CN101125198B - Application of HSP27 in aspect for improving post-ischemic cardiac systolic function - Google Patents
Application of HSP27 in aspect for improving post-ischemic cardiac systolic function Download PDFInfo
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- CN101125198B CN101125198B CN2006100301485A CN200610030148A CN101125198B CN 101125198 B CN101125198 B CN 101125198B CN 2006100301485 A CN2006100301485 A CN 2006100301485A CN 200610030148 A CN200610030148 A CN 200610030148A CN 101125198 B CN101125198 B CN 101125198B
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Abstract
The present invention pertains to the field of biotechnology and medicine and discloses a usage of Heat Shock Protein 27(HSP27) in the heart protection aspect. The present invention firstly reveals that the HSP27 gene can be applied in the specific myofilament protein for stabilizing ischemia, heart failure and other damages of the heart muscle, so as to improve the contractile function of the heart muscle; the present invention also proves that the HSP27 transfected by the adenovirus vector can improve the contractile function of the heart with ischemia and can provide an effective new target for the treatment of heart diseases.
Description
Technical field
The invention belongs to biotechnology or medical domain, more specifically, the present invention relates to the purposes of Heat shock protein 27 HSP27 aspect Cardioprotective.
Background technology
Heart disease is the general name of heart disease, comprises various heart diseases such as rheumatic heart disease, congenital heart disease, hypertensive heart disease, coronary heart disease, cardiomyopathy.Heart disease is one of principal disease of present harm humans health.
The myocardial ischemia pathological changes that is caused by coronary artery pathological changes is the common disease of modern society and the main cause of death.Therefore, how effectively the protection cardiac muscle, improve the resistance of cardiac muscle to stress damages such as ischemias, improve that cardiac insufficiency is international cardiology area research focus behind the myocardial ischemia.
Heat shock protein is the protein of one group of high conservative on evolving, and except that hyperpyrexia, ischemia, tissue injury, virus or bacterial infection etc. also can produce, and it confirms to bring into play various critical functions in a lot of fields.Past people for Heat shock protein 27 HSP27 (Heat Shock Protein27, research HSP27) mainly concentrates on effect (the Small heat shock proteins andprotection against injury.Ann N Y Acad Sci.1999 30 of aspects such as the anti-apoptosis of HSP27, antioxidant radical; 874:66-8.Review); Also HSP27 is not resisted the further investigation of the aspects such as protection mechanism of myocardial ischemia at present.Have bibliographical information HSP27 and myofilament that common positioning phenomenon is arranged in the recent period, and when ischemia, have more HSP27 to insert on the myofilament, but people are not clear to the reason that this phenomenon takes place, the influence and the mechanism of action thereof of myofilament also waited to illustrate from endochylema.
The main composition unit that myofilament shrinks as myocardial cell, irritate the constituent TnT nI (troponin I) of back myofilament and TnT (troponin T) at ischemia/multiple thus can be subjected in various degree damage influence contraction.And whether exist certain relation between HSP27 and TnI and the TnT, thereby and therefore influencing the cellular contraction function brings into play its myocardium protecting action, illustrated as yet.
Therefore, be necessary Heat shock protein 27 HSP27 is carried out deep research, in the hope of new treatment target spot is provided for heart disease.
Summary of the invention
The object of the present invention is to provide the purposes of Heat shock protein 27 HSP27 aspect Cardioprotective.
In a first aspect of the present invention, the purposes of Heat shock protein 27 HSP27 or its encoding gene is provided, be used to prepare the compositions that suppresses TnT nI or TnT degraded; Be used to prepare the medicine of prevention or treatment heart and injury; Or the medicine that is used to screen prevention or treats heart and injury.
In a preference of the present invention, described heart and injury is a myocardial damage.
In another preference of the present invention, the myocardial damage of described heart and injury for being caused by TnT nI or TnT degraded.
In another preference of the present invention, described myocardial damage includes, but is not limited to: a series of ischemic heart desease such as myocardial ischemic injury, heart failure, myocardial infarction, coronary heart disease, angina pectoris, arrhythmia.
In a second aspect of the present invention, the purposes of TnT nT or TnI is provided, be used to screen the medicine of prevention or treatment heart and injury.
In another preference, described TnT nT or TnI are TnT nT or the TnI or their active fragment of total length.In another preference, described TnI can or comprise the TnI fragment of the C-terminal of TnI for the C-terminal of total length TnI.
In a third aspect of the present invention, the method that provides a kind of screening to can be used for preventing or treating the potential drug of heart and injury, described method is:
Drug candidate is contacted with TnT nT or TnI, detect the influence of drug candidate TnT nT or TnI; If described drug candidate can reduce the degraded of TnT nT or TnI, then it is the potential drug that can be used for preventing or treating heart and injury.
In a fourth aspect of the present invention, the method that provides a kind of screening to can be used for preventing or treating the potential drug of heart and injury, described method is:
Drug candidate is contacted with the system of expressing Heat shock protein 27 HSP27, detect the influence of drug candidate Heat shock protein 27 HSP27; If described drug candidate can promote the expression of Heat shock protein 27 HSP27, then it is the potential drug that can be used for preventing or treating heart and injury.
In a fifth aspect of the present invention, the method that provides a kind of screening to can be used for preventing or treating the potential drug of heart and injury, described method is:
Drug candidate is contacted with TnT nT or the interactional reaction system of TnI with Heat shock protein 27 HSP27, detect the interaction situation of Heat shock protein 27 HSP27 and TnT nT or TnI; If described drug candidate can promote the interaction of Heat shock protein 27 HSP27 and TnT nT or TnI, then it is the potential drug that can be used for preventing or treating heart and injury.
In another preference of the present invention, the method that provides a kind of screening to can be used for preventing or treating the potential drug of heart and injury, described method may further comprise the steps:
(a) in the test group, in the reaction system of expressing Heat shock protein 27 HSP27, add material standed for to be screened, and the expression of detection Heat shock protein 27 HSP27, and, in matched group, in not adding reaction system described material standed for, the expression Heat shock protein 27 HSP27, detect the expression of Heat shock protein 27 HSP27
(b) expression with Heat shock protein 27 HSP27 in the expression of Heat shock protein 27 HSP27 in step (a) the test group and the matched group compares,
If the expression of Heat shock protein 27 HSP27 is higher than matched group statistically in the test group, just show that this material standed for is the potential drug that can be used for preventing or treating heart and injury.
More preferably, the myocardial damage of heart and injury for being caused by TnT nI or TnT degraded.
In another preference, described method also comprises step: the degraded situation of TnT nT or TnI in detection test group and the matched group, if the degraded situation of TnT nT or TnI is lower than matched group statistically in the test group, just show that this material standed for is the potential drug that can be used for preventing or treating heart and injury.
