CN102451465B - Use of SIRT1 in preparation of drugs for prevention of diseases caused by endothelial dysfunction - Google Patents

Abstract

The present invention relates to a use of sirtuin 1 (SIRT1) in preparation of drugs for prevention of diseases caused by endothelial dysfunction. Specifically, the present invention relates to a use of the SIRT1 in preparation of drugs for prevention and/or treatment of endothelial dysfunctions of mammals including human, and prevention and/or treatment of diabetic vascular complications of mammals including human.

Description

The purposes of SIRT1 in the medicine of the not normal disease that causes of preparation prevention inner skin cell function

Technical field

The present invention relates to SIRT1 preparation prevent and/or treat mammal comprise human endothelial cell malfunction, prevention/or the treatment mammal comprise purposes in the medicine of people's diabetic vascular complications.

Background technology

Diabetes are a kind of take genetic or posteriori insulin secretion or effect defective as the chronic metabolic disease of characteristics.This defective causes increasing of blood glucose, then damages other target organs of blood circulation and diabetes, such as retina, and kidney and nervous system.At present, the number of patients of diabetes increases rapidly.According to the statistics of World Health Organization (WHO), 2000, there were about 100,000,000 5 thousand ten thousand diabeticss in the whole world, and by inference, by 2010, this numeral will rise to 200,000,000 2 thousand ten thousand (Zimmet et al., 2001).Therefore, diabetes and complication have become very severe public health problem.Cardiovascular complication is the main reason that diabetes disable.Diabetic population cardiovascular disease prevalence is 2-4 times (Fox et al., 2004) of non-diabetic people.The potential mortality rate that is caused by cardiovascular complication accounts for about 80% (Werner and Nickenig, 2006) of the potential mortality rate of diabetics.Because the cardiovascular complication of diabetes can cause huge deadly disability rate, how preventing and treating cardiovascular complication becomes key issue in the treating diabetes.

The vascular complication of diabetes comprises macrovascular complications (coronary heart disease, cerebrovascular disease and peripheral vascular disease etc.) and microvascular complication (nephropathy, neuropathy and retinopathy etc.).A large amount of clinical studies show, no matter be in I type or type ii diabetes, the microvascular complication of diabetes all has extremely strong relatedness with hyperglycemia.And in the pathogenesis of great vessels complication in diabetic patients, hyperglycemia and insulin resistant all have a very important role.Is hyperglycemia how to cause the various blood capillaries of diabetes and macrovascular complications on earth? about the damage mechanism that hyperglycemia is induced, four hypothesis are arranged at present: (1) sorbitol metabolism bypass increases; (2) terminal glycosylation product (advanced glycation end product, AGE) generates increases; (3) protein kinase C activation (PKC); (4) the aminohexose approach increases.Although in the experimental model of cell and animal, more than four approach separately specific inhibitor can both improve the multiple Novel presentation that diabetes cause, but all the time all the clear and definite element of neither one these four different hyperglycemia damage mechanism are connected.And nearest research (Brownlee, 2001) is thought: these four kinds of different mechanisms of causing a disease have all reflected same essential problem---ROS that the mitochondrion electron transport chain produces is superoxide anion (O especially ₂ ^-) increase, and the increase of ROS will cause that inner skin cell function is not normal, and further cause the generation of diabetic vascular complications.

Endotheliocyte is that blood vessel innermost layer a kind of has important biological action, the organ of enormous amount, and to the control blood vessel, even organ dysfunction has played central role.Endotheliocyte is regulated antiotasis and permeability, keeps the balance of blood coagulation and fibrinolytic, and subcutaneous substrate is synthetic in regulating, leukocytic adhesion and with the inflammatory activity of infiltration and whole blood vessel wall.Moreover, endotheliocyte also affects the function of other types cell, such as vascular smooth muscle cell, platelet, leukocyte, kidney mesangial cell etc.The damage of Human Umbilical Vein Endothelial Cells can cause inner skin cell function not normal (endothelial dysfunction), it is characterized by: Endothelial Diastolic Function is impaired, become short scorching state, become coagulant blood state from the anticoagulant state-transition from the antiinflammatory state-transition.Not normal and the most cardiovascular disease of inner skin cell function is all closely related, for example hypertension, atherosclerosis, coronary artery disease, chronic heart failure, sick, the diabetes of peripheral arterial vasculature.And in the pathogenesis of the macrovascular complications of diabetes and little vascular complication, inner skin cell function is not normal to be considered to a very important factor.

