CN108070645A - Stx-t is in prevention and/or treats anaemia or the application of its relevant disease - Google Patents

Abstract

The present invention provides Stx t in prevention and/or to treat anaemia or the application of its relevant disease, specifically, the present invention provides a kind of Stx t genes or the purposes of its albumen or its detection reagent, detection reagent or kit are used to prepare, the detection reagent or kit are used to detect one or more diseases selected from the group below：Anemia, heart disease, fetus at perinatal stage are dead or it is combined.The present invention also discloses Stx t genes for the first time or the expression of its albumen can be as early prediction, the biomarker of diagnosis anemia (especially hypoferric anemia).In addition, the present invention also establishes a kind of anaemia or its relevant disease animal model for the first time, it is mouse or other non-human mammals that Stx t genes were removed or inactivated (including partial inactivation).The animal model of the present invention is a kind of effective anaemia or the animal model of its relevant disease, available for studying anemia disease, heart disease, and/or fetus at perinatal stage death, and can be used for screening and the testing experiment of certain drug.

Description

Stx-t is in prevention and/or treats anaemia or the application of its relevant disease

Technical field

The present invention relates to biomedicine field, in particular it relates to Stx-t prevent and/or treat anaemia or its The application of relevant disease.

Background technology

Asiderosis is that most common nutrient lacks, and accounts for the 50% of global Anemic patients.Known sideropenic anemia with A variety of diseases such as chronic heart failure, chronic kidney disease, cancer and enteritis are closely related.According to the World Health Organization (World Health Organization, WHO) report, hypoferric anemia is most commonly seen in pregnant woman and pediatric population, and wherein hypoferric anemia causes Lethality account for 0.2%, already as a global health problem.

It is about 3-5 grams that adult human body, which stores up iron total amount,.The iron in body circulation is participated in mainly with ferric form and transferrins Compound is combined to form, the compound and the TfR (tranferrin receptor, TFR) on cell membrane are mutual Vesica endocytosis is acted through into intracellular, vesica is in acidity, and ferric iron is in vesica sour environment (pH~5.6-6.2) from endocytosis Separate out in compound is reduced into ferrous iron under the action of metal reductase, and passes through bivalent metal ion passage DMT1 (divalent metal transporter) transports vesica, and the vesica containing TfR is recycled back into cell membrane On.If ferrous iron is free in endochylema, can generate free radicals, so it is very strong by Haber Wei Si Fenton's reactions generation toxicity Hydroxy radical (OH).Therefore, more and more evidences show that ferrous iron is likely to be protected in vesica, and iron content The normal transport of vesica plays an important role to maintenance iron stable state, such as：Outer complex EXOC6 (the exocyst complex of bubble Component 6, is called Sec15l1) participate in the vesica containing TfR bubble spit process；Sorting connection Protein S NX3 (sorting nexin 3) then participates in the sorting of the intracellular of vesica containing TfR and iron absorbs.Except more than animal model Outside, laser co-focusing living cells imaging experiment finds vesica containing TfR and line grain in reticulocyte on cellular level Instantaneous fusion occurs for body, and iron in vesica is conveyed into mitochondria.However, the molecular mechanism of mediation iron content vesica intracellular fusion needs Further research.

Previous research report Syntaxin 12/13 (STX-T, T represent 12 and 13) to be positioned in cell inclusion body, however The physiological function of STX-T is still unclear.

Therefore, there is an urgent need in the art to develop effectively prevent and/or treat anaemia or the drug of its relevant disease.

The content of the invention

It can effectively prevent it is an object of the invention to provide a kind of and/or treat anaemia or the drug of its relevant disease.

Another object of the present invention is to provide a kind of Stx-t genes or the purposes of its albumen or its accelerating agent.

Another object of the present invention is to provide the purposes of the deactivator of a kind of Stx-t genes or its albumen or lower adjustment.

First aspect present invention provides a kind of Stx-t genes or the purposes of its albumen or its detection reagent, is used to prepare One detection reagent or kit, the detection reagent or kit are used to detect one or more diseases selected from the group below：Anaemia Disease, heart disease, fetus at perinatal stage is dead or it is combined.

In another preference, the fetus at perinatal stage death refers to the death before and after fetal birth, specifically refers to pregnancy 28 The death of one week this perinatal important period of thoughtful postpartum.

In another preference, the heart disease includes heart malformations.

In another preference, the detection reagent is used to prepare kit, and the kit is selected from the group for detecting One or more diseases：Anemia, heart disease, fetus at perinatal stage are dead or it is combined.

In another preference, the detection reagent is selected from the group：Antibody, primer, probe, sequencing library, nucleic acid chip (such as DNA chip), protein-chip or its combination.

In another preference, the albumen includes full-length proteins or protein fragments.

In another preference, the Stx-t genes or its albumen source are more preferably derived from and nibbled in mammal Tooth animal (such as mouse, rat), Primate and people.

It is described to be detected as tissue sample detection or Virus monitory in another preference.

In another preference, the Stx-t albumen couplings have or with detectable label.

In another preference, the detectable label is selected from the group：Chromophore, chemiluminescent groups, fluorogen, same to position Element or enzyme.

In another preference, the Stx-t albumen further includes the derivative of Stx-t albumen.

In another preference, the derivative of the Stx-t albumen includes modified Stx-t albumen, amino acid sequence With natural Stx-t albumen homologies and dimer or poly with the protein moleculars of natural Stx-t protein actives, Stx-t albumen Body, the fusion protein containing Stx-t protein amino acid sequences.

In another preference, the modified Stx-t albumen is PEGylated Stx-t albumen.

In another preference, described " amino acid sequence is with natural Stx-t albumen homologies and with natural Stx-t albumen The protein molecular of activity " refers to that its amino acid sequence has >=85% homology compared with Stx-t albumen, preferably >=90% Homology, homology more preferably >=95%, most preferably >=98% homology；And with natural Stx-t protein actives Protein molecular.

Second aspect of the present invention provides a kind of Stx-t genes or the purposes of its albumen or its detection reagent, is used to prepare One detection reagent or kit, the detection reagent or kit are used to screen neonatal one or more diseases selected from the group below Disease：Anemia, heart disease, fetus at perinatal stage are dead or it is combined.

In another preference, the detection reagent is used to prepare kit, and the kit is neonatal for screening One or more disease selected from the group below：Anemia, heart disease, fetus at perinatal stage are dead or it is combined.

Third aspect present invention provides a kind of Stx-t genes or the purposes of its albumen or its accelerating agent, is used to prepare Composition or preparation, the composition or preparation are used to treat one or more diseases selected from the group below：Anemia, heart disease Disease, fetus at perinatal stage are dead or it is combined.

In another preference, the accelerating agent be refer to improve in vivo or in vitro Stx-t genes or its albumen or Or derivatives thereof activity and/or content substance；The substance can be artificial synthesized or natural compound, albumen, Nucleotide etc..

Fourth aspect present invention provides a kind of detection one or more diseases selected from the group below：Anemia, heart disease, The method of fetus at perinatal stage death or its combination, including：

A) subject's test sample is prepared；With

B) expression quantity of Stx-t genes or its albumen in test sample is detected, and by expression quantity testing result and reference value It is compared, the wherein expression quantity of Stx-t genes or its albumen is substantially less than reference value, shows that subject suffers from and is selected from the group One or more diseases：Anemia, heart disease, fetus at perinatal stage be dead or its combination or suffer from it is selected from the group below a kind of or A variety of diseases：Anemia, heart disease, fetus at perinatal stage are dead or its probability combined is higher than normal population.

In another preference, " being substantially less than " refers to the ratio between expression quantity E1 and reference value E0 of test sample≤1/ 2, preferably≤1/3, more preferably≤1/4.

In another preference, the test sample is heart and/or brain tissue sample.

In another preference, the reference value is Stx-t genes or its albumen in normal heart and/or brain tissue Expression quantity.

In another preference, the detecting step (b) includes being detected by RT-qPCR methods or sequencing.

In another preference, the antibody that the detecting step (b) includes the use of anti-Stx-t albumen is detected.

