CN103784944A - Function and application of IRF7 gene in stent and carotid endarterectomy restenosis - Google Patents
Function and application of IRF7 gene in stent and carotid endarterectomy restenosis Download PDFInfo
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- CN103784944A CN103784944A CN201410031620.1A CN201410031620A CN103784944A CN 103784944 A CN103784944 A CN 103784944A CN 201410031620 A CN201410031620 A CN 201410031620A CN 103784944 A CN103784944 A CN 103784944A
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Abstract
The invention discloses function and application of an IRF7 gene in stent and carotid endarterectomy restenosis, belonging to the field of the function and the application of a gene. According to the invention, IRF7 gene knockout mice and wild C57 mice are taken as experimental subjects, a research on vascular injury model mouse intima neogenesis measurement and detection of vascular wall cell proliferation level is carried out through a vascular injury model, and the results show that the IRF7 gene knockout mice realizes more obvious intima neogenesis and cell proliferation in comparison with the wild C57 mice. The invention discloses the function of the IRF7 gene in the stent and carotid endarterectomy restenosis, which mainly means that the IRF7 gene has an effect of inhibiting the restenosis caused by vascular injury, especially the effect that the IRF7 gene can be used for inhibiting the stent and carotid endarterectomy restenosis. According to the abovementioned function, the IRF7 gene can be used for preparing a drug for treating a vascular stenosis disease, especially for preparing the drug for treating the stent and carotid endarterectomy restenosis.
Description
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Technical field
The invention belongs to function and the application of gene, relate to the function and application of a kind of IRF7 gene in support and Endarterectomy postoperative restenosis.
Background technology
At present, the quantity of the cardiovascular disease that angiostenosis causes constantly rises, at present this class disease be there is no to radical cure way, the treatment means of vascular surgery comprises that balloon expandable, support are inserted and the mode such as tremulous pulse bypass, but after reconstructing blood vessel, restenosis has greatly affected therapeutic effect.About the research of vascular restenosis has been carried out for many years, but also not clear and definite up to now.There are some researches show, in the process forming in damage, new intima and middle membrane tissue hyperplasia and the extracellular matrix of simultaneously following form, and are the main pathological basis that causes restenosis.Under physiological status, vascular endothelial cell (vascular endothelialcell, EC) can produce multiple promotion and suppress vascular smooth muscle cell (vascular smooth muscel cell, VSMC) material of growth, and both keep dynamic equilibrium, maintain VSMC in relatively staticizing state.Promote the material of VSMC growth to mainly contain platelet derived growth factor (platelet-derived growth factor, PDGF), Endothelin (endomthelin, and angiotensin U(angiotonin II ET), Ang II) etc., and the material that suppresses VSMC propagation mainly contains crowd nitrogen (nitrogen monoxidum, NO), prostacyclin (prostacyclin, PGl2) etc.After vascular endothelial injury, promote the somatomedin of VSMC propagation increase and suppress the factor minimizing that VSMC breeds, this dynamic equilibrium is broken, and causes a large amount of propagation of VSMC.
The inner membrance new life of blood vessel is the pathological change that blood vessel occurs under various impairment factors stimulate, and is the total pathological process of multiple cardiovascular system diseases.Smooth muscle cell in blood vessel wall plays an important role in this process, and its propagation, apoptosis and phenotypic alternation played the part of important role in the process of neointimal hyperplasia.After blood vessel injury, vascular smooth muscle cell is moved to inner membrance by middle film, the Proliferation and apoptosis loss of equilibrium of smooth muscle cell, and phenotype is changed to synthesis type by shrinkage type, and blood vessel wall discomfort is reinvented, thereby causes neointimal hyperplasia.In recent years, more and more cause people's concern for the research of signal transduction pathway in neointimal hyperplasia process.