(sirtuin 1 for SIRT1; the accession number of Genbank: BC012499) be the III histone deacetylases that depends on NAD+ in the mammal; it is the congener of Sir2 in the lower animal; in many life processes, as all having brought into play important effect in anti-oxidation stress, cell cycle, apoptosis, DNA damage reparation, longevity and the gene silencing.At present research is thought: SIRT1 has mediated the long-lived effect of energy limited (calorie restriction, CR) to biology.CR refers to guaranteeing to reduce the energy absorption of body about 30%～40% under the body basic nutrition demand prerequisite.CR not only significantly increases the life-span (Bordone and Guarente, 2005) of rodent, and the observation in 2 years by a definite date of CR healthy population is found, long-term CR can significantly reduce atherosclerotic incidence rate.The great many of experiments evidence shows, the change of sugar level and cellular metabolism and SIRT1 closely related (Adriano et al., 2006 during CR; Bordone and Guarente, 2005; Moynihan et al., 2005).The change relevant with SIRT1 that CR causes comprises that mainly insulin sensitivity increases and corresponding blood sugar lowering and insulin level (Bordone andGuarente, 2005), adipose cell endocrine characteristic (the Qiao and Shao that changes, 2006), physical activity increases, and the anti-oxidation stress ability strengthens.The inventor infers, crosses expression SIRT1 at endotheliocyte and can protect endotheliocyte, and it is not normal to resist the inner skin cell function that oxidative stress that hyperglycemia causes causes.

Except CR, in existing research, another kind can prolong the means in mammal life-span reliably for knocking out p66shc gene in the body.In mammal, three Shc gene: ShcA, ShcB (Sli) and ShcC (Rai) (Luzi et al., 2000) are arranged.Two kinds of mRNA:p66shc of ShcA gene code and p46/p52shc.P66shc is the important molecule of mediation oxidative stress and apoptosis, and p66shc does not mediate the transmission of Ras signal path, but participates in signal thorough fare in the cell, regulates generation and apoptosis (Migliaccio et al., 1999 of ROS; Trinei et al., 2002).Similar to the CR Mus, the ability that the Mus that p66shc knocks out is resisted oxidative stress strengthens life.At cellular level, p66shc has also participated in the adjusting of apoptosis pathway.P66shc-/-Mus not only the oxidative stress level reduce, the life-span obviously prolongs, it is not normal to resist the inner skin cell function that hyperglycemia causes.

MnSOD is a kind of superoxide dismutase, is important oxygen radical removing molecule.Cross and express the encoding superoxide dismutase and catalase can prolong the life-span of fruit bat, and the gene that knocks out the encoding superoxide dismutase can shorten its life-span; MnSOD lacks the oxidative stress level that increases blood vessel endothelium, causes Endothelial dysfunction.

PAI-1 plasminogen activator inhibitor-1 is one of member of serine protease inhibitor family, is to suppress activator of plasminogen, regulates the key molecule of fibrinolytic.Plasma PAI-1 level rising and heart infarction and apoplexy are closely related, especially concerning diabetics.PAI-1 level in the blood plasma increases not normal relevant with inner skin cell function.

The inventor infers, can protect endotheliocyte if cross expression SIRT1 at endotheliocyte, and it is not normal to resist the inner skin cell function that oxidative stress that hyperglycemia causes causes, and its mechanism may be relevant with the expression of regulating p66shc, PAI-1 and MnSOD.

In sum, reducing the oxidative stress level and be not only the control diabetic vascular complications, also is life-saving, the key point of control diseases associated with senescence.The research of animal and cellular level all points out SIRT1 and oxidative stress closely related, and it is significant to the control of diabetic vascular complications even old and feeble related vascular diseases further to study the effects anb Mechanism of SIRT1 in Endothelial dysfunction due to the diabetes.

Summary of the invention

Therefore, technical problem to be solved by this invention is to probe into the effect of SIRT1 in preventing and/or treating diabetic vascular complications.

Therefore, a first aspect of the present invention relates to SIRT1 and prevents and/or treats purposes in the medicine that mammal comprises the human vascular endothelial malfunction in preparation, and wherein said blood vessel inner skin cell function is not normal to be caused by hyperglycemia.

A second aspect of the present invention relates to SIRT1 and prevents and/or treats purposes in the medicine that mammal comprises people's diabetic vascular complications in preparation.Preferably, described diabetic vascular complications is selected from macrovascular complications or microvascular complication.More preferably, described macrovascular complications is selected from coronary heart disease, cerebrovascular disease or peripheral vascular disease, and described microvascular complication is selected from nephropathy, neuropathy or retinopathy.

Preferably, the medicine in the above-mentioned aspect is the restructuring SIRT1 albumen of purification or the restructuring SIRT1 expression vector that is suitable for expressing in human body, and preferably, the restructuring SIRT1 albumen of described purification utilizes conventional prokaryotic expression system or eukaryotic expression system to obtain.

Preferably, the dosage form of the medicine in the above-mentioned aspect is injection.More preferably, described injection is selected from intradermal injection agent, subcutaneous injection agent, intramuscular injection agent or intravenous injection.

In other words, the present invention detects the variation of SIRT1 expression in the high sugared post-stimulatory Human umbilical vein endothelial cells by the realtimePCR method, and it is lower further to detect the stimulation of height sugar by western blot, crosses expression and disturbs SIRT1 on the impact of p66shc, MnSOD, PAI-1 expression.In order to verify, the not normal impact of inner skin cell function that SIRT1 causes hyperglycemia in vivo, we have at first detected the expression of SIRT1 in the normal C57 Mus aorta with suffering from diabetes, find that the expression of SIRT1 in the diabetic mice aorta significantly reduces; Further set up the special transgenic mice of SIRT1 endothelium, set up the type i diabetes model by the method for lumbar injection streptozotocin (STZ).STZ is after 2 months in injection, and vascular ring tension force detects endothelial function, and SABC detects 8-OH-dG and nitration tyrosine, shows blood vessel oxidative stress level.Detect p66shc, MnSOD, PAI-1 expression with realtimePCR or Western blotting.