In another preference, detecting step (b) is realized by immunohistochemistry or enzyme linked immunoassay method (ELISA method).

In another preference, the antibody of the anti-Stx-t albumen is monoclonal antibody or polyclonal antibody (such as anti-blood Clearly).

In another preference, the method is non-therapeutic and nondiagnostic.

Fifth aspect present invention provides a kind of diagnostic kit, and the kit includes：

(a) Stx-t genes or its albumen；And/or

(b) primer or primer pair of specific amplification Stx-t mRNA or Stx-t cDNA；

And label or specification；

Wherein, the component (a), (b) are located at one or more different containers or respectively in same containers.

In another preference, the component (a) can be used as reference substance or with reference to product.

In another preference, the label or specification indicate the kit and are used for：

(i) detect or diagnose one or more diseases selected from the group below：Anemia, heart disease, fetus at perinatal stage be dead, Or its combination；And/or

(ii) neonatal one or more diseases selected from the group below are screened：Anemia, heart disease, fetus at perinatal stage are dead It dies or it is combined.

Sixth aspect present invention provides the deactivator of a kind of Stx-t genes or its albumen or the purposes of lower adjustment, is used for Prepare structure non-human mammal anaemia or the preparation of its relevant disease animal model.

In another preference, the anaemia or its relevant disease are selected from the group：Anemia, heart disease, perinatal period tire Youngster is dead or it is combined.

In another preference, the deactivator includes inhibitor.

In another preference, the deactivator or lower adjustment refer to the expression decline 50% of Stx-t genes or its albumen, It is preferred that 70%, more preferably, 80%, more preferably, 90%, more preferably, 100%.

In another preference, the deactivator or lower adjustment are selected from the group：Antibody, micromolecular compound, nucleic acid or its Combination.

Seventh aspect present invention provides a kind of purposes of cell, Stx-t genes inactivation in the cell or lowers, and uses In the anaemia or the biological agent of its relevant disease animal model that prepare structure non-human mammal.

In another preference, the biological agent is liquid formulation.

Eighth aspect present invention provides a kind of anaemia of non-human mammal or the preparation of its relevant disease animal model Method comprises the following steps：

(a) cell of non-human mammal is provided, the Stx-t genes in the cell are inactivated, obtains the mistake of Stx-t genes Nonhuman mammalian cells living；With

(b) using the cell of the Stx-t genes inactivation obtained in step (a), the anaemia that Stx-t genes inactivate is prepared Or its relevant disease animal model.

In another preference, in step (a), following steps are further included：

(a1) using DNA homologous recombination technology, by the code area (such as exons 1 to extron 9) in the Stx-t genes Or noncoding region (such as 5 ' UTR or 3 ' UTR) or 3 ' code areas or include sub-district and rejected or gene editing, and use selection markers It replaces, obtains the nonhuman mammalian cells of Stx-t genes inactivation.

In another preference, in step (b), following steps are further included：

(b1) it is prepared using the nonhuman mammalian cells of the Stx-t genes inactivation obtained in step (a) chimeric non- People mammal；

(b2) it is the chimeric non-human mammal and normal wild type mating non-human mammals that are obtained in step (b1) is numerous It educates, the heterozygote non-human mammal of screening acquisition Stx-t gene inactivations in offspring；With

(b3) Stx-t genes inactivation is obtained by the way that the heterozygote non-human mammal obtained in step (b2) is mutually mated Homozygote non-human mammal, so as to obtain Stx-t genes inactivation non-human mammal animal model.

In another preference, in step (b3), step (b4) is further included：The homozygote that Stx-t genes are inactivated is non- People mammal carries out miscellaneous with the brain of same species and/or the specific knockdown instrument non-human mammal of heartspecific It hands over, so as to obtain the animal model of the non-human mammal of the Stx-t genes of brain and/or heartspecific inactivation.

It is described that Stx-t genes inactivation is included into gene knockout, gene disruption or gene insertion in another preference.

In another preference, the gene inactivation includes Stx-t genes and does not express or express inactive Stx-t Albumen.

In another preference, described further include Stx-t genes inactivation declines the expression of Stx-t genes or its albumen 50%, it is preferred that 70%, more preferably, 80%, more preferably, 90%, more preferably, 100%.

In another preference, Stx-t genes inactivation is the Stx-t genes mistake of brain and/or heartspecific Living or whole body Stx-t genes inactivation.

In another preference, the non-human mammal be rodent or primate, be preferably comprised mouse, Rat, rabbit and/or monkey.

In another preference, the non-human mammal is mouse, and Stx-tLoxp/ in step (b4) Loxp mouse are mated with instrument mouse NSE (neuron-specific enolase)-Cre to get in brain and/or heart spy Different in nature Stx-t is because of knock-out mice abbreviation cKO mouse (i.e. brain and/or heartspecific Stx-t inactivated mices).

In another preference, the selection markers are selected from the group：Resistant gene, fluorescence protein gene or its combination.

In another preference, the selection markers include neo genes and/or GFP genes.

In another preference, the animal mould of the non-human mammal of the Stx-t genes inactivation obtained in the step (b) In type, compared with wild-type control animals, there are one or more features selected from the group below：

(t1) sideropenic anemia；

(t2) heart iron deficiency；

(t3) cardiac shape is abnormal；

(t4) cardiomyocyte surface area reduces；

(t5) myocardial mitochondria is abnormal, and the myocardial mitochondria includes extremely：The table of the translocase TOM20 of mitochondrial outer membrane Ridge density is sparse in up to reduction, the increase of mitochondria area, and/or mitochondria；

(t6) full brain iron deficiency.

In another preference, the full brain iron deficiency refers to brain iron deficiency.

Ninth aspect present invention provides non-human mammal model prepared by a kind of eighth aspect present invention the method Purposes, which is used as to the animal model of studying anemia or its relevant disease.

In another preference, the anaemia or its relevant disease are selected from the group：Anemia, heart disease, perinatal period tire Youngster is dead or it is combined.

Tenth aspect present invention provides non-human mammal model prepared by a kind of eighth aspect present invention the method Purposes, wherein, by the model for screening or determine to mitigate or treat anaemia or the substance of its relevant disease (therapeutic agent).

Tenth one side of the invention, which provides, a kind of screens or determines the latent of prevention and/or treatment anaemia or its relevant disease In the method for therapeutic agent, including step：

(a) in test group, in cultivating system, in the presence of compound is tested, the cell one of culture expression Stx-t Section time T1 detects the expression E1 of the Stx-t genes or its albumen in cultivating system described in test group；

And in there is no the test compound and the identical control group of other conditions, culture described in control group is detected The expression E2 of Stx-t genes or its albumen in system；With

(b) E1, the E2 for being detected previous step are compared, so that it is determined that whether the test compound is prevention And/or the potential treatment agent for the treatment of anaemia or its relevant disease；

Wherein, if E1 is significantly higher than E2, then it represents that the test compound is prevention and/or treatment anaemia or it is related The potential treatment agent of disease.

In another preference, " being significantly higher than " refers to E1/E2 >=2, it is preferred that >=3, more preferably, >=4.

In another preference, the method is non-diagnostic and non-therapeutic.

In another preference, the method includes the steps (c), and identified potential treatment agent in step (b) is applied In non-human mammal model prepared by eighth aspect present invention the method, so as to measure its anaemia to the animal model Or the influence of its relevant disease.

The twelfth aspect of the present invention provides a kind of non-human mammal model, model eighth aspect present invention institute It is prepared by the method stated.

In another preference, for Stx-t genes inactivation for, the non-human mammal model be heterozygosis or Homozygous.

In another preference, Stx-t genes inactivation is the Stx-t genes mistake of brain and/or heartspecific Living or whole body Stx-t genes inactivation.

It is to be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment) It can be combined with each other between each technical characteristic of body description, so as to form new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.

