CN105770868A - Application of recombinant lactobacillus and SNase in preparing drug for preventing or treating I type diabetes mellitus - Google Patents

Application of recombinant lactobacillus and SNase in preparing drug for preventing or treating I type diabetes mellitus Download PDF

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CN105770868A
CN105770868A CN201610353343.5A CN201610353343A CN105770868A CN 105770868 A CN105770868 A CN 105770868A CN 201610353343 A CN201610353343 A CN 201610353343A CN 105770868 A CN105770868 A CN 105770868A
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lactis
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吴洁
郎君超
刘坤锋
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China Pharmaceutical University
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Abstract

The invention relates to application of recombinant lactobacillus and SNase in preparing a drug for preventing or treating I type diabetes mellitus. The purpose is to provide new application of a vaccine with recombinant lactobacillus L.lactis NZ9000 p CYT: SNase as a carrier in resisting I type diabetes mellitus. The recombinant lactobacillus can express destination protein staphylococcal nuclease, SNase after being induced by lactose. The L.lactis NZ9000 p CYT: SNase living bacterium vaccine is used for immunity of female NOD/LtJ mice four weeks old in an administering oral medication mode, and a series of pharmacodynamic indexes are evaluated, it is found that morbidity of NOD mouse diabetes can be obviously reduced by orally taking L.lactis NZ9000 p CYT: SNase, blood sugar and body weight of the NOD mice can be controlled well, and the survival rate of the NOD mice can be increased remarkably. Therefore, the recombinant lactobacillus L.lactis NZ9000 p CYT: SNase can obviously restrain occurrence and development of NOD mouse diabetes and can be used for developing drugs for I type diabetes mellitus.

Description

Recombinant lactic acid bacteria and SNase application in preparation prevention or treatment type 1 diabetes medicine
Technical field
The present invention relates to a kind of recombinant lactic acid bacteria with anti-type 1 diabetes effect, and contain the derivant of this recombinant lactic acid bacteria and similar pharmaceutical preparation, the present invention relates to pharmacy and medical science association area.
Background technology
Diabetes (Diabetesmellitus, DM) due to insulin definitely or relative deficiency and cause, with hyperglycemia and multiple complication and the endocrine metabolism disease for feature such as deposit, be one of modal chronic disease.Generally diabetes are divided into two big types, type 1 diabetes (Type1diabetesmellitus, T1DM) and type 2 diabetes mellitus (Type2diabetesmellitus, T2DM).T1DM is typically considered a kind of based on heredity, multifactor common participation, organ specific autoimmune's disease.Patient's beta Cell of islet is subject to the immunologic injury of T cell mediated, causes that insulin secretion is definitely not enough.T1DM is also the result of a large amount of unknown environmental factors combined influence simultaneously, such as viral infection, diet etc., but its mechanism of causing a disease is not yet clear and definite so far.Patient's cardinal symptom is three-many-one-little, namely polydipsia, polyphagia, polyuria, lose weight.At present, the treatment still methods adopting insulin injection for T1DM more, but for Most patients, insulin treatment does not simply fail to constantly maintain in normal range blood glucose level in patients, also has the danger that acute hypoglycemia occurs, more cannot fundamentally improve or terminate the Etiological immunoregulatory disorder of T1DM, and subcutaneous injection frequently, invasive blood sugar monitorings etc. are all the restraining factors of chronic insulin injection for curing, and general immunosuppressant can not directly do hypoglycemic medicine, it is applied to clinic, therefore illustrate that T1DM is a complexity, multifactor cause disease, only immunosuppressant can not play good result, therefore find novel targets, study new Therapeutic Method imperative.