Found that the expression of SIRT1 reduces in the high sugared post-stimulatory Human umbilical vein endothelial cells.Disturb SIRT1 significantly to increase p66shc, PAI-1 expression, reduce the expression of MnSOD; Cross expression SIRT1 and reduce p66shc, PAI-1 expression, the expression of rising MnSOD.The expression of SIRT1 significantly reduces in the diabetic mice aorta; Vascular ring tension force experimental results show that SIRT1 crosses expression and improved the Endothelial dysfunction that diabetes cause, SABC detect to be found the SIRT1 8-OH-dG that causes of diabetes that crossed expression inhibiting and the rising of nitration tyrosine level, has reduced the oxidative stress level of blood vessel; SIRT1 crosses the expression that expression can reduce p66shc, PAI-1 in the diabetic mice tremulous pulse, rising anti-oxidation stress molecule MnSOD level.

The inventor finds first, and in endotheliocyte, SIRT1 may increase the expression of MnSOD by reducing the expression of p66shc, PAI-1, and the inner skin cell function that oxidative stress is caused is not normal to have arrived protective effect; In the type i diabetes model, the transgenic of SIRT1 endothelial-cell specific may increase the expression of MnSOD by reducing the expression of p66shc, PAI-1, has reduced the oxidative stress level of blood vessel, improves the function of endotheliocyte.

The present invention provides the treatment approach of a brand-new mechanism for diabetic vascular complications.

Description of drawings

Fig. 1: high sugar stimulates and reduces SIRT1 expression in the Human umbilical vein endothelial cells.The block diagram data are mean+SD, and the different batches sample repeats 3 times and adds up * .p＜0.05.

Fig. 2: when high sugar stimulated, A. crossed expression SIRT1 reduction p66shc, the protein level of PAI-1, the protein level of increase MnSOD in Human umbilical vein endothelial cells; B. disturb SIRT1, increase p66shc, the protein level of PAI-1, the protein level of reduction MnSOD.

Fig. 3: the expression of SIRT1 significantly lowers in the diabetic mice aorta.The block diagram data are mean+SD, and 5 treated animal western blot results are scanned into gray-scale map, gray value are added up * .p＜0.05vs Normal group.

Fig. 4: SIRT1 endothelium specific transgenic Mus or wild-type mice are made up diabetes model by lumbar injection STZ, Normal group injection citrate buffer.Regularly the mice random blood sugar is monitored by tail vein blood with blood glucose meter weekly.Every group has 13 mices.

Fig. 5: the vascular ring experiment detects the arterial dilation that endothelium relies on.A. the arterial dilation of normal SIRT1 transgenic mouse and the dependence of wild-type mice endothelium; B. the arterial dilation that relies on of diabetes SIRT1 transgenic mouse and wild-type mice endothelium; C. the arterial dilation of normal SIRT1 transgenic mouse and the non-endothelium dependence of wild-type mice; D. the arterial dilation that relies on of the non-endothelium of diabetes SIRT1 transgenic mouse and wild-type mice.* P＜0.05, * * P＜0.01vs, every group of mice has 5～7.

Fig. 6: cross in the SIRT1 body to express and reduce diabetic mice blood vessel oxidative stress level.Cross the level that significantly reduces nitration tyrosine in the aorta of expressing in the A, SIRT1 body.Nitration tyrosine is brown color.Cross in the B, SIRT1 body and express 8-OH-dG positive cell rate in the remarkable descending aorta, the 8-OH-dG positive cell is the cell of nucleus brown color.

Fig. 7: SIRT1 endothelium transgenic, the expression of p66shc and PAI-1 increases the expression of MnSOD in the reduction aorta.A, D block diagram data are mean+SD, 5 treated animal realtimePCR results are added up * * .p＜0.01, * * * .P＜0.0001vs Normal group wild-type mice; #p＜0.05, ##.p＜0.01vs diabetic groups wild-type mice.B, C block diagram data are mean+SD, and 5 treated animal western blot results are scanned into gray-scale map, gray value are added up * * .p＜0.01vs Normal group wild-type mice; ##.p＜0.01vs diabetic groups wild-type mice.

The specific embodiment

The below will further specify the present invention by following non-limiting example, and will be as well known to those skilled in the art, without departing from the spirit of the invention, can make many modifications to the present invention, and such modification also falls into scope of the present invention.

Following experimental technique is conventional method if no special instructions, and employed experiment material all can easily be obtained from commercial company if no special instructions.

Embodiment

Embodiment 1 realtimePCR method detects the variation of SIRT1 expression in the high sugared post-stimulatory Human umbilical vein endothelial cells

For following experiment has been carried out in the effect of pre-test SIRT1 in the Endothelial dysfunction that high sugar is induced.