Perhaps, described method may further comprise the steps:
(i) in the test group; In Heat shock protein 27 and TnT nT or the interactional reaction system of TnI, add material standed for to be screened; And the degraded situation of the interaction situation of detection Heat shock protein 27 and TnT nT or TnI or detection TnT nT or TnI; And; In control group; Do not add described material standed for, in Heat shock protein 27 and TnT nT or the interactional reaction system of TnI; Detect the interaction situation of Heat shock protein 27 and TnT nT or TnI or the degraded situation of detection TnT nT or TnI
(ii) Heat shock protein 27 HSP27 and the interaction situation of TnT nT or TnI or the degraded situation of TnT nT or TnI in the degraded situation of the interaction situation of Heat shock protein 27 HSP27 and TnT nT or TnI in step (i) the test group or TnT nT or TnI and the matched group are compared
If the interaction of Heat shock protein 27 HSP27 and TnT nT or TnI is better than matched group statistically in the test group, or the degraded situation of TnT nT or TnI is lower than matched group statistically in the test group, just shows that this material standed for is the potential drug that can be used for preventing or treating heart and injury.
Perhaps, described method may further comprise the steps:
(1) in the test group, in the system that contains TnT nT or TnI, add material standed for to be screened, and the degraded situation of detection TnT nT or TnI, and, in matched group, in not adding system described material standed for, that contain TnT nT or TnI, detect the degraded situation of TnT nT or TnI
(2) the degraded situation with TnT nT or TnI in the degraded situation of TnT nT or TnI in step (i) the test group and the matched group compares,
If the degraded situation of TnT nT or TnI is lower than matched group statistically in the test group, just show that this material standed for is the potential drug that can be used for preventing or treating heart and injury.
In a sixth aspect of the present invention, also provide a kind of by the prevention of described method acquisition or the medicine of treatment heart and injury.
Aspect of the present invention seven, the method for a kind of prevention or treatment heart and injury is provided, comprise Heat shock protein 27 HSP27 that gives experimenter's effective dose or the carrier or the cell of expressing Heat shock protein 27 HSP27.
In a eighth aspect of the present invention, a kind of purposes of expressing the carrier of Heat shock protein 27 HSP27 is provided, described carrier is used to prepare the medicine for the treatment of heart and injury.
In another preference of the present invention, described carrier is an adenovirus vector, wherein contains the expression cassette of Heat shock protein 27 HSP27.
In a ninth aspect of the present invention, a kind of pharmaceutical composition is provided, contain the Heat shock protein 27 HSP27 of safe and effective amount, TnT nT or TnI, and pharmaceutically acceptable carrier.
More preferably, the ratio of Heat shock protein 27 HSP27 and TnT nT or TnI is 1-5:1-5 (preferred, as to be 1-3:1-3).
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1 has shown that Western blot analyzes at balance and ischemia/when irritating again, the situation of change of the phosphorylation HSP27 of different time points and total HSP27 protein expression.(A) representative Western blot glue figure; (B) statistical result of A.Wherein, BL: balance period; I20: ischemia 20 minutes; R3: irritated again 3 minutes; R30: irritated again 30 minutes.Compare with balance period,
*P＜0.05,
*P＜0.01
Fig. 2 has shown the effect of HSP27 on the myocardial cell contractile function, and improves myocardium shrinkage function after ischemia/multiple filling damage.Wherein, (A) at balance and ischemia/shrinkage amplitude of myocardial cell when irritating again; (B) balance myocardial cell in period myofilament Ca
2+-ATPase activity; (C) ischemia/multiple back myocardial cell myofilament Ca that irritates
2+-ATPase activity; AdGFP contrast: the empty carrier transfection group that has green fluorescence; AdHSP27wt: the adenovirus vector transfection group that has wild type HSP27 expressing gene; AdHSP27mt: the adenovirus vector transfection group that has saltant HSP27 expressing gene.Compare with matched group (AdGFP group)
*P＜0.05,
*P＜0.01.
Fig. 3 shows TnT and the TnI degraded situation at ischemia/when irritating again for Western Blot result.Wherein, (A) be disclosed in balance and ischemia/TnT and TnI master tape and degraded band thereof when irritating again for representational Western Blot result.(B) be statistical result to TnT and TnI degraded band.AdGFP contrast: the empty carrier transfection group that has green fluorescence; AdHSP27wt: the adenovirus vector transfection group that has wild type HSP27 expressing gene; AdHSP27mt: the adenovirus vector transfection group that has saltant HSP27 expressing gene.BL: balance period; I20: ischemia 20 minutes; R3: irritated again 3 minutes; R30: irritated again 30 minutes.Compare with equilibrated AdGFP,
*P＜0.05,
*P＜0.01 He
* *P＜0.001; Compare with the corresponding time point of AdGFP group,
#P＜0.05,
##P＜0.01 He
####P＜0.001.
Fig. 4 has shown observed HSP27 (TRITC, redness under the Laser Scanning Confocal Microscope; A and B) and TnI (FITC, green; A) and TnT (FITC, green; B) common positioning result.Be blue-fluorescence with Hoechst labelling nuclear.Scale: 8 μ m.
Fig. 5 has shown the co-immunoprecipitation result between HSP27 and TnT (B) and the TnI (A).Rat left compartment muscle cell is crossed after the expression HSP27, and cell lysis carries out the co-precipitation test with the antibody of HSP27, TnI, TnT respectively.The interactively of HSP27 and TnI and TnT is analyzed by western blot and is shown.Swimming lane (Lane) 1 is the total protein of handling without co-precipitation (positive control); Lane2 and Lane3 are the product (negative control) that carries out co-precipitation with non-specific rabbit (rabbit)-lgG and goat (goat)-lgG, lane4 is the coprecipitated product with the HSP27 antibodies, lane5 is the coprecipitated product (A) with the TnI antibodies, with the coprecipitated product (B) of TnT antibodies.
Fig. 6 has shown that GST Pull Down analyzes HSP27 and TnI and TnT in the external situation that combines, and definite its calmodulin binding domain CaM.(A) TnI of His labelling, TnT and their saltant expression plasmid structure.The TnI fusion rotein of His-TnI:His labelling; The TnI fusion rotein of the C-terminal disappearance of His-TnIDC:His labelling.The TnI fusion rotein of the N-terminal disappearance of His-TnIDN:His labelling.The TnT fusion rotein of His-TnT:His labelling.The TnT fusion rotein of the C-terminal disappearance of His-TnTDC:His labelling.The TnT fusion rotein of the N-terminal disappearance of His-TnTDN:His labelling.(B) the GST-HSP27 fusion rotein of about 0.5 μ g wild type or saltant His-TnI fusion rotein and 1 μ g carries out external Pull-Down experiment.Pull Down product by the HSP27 antibody test (B, lanes3-5).
Fig. 7 has shown that HSP27 is to the hemodynamic effect of isolated heart.Fan Ying index: left ventricular developed pressure LVDP (B) respectively, the maximum rate of change+dP/dt (C) of left ventricular systolic pressure and left chamber diastolic pressure maximum rate of change-dP/dt (D).(A) balance and the ischemia/initial data of hematodinamics index (representative result) when irritating again for recording.Contrast: injection training liquid matched group; AdGFP contrast: the empty carrier transfection group that has green fluorescence; AdHSP27wt: the adenovirus vector transfection group that has wild type HSP27 expressing gene; Compare with AdGFP,
*P＜0.01.In this test, the inventor has also added one group of sham operated rats (contrast, promptly same process of the test but do not add any viral vector, injection training liquid matched group), to guarantee adding this carrier to not influence of process of the test.