This class disease be there is no to radical cure method at present, the primary treatment means of vascular surgery are inaccessible section reconstructing blood vessels, comprise that balloon expandable, support are inserted and arterial bypass etc., but the incidence rate of restenosis higher (30%～60%) after reconstructing blood vessel, greatly affected therapeutic effect, after reconstructing blood vessel, restenosis is still a clinical difficult problem up to now.
IRF7 is a member in interferon regulatory factor (interferon regulatory factor, IRF) family.Existing research prompting: IRF family member has participated in biological process widely, relates generally to the natural immunity and the acquired immune response, antitumor formation etc.
Summary of the invention
For solving defect and the deficiency of above-mentioned prior art, the object of the present invention is to provide the application of a kind of IRF7 in preparation treatment support and Endarterectomy postoperative restenosis medicine.
Object of the present invention is achieved through the following technical solutions:
The present invention is take wild type C57 mice and IRF7 knock out mice (IRF7-KO mice) as experimental subject, obtain mice blood vessel damage model (vascular injury by the induction of carotid artery seal wire damage model, VI), carry out the research of the detection of the newborn mensuration of blood vessel injury model (VI) mice inner membrance and cells of vascular wall propagation level, result shows: with wild type C57 mice contrast, IRF7 knock out mice shows inner membrance new life and cell proliferation apparently higher than WT mice; IRF7 gene knockout can promote the expression of proliferating cell nuclear antigen (Proliferating Cell Nuclear Antigen, PCNA), can promote propagation and the neointimal hyperplasia of smooth muscle cell.The above results prompting IRF7 gene knockout can aggravate the generation of angiostenosis after damage, and IRF7 gene can suppress the generation of support and Endarterectomy postoperative restenosis.
A new function for IRF7 gene, is embodied in IRF7 and has the function that suppresses inner membrance new life and cell proliferation in support and Endarterectomy postoperative restenosis.
There is the function that suppresses inner membrance new life and cell proliferation for IRF7, the application of IRF7 in the medicine of preparation treatment angiostenosis disease is provided.
A medicine for the treatment of angiostenosis disease, comprises IRF7.
There is the function that suppresses inner membrance new life and cell proliferation for IRF7, the application of IRF7 in the medicine of preparing restenosis after treatment support is provided.
A medicine for the treatment of restenosis after support, comprises IRF7.
There is the function that suppresses inner membrance new life and cell proliferation for IRF7, the application of IRF7 in the medicine of preparation treatment Endarterectomy postoperative restenosis is provided.
A medicine for the treatment of Endarterectomy postoperative restenosis, comprises IRF7.
In the present invention's research, all there is blood vessel injury in wild-type mice and IRF7-KO mice under the induction of blood vessel injury model (VI), and compared with wild-type mice, IRF7-KO mice inner membrance new life and cell proliferation are remarkable.These results suggest, IRF7 has powerful ability of regulation and control to suppressing inner membrance new life and cell proliferation, the ability that has powerful angiostenosis removing and anti-support and Endarterectomy postoperative restenosis to form.The present invention has proved that IRF7 gene has important protective effect in blood vessel injury disease model.
The present invention has following advantage and effect with respect to prior art:
(1) the present invention finds the new function of IRF7 gene, and IRF7 gene has the effect that suppresses support and Endarterectomy postoperative restenosis.
(2) effect in inhibition support and Endarterectomy postoperative restenosis based on IRF7, IRF7 can be used for the medicine of preparation treatment angiostenosis disease, especially for the medicine of preparation treatment support and Endarterectomy postoperative restenosis.
Accompanying drawing explanation
Fig. 1 is HE dyeing and the Intimal area result statistics block diagram of WT and IRF7-KO mice; Wherein, the A:HE colored graph that dyes, B: block diagram.
Fig. 2 is immunofluorescence dyeing and the result statistics block diagram that WT and the postoperative 14d of IRF7-KO mice, the horizontal mark PCNA of 28d cells of vascular wall propagation express; Wherein, A: immunofluorescence dyeing, B: block diagram.