1, the employing conventional method is obtained the human umbilical vein cell and is cultivated.Method is summarized as follows: get neonatal umbilical cord and obtain umbilical vein, after necessity is processed, hatch by perfusion IV collagenase and 37 ℃ and obtain human umbilical vein endothelial cell (HUVEC), the HUVEC cell culture that obtains in culture medium M200 (available from Cascade Biologics Inc., Portland, Oregon, USA., M-200-500) and routinely primitive cell culture method is cultivated and is gone down to posterity;

2, the RNA extracting method by " molecular cloning " second edition Plays extracts RNA and carries out RT-PCR;

3, utilize test kit Quantitect SYBR Green One-Step RT-PCR Kit (available from QIAGEN) (method is carried out according to the test kit operation instruction) to carry out realtimePCR.

Used primer is as follows in the above-mentioned steps:

People SIRT1: positive-sense strand: 5 ' CTT CAG GTC AAG GGA TGG TAT, 3 ' (SEQID NO.1),

Antisense strand: 5 ' GCG TGT CTA TGT TCT GGG TAT, 3 ' (SEQ ID NO.2);

Beta-actin: positive-sense strand: 5 ' TCG TGC GTG ACA TTA AGG AG, 3 ' (SEQ ID NO.3),

Antisense strand: 5 ' GAT GTC CAC GTC ACA CTT CA, 3 ' (SEQ ID NO.4).

Experimental result: stimulate Human umbilical vein endothelial cells take final concentration as the 30mmol/L D-glucose, 0 hour, 2 hours, 6 hours, 12 hours, 24 hours collecting cells of different time points after high sugar stimulates, extract RNA, RT-PCR, realtimePCR detects discovery: high sugar stimulates and reduces SIRT1 mRNA level (Fig. 1) in the Human umbilical vein endothelial cells.Therefore, high sugar stimulates and reduces SIRT1 expression in the Human umbilical vein endothelial cells, infers accordingly the Endothelial dysfunction performance protective effect that SIRT1 induces high sugar.

Embodiment 2 height sugar stimulate lower, cross expression and disturb SIRT1 on the impact of p66shc, MnSOD, PAI-1 expression

The p66shc level reduces can reduce the oxidative stress level, the generation that the inner skin cell function that stops high sugar to cause is not normal; PAI-1 is considered to the marker molecule of endothelial function, and it is bad that it expresses the prompting endothelial function that raises.MnSOD is relevant with oxygen radical removing, and it is strong that its level rising prompting cell is resisted the oxidative stress ability.For the not normal generation of inner skin cell function that whether can stop high sugar to cause at cellular level checking SIRT1, the method that adopts SIRT1 to cross expression and disturb is observed SIRT1 to the impact of p66shc, PAI-1 and MnSOD expression, from the effect of the pros and cons checking SIRT1 the not normal generation of inner skin cell function that the high sugar of prevention causes.

Main experimental procedure is as follows:

1, make up SIRT1 and cross expression adenovirus Ad-SIRT1 and contrast Ad-GFP, method is summarized as follows:

(construction method of this plasmid makes up (Zhang Q J et al according to literature method to use first BamH I and Not I double digestion pcDNA3.1Sirt1 plasmid, 2008), be connected into the pAdTrack-CMV carrier of BamH I and Not I double digestion (available from Qbiogene with downcutting the genes of interest fragment, USA) in, obtain shuttle plasmid pAdTrack-CMV-Sirt1.

2, make up SIRT1 and disturb adenovirus, method is summarized as follows:

(SIRT1 RNA disturbs (RNAi) plasmid to make up (Liu C M et al, 2004) according to literature method with BamH I and Xho I double digestion PBSU6-Sirt1RNAi plasmid.The SIRT1 interference sequence is 5 '-GATGAAGTTGACCTCCTCA-3 ' (SEQ ID NO.5), reference literature synthesizes (Picard F et al, 2004).Concrete construction method is as follows.

The strand of at first synthetic two complementations,

Positive-sense strand is

5′-GGATGAAGTTGACCTCCTCATTCAAGAGATGAGGAGGTCAACTTCATCAAAAAC-3′(SEQ?ID?NO.6)，

Comprising GATGAAGTTGACCTCCTCA interference sequence and its complementary series TGAGGAGGTCAACTTCATC, the centre is that irrelevant sequence is used to form circulus, the AAAAA sequence is the transcription terminator of microRNA, and the C of beginning G and tail end is used for consisting of sticking terminal.Identical with its structure with the sequence of sense chain complementation, two ends form respectively the flat end of ApaI and the sticking end of XhoI.