Lactic acid bacteria prolonged application, in food industry every field, the production of such as fermented product and preservation, has been demonstrated no pathogenicity, is a kind of generally acknowledged safe level (generallyrecognizedassafe, GRAS) microorganism.Currently for lactic acid bacteria, outside carrying out food fermentation other than with genetic engineering bacterium, it has become study hotspot as the function of carrier submission human cytokines or antigen.Lactic acid bacteria NZ9000 is lactococcus lactisLactococcuslactisThe derivative strain of subsp.cremorisMG1363.It is that it is built-up (pepN::nisRnisK) within 1998, to be incorporated in the aminopeptidase N gene (pepN) of MG1363 by Kuipers etc. by controlling gene nisR and the nisK expressing nisin (nisin).Due to its plurality of advantages, now it is widely used in the expression of foreign protein, and as live vector vaccine present antigen.Such as, its Physiology and biochemistry feature is simple；Genetic background is clear；Antigenicity is weak；Thalline self secretory protein is few, reduces the interference to external source secretory protein；Do not produce endotoxin；Do not produce any extracellular protease, it is to avoid extracellular degraded occurs secretory protein；Safety is high, and 2010, Lee etc. have evaluated the safety of NZ9000 first from toxicological point, it was demonstrated that NZ9000, without drug resistance gene, does not produce toxic protein.NZ9000 expression system generally adopts NICE(nisin-controlledexpression, NICE) expression system, the i.e. gene expression system of nisin induction.The application of this expression system is wide, and controllability is strong.Effective NICE system mainly includes the Host Strains with nisRK gene, plasmid containing PnisA or PnisF promoter fragment (multiple clone site containing can be inserted into genes of interest) and as the nisin of inducer or its analog.
PCYT:SNase(document is pCYT:NUC) it is the conventional nisin exogenous gene expression carrier induced, it is with chlorampenicol resistant, containing PnisA promoter, if inserting genes of interest in its polyclone restriction enzyme site, it becomes possible to carry out the expression of external source destination protein under nisin induces.The SNase of nuc gene code therein is often taken as reporter protein (reporter) and monitors albumen and target and conformational state.SNase molecular weight is little, and genetic background and biochemical property are clear, good stability, not changeableness, activity is prone to detection, when as when expressing label with destination protein amalgamation and expression, on the one hand without influence on the activity of destination protein, the endocellular enzyme Degradation to destination protein can be reduced on the other hand.
Described SNase is staphylococcal nuclease (Staphylococcalnuclease), belong to the non-specific ribonuclease of class found the earliest, the SNase of mature form is made up of 149 amino acid residues, wherein homologous sequence SNc contains 135 amino acid residues, catalytic site contains 67 amino acid residues, can non-specifically degradation of dna and RNA, and discharge 3 '-mono phosphoric acid ester nucleotide and dinucleotide, it has excision enzyme and endonuclease activity simultaneously, additionally also there is molecular weight compared with other DNase little, Heat stability is good, the advantages such as pH field of activity wide (pH4 ~ 10).
Above-mentioned pCYT:SNase is conventional carrier, does not have any hypoglycemic report at present.
Present invention firstly discovers that SNase or the lactococcus lactis expression strain containing SNase namelyL.lactisNZ9000pCYT:SNase has hypoglycemic activity, and lactococcus lactis expression strain is namelyL.lactisNZ9000pCYT:SNase can be administered orally, and overcomes the difficult problems such as insulin etc. can not be administered orally.
Summary of the invention
Goal of the invention:
It is an object of the invention to provide the recombinant lactic acid bacteria expressing SNaseL.lactisThe new application of NZ9000pCYT:SNase.
Technical scheme:
SNase application in preparation prevention or treatment type 1 diabetes medicine.
A kind of recombinant lactic acid bacteriaL.lactisNZ9000pCYT:SNase application in preparation prevention or treatment type 1 diabetes medicine.
The recombinant lactic acid bacteria that the present invention relates toL.lactisNZ9000pCYT:SNase, i.e. lactococcus lactis NZ9000pCYT:SNaseLactococcuslactisNZ9000pCYT:SNase is preserved in China typical culture collection center, address: Wuhan, China, Wuhan University;Preserving number is CCTCCNO:M2016084, and the preservation time is on March 4th, 2016.
Described be effective ingredient with recombinant lactic acid bacteria or SNase preparation be containing recombinant lactic acid bacteria and the injection of SNase, slow releasing agent, subdermal implants, tablet, powder, granule, capsule or oral liquid.
A kind of detection kit, its feature contains described recombinant lactic acid bacteria or SNase.
Recombinant Lactococcus lactis provided by the present invention can beLactococcuslactisNZ9000 type, it is possible to for the lactic acid bacteria that other license uses at human body.
Expression vector provided by the present invention is alternatively the lactic acid bacteria expression vectors that other license uses at human body.
Beneficial effect:
1, the present invention adopts staphylococcal nuclease (Staphylococcalnuclease, SNase) to find that it has hypoglycemic activity first.And find recombinant lactic acid bacteriaL.lactisNZ9000pCYT:SNase also has blood sugar reducing function, and can be taken orally, and the present invention relates to its new application.