The specific embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
Animal for research and raising:
Laboratory animal kind, sex, week age and source: C57BL/6 mice (WT mice) and IRF7 knock out mice (IRF7-KO mice), male, 8-10 age in week, body weight 24-27g, C57BL/6 mice is purchased from magnificent Fukang, Beijing bio tech ltd; IRF7 knock out mice (IRF7-KO, C57BL/6J background) is purchased from RIKEN BRC company, and BRC numbers: RBRC01420.
Animal feeding and environmental condition: all experiment mices are all raised at the SPF of angiocardiopathy institute of Wuhan University level Animal House (credit number: SYXK(Hubei Province): 2009-0053).Alternately illumination in every 12 hours, 24 ± 2 ℃ of temperature, humidity 40%-70%, the mice feed of freely drinking water.
Embodiment 1 mice blood vessel damage model (VI) obtains
1. laboratory animal grouping: use 8-10 age in week, the WT of body weight 24-27g and IRF7-KO mice, be divided into four groups: WT blood vessel injury group; WT sham operated rats; IRF7-KO blood vessel injury group; IRF7-KO sham operated rats, every group of each 60 mices.20 mices of operation every group of each execution in latter 14 days, 28 days, get damage segmental vessels and analyze respectively.
2. mice blood vessel damage model operating process:
1) under dynamic mode, accurately take Mouse Weight (g) with electronic balance, with accurately configuration 3% pentobarbital sodium solution of distilled water, shake is fully dissolved it gently, adopt 80mg/kg body weight dosage, calculate after required pentobarbital sodium liquor capacity and accurately extract respective volume solution with 1mL syringe, row intraperitoneal injection of anesthesia mice, after mice is fully anaesthetized down (about 3min), 8% sodium sulfide cervical region depilation.
2) separate in neck and external carotid artery.
3) prick external carotid artery at internal carotid artery and external carotid artery crotch with 8-0 toe-in, use the temporary blocking-up internal carotid artery of vascular clamp (WPI, 501784-G) and common carotid artery blood supply simultaneously.
4) with microscissors (WPI, 501839) osculum of Transverse Shear above ligation of external carotid artery line.Insert the seal wire (No. C-SF-15-15, Cook, Bloomington, Indiana) of 0.015 inch of diameter through this blood vessel otch, rotation seal wire advance and retreat 5-6 time.
5) at otch proximal part ligation external carotid artery, unclamp in neck and common carotid artery is put the vascular clamp staying, cut off the end of a thread, cleaning visual area, sews up cervical incision (sham-operation is except not carrying out seal wire insertion and rotation advance and retreat, and other operations are all identical).
Embodiment 2 blood vessel injury model (VI) mice inner membrances are newborn to be measured
1. mice is drawn materials
1) anesthetized mice, breaks heart blood-letting.
2) cut carotid artery from the nearly crotch of carotid artery, get 0.5-0.6cm long, retain external carotid artery toe-in.
3) carotid artery is put into PBS, softly drain intraluminal residual blood with microforceps.
4) blood vessel is put into that the 1.5mL EP pipe of 1mL 4% paraformaldehyde is housed is fixing.
2. pathology detect
2.1 prepare paraffin specimen section
Prepare paraffin specimen section by laboratory specialty pathology staff, main operation sequence comprise the flushings → dehydration → transparent → waxdip → embedding → section of pruning heart → embedding frame processing → flowing water (3 μ m) → spread out sheet → dry or toast rear for subsequent use.