Fig. 8 shown systemic heart cross express the HSP27 gene after to the influence of heart infarction area and cardiac function.(A) the section cardiac muscular tissue behind representative schematic diagram Eveas indigo plant and the TTC dyeing heart infarction, matched group (left side) and mistake expression HSP27 group (right side).The below is the statistical result of heart infarction area.At the body hematodinamics maximum rate of change+dP/dt (C) of left ventricular developed pressure LVDP (B) and left ventricular systolic pressure as a result.Contrast: injection training liquid matched group; AdGFP: the empty carrier transfection group that has green fluorescence; AdHSP27wt: the adenovirus vector transfection group that has wild type HSP27 expressing gene.Before the ischemia: handle without ischemia; R120: irritated again 120 minutes.Compare with balance matched group in period
*P＜0.05; Compare with the matched group after ischemia/multiple filling:
#P＜0.05; Compare with the AdGFP group after ischemia/multiple filling:
P＜0.05.
The specific embodiment
The inventor has proved Heat shock protein 27 HSP27 (HSP27) first and can prevent or treat heart and injury (particularly myocardial damage) through extensive and deep research.Finished the present invention based on this.
Particularly, the inventor has found HSP27 and to keep myocardium normal contraction function relevant first; HSP27 can be to ischemia resisting/multiple degraded of irritating myofilament protein (as TnI, TnT), and can protect myocardium shrinkage function; Found HSP27 can with the calmodulin binding domain CaM that has combined and found TnI and HSP27 of specific myofilament protein TnI, TnT; In the protective effect that has further proved myocardium shrinkage function after HSP27 is to ischemic injuries on the basis of these discoveries in integral level.
Heat shock protein 27 HSP27 (HSP27)
In the present invention, used Heat shock protein 27 HSP27 can be naturally occurring, such as its can be separated or purification from mammal.In addition, described Heat shock protein 27 HSP27 also can be an artificial preparation, such as producing the reorganization Heat shock protein 27 HSP27 according to the gene recombination technology of routine.Preferably, the present invention adopts the Heat shock protein 27 HSP27 of reorganization.
Any suitable Heat shock protein 27 HSP27 all can be used for the present invention.Described Heat shock protein 27 HSP27 comprises Heat shock protein 27 HSP27 or its bioactive fragment of total length.
The aminoacid sequence of the Heat shock protein 27 HSP27 that passes through replacement, disappearance or the interpolation of one or more amino acid residues and form is also included among the present invention.Heat shock protein 27 HSP27 or its bioactive fragment comprise the alternative sequence of a part of conserved amino acid, and described sequence of replacing through aminoacid does not influence its activity or kept the activity of its part.Suitably replacing aminoacid is the technique known of this area, and described technology can be implemented and guarantee not change the biological activity of gained molecule at an easy rate.These technology are recognized people, in general, can not change biological activity basically at the inessential area change single amino acids of a peptide species.See Molecular Biology of The Gene such as Watson, the 4th edition, 1987, The Benjamin/CummingsPub.Co.P224.
The bioactive fragment of any Heat shock protein 27 HSP27 can be applied among the present invention.Here, the implication of the bioactive fragment of Heat shock protein 27 HSP27 is meant that as a peptide species it still can keep all or part of function of the Heat shock protein 27 HSP27 of total length.Generally, described bioactive fragment keeps the activity of 50% total length Heat shock protein 27 HSP27 at least.Under preferred condition, described active fragment can keep 60%, 70%, 80%, 90%, 95%, 99% or 100% activity of total length Heat shock protein 27 HSP27.
The present invention also can adopt Heat shock protein 27 HSP27 modified or improvement, such as, can adopt the Heat shock protein 27 HSP27 of being modified or improveing in order to promote its half-life, effectiveness, metabolism and/or proteic effectiveness.Described can be a kind of conjugate of Heat shock protein 27 HSP27 through the Heat shock protein 27 HSP27 of modifying or improve, or it can comprise substituted or artificial aminoacid.Described can be to have less common ground with naturally occurring Heat shock protein 27 HSP27 through the Heat shock protein 27 HSP27 of modifying or improve, but also can prevent or treat mammiferous heart and injury (especially myocardial damage), and can not bring other harmful effect or toxicity.That is to say that any bioactive version that does not influence Heat shock protein 27 HSP27 all can be used among the present invention.
In a kind of mode of the present invention, described Heat shock protein 27 HSP27 directly can be delivered medicine to mammal (such as the people), perhaps, the gene of coding Heat shock protein 27 HSP27 can be cloned in the appropriate carriers (as a kind of adenovirus vector) by the method for routine, described carrier is imported in the cell that can express described Heat shock protein 27 HSP27, make described cellular expression Heat shock protein 27 HSP27.Can realize the expression of Heat shock protein 27 HSP27 in the body by an amount of described cell being incorporated into the suitable position of heart.
Preferably, the aminoacid sequence of described naturally occurring Heat shock protein 27 HSP27 can with the GenBank accession number: the sequence shown in the NM_031970 is substantially the same.
Preferably, the gene of coding Heat shock protein 27 HSP27 or the carrier that the carries described gene method by routine can be incorporated into the expression that realizes Heat shock protein 27 HSP27 in target cell or the target tissue.Described target cell includes but not limited to: myocardial cell, myofilament cell etc.; Described cell can be transferred in the suitable position of heart.
The purposes of Heat shock protein 27 HSP27
The invention provides the purposes of Heat shock protein 27 HSP27, be used to prepare the medicine of prevention or treatment heart and injury; Or the medicine that is used to screen prevention or treats heart and injury.
Preferred, described heart and injury is a myocardial ischemic injury.
In another optimal way, described heart and injury is the myocardial damage that damage caused by TnT nI or TnT (both are the mark of myocardial damage).Preferred, the damage of described TnI or TnT is the damage that the degraded of TnI or TnT causes.
In order to prove Heat shock protein 27 HSP27 for myocardial protective effect; the inventor is animal model with the rat; research detected HSP27 in the expression of normal and ischemia/multiple single adult rat myocardial cell of irritating, compared empty carrier and Adenovirus Transfection wild type hsp27 gene or saltant hsp27 gene after the kinetics and the contractile protein stability of ischemia/multiple single myocardial cell cellular contraction of irritating, and then identified at ischemia/HSP27 and the common location of relevant myofilament protein and myofilament protein that may combine when irritating again with HSP27.Result proof is expressed constant at the HSP27mRNA of ischemia/when irritating again and total protein, but phosphorylation HSP27 protein expression increases.