2, the prior art indicate that many albumen such as HSP65 etc can be used for the preventing and treating of type 1 diabetes, engineering bacteria has broken its yoke being difficult to produce in enormous quantities for the expression of this albuminoid, but the problem that there is also other, the such as degraded of fusion protein in separating purge process, control manipulation condition is strict, and purification efficiency is too low.This invention adopts lactococcus lactis to deliver destination protein, albumen is expressed in body, evade protein degradation and the problem of purification steps troublesome complexity dexterously, save production cost, significant for the preventing and treating produced on a large scale for type 1 diabetes.
Traditional vaccine is administered generally by hypodermic mode, but subcutaneous injection is excessively painful for sufferer, so being difficult to be accepted, it addition, this administering mode there is also serious safety issue.And the recombinant lactic acid bacteria involved by this patent is by oral administration, the mode of mucosal absorption, overcome above-mentioned all limitations, it is easy to accept for sufferer.
Lactic acid bacteria prolonged application, in food industry every field, has been demonstrated no pathogenicity, is a kind of generally acknowledged safe level (generallyrecognizedassafe, GRAS) microorganism.Lactococcus lactis is the type strain of lactic acid bacteria, it has the advantage of its uniqueness as the ideal carrier expressing foreign protein: first, conventional vaccine carrier, such as Salmonella, escherichia coli and poxvirus etc., great majority are pathogen, there is certain risk, and lactococcus lactis is a kind of food-grade microorganisms, has better safety especially for specific crowd such as old people, infant；Secondly lactococcus lactis is as a kind of gram positive bacteria, it does not have this proinflammatory substance of lipopolysaccharide, therefore has poor antigen, and it will not cause strong immunne response as carrier itself；Furthermore, lactococcus lactis self secretory protein is less, thus decreasing the interference to external source secretory protein, and does not produce extracellular protease, thus ensureing that secretory protein is not degraded, it is ensured that the integrity of its 26S Proteasome Structure and Function；Additionally lactococcus lactis all has metabolic activity at all intestinal segments, directly contacts intestinal mucosa, promotes foreign protein presenting to mucosa；Lactococcus lactis provides a kind of encapsulated protective barrier can also to Exogenous therapies albumen; protect its be not degraded in the gastrointestinal tract (90% ~ 98% survival rate); successfully arrive at the release of intestinal mucosa place, and study and show that lactococcus lactis is only planted short-and-medium the fixing tentatively of intestinal, will not endanger
Normal probiotic bacteria or cause normal flora disorderly in intestinal.
Compared with other expression system, NICE expression system is with the obvious advantage: first its induced efficiency is high, up to more than 1000 times；Secondly inducer nisin is the bio-safety peptide of a kind of food stage, can as antistaling agent in the food industry of some countries；Controlled strictly when non-induced, destination protein is not expressed or expression is few, makes the expression phase of bacterial growth and exogenous gene separate, thus alleviating the metabolic burden of Host Strains when destination protein is expressed.Additionally while nisin is a kind of bacteriocin, most of gram positive bacterias can be played inhibitory action, but the nisin amount needed for this inhibitory action is that Gamma Magnitude is other, and has only to the amount of nanogram rank as derivant, and therefore it is less to the inhibitory action of lactococcus lactis as derivant.
Accompanying drawing explanation
Fig. 1 pCYT:SNase plasmid composition schematic diagram
Genes of interest SNase clip size in plasmid is 516bp, altogether 171 amino acid residues of coding, containing SNc(135 amino acid residue of homeodomain), the latter includes catalytic site (67 amino acid residues).
The agarose gel electrophoresis testing result of Fig. 2 pCYT:SNase plasmid
M:marker；Lane1: purpose band, i.e. pCYT:SNase plasmid band.
Fig. 3 SNase protein SDS-PAGE electrophoresis detection result
M:marker；Lane1: before lactose-inducedL.lactisThe total protein of NZ9000pCYT:SNase；Lane2 ~ 6: 0.5h, 1h, 1.5h, 2h and 2.5h after inductionL.lactisThe total protein of NZ9000pCYT:SNase.
The impact on NOD mouse invasion rate of Fig. 4 Recombinant Lactococcus lactis vaccine
The impact on NOD mouse blood sugar of Fig. 5 Recombinant Lactococcus lactis vaccine
The impact on NOD Mouse Weight of Fig. 6 Recombinant Lactococcus lactis vaccine
The impact on NOD mouse survival ability of Fig. 7 Recombinant Lactococcus lactis vaccine
Wherein in Fig. 4-7, LL-pCYT:SNase isL.lactisNZ9000pCYT:SNase group.