2.2 hematoxylin-eosins (HE) dyeing
Key step is: 55 ℃ of baking 30min → dimethylbenzene 5min, 3 times → 100% ethanol 1min → 95% ethanol 1min → 70% ethanol 1min → distilled water 1min → haematoxylin solution (Zhuhai shellfish rope, BA-4021) 5min → washing 1min → 1% hydrochloride alcohol (getting 3mL concentrated hydrochloric acid fully mixs homogeneously with 297mL 70% ethanol) 1-3s → washing 1min → Scott liquid (sodium bicarbonate 0.35g, magnesium sulfate 2g, distilled water 100mL) 1min → washing 1min → Yihong solution (Zhuhai shellfish rope, BA-4024) 3-5min → distilled water washes away loose colour → 70% ethanol 1s → 95% ethanol 1s → 100% ethanol 30s, 3 times → dimethylbenzene 2min, 3 times → take advantage of in the not dry mounting → fume hood immediately of dimethylbenzene and dry up, microscope is taken pictures.
Take elastic fibers in blood vessel and outer elastic fibers as boundary, interior elastic plate, take interior as tunica intima, is tunica adventitia beyond outer elastic plate, is tunica media between inside and outside elastic plate.Enclose respectively each vessel lumen area with Image-Pro Plus 6.0 softwares.
The calculating of Intimal area size is as follows with reference to formula:
New intima area=interior elastic plate area-tube chamber area;
Media area=outer elastic plate area-Nei elastic plate area.
The result of tunica intima new life after mice HE dyeing is as Fig. 1.Dye and can observe by HE, sham operated rats (Sham group) blood vessel wall structural integrity, marshalling, tunica intima is monolayer endothelial cell, structural integrity, middle film smooth muscle cell marshalling.Blood vessel injury group (VI group) blood vessel wall structure is imperfect, vascular endothelial cell disappearance, and new intima hypertrophy is obvious, and with a large amount of cell infiltration; IRF7-KO organizes that 14d new intima area is obviously high than WT mice after surgery, and it is more obvious that this deterioration acts on postoperative 28d.Equally, the ratio of Intimal area/media area will be higher than WT group in the postoperative IRF7-KO group of VI, and this to act on 28d more remarkable.
The detection of embodiment 3 cells of vascular wall propagation levels
Immunofluorescence dyeing detects the expression of proliferating cell nuclear antigen (Proliferating Cell Nuclear Antigen, PCNA).Required primary antibodie information: PCNA (#2586; 1:100; Mouse; Cell Signaling Technology); Required two anti-information: Alexa Fluor 568-conjugated goat anti-mouse IgG (A11004; Invitrogen, Carlsbad, 150 d, CA).
Key step is:
1) roasting sheet: more than paraffin section is placed in to baking box 30min.
2) dewaxing: dimethylbenzene 5min × 3.
3) hydration: 100% ethanol 5min × 2; 95% ethanol 5min; 70% ethanol 5min; ddH
2o embathes 5min × 2.
4) citrate tissue antigen recovery (Pressure method): get a certain amount of pH6.0 citrate antigen retrieval working solution in repairing box, necessary enough whole sections of submergence of amount of repair liquid, put into by repairing box the pressure cooker that adds appropriate tap water, big fire is heated to boiling, tissue slice after dewaxing hydration is placed on high temperature resistant staining rack, again staining rack is slowly put into and repaired box, cover pot cover, buckle pressure valve, continue to be heated to jet, start after timing 5min pressure cooker deenergization, go valve to uncap, take out and repair box; Room temperature is taken out section after placing 20min natural cooling.
5) ddH
2o rinsing 5min × 2 time, PBS rinsing 5min × 2 time.
6) groupization stroke circle, drips 10% sheep blood serum (GTX27481, GeneTex) sealing, 37 ℃ of sealing 60min in wet box.
7) abandon confining liquid, drip the primary antibodie of proper proportion dilution, 4 ℃ of overnight incubation, 37 ℃ of rewarming 30min, discard primary antibodie, and PBS washes 10min × 3 time.
8) drip two and resist, in wet box, hatch 60min for 37 ℃, discard two and resist, PBS embathes 5min × 3 time.
9) SlowFade Gold antifade reagent with DAPI(S36939, Invitrogen) mounting.
10) fluorescence Microscopic observation, takes pictures.Preserve 4 ℃ of preservations in dark wet box if need.