The antisense strand sequence is

5-TCGAGTTTTTGATGAAGTTGACCTCCTCATCTCTTGAATGAGGAGGTCAACTTCATCC-3′(SEQ?ID?No.7)。

Article two, after chain synthesizes, each OD is with 11.25 μ l sterilization deionized water dissolving, the lysate of two chains is merged mixing, add 2.5 μ l annealing buffers (1M NaCl, 100mM TrispH7.4), in boiling water bath, boiled 5 minutes, powered-down, the annealing product is changed in the heat-preserving container that fills boiling water fast, add a cover rearmounted cold house and spend the night, make its temperature slowly be down to room temperature and form stable two strands.Product one end of annealing this moment is equivalent to ApaI to be cut and scabbled, and an end is equivalent to XhoI and cut.Before being cloned into carrier, the two strands that needs annealing is formed is done suitably dilution, and the scope of dilution can be larger, can accept between 50 to 1000 times.Then the two strands that forms of will annealing at first is cloned into the pSilencerU6 carrier, detailed process is with pSilencerU6 carrier ApaI enzyme action, scabbles with Klenow, uses the XhoI enzyme action again, be connected with annealing is double-stranded after reclaiming, the clone of formation is the interference sequence expression cassette that links to each other with the U6 promoter.) and control plasmid PBSU6 (available from Qbiogene, USA).Be connected in the pAdTrack carrier of Bgl II and Xho I double digestion downcutting fragment.PAdTrack-U6 and pAdTrack-Sirt1 RNAi shuttle plasmid have been made up.

3, with the plasmid that obtains in step 1 and 2 with the PmeI linearization for enzyme restriction and utilize conventional gel absorption method to carry out purification, with the linearized vector of recovery and pAdEasy-1 in molar ratio about 10: 1 ratio corotation dissolve the BJ5183 electricity and turn competent cell available from (Stratagene company).The preparation method of competent cell is: with bacillus coli DH 5 alpha list colony inoculation to 5ml LB culture medium (1% tryptone, 0.5% yeast extract, 1%NaCl, pH7.0) in, 37 ℃ of joltings are spent the night, get the 1/50 volume bacterium liquid that spends the night next day and be inoculated in the 50-100ml LB culture medium, bacterium liquid OD is treated in 37 ℃ of joltings _600nm(need approximately 2-3 hour) during near 0.3-0.4, in 4 ℃ 4, centrifugal 10 minutes of 000rpm collects thalline, gets the 0.1MMgCl of 1/5 volume pre-cooling of original bacteria liquid volume ₂Solution adds in the thalline gently, pressure-vaccum MgCl gently on the ice bath ₂Solution slowly scatters thalline, leaves standstill in the ice bath 20 minutes, and 4 ℃ centrifugal, and condition is the same, slowly adds the 0.1M CaCl of the pre-cooling of 1/20 volume in the thalline ₂Solution behind the mixing, left standstill in the ice bath 20 minutes gently, behind the centrifugal collection of the similarity condition bacterium, added the 0.1MCaCl of 1/50 volume pre-cooling in the thalline ₂, mixing leaves standstill in the ice bath after 12 hours for transforming.Preserve competent cell such as need, can add immediately the glycerol of 1/5 volume after preparation is complete, packing is stored in-70 ℃, obtains positive recombinant bacterium.Utilize described positive recombinant bacterium to prepare in a large number plasmid and for subsequent use with purification behind the PacI linearization for enzyme restriction.Use LipofectAmine 2000 transfected HEK 293s (available from preclinical medicine institute of Chinese Academy of Medical Sciences cell centre) to obtain the recombinant adenovirus of packing above linearizing adenoviral plasmid.

4, use adenovirus infection HUVEC, extract albumen and carry out western blot, concrete steps are by the correlation technique of " molecular cloning " second edition.

Experimental result: A. infected Human umbilical vein endothelial cells 24 hours with adenovirus Ad-GFP and Ad-SIRT1 respectively, D-glucose take final concentration as 30mmol/L was processed 24 hours, collecting cell extracts albumen, wesren blot detects the discovery endotheliocyte and crosses the protein level of expressing SIRT1 reduction p66shc and PAI-1, increases the protein level of MnSOD; B is respectively with disturbing adenovirus Ad-U6 and Ad-SIRT1RNAi to infect Human umbilical vein endothelial cells 24 hours, D-glucose take final concentration as 30mmol/L was processed 24 hours, collecting cell extracts albumen, wesren blot detects and finds to disturb endotheliocyte SIRT1, the protein level of p66shc and PAI-1 in the increase endothelium, reduce the protein level (Fig. 2, A, B) of MnSOD.That is, the present invention confirms that at cellular level SIRT1 can regulate the level of the albumen relevant with endothelial function: p66shc, PAI-1 and MnSOD, the not normal performance protective effect of inner skin cell function of inducing at high sugar.

The expression of SIRT1 reduces in the C57 Mus aorta of embodiment 3 diabetes

The present invention has studied SIRT1 in the diabetic mice tremulous pulse by making up the type i diabetes mouse model expression and SIRT1 have or not the performance protective effect in the diabetic vascular course of damage.Method is summarized as follows.

1, the STZ intraperitoneal injection makes up the type i diabetes model, and method detailed is referring to J.Clin.Invest.112:725-735 (2003) (incorporating by reference in full it into this paper), and STZ is available from (Sigma-Aldrich, S0130);

2, protein extraction and western blot see the relevant portion of " molecular cloning " second edition in detail.Used antibody is respectively anti--SIRT1 (Upstate, 07-131) in the experiment, and is anti--actin (Sigma, A5376).