Detailed description of the invention
The extraction of embodiment 1:pCYT:SNase plasmid and agarose gel electrophoresis checking
The little extraction reagent kit of plasmid (Beijing Tian Gen biochemical technology company limited) is used to carry out plasmid extraction, process is as follows: in adsorption column CP3, (adsorption column is put in collecting pipe) adds the balance liquid BL of 500 μ L, 12000rpm is centrifuged 1min, outwell the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe；Taking the bacterium solution of 5mL incubated overnight, add in centrifuge tube, 12000rpm is centrifuged 1min, as far as possible sucking-off supernatant；In the centrifuge tube leave bacterial sediment, add 250 μ L solution P1(add RNaseA and lysozyme in advance), use liquid-transfering gun thoroughly to suspend bacterial sediment, be placed in 37 DEG C of water-baths cracking 40min；In centrifuge tube, add 250 μ L solution P2, leniently spin upside down 6 ~ 8 times and make thalline fully crack；Adding 350 μ L solution P3 in centrifuge tube, leniently spin upside down 6 ~ 8 abundant mixings immediately, white flock precipitate now occur, 12000rpm is centrifuged 10min；Being transferred to by supernatant (adsorption column is put in collecting pipe) in adsorption column CP3, avoid sucking-off to precipitate as far as possible, 12000rpm is centrifuged 30 ~ 60s, outwells the waste liquid in collecting pipe, is put in collecting pipe by adsorption column；In adsorption column, add 600 μ L rinsing liquid PW(add dehydrated alcohol in advance), 12000rpm is centrifuged 30 ~ 60s, outwells the waste liquid in collecting pipe, is put in collecting pipe by adsorption column, and this step is repeated once；Being put into by adsorption column in collecting pipe, 12000rpm is centrifuged 2min, is removed the rinsing liquid remained in adsorption column；Being put into by adsorption column in a clean centrifuge tube, drip 70 μ L elution buffer EB to adsorbed film mid portion, room temperature places the centrifugal 2min of 2min, 12000rpm, is collected in centrifuge tube by plasmid solution.With agarose gel electrophoresis, plasmid is verified, as shown in Figure 2.
Embodiment 2: induction Recombinant Lactococcus lactis bacterial strainL.lactisNZ9000pCYT:SNase expresses SNase.
By Recombinant Lactococcus lactisL.lactisNZ9000pCYT:SNase, the method provided according to document " LuisG.Berm ú dez-Humar á netal.Controlledintra-orextracellularproductionofstaphyl ococcalnucleaseandovineomegainterferoninLactococcuslacti s [J] .FEMSMicrobiologyLetters; 224 (2003): 307-313 " prepares, called after pCYT:NUC in the literature.Described recombinant lactic acid bacteriaL.lactisNZ9000pCYT:SNase, i.e. lactococcus lactis NZ9000pCYT:SNaseLactococcuslactisNZ9000pCYT:SNase is preserved in China typical culture collection center, address: Wuhan, China, Wuhan University;Preserving number is CCTCCNO:M2016084, and the preservation time is on March 4th, 2016.
By Recombinant Lactococcus lactisL.lactisNZ9000pCYT:SNase is inoculated in GM17 culture medium, 30 DEG C of incubated overnight；Incubated overnight liquid 1: 100 switching GM17 culture medium, 30 DEG C of amplification culture, derivant nisin(50ng/mL is added) when bacteria growing to OD600 value is 0.4～0.6, continue to cultivate a period of time, start to receive bacterium until OD600 value reaches 1.2, receive bacterium process as follows: 4000rpm is centrifuged 5min, abandons supernatant；Wash thalline twice (4000rpm is centrifuged 5min) with sterilizing PBS, abandon supernatant；With the resuspended thalline of sterile PBS buffer；Adjust thalline final concentration of 2 × 1010CFU/mL, gained viable bacteria re-suspension liquid is immediately available for immunity.
Embodiment 3: Recombinant Lactococcus lactisL.lactisThe anti-type 1 diabetes vaccine pharmacodynamic evaluation of NZ9000pCYT:SNase.