Description of the drawings

Fig. 1 shows interior death when Stx-t knock out mice birth 12 is small.Wherein, (a), P (postnatal) 0 day is same Tire cub, the left side are typical Stx-t^-/-Knock out mice homozygote, the right are normal wild-type mice.And wild type Mouse is compared, Stx-t^-/-Knock out mice body is less than normal and stiff.(b), the box figure of merging and scatter diagram show Stx- t^-/-Cub (homozygote) weight (unit for gram/g, mean+SD, t examine) no matter embryo 18.5 days (E18.5) also Be first day of life (P0) it is more relatively low than wild-type mice or hybrid mice (homozygote, n=16,0.95 ± 0.08；Heterozygosis Son, n=27,1.16 ± 0.07, P=4.3x10^-10；Wild type, n=13,1.17 ± 0.12, P=5.7x10^-6) and P0 (homozygosis Son, n=17,0.99 ± 0.14；Heterozygote, n=32,1.35 ± 0.12, P=5.3x10^-14；Wild type, n=22,1.36 ± 0.15, P=1.4x10^-9).(c), STX-T western blot analysises.The albumen sample of preparation comes from P0 days mouse, shown above STX-T is below control tubulin Tubulin.(d), the genotype distribution of heterozygosis gametic copulation filial generation.All data come from embryo The genotype of tire period and mouse post-natal, wild-type mice, hybrid mice and homozygote mouse embryo 18.5 days when Time meets mendel's law ratio (1：2：1) (P=0.19, χ²), but do not met (P=2.1x10 at P0 days^-12, χ²), And Stx-t^-/-Homozygote (*, n=43) interior death when 12 is small after birth.

Fig. 2 shows that Stx-t knock out mice suffers from hypoferric anemia.Wherein, (a) and (b) represents wild type respectively Mouse and Stx-t gene knockout homozygote mouse, scale represent 20 μm.(c), wild-type mice, hybrid mice and homozygote The whole blood of mouse.Data are represented with mean+SD.(d) and (e), the scatter diagram of merging and box chart Stx- t^-/-The homozygous total iron content of cub is more relatively low than wild-type mice or hybrid mice in heart and brain tissue, but Without significant change in liver, in addition, the copper contents of three above mouse genotypes is in heart, brain and hepatic tissue also without bright Aobvious variation.All data are explained further in supplementary tables 1, and * represents that P values are less than 0.05.

Fig. 3 shows Stx-t knock out mice heart malformations.Wherein, (a), mouse heart body formula imaging in P0 days, the left side For normal wild-type mice heart, the right is homozygote mouse deformity heart, and scale represents 500 μm；(b) revive with (c), heart Lignin Yihong (Haematoxylin and eosin, HE) is dyed, and longitudinal section (b) and horizontal section (c), the left side is normal Wild-type mice, the right are homozygote mouse, and scale represents 500 μm；(e), tritin (WGA) dye of heart transverse direction section Color, the left side are normal wild-type mice, and the right is homozygote mouse, and scale represents 5 μm；(f), wild-type mice, heterozygote TfR TFR1, the western blot analysis of ferroportin in the heart tissue of mouse and homozygote mouse.It prepares Albumen sample comes from P0 days mouse heart tissues, and antibody shown above is that specific iron-resistant produces albumen ferroportin, intermediate It is specific anti-rotation Human Placental Ferritin Receptor TFR1 to represent antibody, and bottom is control actin aActin.

Fig. 4 shows Stx-t knock out mice myocardial mitochondria exception.Wherein, (a), E18.5 days Stx-t clpp genes Except the myocardial mitochondria electron microscope of mouse exception, the left side is normal wild mouse, and the right side is homozygote mouse, above scale Represent 1 μm, below scale represent 0.5 μm.(b), Stx-t^-/-Knock out mice mitochondria (361 from three mouse) Area (0.61 ± 0.68) is than wild-type mice (3232 from two mouse) area (0.28 ± 0.15) bigger and distribution More uneven (P=1.3x10^-17, t inspections) and (such as figure .4a, 4b)；Ridge density in mitochondria (unit is 1/ micron, average ± Standard deviation) quantitatively find Stx-t^-/-Ridge (20 ± 3) is more sparse than wild-type mice (27 ± 3) in gene knockout homozygote mouse (P=1.7x10^-22, t inspections).(c), the immunoblotting assay of myocardial mitochondria GAP-associated protein GAP, including TOM20, OPA1, ATP5B And HSP60, albumen sample come from the heart tissue of P0 days wild-type mices, hybrid mice and homozygote mouse.(d), TOM20 Respectively wild-type mice, hybrid mice and homozygote mouse expression comparison.

Fig. 5 shows 11 Qa-SNAREs albumen Multiple sequence alignments.Wherein, Syntaxin-T represents syntaxin12/ 13.Amino acid similarity is higher than 80% highlighted presentation, wherein similar amino acid gray background, same amino acid is with pink Color background.Red asterisk refers to participating in the conservative glutaminic acid residue in composition fusion compound helical bundle region, C-terminal trans-membrane region It is marked with horizontal line.Sequence number of 11 people source Qa-SNARE family members in gene pool is as follows：Syntaxin-1A, NP_ 004594.1；Syntaxin-2, NP_919337.1；Syntaxin-3, NP_004168.1；Syntaxin-4, NP_ 004595.2；Syntaxin-5, NP_003155.2；Syntaxin-7, NP_003560.2；Syntaxin-11, NP_ 003755.2；Syntaxin-T, NP_803173.1；Syntaxin-16, NP_001001433.1；Syntaxin-17, NP_ 060389.2；Syntaxin-18, NP_058626.1.

Fig. 6 show Stx-t knock out mice structure and STX-T each tissue expression.Wherein, (a), Western blot analysis of the STX-T in 18.5 days mouse of embryo E (embryonic) respectively tissue.The albumen sample of preparation comes From E18.5 days mouse, antibody was the anti-STX-T of specificity, showed that STX-T is expressed in each tissue in ubiquitination, and in brain and the heart Dirty middle high expression；(b), Stx-t knock out mice structure schematic diagram.For the expression of silence STX-T, originated in Stx-t genes Insertion carries GFP (the green fluoresence protein) genes of terminator codon to sink at codon ATG upstream 10bp The expression of silent STX-T, the carrier that targets being injected into embryonic stem cell generate Stx-t knock out mice by homologous recombination, Removal Neo genes are further handed over FLP instruments mouse on this basis.Neo (Neomycin resistance gene) is Neomycin Neomycin resistant genes；FRT (flippase recognition target) identifies target sequence for flippase； FLP (flippase), flippase；LoxP (locus of X-over P1), P1 transformer seats；(c), GFP western blots point Analysis.The albumen sample of preparation 0 day Mice brain tissues of P (postnatal) after birth, antibody shown above are anti-for specificity GFP albumen is below control tubulin Tubulin.

Fig. 7 shows that STX-T is imaged with TFR common locations.Wherein, (a)-(c), middle STX-T (red, with red fluorescence egg White RFP) it is thin in H9C2 (2-1) cell lines (a) and primary neuron respectively with TFR (green, merged with green fluorescent protein GFP) Born of the same parents (b) common location is imaged, and scale represents 5 μm.(c), the scatter diagram of merging and box figure represent STX-T and TFR respectively in nerve In first (0.92 ± 0.03, n=6) and H9C2 cell lines (0.87 ± 0.05, n=9) common location related coefficient (with average value ± Standard deviation represents)

Fig. 8 shows brain, liver, spleen, and haematoxylin Yihong (HE) of kidney and lung tissue is coloured to picture.Wherein, the left side is homozygosis Sub- mouse, the right are normal wild-type mice, and the scale of brain tissue represents 500 μm, and other tissue scales represent 20 μm.

Fig. 9 shows brain, the mitochondria electron microscope of liver organization and the functional examination of myocardial mitochondria enzyme.Wherein, (a) and (b), the Electronic Speculum Mitochondrial Shape in the hippocampal tissue (a) of E18.5 days mouse brains and hepatic tissue (b), the left side are normal heterozygosis Sub- mouse, the right side are homozygote mouse, and scale represents 0.5 μm；(c) and (d), P0 days mouse cardiac muscle cyclophorase immunohistochemistry Analysis, cytochrome c oxidase COX (cytochrome c oxidase) (c, light brown) and succinate dehydrogenase SDH (succinate dehydrogenase) (d, navy blue), the left side are normal wild-type mice, and the right side is small for homozygote Mouse, scale represent 0.5 μm.