And, the inventor also finds, there is location altogether in the HSP27 protein expression at ischemia/irritate again process neutralizer fibroin, and has confirmed first that by the protein-interacting experiment HSP27 and TnT nI (Troponin I) exist interaction relationship with TnT (Troponin T) and searched out the calmodulin binding domain CaM of HSP27 and TnI.
In addition, the inventor has also proved in the myocardial cell TnI of transfection wild type hsp27 gene and the degraded of TnT in ischemia/filling process again and has been starkly lower than ischemia/irritate matched group (P＜0.01) again, and has been accompanied by improve (P＜0.01) of myocardial cell contractility; And behind the transfection saltant hsp27 gene, the degraded showed increased of TnI and TnT is also followed go down (P＜0.01) of contractile function.
In addition, after body is crossed expression HSP27, find that HSP27 has significant protective effect equally to post-ischemic cardiac systolic function at adult rat.The discovering of the inventor proved to cross expresses the myocardium shrinkage function that external source HSP27 gene can obviously improve the phase of irritating again behind the ischemia, this effect combines TnI, TnT with HSP27, thus enhancing TnI and TnT the stability of ischemia/when irritating again, suppress them damaging degraded relevant.These result of study promptings hsp27 gene has significant values in the clinical treatment myocardial ischemic injury.
Pharmaceutical composition
The present invention also provides a kind of pharmaceutical composition, and it contains the described Heat shock protein 27 HSP27 of effective dose (0.0001-50%), and pharmaceutically acceptable carrier.
Preferred, described pharmaceutical composition contains the Heat shock protein 27 HSP27 of effective dose, TnT nT or TnI, and pharmaceutically acceptable carrier.
Usually, these materials can be formulated in nontoxic, the inert and pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably, pH is about 6-8.
Among the present invention, term " contains " the various compositions of expression and can be applied to together in mixture of the present invention or the compositions.Therefore, term " mainly by ... form " and " by ... composition " be included in during term " contains ".
As used herein, term " effective dose " or " effective dose " are meant and can produce function or amount active and that can be accepted by people and/or animal to people and/or animal.
In the present invention, the composition of " pharmaceutically acceptable " is applicable to people and/or mammal and does not have excessive bad side reaction (as toxicity, stimulation and allergy), promptly has the material of rational benefit/risk ratio.Term " pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier of agent administration, comprises various excipient and diluent.This term refers to some medicament carriers like this: they itself are not necessary active component, and do not have undue toxicity after using.Suitable carriers is well known to those of ordinary skill in the art.At Remington " (Mack Pub.Co. can find proving absolutely about pharmaceutically acceptable carrier in N.J.1991) to s Pharmaceutical Sciences.Acceptable carrier can contain liquid on combination of Chinese medicine is learned, as water, saline, glycerol and ethanol.In addition, also may there be complementary material in these carriers, as lubricant, fluidizer, wetting agent or emulsifying agent, pH buffer substance etc.
Pharmaceutical composition of the present invention can be directly used in the treatment of heart disease (particularly myocardial damage).In addition, also can unite use with other therapeutic agent or adjuvant simultaneously.
Pharmaceutical composition of the present invention contains the Heat shock protein 27 HSP27 and the pharmaceutically acceptable carrier of safe and effective amount.This class carrier comprises (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof.Usually pharmaceutical preparation should be complementary with administering mode, and pharmaceutical composition of the present invention can be made into the injection form, for example with normal saline or contain glucose and the aqueous solution of other adjuvant is prepared by conventional method.Described pharmaceutical composition should be made under aseptic condition.The dosage of active component is the treatment effective dose.Pharmaceutical preparation of the present invention also can be made into slow releasing preparation.
The adenovirus administration
After the proteic purposes of the described HSP27 of cicada, can adopt several different methods well known in the art that described HSP27 albumen or its encoding gene or its pharmaceutical composition are delivered medicine to mammal.Preferably, can adopt the means of gene therapy to carry out, such as can be directly with HSP27 albumen by delivering medicine to the experimenter such as methods such as injections; Perhaps, can will carry hsp27 expression of gene unit (such as expression vector or virus etc.) by certain approach and be delivered on the target spot, and make it the HSP27 albumen of expression activity.
In a kind of optimal way of the present invention, adopt the method for Adenovirus Transfection to realize that HSP is proteic and send and express.
Adenovirus is a nonenveloped virus, double-stranded DNA is about 36Kb, genome is made up of nine districts, four expression are called district early morning (E1-E4), five expression later (L1-L5) are called late district, express to be subjected to cell transcription factor and the control of E1 district the main dna replication dna of regulating in E2 district, E3 district gene outcome is relevant with the removing that virus is escaped host immune system, and the product in E4 district participates in activities such as viral dna replication, district's gene expression in evening and transcript splicing.
Adenovirus enters cell by taking the photograph approach in the receptor-mediated cell, breaks after the endosome acidify, and endochylema inner virus DNA disengaged before lysosome degraded, enters nucleus then and becomes the attachment state.
The inventor finds, when the transfection that is used for cardiac muscle, it is very effective adopting adenovirus to send HSP27 as carrier.Compare with general plasmid vector, adenovirus is wanted high 10-1000 times or higher to the transfection efficiency of cardiac muscle; More preferably 50-500 times, as 100 times, 200 times etc.
When carrying out administration, can adopt out modes such as breast cardiac muscle direct injection and the interior release of percutaneous catheter coronary artery to give to express the adenovirus of hsp27 gene.Method such as can directly adopting injection in the cardiac muscle will carry the Adenovirus Transfection myocardial cell of genes of interest (that is: hsp27), and make in the HSP27 albumen myocardial cell by high level expression.
The adenovirus administration has good safety, and the adenovirus DNA unconformity is to the target cell genome.
The method of prevention or treatment heart and injury
The invention provides the method for a kind of prevention or the damage of treatment mammalian heart, comprise that the Heat shock protein 27 HSP27 that gives experimenter's effective dose maybe can express the cell of Heat shock protein 27 HSP27.When being used for the treatment of the human heart damage, the preferred Heat shock protein 27 HSP27 that adopts reorganization.
Should be understood that the effective dose of used Heat shock protein 27 HSP27 can change with the order of severity of object to be treated when being used for the treatment of the mammalian heart damage.Concrete condition decides according to experimenter's individual instances, and this is in the scope that skilled practitioners or nutritionist can judge.
The pharmaceutical methods of screening protection heart
After getting the proteic purposes of the described HSP27 of cicada, can adopt several different methods well known in the art to screen the medicine of protection heart and injury (particularly myocardial damage).
In a kind of optimal way of the present invention; the method of the medicine of a kind of screening protection (as prevention, improvement or treatment) heart and injury is provided; described method comprises: add material standed for to be screened in the reaction system of expressing Heat shock protein 27 HSP27, and detect the expression of Heat shock protein 27 HSP27.If after adding drug candidate, the expression of Heat shock protein 27 HSP27 is higher than statistically and (preferably significantly is better than, as higher by 20% than matched group; More preferably high by 40% than matched group; Most preferably than matched group high 60% or higher) matched group, just show that this material standed for is the medicine of prevention or treatment heart and injury.