Experimental result: adopt continuously 5 days method of lumbar injection structure type i diabetes model mouse of STZ (50mg per kilogram of body weight) with C57/BJ Mus (available from Chinese military medicine academy of science animal center).After 8 weeks of modeling, process mice, get aorta, Western blotting detects discovery: with cotemporary normal wild type C57 Mus, compare, the expression of SIRT1 significantly reduces (Fig. 3) in the diabetes C57 Mus blood vessel.Therefore, SIRT1 may have protective effect to the blood vessel injury that the diabetes hyperglycemia causes

Embodiment 4 sets up the special type i diabetes model of SIRT1 endothelium

Main experimental procedure is as follows.

1, the special SIRT11 transgenic mouse of endothelium makes up, the detailed construction method of this transgenic mouse is asked for an interview Zhang QJ, Wang Z, Chen HZ, Zhou S, Zheng W, Liu G, Wei YS, CaiH, Liu DP, Liang CC.Endothelium-specific overexpression of class IIIdeacetylase SIRT1 decreases atherosclerosis in apolipoprotein E-deficientmice.Cardiovasc Res.2008; 80 (2): 191-199. (incorporating by reference in full it into this paper).

2, the STZ intraperitoneal injection makes up the type i diabetes model, the detailed construction method of this model sees also J.Clin.Invest.112:725-735 (2003) (incorporating by reference in full it into this paper), STZ is available from (Sigma-Aldrich, S0130).

Experimental result: in rear 8 weeks of modeling success, monitor weekly the mice random blood sugar: the average blood sugar of diabetic groups mice namely rose to rapidly about 20mmol/l after STZ injection in the 1st week, later 3 weeks are gradually ascendant trend, rising to the about 28mmol/l of maximum the 4th week, 4 all blood glucose after this all maintain about 25mmol/l, within the detection time in whole 8 weeks, significant difference does not appear in the SIRT1 transgenic mouse in the diabetic groups and the blood sugar level of wild-type mice.Transgenic mouse in the Normal group and the blood sugar level of wild-type mice do not have significant difference during this period.The result sees also Fig. 4.What therefore, the present invention had set up successfully that diabetic mice causes phenotypic difference side by side except the difference of the transgenic mouse on the same group and wild-type mice blood glucose may.

The experiment of embodiment 5 vascular rings detects the arterial dilation that endothelium relies on

Vascular ring experiment is the Classic Experiments means that detect inner skin cell function, the present invention by this experimental verification in the type i diabetes model mouse, whether SIRT1 endothelium transgenic the protective effect of Human Umbilical Vein Endothelial Cells function.

The detailed step of vascular ring test method sees also J.Clin.Invest.112:725-735 (2003) (incorporating by reference in full it into this paper); Phenylephrine is available from Sigma-Aldrich, and catalog number (Cat.No.) P8155, acetylcholine are available from Sigma-Aldrich, and catalog number (Cat.No.) A6500, sodium nitroprusside are available from Sigma-Aldrich, and catalog number (Cat.No.) is S0501.

Experimental result: in the Normal group, the SIRT1 transgenic mouse is compared with wild-type mice, and the arterial dilation that endotheliocyte relies on is without significant difference; The wild-type mice of diabetes is compared with the wild-type mice of Normal group, and the arterial dilation that endotheliocyte relies on significantly reduces.And in the diabetic groups, transgenic mouse is compared with wild-type mice, and the arterial dilation that endotheliocyte relies on has obtained remarkable improvement.For the arterial dilation that non-endotheliocyte relies on, transgenic mouse is compared all without significant difference with wild-type mice in diabetic groups and the Normal group.Experimental result is seen Fig. 5, A-B.This shows that diabetes significantly reduce inner skin cell function, and endotheliocyte is crossed and is expressed the endothelial injury that SIRT1 causes diabetes remarkable protective effect is arranged.

Cross in the embodiment 6SIRT1 body to express and reduce diabetic mice blood vessel oxidative stress level

In order to verify whether SIRT1 endothelium transgenic can reduce blood vessel oxidative stress level, the present invention has just studied the height of oxidative stress level by detecting 8-hydroxyl-2 ' NSC 22837 (8-hydroxy-2 '-deoxyguanosine, 8-OH-dG) and nitration tyrosine (nitrotyrosine) level.

Main experimental procedure is as follows.

SABC (two step method)

1) dewaxing

Place dimethylbenzene to soak 20min paraffin section, every 20min changes a dimethylbenzene, changes continuously 3 times.After the dimethylbenzene dewaxing, enter 100% → 100% → 95% → 85% → 70% gradient ethanol, in each concentration, soak 2min.PBS and wash 3 times, each 5min.

2) eliminate endogenous peroxydase

Section enters 3% hydrogen peroxide, places room temperature 10min, and PBS washes 3 times, each 5min.

3) the hot antigen retrieval of high-pressure process

Section is immersed in the citric acid repair liquid of PH6.0, puts into pressure cooker after will being placed with again the glass jar cover lid of section.Be heated to the rear valve-off that steams and begin supercharging, when be pressurized to begin to be incubated the hot antigen retrieval of 10min. when temperature reaches 121 ℃ and finish after, allow it naturally cool to room temperature.PBS washes 3 times, each 5min.