Figure 10 shows the cSNPs collection of illustrative plates of people source Stx-t gene coding regions.Wherein, (a), STX-T albumen in people source is by one C-terminal transmembrane region TM (transmembrane domain, green), a SNARE area (SNARE, red) and a N-terminal control region (Habc, blueness) composition；(b), the cSNP missense mutation of STX-T.Asterisk * represents that terminator codon is mutated, insertion symbol ^ Represent frameshift mutation, while missense mutation correct amino acid sequence it is identified below out and same sense mutation does not mark additionally.

Specific embodiment

The present inventor is by in-depth study extensively, for the first time it was unexpectedly observed that Stx-t gene expressions in brain or heart After level reduces, mouse can be caused to suffer from fe-deficiency anaemia, heart malformations, myocardial mitochondria abnormal.Therefore, Stx-t genes or The expression of its albumen and the morbidity (or neurological susceptibility) of anemia (especially hypoferric anemia), can conducts there are strong correlation The biomarker of early prediction, diagnosis anemia (especially hypoferric anemia).

In addition, the present invention also establishes a kind of anaemia or its relevant disease animal model for the first time, it is that Stx-t genes are picked Remove or inactivate mouse or other non-human mammals of (including partial inactivation).The animal model of the present invention is a kind of effective poor The animal model of blood or its relevant disease available for diseases such as studying anemia, heart disease, and/or fetus at perinatal stage death, is gone back It can be used for the diseases such as screening or the neonatal anaemia of examination, heart disease, and/or fetus at perinatal stage death, and can be used for The screening of certain drug and testing experiment.On this basis, the present inventor completes the present invention.

Sample

Term " sample " used herein or " sample " refer to subject's specifically associated material, from wherein may be used To determine, calculate or be inferred to the specific information related with subject.Sample can be completely or partially by the life from subject Object material is formed.Sample can also be the material contacted in some way with subject, and this way of contact causes to sample The test of progress can provide the information related with subject.Sample can also be the material contacted with other materials, This other materials are not subjects, but the first material can be made then tested with the definite letter related with subject Breath, such as sample can be the cleaning solution of probe or scalpel.Sample can be the biomaterial source outside contact subject, only The professional of the art is wanted to remain able to determine the information related with subject just from sample.

Expression

As used herein, term " expression " includes generations of the mRNA from gene or Gene Partial, and including by RNA or base The generation of cause or the encoded protein of Gene Partial further includes the appearance with expressing relevant detection substance.For example, cDNA, Binding partner (such as antibody) and gene or other oligonucleotides, the combination of protein or protein fragments and binding partner it is aobvious Color part is included in the range of term " expression ".Therefore, in the increase of Western blotting such as western traces upper half dot density It is also in the range of the term based on biological molecule " expression ".

Reference value

As used herein, term " reference value " refers to relevant with particular result statistics when compared with analysis result Value.In preferred embodiments, reference value is according to comparing the expression of Stx-t albumen and the research of known clinical effectiveness The statistical analysis of progress comes definite.Some such researchs are shown in the embodiments herein part.But from text The research offered and the user experience of method disclosed herein can also be used for producing or adjusting reference value.Reference value can also be by examining The situation especially relevant with the medical history of patient, science of heredity, age and other factors and result are considered to determine.

Stx-t genes

As used herein, term " Stx-t genes ", " Syntaxin 12/13 ", " STX-T " are used interchangeably.

There are two the generally acknowledged english names of the Stx-t genes, is respectively Syntaxin12 and Syntaxin13, in order to Writing is easy, with Stx-t instead of syntaxin12/13.

It is to be understood that term " Stx-t " further includes the variant form of various naturally occurring Stx-t genes.Representative example Attached bag includes：The nucleotide sequence of the Stx-t albumen identical with wild type, encoding wild type are encoded due to the degeneracy of codon The nucleotide sequence of conservative variation's polypeptide of Stx-t albumen.In addition, during for other mammals outside mouse, the art Language refers to homologue of the Stx-t genes in the mammal.Such as people, which refers to Stx-t (the known mouse of people Stx-t genes and the homologous degree of cDNA of mankind's Stx-t genes are 90%%, 96%) the homologous degree of amino acid sequence is.Mouse The accession number of stx-t genes：NCBI Gene ID:100226；The accession number of mouse stx-t albumen：NCBI NP_598648.1, The accession number of the stx-t genes of people：NCBI Gene ID:23673；The accession number of people's stx-t albumen：NCBI NP_ 803173.1。

In the present invention, the shortage of STX-T can cause mouse to be born lethal (embryo survival), and adjoint hypoferric anemia, The exception of brain and heart asiderosis, heart malformations and myocardial mitochondria.

Prediction or diagnostic application

The present invention provides a kind of prediction or diagnosis (especially early stage complementary prediction or diagnosis) anaemias or its related disease Disease or the method for its neurological susceptibility and corresponding reagent box.

The method of the present invention make use of detection object brain or heart tissue in stx-t expressions and anaemia or its is related Correlation between the Expectancy of disease.

The research of the present invention shows the expression of stx-t and anaemia in brain or heart tissue or its relevant disease There are significant correlation between Expectancy, thus the complementary prediction of early stage particularly suitable for anaemia or its relevant disease or Diagnose the biomarker of (the especially early screening of neonatal anemia or its relevant disease).

The detection method of the present invention can be based on the mRNA expressions of stx-t or the protein expression level of stx-t.

In the present invention, stx-t genes include the various nucleotide sequences of stx-t, such as DNA sequence dna or mRNA sequence.

A kind of preferred detection method includes step：

A) subject's test sample is prepared；

B) expression quantity of Stx-t genes or its albumen in test sample is detected, and by expression quantity testing result and reference value It is compared, the wherein expression quantity of Stx-t genes or its albumen is substantially less than reference value, shows that subject suffers from and is selected from the group One or more diseases：Anemia, heart disease, and/or fetus at perinatal stage are dead or suffer from one or more selected from the group below Disease：The probability of anemia, heart disease, and/or fetus at perinatal stage death is higher than normal population.

In another preference, " being substantially less than " refers to the ratio between expression quantity E1 and reference value E0 of test sample≤1/ 2, preferably≤1/3, more preferably≤1/4.

The deactivator or lower adjustment of stx-t genes or its albumen

In the present invention, the deactivator of the stx-t includes all inactivation or partial inactivation.

The deactivator of the stx-t albumen of the present invention includes (a) inhibitor, and the example of the inhibitor includes (but and unlimited In)：Micromolecular compound, antibody, antisensenucleic acids, miRNA, siRNA or its combination；And/or the knockout of (b) stx-t genes Agent.

In the present invention, the deactivator or lower adjustment refer to declines 50% by the expression of Stx-t genes or its albumen, preferably Ground, 70%, more preferably, 80%, more preferably, 90%, more preferably, 100%.

Anaemia or its relevant disease

In the present invention, anaemia or its relevant disease include but is not limited to：Anemia, heart disease, and/or perinatal period Foetal death.Wherein, heart disease includes (but being not limited to)：Heart malformations.

Also, the fetus at perinatal stage death refers to the death before and after fetal birth, specifically refers to and specifically refers to pregnancy 28 weeks To the death of one week this perinatal important period of postpartum.

Gene inactivates

Many methods can be used for the research of Unknown Function gene, such as inactivate the gene to require study, analyze institute The character mutation of the genetic modification obtained, and then obtain the functional information of the gene.Another advantage of this research method is can be with Gene function and disease are associated, so as to can also be obtained while gene function is obtained the gene as potential drug or The disease information and disease animal model that person's drug target can treat.The method of gene inactivation can pass through gene knockout, gene It interrupts or the mode of gene insertion is completed.Wherein, gene knochout technique is the non-of function of the research human gene in entirety Normal strong means.

In the present invention, gene inactivation is further included declines 50% by the expression of Stx-t genes or its albumen, it is preferred that 70%, more preferably, 80%, more preferably, 90%, more preferably, 100%.