In another kind of optimal way of the present invention; the method of the medicine of a kind of screening protection (as prevention, improvement or treatment) heart and injury also is provided; described method comprises: add material standed for to be screened in Heat shock protein 27 HSP27 and the interactional reaction system of TnT nT (or TnI), and detect the interaction situation of Heat shock protein 27 HSP27 and TnT nT (or TnI).If after adding drug candidate, the interaction of Heat shock protein 27 HSP27 and TnT nT (or TnI) is better than statistically and (preferably significantly is better than, as stronger by 20% than matched group; More preferably strong by 40% than matched group; Most preferably than matched group strong 60% or stronger) matched group, just show that this material standed for is the medicine of prevention or treatment heart and injury.
In another kind of optimal way of the present invention; the method of the medicine of a kind of screening protection (as prevention, improvement or treatment) heart and injury also is provided; described method comprises: add material standed for to be screened in Heat shock protein 27 HSP27 and the interactional reaction system of TnT nT (or TnI), and detect the degraded situation of TnT nT or TnI.If after adding drug candidate, the degraded of TnT nT or TnI is lower than statistically and (preferably significantly is lower than, as hanging down 20% than matched group; More preferably than matched group low 40%; Most preferably than matched group low 60% or lower) matched group, just show that this material standed for is the medicine of prevention or treatment heart and injury.
In the present invention, described system or reaction system include but not limited to solution system, intracellular system etc.
The material that these Preliminary screening go out can constitute a screening storehouse so that people finally can therefrom filter out can be for the useful medicine of protection heart (particularly cardiac muscle).
Therefore, the present invention also comprises the medicine that obtains by described screening technique, and described medicine can be used for protecting heart (particularly cardiac muscle).
Major advantage of the present invention is:
(1) the present invention has proved the protective effect of HSP27 for myocardium shrinkage function first, and it is directly related to have disclosed this protective effect and its Stabilization to specific myofilament protein simultaneously.And verified on the whole animal acute myocardial infarction model improve HSP27 expression to ischemia after the influence of contractile function of heart.
(2) the present invention has disclosed the HSP27 gene first and can be applicable to stablize the specific myofilament protein of ischemia, heart failure equivalent damage cardiac muscle, and has verified its protection to the damaged heart function, for heart disease provides effective treatment novel targets.
I. main experimental methods
Conventional PCR, enzyme action, connection, operation such as the preparation of competent cell, plasmid conversion and extraction, immunoprecipitation also can be with reference to molecular cloning experiment guide (Sambrook J., Russell DW: molecular cloning: lab guide, Cold Spring Harbor, NY.:Cold Spring Harbor Laboratory Press; 2001.) described in.
1. cross the structure of the expression vector of expressing wild type HSP27 and saltant HSP27 in the body, and the foundation of damage model
(1) structure of adenovirus expression carrier
Wild type expresses HSP27 plasmid (being pSV27L1) and saltant is expressed HSP27 plasmid (being pSV27L1mt) available from the University of Texas.The inventor used AdEasyTM Vector System (available from Promage company) with two cDNA fragments (be respectively the HSP27cDNA plasmid pSV27L1 of 1. people source wild type, corresponding A dHSP27wt; 2. the saltant HSP27cDNA plasmid pSV27L1mt in people source, corresponding A d-HSP27DN; The GenBank accession number: NM_001540) be cloned into respectively on pAdEasy (Promage company) plasmid that can be expressed as adenovirus, obtain respectively expressing green fluorescence and genes of interest adenovirus vector Ad-GFP, can express wild type HSP27 adenovirus vector AdHSP27wt, can express the adenovirus AdHSP27mt of saltant HSP27.
Concrete clone's step is as follows: because wild type and saltant HSP27 place carrier do not have suitable site and adenovirus shuttle plasmid to match, so the inventor makes up by using some transition vectors:
1) with PstI genes of interest is downcut (restriction enzyme site of wild type and saltant is identical) by SacI, be connected on the pEGFp-N1 carrier (available from Clontech company);
2) downcut HSP27cDNA by XhoI and PstI, be connected on the pBSK carrier (available from Clontech company);
3) downcutting genes of interest by XbaI and XhoI is connected on the adenovirus shuttle plasmid pTrack (available from Promage company);
4) with the PacI restriction endonuclease pTrack-HSP27wt and the pTrack-HSP27mt plasmid that build of linearisation respectively, obtain pAdEasy-HSP27wt and pAdEasy-HSP27mt expression plasmid then with behind the adenovirus skeleton plasmid homologous recombination.
And, by rotaring redyeing 293 cell (available from ATCC), obtain expressing the cell of the gland-containing virus carrier (AdGFP) of green fluorescence and genes of interest, and the cell that contains the adenovirus AdHSP27mt carrier of the adenovirus AdHSP27wt carrier of wild type HSP27 and saltant HSP27.Through the dialysis purification after in-80 ℃ of preservations.
(2) separation and Culture of myocardial cell and in-vitro transfection
The inventor uses the enzyme digestion of standard (with reference to Relaxation in rabbit and rat cardiaccells:species-dependent differences in cellular mechanisms.J Physiol (Lond) .476:279-293; 1994), use the II Collagen Type VI enzyme (Sigma company) of 1mg/ml, digested about 15 minutes, obtain the adult male SD rats left compartment muscle cell about 300 grams, used apparatus installs equal sterilization treatment.Obtaining shaft-like survivaling cell rate〉90% o'clock, be used for cell culture.The culture fluid that experiment is used is the M199 of Sigma company, and contains 10% standard hyclone (GIBCO company) (Adult ratventricular myocytes cultured in defined medium:phenotype and electromechanicalfunction.Am J Physiol.265 (2Pt2): H747-54; 1993).
With 5 * 10
6Cell inoculation after adherent the stablizing, changes liquid and pair cell and carries out Adenovirus Transfection in the Tissue Culture Dish of handling with 10% laminine (laminine), uses the M199 culture fluid of serum-free this moment and makes its volume reach minimum.Act on and change liquid once more after 2 hours.Carried out function and biochemistry detection after the transfection in 48 hours.
(3) ischemia/irritate again damage model
Damage model design main reference p38Mitogen-activated protein kinase mediates anegative inotropic effect in cardiac myocytes.Circ Res.8; 90 (2): 190-6; 2002; Or ATP-dependent potassium channels involved in the cardiac protection induced byintermittent hypoxia against ischemia/reperfusion injury.Life Sciences73:1275-1287; Method described in 2003.
Myocardial cell after the cultivation used the middle balance of Krebs-Henseleit (K-H) earlier 20 minutes, changed simulation ischemia liquid (mM of unit) then: 123.0NaCl, 8.0KCl, 6.0NaHCO
3, 0.9NaH
2PO
4, 0.5MgSO
4, 20.0 sodium lactates (Na-lactate), 1.8CaCl
2, fill 95%N simultaneously
2/ 5%CO
2, stable pH value is 6.8, acts on 20 minutes.Irritated again 30 minutes with K-H liquid subsequently.