By 24 the 4 female NOD/LtJ mices of week old (purchased from Beijing HFK Bio-Technology Co., Ltd., credit number: SCXK(capital) 2012-0004) it is randomly divided into 2 groups, be respectivelyL.lactisNZ9000 group andL.lactisNZ9000pCYT:SNase group, often group 12, starts gastric infusion when mice is five week old, first week every day gavage once, after administration, second week starts weekly gastric infusion once, until terminating during mice 20 week old to be administered.Before gavage, adjusting often group viable bacteria bacteria concentration is 2 × 1010CFU/ml, dosage is 4 × 109CFU/200 μ L/ time/.
(1) Recombinant Lactococcus lactisL.lactisThe sickness rate of NOD mice is affected by NZ9000pCYT:SNase vaccine.
From experiment starts, within every two weeks, take blood once from NOD mouse orbit venous plexus, and detect the blood sugar level of mice with blood-sugar detecting instrument, weigh its body weight simultaneously.The situation hyperglycemia value of onset diabetes represents, double blood glucose value is higher than 11.1mmol/L, and is then judged to morbidity with polyphagia, polydipsia, polyuria, the situation such as lose weight.As shown in Figure 4, with matched group (L.lactisNZ9000 group) compare, administration group (L.lactisPCYT:SNase group) mouse invasion situation is substantially suppressed (P=0.002, n=12).When 4 week old, each group mice is all healthy and does not fall ill, and from 12 week old, namely matched group NOD mice falls ill successively, and when 16 week old, sickness rate substantially rises；At 20 weeks, when last administration terminates, experimental group sickness rate only only has 8.3%(1), and now the sickness rate of matched group has only reached 66.7%(8)；When the observation phase terminates, matched group accumulation has 91.7%(11 only) NOD mouse invasion, and the sickness rate of administration group be 50%(6 only).Therefore it is shown that Recombinant Lactococcus lactis vaccine can substantially suppress NOD diabetes mice to develop.
(2) Recombinant Lactococcus lactisL.lactisThe impact of the NZ9000pCYT:SNase vaccine blood glucose on NOD mice and body weight.
During observation, administration group and control group mice change of blood sugar as it is shown in figure 5, body weight change as shown in Figure 6.Control group mice average blood sugar maximum reaches 13.63mmol/L, and administration group mice average blood sugar is in below 11.1mmol/L all the time, and peak is 10.29mmol/L, less than matched group.In the observation phase, each group average mice body weight is fallen after rising, when stopping administration,L.lactisPCYT:SNase group average mice body weight is significantly higher than matched group (P < 0.05), and latter stage at end, body weight was also above matched group.
(3) Recombinant Lactococcus lactisL.lactisThe impact on NOD mouse survival ability of the NZ9000pCYT:SNase vaccine.
The survival state of every mice often organized in record, until the observation phase terminates.The survival state of mice is as shown in Figure 7:L.lactisNZ9000 group mice started dead successively from 14 weeks, and when within 20 weeks, administration terminates, dead three, survival rate is 75%, and when within 32 weeks, the observation phase terminates, mice dead 7, survival rate is 41.7%；AndL.lactisPCYT:SNase group mice is before 24 weeks, and survival rate maintains 100% always, and administration terminated after six weeks, and mice starts death occur, but to the observation phase terminate time, survival rate is still 66.7%.Test result indicate that,L.lactisPCYT:SNase can significantly increase the survival ability of NOD mice.
Conclusion: it follows that Recombinant Lactococcus lactisL.lactisNZ9000pCYT:SNase can substantially suppress NOD diabetes mice to develop, and better controls blood glucose and the body weight of NOD mice, it is possible to significantly increase the survival ability of NOD mice, can be used for the exploitation of type 1 diabetes medicine.
What finally illustrate is, above example is only in order to illustrate technical scheme and unrestricted, although by referring to some preferred embodiment invented, invention has been described, but it will be understood by those within the art that, in the form and details it can be made various change, without departing from the spirit and scope of appended claims invention defined.