4) antibody hybridization

Firmly organize with tissue penization circle circle, be added drop-wise to tissue after primary antibodie is diluted to suitable proportion with PBS.Section places wet box, and 4 ℃ are spent the night.

Add 0.2%Tween20 in PBS, section enters among this PBS to wash 3 times, each 5min.

With the two anti-tissues that are added drop-wise to, section places wet box, hatches 30min for 37 ℃.PBS washes 3 times, each 5min.

5) DAB colour developing is redyed

(1) DAB colour developing 1-2min grasps dye levels at microscopically.Tap water flushing 2-3min, cessation reaction.

(2) the Harris haematoxylin is redyed nuclear, dyeing 3min, and tap water flushing 5min returns indigo plant.

(3) section enters 0.1% acidic alcohol differentiation liquid (99.9ml 70% ethanol, 100ul concentrated hydrochloric acid), differentiation 10s.Tap water flushing 20min.

6) mounting

Section enters 70% → 85% → 95% → 100% dehydration, and it is transparent to reenter dimethylbenzene immersion 5min, uses at last the resinene mounting.

Wherein, the primary antibodie of using in the above-mentioned steps is that anti-mice 8-OH-dG monoclonal antibody is (available from Japan Institute for the control of Aging, MOG-020P), two anti-are anti-nitrotyrosine rabbit polyclonal antibody (available from Upstate, 06-284).

Experimental result: A. diabetic groups mice is compared with Normal group, and endotheliocyte nitrotyrosine dyeing obviously strengthens, and the transgenic mouse in the diabetic groups is compared nitrotyrosine dyeing and obviously reduced with wild-type mice.B. the diabetic groups mice is compared with Normal group, and the cell that 8-OH-dG dyeing is positive in endotheliocyte and the whole vascular cell obviously increases, and the dyeing of positive cell obviously strengthens.Transgenic mouse in the diabetic groups is compared with wild-type mice, and the cell number that 8-OH-dG dyeing is positive obviously reduces, and the dyeing of positive cell obviously reduces.Experimental result is seen Fig. 6, A, B.This shows the oxidative stress level of endothelium and even whole blood vessel when SIRT1 endothelium transgenic can reduce diabetes.

Embodiment 7 usefulness realtimePCR and Western blotting detect p66shc, MnSOD, PAI-1 expression

For zoopery horizontal detection SIRT1 endothelium transgenic on blood vessel in the impact expressed of p66shc, MnSOD, PAI-1 carried out following experiment.

1, the method detailed of blood vessel RNA extraction and realtimePCR is referring to the relevant portion of " molecular cloning " second edition, and Trizol is available from invitrogen company, and catalog number (Cat.No.) is that 15596-026realtimePCR the primer sequence is:

P66shc: positive-sense strand: 5 ' AAG TAC AAC CCA CTT CGG AAT GA, 3 ' (SEQ ID NO.8),

Antisense strand: 5 ' GGG TCC CAG GGATGAAG 3 ' (SEQ ID NO.9);

PAI-1: positive-sense strand: 5 ' CTC CGA GAA TCC CAC ACA G, 3 ' (SEQ IDNO.10),

Antisense strand: 5 ' ACT TTG AAT CCC ATA GCA TC, 3 ' (SEQ ID NO.11);

Beta-actin: positive-sense strand: 5 ' TCG TGC GTG ACA TTA AGG AG, 3 ' (SEQ ID NO.12),

Antisense strand: 5 ' GAT GTC CAC GTC ACA CTT CA, 3 ' (SEQ IDNO.13).

2, the detailed step of protein extraction and western blot sees also the relevant portion of " molecular cloning " second edition.Used associated antibodies is that anti--SIRT1 is (available from Upstate in this experiment, 07-131), anti--Shc is (available from Santa Cruz Biotechnology, sc-1695), anti--MnSOD is (available from BD Transduction Laboratories, M99920-050), anti-beta-actin (available from Sigma, A5376).

Experimental result: the aorta of 8 all diabetic mice and normal control Mus extracts RNA or albumen, detect discovery with realtimePCR or Western blotting: p66shc mRNA and protein level in 8 all diabetes wild-type mice tremulous pulsies significantly raise than matched group, and the p66shcmRNA in the genetically modified diabetic mice tremulous pulse of SIRT1 compares with matched group without obviously raising with protein level, compares with the diabetes wild-type mice then and obviously reduces.The protein level of MnSOD is compared with matched group extremely obviously and is reduced in the diabetes wild-type mice tremulous pulse, and the MnSOD protein level in the genetically modified diabetic mice tremulous pulse of SIRT1 is compared with matched group without obviously reduction, and is significantly higher than the diabetes wild-type mice.The mRNA level of PAI-1 is compared with matched group extremely obviously in the diabetes wild-type mice tremulous pulse increases, and the PAI-1 protein level in the genetically modified diabetic mice tremulous pulse of SIRT1 is compared with matched group without obviously reduction, and significantly is lower than the diabetes wild-type mice.The result sees also Fig. 7, A-D.Also namely, the present invention reconfirms that in the animal level SIRT1 can regulate the level of the albumen relevant with endothelial function: p66shc, PAI-1 and MnSOD, and this is the mechanism of SIRT1 performance anti-diabetic blood vessel injury effect.