(4) detection of cellular contraction
The inventor uses fluorescence and cellular contraction synchronous recording system (IonOptix Co., MA, USA), synchronous monitoring has been analyzed cell at ischemia/irritate the contraction situation of myocardial cell in the damage process again (with reference to Simulated ischaemia and reperfusion in isolated guinea pig ventricular myocytes.Cardiovascular Research28:1794-1802; 1994).The cell that to cultivate on the slide of 25mm with PBS places the perfusion groove, balance 20 minutes.In balance period with the filling phase makes cell be subjected to the electricity irritation of 0.5Hz again.
(5) myocardium myofilament Ca
2+-ATPase is active to be detected
Collect myocardial cell, extract the myofilament tissue, decide protein concentration with the Brandford method, it is standby that packing is stored in-70 ℃ of refrigerators.The description that active detecting operation step is built up the test kit that company provides by Nanjing is carried out.
Calculate: Ca
2+-ATPase activity=total enzyme activity-no Ca
2+The time ATPase activity.Try to achieve the Pi value X of sample according to the standard curve y=a+bx of phosphorus.
2. adopt Western Blot method, after analyzing enhancing and suppressing the HSP27 function, the degraded situation of myofilament protein
List of references Confocal microscopic localization of constitutive and heatshock-induced proteins HSP70 and HSP27in the rat heart.Circulation.20003; 102 (14): 1703-9.Intermittent high altitude hypoxia protects the heart against lethalCa
2+Overload injury.Life Sciences76:559-572; The method of describing in 2004, the inventor uses the method for Western Blot to detect HSP27 (1:2000, Santa Cruz), TnI (1:500, Santa Cruz), TnT (1:500, Santa Cruz) and Actin (1:2500, Sigma) expression in ischemia/filling process again.
Applied sample amount is 30 μ g, and changeing the film condition is 150mA4 hour, and with 5% skim milk (available from Nestle SA) room temperature sealing 1.5 hours, and one anti-(anti-HSP27, anti-TnI, anti-TnT are all available from santa cruz company; Anti-actin is available from sigma company) 4 ℃ of effects 16 hours.Use two anti-be that (1:2500, Sigma) and the anti-goat IgG of rabbit (1:5000, Santa Cruz), effect is 2 hours under the room temperature for the goat anti-mouse IgG of horseradish peroxidase-labeled.What developer used is that (analytical system is Gel Doc2000 (available from Bio-Rad) to enhanced chemiluminescence for enhanced chemiluminescence, ECL) detectable (available from Amersham PharmaciaBiotech).
3. adopt the interactively of co-immunoprecipitation, immune location altogether and GST-Pull Down experimental verification HSP27 and myofilament target protein, and calmodulin binding domain CaM
(1) cellular immunization groupization
Have the slide of cell to take out cultivation and wash 3 times with PBS, fix 20 minutes with 4% paraformaldehyde then, PBS cleans 3 times, after each 10 minutes.The Triton-X100 rupture of membranes of reuse 0.3% 15 minutes, afterwards reuse PBS clean 3 times each 10 minutes.
In order to reduce nonspecific combination, the inventor has also used 10% lowlenthal serum, and (available from Jackson, USA) sealing is 30 minutes.
One is anti-: anti-HSP27 (anti-HSP27,1:100 is available from Santa Cruz), anti-TnT (anti-TnT) 1:50 is available from Santa Cruz), anti-TnI (anti-TnI) 1:100 is available from Santa Cruz), 4 ℃, act on 16 hours.
Two anti-mouse-anti sheep IgG (1:200 is available from Jackson) for goat anti-rabbit igg of red fluorescence TRITC labelling (1:200 is available from Jackson) and green fluorescence FITC labelling act on 1 hour 30 minutes at room temperature.
Nucleus is dyeed by Hoechst No.33342 dyestuff (1:150 is available from Sigma).
Dyeing finishes, and the glycerol mounting is also observed under Laser Scanning Confocal Microscope (Leica).
(2) co-immunoprecipitation analysis
Use the RIPA lysate, 293 cells of expressing HSP27 and TnI, TnT are crossed in cracking.Measure protein concentration, carry out the co-immunoprecipitation experiment by laboratory manual available from the Co-IP Protocol of Santa Cruz.And detect the protein binding situation with the method for Western Blot.
And, adopt non-specific IgG (2 μ g are available from Santa Cruz) as the negative control in this experiment.
(3) GST Pull Down analyzes
The full length amino acid sequence of the TnI that adopts in this example is referring to GenBank accession number X58499; The full length amino acid sequence of TnT is referring to GenBank AY334080.
Make up 17 aminoacid sequences of disappearance N-terminal respectively, the TnI of 30 aminoacid sequences of C-terminal and the His-fusion protein expression plasmid of TnT: the in-vitro expression plasmid pGEX4T1-HSP27 of pET30a-TnI, pET30a-TnIN, pET30a-TnIC, pET30a-TnT, pET30a-TnTN, pET30a-TnTC and HSP27-GST fusion rotein sees Table 1.Wherein, pET30a is available from Novagen company, and exogenous gene inserts its SalI and BamHI restriction enzyme site; PGEX4T1 is available from available from Amersham Pharmacia company, and exogenous gene inserts its NotI and BamHI restriction enzyme site.
Table 1
| pET30a-TnI | Express total length TnI |
[0140]?
| pET30a-TnIN | 17 amino acid whose TnI of expression deletion N-terminal |
| pET30a- |
30 amino acid whose TnI of expression deletion C-terminal |
| pET30a-TnT | Express total length TnT |
| pET30a-TnTN | 97 amino acid whose TnT of expression deletion N-terminal |
| pET30a-TnTC | 190 amino acid whose TnT of expression deletion C-terminal |
| pGEX4T1-HSP27 | Express the HSP27-GST fusion rotein |
Express in the saltant plasmid transformation escherichia coli JM109 competent cell that order-checking is finished (giving birth to worker's biological engineering company limited) available from Shanghai, shaking bacterium next day spends the night, and be forwarded in the 50mlLB fluid medium with the switching amount of 1-2%, 37 ℃ of concussions were cultivated 2-3 hour, made bacterium liquid OD
600Adding IPTG when value reaches 0.7-0.9 is 0.1-1mmol/L to final concentration, collection bacterium liquid after 4 hours.
4 ℃ of centrifugal 5000rpm * 5min collect thalline, resuspended with 5mL1 * PBS, and add 1 μ L beta-mercaptoethanol (the about 2mmol/L of final concentration) and 25 μ L100mmol/L PMSF (final concentration is 0.5mmol/L), 0 ℃ of carrying out ultrasonic bacteria breaking 20min, 4 ℃ of centrifugal 15000rpm * 10min afterwards, cleer and peaceful precipitation in the acquisition will precipitate with 3～4mL1 * PBS suspension.