SEQUENCELISTING
<110>China Medicine University
<120>recombinant lactic acid bacteria and SNase application in preparation prevention or treatment type 1 diabetes medicine
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<170>PatentInversion3.3
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MetAsnAlaSerGlnThrAspAsnGlyValAsnArgSerGlySerGlu
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AspProThrValTyrSerAlaThrSerThrLysLysLeuHisLysGlu
202530
ProAlaThrLeuIleLysAlaIleAspGlyAspThrValLysLeuMet
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TyrLysGlyGlnProMetThrPheArgLeuLeuLeuValAspThrPro
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GluThrLysHisProLysLysGlyValGluLysTyrGlyProGluAla
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SerAlaPheThrLysLysMetValGluAsnAlaLysLysIleGluVal
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GluPheAspLysGlyGlnArgThrAspLysTyrGlyArgGlyLeuAla
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TyrIleTyrAlaAspGlyLysMetValAsnGluAlaLeuValArgGln
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GlyLeuAlaLysValAlaTyrValTyrLysProAsnAsnThrHisGlu
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GlnHisLeuArgLysSerGluAlaGlnAlaLysLysGluLysLeuAsn
145150155160
IleTrpSerGluAspAsnAlaAspSerGlyGln
165170

Claims (4)

1.SNase application in preparation prevention or treatment type 1 diabetes medicine, the nucleotides sequence of described SNase is classified as SEQNO.1.
2. a recombinant lactic acid bacteriaL.lactisNZ9000pCYT:SNase application in preparation prevention or treatment type 1 diabetes medicine；Described recombinant lactic acid bacteriaL.lactisNZ9000pCYT:SNase, i.e. lactococcus lactis NZ9000pCYT:SNaseLactococcuslactisNZ9000pCYT:SNase is preserved in China typical culture collection center, address: Wuhan, China, Wuhan University；Preserving number is CCTCCNO:M2016084, and the preservation time is on March 4th, 2016.
3. a recombinant lactic acid bacteria as claimed in claim 2 or as claimed in claim 1 SNase as the preparation of effective ingredient, it is characterised in that the dosage form of described preparation is injection, slow releasing agent, subdermal implants, tablet, powder, granule, capsule or oral liquid.
4. a detection kit, its feature contains the recombinant lactic acid bacteria described in claim 2 or claim 1 gained SNase.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109608526A (en) * 2019-01-25 2019-04-12 中国药科大学 Application of the SNase in preparation treatment inflammatory bowel medicine
CN112023031A (en) * 2020-10-16 2020-12-04 中国药科大学 Application of SNase in preparation of medicine for promoting diabetic wound healing

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7563885B1 (en) * 2004-05-24 2009-07-21 Isis Pharmaceuticals, Inc. Modulation of Tudor-SN expression
CN105255800A (en) * 2015-10-08 2016-01-20 中国药科大学 Recombination lactic acid bacteria and application thereof
CN105296408A (en) * 2015-04-01 2016-02-03 中国药科大学 Novel recombinant lactobacillus resisting diabetes mellitus type 1 and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7563885B1 (en) * 2004-05-24 2009-07-21 Isis Pharmaceuticals, Inc. Modulation of Tudor-SN expression
CN105296408A (en) * 2015-04-01 2016-02-03 中国药科大学 Novel recombinant lactobacillus resisting diabetes mellitus type 1 and application thereof
CN105255800A (en) * 2015-10-08 2016-01-20 中国药科大学 Recombination lactic acid bacteria and application thereof

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
EVELIEN T.M. BERENDS ET AL.: "Nuclease Expression by Staphylococcus aureus Facilitates Escape from Neutrophil phylococcus Extracellular Traps", 《JOURNAL OF INNATE IMMUNITY》 *
KAI KESSENBROCK ET AL.: "Netting neutrophils in autoimmune small-vessel vasculitis", 《NATURE MEDICINE》 *
SHORTLE,D.: "nuclease,staphylococcal，Accession NO：1109959A", 《GENBANK》 *
YUDONG WANG ET AL.: "Increased Neutrophil Elastase and Proteinase 3 and Augmented NETosis Are Closely Associated With β-Cell Autoimmunity in Patients With Type 1 Diabetes", 《DIABETES》 *
刘义庆等: "中性粒细胞的功能及其与自身免疫性疾病关联性研究进展", 《中国免疫学杂志》 *
吴洁等: "重组HSP65—6P277乳酸乳球菌疫苗对链脲佐菌素诱导的1型糖尿病小鼠的降糖作用", 《中国药科大学学报》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109608526A (en) * 2019-01-25 2019-04-12 中国药科大学 Application of the SNase in preparation treatment inflammatory bowel medicine
CN112023031A (en) * 2020-10-16 2020-12-04 中国药科大学 Application of SNase in preparation of medicine for promoting diabetic wound healing

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