Animal model

In the present invention, a kind of very effective anaemia or the non-human mammal model of its relevant disease are provided.

In the present invention, the example of non-human mammal includes (but being not limited to)：Mouse, rat, rabbit, monkey etc., more preferably Ground is rat and mouse.

As used herein, term " stx-t genes inactivation " includes the situation that one or two stx-t gene is deactivated, i.e., And it inactivates homozygously including stx-t genetic heterozygosis.For example, stx-t genes inactivation mouse can be heterozygosis or homozygosis it is small Mouse.

In the present invention, can gene knockout or be transferred to foreign gene (or segment) and make stx-t genes inactivate the methods of system The non-human mammal (such as mouse) of standby stx-t genes inactivation.In the art, by gene knockout or be transferred to foreign gene and The technology that target gene inactivates is made to be known, these routine techniques can be used in the present invention.

In another preference of the present invention, the inactivation of stx-t genes is realized by gene knockout.

In another preference of the present invention, the inactivation of stx-t genes is by being inserted into foreign gene in stx-t genes (or segment) and realize.

In the specific example of the present invention, a construction containing external source Insert Fragment can be built, which contains The homologous homology arm with the flanking sequence of the both sides of the insertion point of target gene (stx-t), so as to pass through homologous recombination height Frequently external source Insert Fragment (or gene) is inserted into stx-t genome sequences (especially exon region), causes mouse The frameshit of stx-t genes is terminated or knocked out in advance, so as to cause stx-t gene delections or inactivation.

The homozygosis obtained with the method for the present invention or the mouse of heterozygosis are fertile.The stx-t genes of inactivation can be with Mendel's rule Entail progeny mice.

In a preference, the present invention provides a kind of Mice homozygous animal patterns for lacking stx-t genes.

Drug candidate or therapeutic agent

In the present invention, a kind of animal model using the present invention, screening treatment anaemia or its relevant disease are additionally provided Drug candidate or therapeutic agent method.

In the present invention, drug candidate or therapeutic agent refer to it is known with certain pharmacological activity or be detected can Can have the substance of certain pharmacological activity, including but not limited to nucleic acid, albumen, carbohydrate, the small molecule of chemical synthesis or big point Sub- compound, cell etc..The administering mode of drug candidate or therapeutic agent can be taken orally, be injected intravenously, being injected intraperitoneally, subcutaneously noting It penetrates, canalis spinalis is administered or direct intracerebral injection.

Drug screening method

The present invention also provides the methods that drug screening is carried out based on stx-t.A kind of method is that first screening influences (enhancing) Stx-t is expressed or the compound of activity, then further tests the compound filtered out it to suffering from anaemia or its related disease The therapeutic effect of sick animal model mouse.

Screening prevention provided by the invention and/or treatment anaemia or the method for its treating correlative diseases agent, based on the chemical combination Object includes step to the expression quantity of stx-t and/or the influence of activity, a kind of typical screening technique：

(a) in test group, in cultivating system, in the presence of compound is tested, the cell one of culture expression Stx-t Section time T1 detects the expression E1 of the Stx-t genes or its albumen in cultivating system described in test group；

And in there is no the test compound and the identical control group of other conditions, culture described in control group is detected The expression E2 of Stx-t genes or its albumen in system；With

(b) E1, the E2 for being detected previous step are compared, so that it is determined that it is described test compound whether be prevention or Treat anaemia or the potential treatment agent of its relevant disease；

Wherein, if E1 is significantly higher than E2, then it represents that the test compound is prevention or treatment anaemia or its related disease The potential treatment agent of disease.

In a preferred embodiment, the method includes the steps (c), by identified potential treatment agent in step (b) Be applied to the method for the invention preparation non-human mammal model, so as to measure its to the anaemia of the animal model or its The therapeutic effect of relevant disease.

The expression of stx-t can be carried out in mRNA level in-site or protein level, such as by conventional method or commercially available Equipment and reagent (such as antibody, primer) carry out.

Main advantages of the present invention include：

(1) provide for the first time it is a kind of be present in it is in brain, heart tissue, be easy to early prediction or early diagnosis anaemia or The objective biomarker of its relevant disease (especially hypoferric anemia).The present invention confirm for the first time the mRNA of stx-t with The apparent correlation of anaemia or its relevant disease (especially hypoferric anemia), so that it is determined that stx-t mRNA level in-sites can be made For diagnosis anaemia or the molecular biology foundation of its relevant disease (especially hypoferric anemia).

(2) present invention firstly discovers that stx-t is to maintaining intracellular iron stable state to play an important roll, and stx-t is found for the first time (Syntaxin12/13) shortage can cause mouse to be born lethal (embryo can survive), and with hypoferric anemia, brain and the heart The exception of dirty asiderosis, heart malformations and myocardial mitochondria, these discoveries can be applied to pre-natal diagnosis, and prevention and reduction birth lack It falls into.

(3) present invention builds a kind of anaemia of non-human mammal or the animal model of its relevant disease for the first time, and can profit With model Effective selection prevention and/or treat anaemia or the therapeutic agent of its relevant disease.

With reference to specific embodiment, the present invention is further explained.It is to be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.The experimental method of actual conditions is not specified in the following example, usually according to conventional strip Part such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in or according to the condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and Number is calculated by weight.

Unless otherwise instructed, all material used in embodiment and reagent are commercial product.

Experimental method

Build Stx-t knock out mice

Stx-t targets carrier and is inserted into insertion gene sequence at Stx-t gene start codon ATG upstream 10bp including one Loxp-GFP-PolyA-loxp is arranged, the GFP of the sequence contains terminator codon and neomycin resistance gene, and targeting vector electricity turns The gene order targeted into embryonic stem cell (ES) and by homologous recombination reaction displacement.ES cells (are purchased from Shanghai south mould Formula biotechnology Development Co., Ltd) electroporation for 24 hours with changed respectively after 48h containing selection drug G418 it is (final concentration of 300mg/L) and culture solution the making choice property culture of proganoside (final concentration of 2umol/L), then select resistance clone after Continuous culture targets whether carrier is correctly integrated by PCR (polymerase chain reaction) amplifications and sequence verification Genome.It is allophenic mice that the blastaea being proved to be successful, which is transplanted to the offspring given birth in false pregnancy female rat uterus, Ran Houyu C57BL/6J (being purchased from Shanghai Nanfangmoshi Biological Sci-Tech Dev Co., Ltd.) backcrossing breeding expands population, GFP gene knock-ins Positive hybrid mice selfing generates Stx-t knock out mice.It is considered as pregnancy when usually checking female rat vaginal plug 0.5 day i.e. E0.5, the birth mouse of dissection are handled with isoflurane anesthesia.

Western blot

The mouse tissue removed is put into precooling lysate (the 1%Triton X- for having added in phosphatase and protease inhibitors 100 and 1%DOC is in Tris buffer solutions) in, after being ground with microcomponent's electric grinder (Kimble 749540-0000, USA) Being incubated 30 minutes on ice makes cell fully crack, and 12,000rpm, 4 DEG C of centrifugations take supernatant for 15 minutes and with BCA protein determinations Kit (CWBIO, Cat NO:CW0014 protein concentration) is measured.The albumen of extraction is separated with 4~20% Gradient Gel Electrophoresis, 1.5 hours of 120 volts of constant pressure transferring films (PerkinElmer), then 5% skim milk room temperature, 1 hour of closing, 4 DEG C light overnight It is gently incubated primary antibody with rocking, is resisted with washing after three times (10 minutes every time) to continue to be incubated at room temperature containing 0.05% polysorbas20 Tris buffer solutions Rabbit secondary antibody (GE, 1:2000, Cat No:NA934) or anti-mouse secondary antibody (Cell Signaling, 1:2000, Cat No:7076S)1 Hour, finally three times (10 minutes are every time) post-exposure (GE, ImageQuant are washed again with containing 0.05% polysorbas20 Tris buffer solutions LAS 4000mini).Primary antibody include Syntaxin12 (Abcam, 1:1000, Cat No:ab13261),β-Tubulin (Sigma, 1:2000, Cat No:T4026), GFP (Abmart, 1:2000, Cat No:P30010), TFR1 (Abcam, 1: 1000, Cat No:Ab84036), Ferroportin (Novus, 1:500, Cat No:NBP1-21502), TOM20 (Santa, 1:200, Cat No:SC11415), OPA1 (BD Biosciences, 1:500, Cat No:612607), ATP5B (Abcam, 1: 400, Cat No:Ab14730), HSP60 (Stressgen, 1:1000, Cat No:ADI-SPA-806-F), α-Actin (Abmart, Cat No:M20010)