Precipitation on getting in right amount after the cleer and peaceful suspension, each adds the last sample Buffer of equal-volume 2 * SDS, 95 ℃ of heating 5min, room temperature 13000rpm * 10min is centrifugal, gets supernatant and carries out 10% polyacrylamide denaturant gel (SDS-PAGE) electrophoresis.
Electrophoresis finishes, and gel with protein staining liquid and destaining solution dyeing and decolouring, is observed derivative thalline and do not added the inductive thalline of IPTG (negative control group).
Evaluation finishes and increases in a large number and carry out purification experiment (Novagen tests handbook), with the His-fusion rotein of the TnI of 500ng and TnT and deletant thereof respectively with the GST-HSP27 fusion rotein of 500ng purification, hatch 2h for GST albumen and 50ul glutathion-Sepharose4B4 ℃, centrifugal collection Sepharose4B granule, with lavation buffer solution (washing buffer) (142.2mM KCL, 5mM MgCl2,10mM HEPES, pH7.61mM EDTA, 0.2%NP40) washing 5 times after, add 2 * SDS-PAGE sample-loading buffer, boil 5min, getting supernatant after centrifugal carries out the 12%SDS-PAGE electrophoresis.
On the whole animal model checking HSP27 in the function of protection aspect myocardium
Mainly take after body transfection adenovirus is crossed expression HSP27, give, detect its contraction of indices and heart infarction area with stripped Langedorff perfusion with behind the body acute myocardial infarction.
(1) in the body Adenovirus Transfection
The experimental technique that adopts is with reference to Translocation of HSP27to sarcomere induced byischemic preconditioning in isolated rat hearts.Biochem Biophys Res Commun.269 (1): 137-42; Carrying out described in 2000.Use apparatus to carry out sterilization treatment, and carry out the sterile working.Male SD rat about anesthesia 300 grams, connecing the respirator frequency is 70 times/minute, opens breast and pleura needled and will contain 200 μ l adenoviruss (8 * 10
10Pfu/ml) conduit inserts from the left ventricle apex of the heart, and extends to aortic root.Aorta and pulmonary artery clamped inject adenovirus 10 seconds simultaneously.Close breast, detect the hematodinamics index after 3 days.
(2) Langendorff cardiac perfusion
Male SD rat urethane 1.2g/Kg body weight intraperitoneal injection of anesthesia, it is fixing to face upward the position, open breast, take out heart rapidly, place 0 ℃ K-H liquid to cut off pericardium, separate aorta, the extruding heart is discharged residue blood in the heart, and the sleeve pipe that will be full of perfusate inserts the aorta ligation and fixes, and the aorta sleeve pipe is connected to the Langendorff perfusion device, cut an osculum at the left atrium sidewall, little latex water pocket is inserted left ventricle by atrioventricular valves (A V valves).The conduit that links to each other with the latex water pocket is connected pressure converter by tee T, and pressure transducer signal is imported polygraph (Powerlab/8s, Australia) by bridge amplifier.Finish real-time signal acquisition and processing by computer run chart software.
(3) foundation of rat heart infarction model
The male SD rat lumbar injection of getting about 300 grams does not have the anesthesia of barbital sodium 1g/Kg body weight, faces upward the position and fixes, and connects toy respirator (respiratory frequency 90/min) with tracheal intubation.Open the thoracic cavity, the back exposes heart, and the ligation left anterior descending coronary artery was poured into after 45 minutes 2 hours with ischemia again.
(4) detection of heart infarction area
With transfection empty plasmid matched group with cross expression HSP27 group and win heart to go urethane 1.2g/Kg body weight intraperitoneal injection of anesthesia, flush away blood is tightened suture, along aortic root retroperfusion 5%Evans indigo plant.Cut off atrium and right ventricle, keep interventricular septum.Perpendicular to left chamber major axis the thick piece of tissue of about 1～2mm is cut in left chamber, separation indigo plant is dyed district cardiac muscular tissue and is used to weigh; Remainder is the ischemic region cardiac muscle, and specimen is inserted in the 1%TTC solution, and 30min is hatched in 37 ℃ of water-baths, does not wherein dye the district and is infarcted region, weighs respectively for non-infarcted region in the red district of dying.
(5) in the body-centered Function detection
After rats by intraperitoneal injection pentobarbital sodium (1g/Kg) anesthesia, in the right carotid artery sheath, isolate common carotid artery,, make fixedly intubate pipe shaft of ligation with line with the heparin intubate got ready incision about 4cm in the heart direction is inserted blood vessel from then on.Trace Blood pressure of carotid artery earlier, (about 2min) rotates intubate gently after a period of stabilisation.Make it to enter left ventricular cavity through aortic valve along the blood vessel path.At this moment, screen is traced curve and is transferred the ventricular beat waveform of big spoke degree lifting to by the blood pressure waveform of little amplitude, deposits behind about 2min of the time of tracing.
Measure the every index of left ventricular function, specific targets comprise: left ventricle shrinks that peak pressure (LVSP), left ventricular end diastolic are pressed (LVEDP), left ventricular pressure rises and the decline maximum rate (+dp/dt ,-dp/dt), the time constant (T) that descends of heart rate (HR) and isovolumic relaxation phase intraventricular pressure etc.
Before the experiment, debugged PowerLab bio signal acquisition processing system (available from Australian Ai De company), be used for measuring.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.
II. specific embodiment
Embodiment 1HSP27 is in ischemia/irritate the again expression of myocardial cell
In the present embodiment, adopt Western Blot test determination HSP27 at ischemia/irritate the again expression of myocardial cell.
Western Blot the results are shown in Figure 1A and Figure 1B, and the result shows, ischemia 20 minutes, and the HSP27 total protein did not have significant difference than the expression of balance period when irritating 3 minutes, 30 minutes again, and the expression of phosphorylated protein is significantly increased (P＜0.01).
Embodiment 2
Cross the contractility of expressing after wild type hsp27 gene can improve myocardial cell ischemia/multiple filling
In order to determine whether HSP27 has relation with myocardial contraction, the inventor at first detects its influence to contractile function on the myocardial cell level.
The carrier that experiment is adopted is divided into following 3 groups:
(1) transfection myocardial cell contrast empty carrier AdGFP (being matched group (CON));
(2) the AdHSP27wt carrier of expression wild type hsp27 gene; With
(3) the AdHSP27mt carrier of expression saltant hsp27 gene.
The result shows, irritated the phase again in 30 minutes after the simulation ischemia in 20 minutes having passed through, the shrinkage amplitude of the cellular contraction amplitude of AdGFP group before than ischemia obviously descends, the cellular contraction amplitude of AdHSP27wt group then obviously is better than matched group (P＜0.01 irritating the later stage again, Fig. 2 A), but the AdHSP27mt group all is less than normal control group (P＜0.01, Fig. 2 A) all the time in the cellular contraction amplitude of irritating the phase again.
Simultaneously, in order further to determine the influence of HSP27 to the myofilament contractile function, the inventor has also studied myofilament ATPase activity, the result shows that ischemia/multiple back myofilament activity after suppressing the HSP27 function of irritating obviously reduces (P＜0.05 than matched group and AdHSP27wt group, Fig. 2 C), and the recovery situation that AdHSP27wt organizes than matched group has trend of rising, but does not have statistical significance (P＜0.05, Fig. 2 C).