List of references:

Zimmet，P.，Alberti，K.G.，and?Shaw，J.(2001).Global?and?societalimplications?of?the?diabetes?epidemic.Nature?414，782-787.

Fox，C.S.，Coady，S.，Sorlie，P.D.，Levy，D.，Meigs，J.B.，D′Agostino，R.B.，Sr.，Wilson，P.W.，and?Savage，P.J.(2004).Trends?in?cardiovascularcomplications?of?diabetes.JAMA?292，2495-2499.

Werner，N.，and?Nickenig，G.(2006).Clinical?and?therapeuticalimplications?of?EPC?biology?in?atherosclerosis.J?Cell?Mol?Med?10，318-332.

Brownlee，M.(2001).Biochemistry?and?molecular?cell?biology?of?diabeticcomplications.Nature?414，813-820.

Bordone，L.，and?Guarente，L.(2005).Calorie?restriction，SIRT1?andmetabolism：understanding?longevity.Nat?Rev?Mol?Cell?Biol?6，298-305.

Adriano，M.A.，Vergnes，B.，Poncet，J.，Mathieu-Daude，F.，da?Silva，A.C.，Ouaissi，A.，and?Sereno，D.(2006).Proof?of?interaction?betweenLeishmania?SIR2RP1?deacetylase?and?chaperone?HSP83.Parasitol?Res.

Moynihan，K.A.，Grimm，A.A.，Plueger，M.M.，Bernal-Mizrachi，E.，Ford，E.，Cras-Meneur，C.，Permutt，M.A.，and?Imai，S.(2005).Increased?dosageof?mammalian?Sir2?in?pancreatic?beta?cells?enhances?glucose-stimulatedinsulin?secretion?in?mice.Cell?Metab?2，105-117.

Qiao，L.，and?Shao，J.(2006).SIRT1?Regulates?Adiponectin?GeneExpression?through?Foxo1-C/Enhancer-binding?Protein?TranscriptionalComplex.J?Biol?Chem?281，39915-39924.

Luzi，L.，Confalonieri，S.，Di?Fiore，P.P.，and?Pelicci，P.G.(2000).Evolutionof?Shc?functions?from?nematode?to?human.Curr?Opin?Genet?Dev?10，668-674.

Migliaccio，E.，Giorgio，M.，Mele，S.，Pelicci，G.，Reboldi，P.，Pandolfi，P.P.，Lanfrancone，L.，and?Pelicci，P.G.(1999).The?p66shc?adaptor?proteincontrols?oxidative?stress?response?and?life?span?in?mammals.Nature?402，309-313.

Trinei，M.，Giorgio，M.，Cicalese，A.，Barozzi，S.，Ventura，A.，Migliaccio，E.，Milia，E.，Padura，I.M.，Raker，V.A.，Maccarana，M.，et?al.(2002).Ap53-p66shc?signalling?pathway?controls?intracellular?redox?status，levels?ofoxidation-damaged?DNA?and?oxidative?stress-induced?apoptosis.Oncogene21，3872-3878.

Alp?NJ，Mussa?S，Khoo?J，et?al.Tetrahydrobiopterin-dependentpreservation?of?nitric?oxide-mediated?endothelial?function?in?diabetes?bytargeted?transgenic?GTP-cyclohydrolase?I?overexpression.J?Clin?Invest.Sep?2003；112(5)：725-735.

Zhang?QJ，Wang?Z，Chen?HZ，et?al.Endothelium-specific?overexpressionof?class?III?deacetylase?SIRT1?decreases?atherosclerosis?in?apolipoproteinE-deficient?mice.Cardiovascular?Research(2008)80，191-199.

Picard?F，Kurtev?M，Chung?N，et?al.Sirt1?promotes?fat?mobilization?inwhite?adipocytes?by?repressing?PPAR-gamma.Nature?2004?Jun?17；429(6993)：771-6.

Liu?CM，Liu?DP，Dong?WJ，et?al.Retrovirus?vector-mediated?stable?genesilencing?in?human?cell.Biochem?Biophys?Res?Commun.2004?Jan16；313(3)：716-20。

Claims (4)

1.SIRT1 prevent and/or treat purposes in the medicine that mammal comprises people's diabetic vascular complications in preparation, wherein said diabetic vascular complications is selected from nephropathy or retinopathy.

2. purposes according to claim 1, wherein said medicine is the restructuring SIRT1 albumen of purification or the restructuring SIRT1 expression vector that is suitable for expressing in human body, preferably, the restructuring SIRT1 albumen of described purification utilizes conventional prokaryotic expression system or eukaryotic expression system to obtain.

3. purposes according to claim 1 and 2, the dosage form of wherein said medicine is injection.

4. purposes according to claim 3, wherein said injection is selected from intradermal injection agent, subcutaneous injection agent, intramuscular injection agent or intravenous injection.

Images