Fluorescence statistical method: PCNA immunofluorescence dyeing statistics adopts IPP software counting, total DAPI number * 100% of PCNA positive cell percentage=PCNA positive cell number/(inner membrance+middle film);
Immunofluorescence is sent out and is observed the expression variation of PCNA after WT and IRF7-KO mice blood vessel injury, the results are shown in Figure 2.PCNA has expression in vascular tissue, and the IRF7-KO mice after surgery positive cell number of 14d, 28d PCNA is greater than WT mice on the same group, shows that IRF7 gene knockout can promote the expression of PCNA, can promote the propagation of smooth muscle cell.
The demonstration of above-described embodiment result, under the induction of blood vessel injury model (VI), all there is blood vessel injury in wild-type mice and IRF7-KO mice, and compared with wild-type mice, IRF7-KO mice inner membrance new life and cell proliferation are remarkable.These results suggest, IRF7 has powerful ability of regulation and control, the ability that has powerful angiostenosis to remove to suppressing inner membrance new life and cell proliferation.Prove that IRF7 gene has important protective effect in blood vessel injury disease model, can be used for the medicine of preparation treatment angiostenosis disease.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (6)
- The application of 1.IRF7 in the medicine of preparation treatment angiostenosis disease.
- 2. a medicine for the treatment of angiostenosis disease, comprises IRF7.
- Application in the medicine of 3.IRF7 restenosis after preparation treatment support.
- 4. a medicine for the treatment of restenosis after support, comprises IRF7.
- The application of 5.IRF7 in the medicine of preparation treatment Endarterectomy postoperative restenosis.
- 6. a medicine for the treatment of Endarterectomy postoperative restenosis, comprises IRF7.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106390140A (en) * | 2016-10-11 | 2017-02-15 | 武汉大学 | Interferon regulatory factor 6 and function and application of inhibitor thereof in treating vascular injury restenosis |
| CN106390142A (en) * | 2016-10-11 | 2017-02-15 | 武汉大学 | Function and application of IRF5 (interferon regulatory factor-5) and IRF5 inhibitor in treatment of restenosis after VI (vascular injury) |
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| WO2007013391A1 (en) * | 2005-07-26 | 2007-02-01 | Japan Science And Technology Agency | Interferon-inducible molecule ips-1 |
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2014
- 2014-01-23 CN CN201410031620.1A patent/CN103784944A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2007013391A1 (en) * | 2005-07-26 | 2007-02-01 | Japan Science And Technology Agency | Interferon-inducible molecule ips-1 |
Non-Patent Citations (4)
| Title |
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| CLAUDIA BUSS等: "Essential Role of Mitochondrial Antiviral Signaling, IFN Regulatory Factor (IRF)3, and IRF7 in Chlamydophila pneumoniae-Mediated IFN-b Response and Control of Bacterial Replication in Human Endothelial Cells", 《THE JOURNAL OF IMMUNOLOGY》 * |
| DE VRIES, M等: "The TLR3 Pathway Including Downstream Interferon Regulating Factors, Regulates Vein Graft Remodeling", 《CIRCULATION》 * |
| XIN-AN WANG等: "Interferon regulatory factor 7 deficiency prevents diet-induced obesity and insulin resistance", 《AM J PHYSIOL ENDOCRINOL METAB》 * |
| 缪倩等: "IRF7表达与系统性红斑狼疮的相关性", 《华东师范大学学报(自然科学版)》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106390140A (en) * | 2016-10-11 | 2017-02-15 | 武汉大学 | Interferon regulatory factor 6 and function and application of inhibitor thereof in treating vascular injury restenosis |
| CN106390142A (en) * | 2016-10-11 | 2017-02-15 | 武汉大学 | Function and application of IRF5 (interferon regulatory factor-5) and IRF5 inhibitor in treatment of restenosis after VI (vascular injury) |
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Application publication date: 20140514 |