In addition, under normal circumstances, the ATPase activity of the myofilament of three groups of cells does not have notable difference, sees Fig. 2 B.
Embodiment 3HSP27 irritates the TnI that causes and the influence of TnT degraded to ischemia/multiple
Seeking the action target spot on the myofilament of acting on of HSP27, the inventor finds that ischemia/multiple irritates the degraded that damage can cause myocardial cell TnI and TnT.
Western Blot result shows, transfection in the cell of AdHSP27wt group, the degradation of small molecular albumen of TnI and TnT is starkly lower than matched group (P＜0.01); Transfection TnI in the cell of AdHSP27mt group and TnT palliating degradation degree then apparently higher than matched group (P＜0.01, Fig. 3).
The common positioning analysis of embodiment 4TnI and TnT and HSP27
In order to determine whether HSP27 exists interactively with TnI and TnT, the inventor carries out an anti-labelling and the two dyeing of fluorescence to TnI (green) and HSP27 (redness) and TnT (green) with HSP27 (redness) respectively, and Laser Scanning Confocal Microscope is observed its common positioning phenomenon down.
The result as shown in Figure 4, TnI and TnT and HSP27 be at intracellular position basically identical, is striation and distributes, and exists certain interaction between this altogether localized phenomenon prompting TnI and TnT and the HSP27.
The interaction of embodiment 5TnI and TnT and HSP27
Because the result of SABC can only illustrate the interactively that exists between TnI and TnT and the HSP27 indirectly, whether there is interaction in order further to confirm between them, the inventor carried out expression HSP27 to rat myocardial cell, and carried out lysis and the test of intravital co-immunoprecipitation.
Western Blot the analysis showed that, HSP27 and TnI and TnT can both in conjunction with, negative control IgG does not then demonstrate band (seeing 5A, Fig. 5 B), illustrates that HSP27 can carry out specific the combination with TnI and TnT respectively.
The interactional zone of action of embodiment 6TnI and TnT and HSP27
In addition, in order to confirm further whether the effect between HSP27 and TnI, the TnT is direct, and the inventor has adopted the experiment of external GST Pull Down, has confirmed to exist between HSP27 and TnI, the TnT to mutually combine under external condition.
Simultaneously, in order to seek the calmodulin binding domain CaM between HSP27 and TnI, the TnT, again the GST-HSP27 fusion rotein has been carried out Pull Down experiment with His-TnIDN, His-TnIDC, His-TnTDN, His-TnTDC respectively.
Found that the calmodulin binding domain CaM of HSP27 and TnI is at the C-terminal of TnI, shown in Fig. 6 B.
Therefore as seen, HSP27 by with the combining of TnI or TnT, can suppress the degraded of TnI or TnT, thereby play a protective role.
For further determining the protective effect of HSP27 to the Ischemic Heart contractile function, the inventor has studied after body is crossed expression HSP27 heart ischemia/multiple influence of irritating the damage contractility.
Langendorff cardiac perfusion result shows, heart cross expression HSP27 group left ventricular developed pressure LVDP with and the recovery situation of rate of change ± dp/dt will be significantly better than the empty carrier matched group, as shown in Figure 7, (P＜0.01).
Embodiment 8 crosses at systemic heart and expresses the protective effect of HSP27 to the Ischemic Heart contractile function
Except the checking of whole cardiac function, the inventor is also further in confirmed the protective effect of HSP27 to cardiac systolic function under the body situation.
Found that of body-centered Function detection, coronary ligation 45 minutes, after irritating 2 hours again, heart cross express HSP27 group left ventricular developed pressure LVDP with and the recovery situation of contraction rate+dP/dt will be significantly better than the empty carrier matched group, shown in Fig. 8 B, Fig. 8 C (P＜0.05).
In addition, cross and to express the heart infarction area that has also obviously reduced after the HSP27 after ischemia is irritated again, shown in Fig. 8 A (P＜0.01).
The method of the medicine of embodiment 9 screening preventions or treatment heart and injury
Method 1:
The expression system of Heat shock protein 27 HSP27: the method described in employing embodiment 1 and the aforesaid main test method, set up the myocardial cell of expressing Heat shock protein 27 HSP27.
Test group: in the expression system of aforesaid Heat shock protein 27 HSP27, add drug candidate.
Matched group: in the expression system of aforesaid Heat shock protein 27 HSP27, do not add drug candidate.
Detect the expression of the Heat shock protein 27 HSP27 in the test group myocardial cell, and compare with the expression of Heat shock protein 27 HSP27 in the matched group myocardial cell, if the expression of Heat shock protein 27 HSP27 is better than matched group statistically in the test group, just show that this material standed for is the medicine of prevention or treatment heart and injury.
Method 2:
Heat shock protein 27 HSP27 and TnT nT or the interactional reaction system of TnI can adopt as the co-immunoprecipitation system described in the embodiment 5.
Test group: in aforesaid co-immunoprecipitation system, add drug candidate;
Matched group: in aforesaid co-immunoprecipitation system, do not add drug candidate.
Detect the interaction situation of test group Heat shock protein 27 HSP27 and TnT nT or TnI, and compare with the interaction situation of TnT nT or TnI with the matched group Heat shock protein 27 HSP27, if the interaction of Heat shock protein 27 HSP27 and TnT nT or TnI significantly is better than matched group statistically in the test group, just show that this material standed for is the medicine of prevention or treatment heart and injury.
Method 3:
Heat shock protein 27 HSP27 and TnT nT or the interactional reaction system of TnI can adopt as the co-immunoprecipitation system described in the embodiment 5.
Test group: in aforesaid co-immunoprecipitation system, add drug candidate;
Matched group: in aforesaid co-immunoprecipitation system, do not add drug candidate.
Detect the degraded situation of test group TnT nT or TnI, and compare with the degraded situation of matched group TnT nT or TnI, if the degraded of TnT nT or TnI significantly is lower than matched group statistically in the test group, just show that this material standed for is the medicine of prevention or treatment heart and injury.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (3)
1. the purposes of Heat shock protein 27 HSP27 or its encoding gene is characterized in that, is used to prepare the medicine that suppresses TnT nI or TnT degraded.
2. a screening can be used for preventing or treating the method for the potential drug of heart and injury, it is characterized in that described method is:
Drug candidate is contacted with TnT nT or the interactional reaction system of TnI with Heat shock protein 27 HSP27, detect the interaction situation of Heat shock protein 27 HSP27 and TnT nT or TnI; If described drug candidate can promote the interaction of Heat shock protein 27 HSP27 and TnT nT or TnI, then it is the potential drug that can be used for preventing or treating heart and injury.
3. a pharmaceutical composition is characterized in that, contains the Heat shock protein 27 HSP27 of safe and effective amount, TnT nT or TnI, and pharmaceutically acceptable carrier.
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