Plasmid construction, cell culture and transient transfection

Tfr the and Stx12 genetic fragments from rat brain complementary genes group are expanded, are then inserted respectively into pEGFP-N1 (Invitrogen) in and TagRFP-T-N1 carriers (being obtained from Roger Y.Tsien laboratories)；Primary neuronal culture method With reference to report (Kang et al., 2008) before, H9C2 (2-1) cell line is obtained from the Chinese Academy of Sciences (Shanghai, China) cell Storehouse, condition of culture be 10% hyclone culture medium, 37 DEG C and 5%CO2；Phosphoric acid is used during TFR-GFP and STX12-RFP corotation Calcium robin, and it is about 1 × 10 to require cell density⁴A cell is every square centimeter, continue after transfection culture 48 it is small when after be copolymerized it is burnt It is imaged (FV1000；Olympus), the plug-in unit of the common location situation software imageJ of TFR-GFP and STX12-RFP " colocalization Indices " is analyzed, and result is represented with related coefficient.

Tissue morphology and immunohistochemical staining

It dissects obtained tissue and is directly placed into the 0.01M phosphate buffers of precooling (137mM sodium chloride, 2.7mM chlorinations Potassium, 10mM disodium hydrogen phosphate dodecahydrates, 1.76mM potassium dihydrogen phosphates are dissolved in 1 liter of distilled water) rinse after be put into 4% poly Formalin (Sigma, Cat No:158127) 4 DEG C of overnight fix (need when configuring paraformaldehyde solution with phosphate buffer in Sodium hydroxide solution hydrotropy is added dropwise) frozen section or paraffin section afterwards.It is molten to continue 30% sucrose for frozen section heart tissue OCT embedding mediums (Sakura, Cat No is used after the dehydration overnight of 4 DEG C of liquid:4583) in the isopentane of Liquid nitrogen precooler frozen embedding into Block, about 10 microns of thickness of then cutting into slices；For the brain of paraffin section, liver, spleen, kidney and lung tissue are (including Stx-t knock out mice And wild-type mice) then continue alcohol serial dehydration (including 70% × 2 time, 80% × 2 time, 95% × 2 time, 100% × 3 It is secondary), then three times (1 hour every time), infiltration is (1.5 small every time twice in the paraffin (62 DEG C) of melting for pure dimethylbenzene infiltration When) paraffin mass is embedded into afterwards, about 3 microns of thickness of then cutting into slices are used for haematoxylin eosin stains.Crosscutting heart frozen section is used for Tritin (WGA) dyeing (Invitrogen, 1:400, Cat No:W21405), condition is incubated overnight for 4 DEG C, 0.01M phosphorus Acid buffer washes (every time 15 minutes) three times, then with DAPI (Invitrogen, 1:2000, Cat No:D1306 after) redying again It is washed three times with phosphate buffer, finally with anti-quenching fluorescence agent (Southern Biotech, Cat No:0100-01) mounting in Co-focusing imaging (FV1000 under 100 times of oil mirrors；Olympus).Cardiac muscle cell from ventricle is (small including Stx-t gene knockouts Mouse and wild-type mice) horizontal area with the plug-in unit WGA v3.1 of software I mageJ (Dr.Kees Straatman, University of Leicester) analysis, for analysis every pictures should at least containing the cardiac muscle cell of 100 or more, Each phenotype at least needs the picture that four hearts and each heart include at least five isolated areas；The density of cardiac muscle cell is used Cell number in unit area represents, cardiac muscle cell must be full of for the picture of analysis, including 4 homozygotes and 6 Each 57 pictures of wild-type mice.Brain, liver, spleen, kidney and lung tissue paraffin section haematoxylin eosin stains reference standard method, Loose heart frozen section cytochrome c oxidase (COX) and the immunohistochemistry step reference of succinate dehydrogenase (SDH) Kit protocol (GENMED, USA, GMS80086.1).HE is dyed and the immunohistochemistry slice, thin piece of enzyme is imaged with inverted microscope (Olympus IX71), heart living imaging is with Stereo microscope (AxioCam, Carl Zeiss).

Transmission electron microscope is tested

Tissue is taken out as quickly as possible after dissection mouse, it is solid after washing out extra watery blood with the 0.01M phosphate buffers of precooling It is scheduled on 2.5% glutaraldehyde (Electronic Speculum rank, Cat No:16220), 4 DEG C of refrigerators fix 2h, are rinsed 3 times with 0.01M phosphate buffers (10 minutes every time), 1% osmic acid fixer fixes 1.5h, rinses 3 times and then carries out alcohol serial dehydration again, tissue is placed on Pure acetone and epoxy resin (1：1) it is put into pure epoxy resin and permeates after when the pre- infiltration 12 of room temperature is small in mixed liquor, embeds and gather It closes and (is stayed overnight in 37 DEG C of baking ovens, 12h in 45 DEG C of baking ovens, 48h in 60 DEG C of baking ovens) into module, finally with ultramicrotome EM UC6 (Leica) thin slice that module is cut into thickness about 70nm carries out acetic acid uranium and the double dyeing of lead citrate, transmission electron microscope Tecnai G2Spirit Bio-Twin (FEI) film making imagings.The area of mitochondria is analyzed with software image J, interior ridge density image Plug-in unit " Plot Profile " unit of account length of J intersects points, and each two crosspoint represents an interior ridge.

The measure of micro- (iron and copper)

The tissue of known weight in wet base is put into the pure nitric acid of 5ml progress microwave digestion (800 after 1 hour of room temperature predigestion Watt, 20 minutes；1600 watts, 40 minutes), it is then diluted with 2% nitric acid, drives extra acid (100 DEG C, 4 hours) away, continue to use 2% nitric acid is diluted to 2ml, is finally surveyed with inductive coupling Plasma Mass Spectrometry (ICP-MS) (Agilent 7700X series) Determine liver, heart, total iron of brain tissue and total copper content, unit is every gram of wet tissue weight of microgram.

Whole blood collection and analysis

One is bled to be dropped in one end of slide and then equably spread into the other end with another slide and forms blood film, Rui Shi Giemsa stainings kit (Solarbio, Cat No is used after being spontaneously dried in air:1020) dye；By embryo 18.5 days Tire mouse break and collect 20 μ l whole bloods after neck, be then finally diluted to 200 μ l and divided in blood automatic analyzer (Sysmex) Analysis.

Single nucleotide polymorphism snp analysis

Most genome mutations be by single nucleotide polymorphism (single nucleotide polymorphisms, SNPs, the especially mononucleotide positioned at protein coding DNA sequence, are called cSNPs) caused by.

The small-scale accidental data of known single nucleotide polymorphism (SNPs) and Stx-t genes of the present invention derives from DbSNP databases (NCBI, build 148).

Data analysis

The data of collection have independent repetition above three times to test, and the definite of sample size does not use statistical method It derives, more detailed experimental procedure can be found in supplementary data, and all data all use average value ± mark in text or in picture Accurate poor (Mean ± SD) is represented, the progeny genotypes analysis double tail t of Chi-square Test others statistical analysis of heterozygosis gametic copulation It examines, being represented when P values are less than 0.05 has notable difference.

The postnatal survival of 1 Stx-t gene pairs mouse of embodiment is essential

The human genome SNAREs genes being currently known totally 35, wherein 11 belong to Qa-SNAREs families (Fig. 5), And STX-T is the albumen that only one and TfR TFR have common location in Qa-SNAREs families.Promise as in the previous The plucked instrument marking hybridizes as Western blot analysis, and STX-T has expression (Fig. 6 a) in most of tissues.

