Claims (19)
1. separated microorganism strain isolated BR101 is characterized in that it is one can produce the strain isolated of high anti-inflammation cytohormone, and it is deposited at Foodstuff Industrial and Development Inst., deposits to be numbered BCRC910512.
2. strain isolated according to claim 1 is characterized in that wherein this strain isolated is that screening is from an infant faeces corpse or other object for laboratory examination and chemical testing.
3. strain isolated according to claim 1 is characterized in that wherein this strain isolated is to compare through the 16SrDNA gene fragment order to be accredited as Lip river moral lactobacillus bacterial classification.
4. strain isolated according to claim 1 is characterized in that wherein this strain isolated has Lip river all characteristics of being known by mirror of moral lactobacillus bacterial classification.
5. strain isolated according to claim 1 is characterized in that wherein this strain isolated is is Gram-positive bacillus through the gram stain microscopy observations.
6. strain isolated according to claim 1 is characterized in that wherein this strain isolated has catalase.
7. strain isolated according to claim 1 is characterized in that wherein this strain isolated does not have oxydase.
8. strain isolated according to claim 1 is characterized in that wherein this strain isolated does not have mobility.
9. strain isolated according to claim 1 is characterized in that wherein this strain isolated all can be grown in the anaerobic-aerobic environment.
10. strain isolated according to claim 1 is characterized in that wherein this strain isolated can ferment carbohydrate arbutin, Horse Chestnut Extract glycosides Tang and D-lyxose.
11. strain isolated according to claim 1, it is characterized in that its its have an ability that stimulating human peripheral blood glomus cell produces the anti-inflammation cytohormone.
12. strain isolated according to claim 1 is characterized in that wherein this strain isolated can produce the anti-inflammation cytohormone high than other probiotic bacterium kinds.
13. strain isolated according to claim 12 is characterized in that 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.
14. strain isolated according to claim 1, it is characterized in that this anti-inflammation cytohormone wherein be between white plain 10 and group that tumour necrosis factor-β formed.
15. strain isolated according to claim 1 is characterized in that wherein this anti-inflammation cytohormone detecting is for utilizing the ferment immune analysis method to analyze.
16. strain isolated according to claim 15 is characterized in that wherein said ferment immune analysis method, comprises the method that ELISA assay, ELISPOT assay and other application ferment immunity principles are formed.
17. but the composition that the irritation cell hormone produces is characterized in that it comprises:
Provide a significant quantity as microorganism strain isolated BR101 according to claim 1; And with filled capsules behind this strain isolated interpolation excipient.
18. composition according to claim 17 is characterized in that its indication is an autoimmune disorders.
19. composition according to claim 18 is characterized in that wherein this autoimmune disorders comprises the group that rheumatoid arthritis, ankylosing spondylosis and lupus erythematosus are formed.
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| CN1696281A (en) * | 2004-05-10 | 2005-11-16 | 景岳生物科技股份有限公司 | New microbe gene stock similar to lactobacillus casei GM-O80, and application for treating disease related to irritability |
| CN101503662A (en) * | 2008-02-05 | 2009-08-12 | 光晟生物科技股份有限公司 | Microbial strain for antianaphylaxis symptom, composition thereof and method for producing interferon-gamma by using the microbial strain irritation cell |
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| CN1696281A (en) * | 2004-05-10 | 2005-11-16 | 景岳生物科技股份有限公司 | New microbe gene stock similar to lactobacillus casei GM-O80, and application for treating disease related to irritability |
| CN101503662A (en) * | 2008-02-05 | 2009-08-12 | 光晟生物科技股份有限公司 | Microbial strain for antianaphylaxis symptom, composition thereof and method for producing interferon-gamma by using the microbial strain irritation cell |
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