To study the physiological function of STX-T, the present invention at Stx-t gene start codon ATG upstream 10bp by inserting Enter with terminator codon GFP (green fluoresence protein) gene come silence STX-T expression (Fig. 6 b, 6c).Compared to wild-type mice, Stx-t knock out mice build is apparent (Fig. 1 a) less than normal, embryo 18.5 days and birth first It weight significantly reduces (Fig. 1 c), when birth 12 is small interior lethal (Fig. 1 d).

2 STX-T of embodiment shortages cause mouse to suffer from sideropenic anemia disease

STX-T protein expressions in brain and heart tissue are relatively high (Fig. 6 a), further check STX-T primary It is found after the positioning scenarios of neuronal cell and heart sarcoblast system H9C2 (2-1) (being purchased from Chinese Academy of Sciences's cell bank), STX-T and TFR has very strong common location (Fig. 7a-7c).

Blood film and whole blood have been carried out afterwards, it is found that Stx-t knock out mice erythrocyte numbers substantially reduce (figure 2a, 2b, 2c), this shows that STX-T plays an important role to RBC acceptor garland rate.And the sub- mouse of Stx-t genetic heterozygosis has normally Blood parameters (Fig. 2 c).

In addition to red blood cell number is reduced, STX-T lacks the hematocrit of mouse and hemoglobin is significantly reduced (figure 2c), illustrate that STX-T lacks mouse and suffers from hypoferric anemia.Further to verify the function of STX-T, the present invention also determine brain, The content (Fig. 2 d, 2e and table 1) of the trace elements iron and copper of liver and heart tissue is finding three above tissue copper content just Often, brain and heart iron content are reduced, and liver as main storage ironware official iron content without significant change, this to a certain extent and STX-T albumen is consistent (Fig. 2 a) in each tissue expression.These results indicate that STX-T is steady not only for iron in brain and heart tissue State plays an important roll, and also demonstrating the tissues such as liver and erythrocyte, brain, heart from side has the intracellular of different approaches Iron transport mechanism.

1 wild-type mice of table, hybrid mice and homozygote mouse in heart, brain and hepatic tissue trace element (iron and Copper) content

Compared to wild-type mice or hybrid mice, the reduction of homozygote mouse iron content (unit is every gram of μ g/g of microgram, Mean+SD, t are examined) and in heart (homozygote, n=11,178.91 ± 52.02；Heterozygote, n=12,268.60 ± 94.34, P=0.01；Wild type, n=12,255.97 ± 66.44, P=5.7x10^-3) and brain tissue (homozygote, n=12, 16.39±3.11；Heterozygote, n=11,18.13 ± 3.54, P=0.22；Wild type, n=12,19.65 ± 2.95, P= 0.01), but in liver without significant change (homozygote, n=13,151.29 ± 30.60, heterozygote, n=12,168.22 ± 28.09 P=0.16；Wild type, n=12,154.08 ± 31.56, P=0.82) and heart in copper content (homozygote, n =12,3.95 ± 0.58；Heterozygote, n=12,3.79 ± 0.66, P=0.57；Wild type, n=12,3.65 ± 0.54, P= 0.25), the copper content in brain tissue (homozygote, n=11,1.29 ± 0.17；Heterozygote, n=12,1.27 ± 0.21, P= 0.80；Wild type, n=12,1.39 ± 0.21, P=0.20) in liver copper content (homozygote, n=13,29.70 ± 13.71；Heterozygote, n=12,26.36 ± 7.18, P=0.46；Wild type, n=12,24.18 ± 9.48, P=0.26) also without Significant change.

3 STX-T of embodiment lacks mouse heart deformity

Deeply to probe into the physiological function of STX-T, the form of brain, heart, liver, spleen, lung and nephridial tissue is carried out point It analyses (Fig. 3 and Fig. 8).Compared to wild-type mice, STX-T lacks mouse and heart malformations is presented, and other each tissues are less than normal except volume Outer and no significant difference (Fig. 8).

STX-T lack mouse except cardiac shape extremely in addition to (Fig. 3 a, 3b, 3c), H/BW mass ratio also significantly increases Add (Fig. 3 d), this may be caused by cardiomegaly or weight loss (Fig. 1 b).To distinguish true cause, pass through malt The size that agglutinin dyeing (wheat germ agglutinin staining, WGA staining) measures cardiac muscle cell is (single Microns square is in position, average ± standard deviation) (Fig. 3 e).With wild-type mice (6.96 ± 7.29, n_Mouse=6, n_Area=3176) It compares, STX-T lacks mouse cardiac myocytes area (5.87 ± 6.87, n_Mouse=4, n_Area=2734) (P=4.2x10- less than normal⁹, t Examine), should the result shows that, STX-T shortage do not cause myocardial hypertrophy, increased H/BW mass ratio is subtracted by weight Caused by light.In addition, also calculate unit area myocardium cell number i.e. cardiac muscle cell's density (unit 10⁴/mm², it is average Number ± standard deviation), find wild-type mice (2.9 ± 0.6, n_Mouse=6, n_Picture=49) with STX-T lack mouse (3.0 ± 0.7, n_Mouse=4, n_Picture=57) no significant difference between (P=0.624, T are examined).Result of study shows that STX-T shortages cause mouse core Dirty iron deficiency and paramophia.

There are two the reason for Stx-t gene knockouts cause heart iron content to reduce is possible, one is that STX-T lacks so that born of the same parents The disorder of intracellular vesicle transport or be that STX-T shortages affect indirectly iron and rotate into albumen TFR1 (tranferrin Receptor 1) and iron transport the expression of albumen Ferropertin, protein blot experiment shows TFR1 and Ferropertin tables Up to have almost no change (Fig. 3 f).Known STX-T is one of member for mediating intracellular Vesicle fusion Qa-SNAREs families, therefore As a result prompt, STXT-T takes part in maintenance intracellular iron stable state by influencing the fusion process of iron content vesica.

4 STX-T of embodiment lacks mouse cardiac muscle abnormalities

In addition, mitochondria is for maintaining intracellular iron stable state (for example iron is transported, and is utilized, storage and output) to have important work With, therefore the Mitochondrial Shape by transmission electron microscope Germicidal efficacy, and the immunohistochemical experiment for passing through enzyme determines mitochondria work( Energy (Fig. 4 and Fig. 9).

Transmission electron microscope experiment the result shows that, compared to normal wild-type mice, STX-T lacks mouse cardiac muscle mitochondria Expand form (Fig. 4 a, 4b) in irregular, and brain and liver mitochondrion form are normal (Fig. 9 a, 9b).Myocardial mitochondria area is (single Microns square is in position, average ± standard deviation) it is quantitative find, Stx-t^-/-Knock out mice mitochondria area (0.61 ± 0.68) it is more uneven (P=1.3x10 than wild-type mice (0.28 ± 0.15) bigger and distribution^-17, t inspections) and (Fig. 4 a, 4b)； Ridge density (unit is 1/ micron, average ± standard deviation) quantitatively finds Stx-t in mitochondria^-/-Ridge (20 in knock out mice ± 3) it is more sparse (P=1.7x10 than wild-type mice (27 ± 3)^-22, t inspections).

Cardiac muscle cell pigment c oxidizing ferment (cytochrome c oxidase, COX) and succinate dehydrogenase (succinate Dehydrogenase, SDH) immunohistochemical experiment can be used for assessment measure mitochondrial function, the results showed that, Stx-t^-/-Clpp gene Except mouse and normal wild type mouse and no significant difference (Fig. 9 c, 9d), which implies that mitochondria can be otherwise Iron is obtained to maintain the normal function of cyclophorase COX and SDH, as ferrous ion can be directly from Tie Tong road albumen Mitoferrin enters mitochondria.Due to the toxicity of ferrous ion, the concentration of free ferrous ion is subject to strictly in endochylema Regulation and control so that this iron transfer approach function of mitoferrin is limited.

In addition, the expression of myocardial mitochondria GAP-associated protein GAP has been also checked for, such as positioned at the displacement of mitochondrial outer membrane Enzyme TOM20 (translocase of outer mitochondrial membrane 20), the optic nerve of mitochondrial membrane space Atrophy GAP-associated protein GAP OPA1 (optic atrophy 1), ATP synzyme ATP5B (the ATP synthase of mitochondrial inner membrane Subunit beta), positioned at the heat shock protein HSP60 (heat shock protein 60) and endochylema of mitochondrial matrix Actin actin.For wild-type mice, only to find that TOM20 albumen is pure in Stx-t genes in last albumen Expression quantity is substantially reduced in zygote and hybrid mice, and other expressing quantities do not have notable difference (Fig. 4 c, 4d)

The nucleotide polymorphisms analysis of embodiment 5Stx-t genes

The heart of asiderosis most serious is likely to Stx-t^-/-The main cause of death of knock out mice.Neonatal lethality It is prompted with the phenotype of heart asiderosis, Stx-t^-/-It may be one of gene the defects of causing hypoferric anemia.

The present inventor carries out different cdna samples (healthy population/patient) based on conventional SNP atlas analysis technology Analysis, and people source Stx-t gene coding regions cSNPs collection of illustrative plates is analyzed, it turns out that, at least dash forward there are five known frameshit Become and terminator codon obtains mutation and may cause that STX-T functions completely lose and about 90 may influence STX-T functions Missense mutation (Figure 10 a-10b), therefore the snp analysis of Stx-t genes has for the pre-natal diagnosis for preventing and reducing inborn defect There is important practical significance.

It discusses

2/3rds of the total iron of human body are present in hemoglobin, and ferroheme is the important component of hemoglobin, main Hemoglobin Dynamic Oxygen is mediated to close.The last one step of ferroheme synthesis is completed in mitochondria, ferrochelatase, one Iron is inserted into protoporphyrin IX and forms ferroheme by a enzyme containing sub- iron-sulfur cluster, and therefore, most iron need first to be transported to line grain For synthesis ferroheme in body.The ferrous ion being protected in inclusion body can be direct by iron by " kiss-and-run " model It is transported in mitochondria, but the molecular mechanism that iron content inclusion body is mediated to be merged with mitochondrial membrane is still unclear.

The study find that STX-T shortages cause the reduction of red blood cell number and the reduction (Fig. 2 a-2c) of content of hemoglobin, The reduction of the two parameters often means that hypoferric anemia, show Qa-SNARE STX-T for red blood cell iron stable state to close weight Will, while also prompt, STX-T may participate in " kiss " process as a Vesicle fusion albumen.In addition, mitochondrial outer membrane protein The reduction of TOM20 protein expression levels shows that STX-T is also possible to the dynamic with mitochondrial outer membrane in addition to participating in intracellular iron stable state It balances related.

Iron usually exists in mitochondria with iron-sulfur cluster and hemo- protein forms, is the gold that content is most abundant in mitochondria Belong to one of ion.In mitochondrial complex I-III containing iron-sulfur cluster and the albumen containing hemo- Complex II-IV participates in breathing Electronics transfer and oxidative phosphorylation reaction in chain.Heart as body kinetic pump, it is necessary to by a large amount of mitochondrias generate foot Enough ATP (adenosine triphosphate) maintain normal operating, therefore are also required to utilize substantial amounts of ferro element (figure 2d).Myocardial mitochondria is when entire cell volume is than being born about 20%, and ratio is increased to 40% after adult, therefore compared with Other organ, heart are more sensitive for asiderosis.STX-T shortages cause Stx-t^-/-Knock out mice heart iron content drops Low about 30% (Fig. 2 d and table 1), further results in heart malformations (Fig. 3) and myocardial mitochondria paramophia (Fig. 4).These results Show, the Vesicle fusion of STX-T mediations is for maintaining the normal iron source of Heart mitochondria most important.

STX-T shortages cause Stx-t^-/-The iron content of knock out mice brain reduces about 19% (Fig. 2 d and table 1).

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To be made various changes or modifications to the present invention, such equivalent forms equally fall within the model that the application the appended claims are limited It encloses.

Bibliography

1.Kang,J.-S.,Tian,J.-H.,Pan,P.-Y.,Zald,P.,Li,C.,Deng,C.,and Sheng,Z.- H.(2008).Docking of Axonal Mitochondria by Syntaphilin Controls Their Mobility and Affects Short-Term Facilitation.Cell 132,137–148。

Claims (11)

1. a kind of Stx-t genes or the purposes of its albumen or its detection reagent, which is characterized in that be used to prepare a detection reagent Or kit, the detection reagent or kit are used to detect one or more diseases selected from the group below：Anemia, heart disease Disease, fetus at perinatal stage are dead or it is combined.

2. a kind of Stx-t genes or the purposes of its albumen or its detection reagent, which is characterized in that be used to prepare a detection reagent Or kit, the detection reagent or kit are used to screen neonatal one or more diseases selected from the group below：Anemia, Heart disease, fetus at perinatal stage are dead or it is combined.

3. a kind of Stx-t genes or the purposes of its albumen or its accelerating agent, which is characterized in that be used to prepare composition or system Agent, the composition or preparation are used to treat one or more diseases selected from the group below：Anemia, heart disease, perinatal period tire Youngster is dead or it is combined.

4. a kind of detection one or more diseases selected from the group below：Anemia, heart disease, fetus at perinatal stage death or its group The method of conjunction, which is characterized in that including：

A) subject's test sample is prepared；With

B) expression quantity of Stx-t genes or its albumen in test sample is detected, and expression quantity testing result and reference value are carried out Compare, the wherein expression quantity of Stx-t genes or its albumen is substantially less than reference value, shows that subject suffers from selected from the group below one Kind or a variety of diseases：Anemia, heart disease, fetus at perinatal stage are dead or it combines or suffer from one or more selected from the group below Disease：Anemia, heart disease, fetus at perinatal stage are dead or its probability combined is higher than normal population.

5. a kind of diagnostic kit, which is characterized in that the kit includes：

(a) Stx-t genes or its albumen；And/or

(b) primer or primer pair of specific amplification Stx-t mRNA or Stx-t cDNA；

And label or specification；

Wherein, the component (a), (b) are located at one or more different containers or respectively in same containers.

6. the purposes of the deactivator or lower adjustment of a kind of Stx-t genes or its albumen, which is characterized in that it is inhuman to be used to prepare structure The preparation of mammal anaemia or its relevant disease animal model.

7. a kind of purposes of cell, which is characterized in that Stx-t genes inactivation or downward in the cell are used to prepare structure The biological agent of the anaemia of non-human mammal or its relevant disease animal model.

8. the preparation method of a kind of anaemia of non-human mammal or its relevant disease animal model, which is characterized in that including with Lower step：

(a) cell of non-human mammal is provided, the Stx-t genes in the cell are inactivated, obtains Stx-t genes inactivation Nonhuman mammalian cells；With

(b) using the cell of the Stx-t genes inactivation obtained in step (a), be prepared Stx-t genes inactivation anaemia or its Relevant disease animal model.

9. the purposes of non-human mammal model prepared by a kind of claim 8 the method, which is characterized in that use the model Make studying anemia or the animal model of its relevant disease.

10. the purposes of non-human mammal model prepared by claim 8 the method a kind of, wherein, which is used to sieve Choosing determines to mitigate or treats anaemia or the substance of its relevant disease (therapeutic agent).

11. a kind of method screened or determine prevention and/or the potential treatment agent for the treatment of anaemia or its relevant disease, including step Suddenly：

(a) in test group, in cultivating system, in the presence of compound is tested, during one section of the cell of culture expression Stx-t Between T1, detect the expression E1 of the Stx-t genes or its albumen described in test group in cultivating system；

And in there is no the test compound and the identical control group of other conditions, detect cultivating system described in control group In Stx-t genes or its albumen expression E2；With

(b) E1, the E2 for being detected previous step are compared, so that it is determined that it is described test compound whether be prevention and/or Treat anaemia or the potential treatment agent of its relevant disease；

Wherein, if E1 is significantly higher than E2, then it represents that the test compound is prevention and/or treatment anaemia or its relevant disease Potential treatment agent.