CN103520704A - Application of lampetra japonica genetic recombinant protein rLj-RGD3 in preparation of cerebral ischemia reperfusion protective medicament - Google Patents
Application of lampetra japonica genetic recombinant protein rLj-RGD3 in preparation of cerebral ischemia reperfusion protective medicament Download PDFInfo
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- CN103520704A CN103520704A CN201310407898.XA CN201310407898A CN103520704A CN 103520704 A CN103520704 A CN 103520704A CN 201310407898 A CN201310407898 A CN 201310407898A CN 103520704 A CN103520704 A CN 103520704A
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Abstract
The invention discloses application of lampetra japonica genetic recombinant protein rLj-RGD3 in preparation of a cerebral ischemia reperfusion protective medicament. Experimental results show that the lampetra japonica genetic recombinant protein rLj-RGD3 can be used for reducing nerve function deficits in a dose-dependent manner, reducing the infarction area ratio in a dose-dependent manner and improving brain tissue pathological morphological changes. The lampetra japonica genetic recombinant protein rLj-RGD3 has a very good protective effect on brain injuries caused by ischemia reperfusion, and the protective effect is superior to that of edaravone and eptifibatide, so that the lampetra japonica genetic recombinant protein rLj-RGD3 can be applied to the preparation of the cerebral ischemia reperfusion protective medicament.
Description
Technical field
The present invention relates to the new purposes of a kind of jamprey-ell gene recombinant protein rLj-RGD3, the especially application of jamprey-ell gene recombinant protein rLj-RGD3 in preparing cerebral ischemia reperfusion protection medicine.
Background technology
The health that the mankind in cerebral ischemia diseases serious threat is life even, and ischemia apoplexy has become one of mankind's main causes of death.Ischemia apoplexy, claims again cerebral infarction, is a kind of because local brain tissue ischemia, anoxia cause cerebral tissue softening, downright bad, and then shows as clinically the disease of corresponding neurological deficit symptom.
Integrin family is the important member of cell adhesion molecule family, mutually sticking between main mediated cell and cell, cell and extracellular matrix (ECM), and the conduction of the two-way signaling between mediated cell and ECM.The adhesion of mediated by integrin is regulating many cell functions, comprising apoptosis, cell proliferation, cell adhesion and migration, lymphocyte homing etc.At present, the specific recognition site of extracellular matrix protein and integrin receptor combination is determined, for arginine-glycine-aspartic acid (Arg-Gly-Asp, RGD) sequence, thus the polypeptide that contains RGD sequence can be identified integrin receptor albumen and with it in conjunction with the biological function that affects cell.
China Patent No. is that the patent of invention that ZL200510083437.7, name are called " gene cloning and expression of the Japanese lamprey oral gland RGD die body albumen of tool antitumor action " discloses three kinds of jamprey-ell gene recombinant proteins, comprises jamprey-ell gene recombinant protein rLj-RGD3.RLj-RGD3cDNA sequence 351bp is long, its protein is comprised of 117 aminoacid, contain three RGD die bodys, what it take that NdeI and HindIII restriction enzyme site be implemented in pET23b vector expression is 11.934 kDa with six histidine-tagged gene recombinant protein molecular weight, can obtain by molecular biology method.At present, there are some researches show that jamprey-ell gene recombinant protein rLj-RGD3 has the activity of angiogenesis inhibiting, antiplatelet aggregation, but the protective effect to acute cerebral ischemic reperfusion injury there is no research report to it.
Summary of the invention
The present invention has found that jamprey-ell gene recombinant protein rLj-RGD3 causes brain and is far apart organ injury cerebral ischemic reperfusion in rats and has protective effect, thereby has invented the application of jamprey-ell gene recombinant protein rLj-RGD3 in preparing cerebral ischemia reperfusion protection medicine.
Technical solution of the present invention is the application of a kind of jamprey-ell gene recombinant protein rLj-RGD3 in preparing cerebral ischemia reperfusion protection medicine.
The results show jamprey-ell gene recombinant protein rLj-RGD3 is dose dependent to ischemia-reperfusion and reduces nervous function damage, is dose dependent and reduces infarct size and when can improve histopathology morphological change; jamprey-ell gene recombinant protein rLj-RGD3 has good protective effect to the caused brain injury of ischemia-reperfusion; its protective effect is better than Edaravone and eptifibatide, can be applicable to prepare cerebral ischemia reperfusion protection medicine.
Accompanying drawing explanation
Fig. 1 is TTC coloration result figure after different experiments group rat MCAO/RF middle cerebral artery occlusion/pour into again 24 h.
Fig. 2 is the affect schematic diagram of different experiments group on rat MCAO/RF cerebral infarction volume.
Fig. 3 is different experiments group rat MCAO/RF Brain Injury Tissue HE colored graph.
The specific embodiment
1. experimental group grouping and administration: 48 of healthy male SD rats, body weight 280～320 g, laboratory condition adaptability is fed one week, preoperative fasting 12 h, freely drink water, be divided at random following A ~ F group (8 every group): A-sham operated rats, B-model group, high (the 100.0 μ gkg of C-rLj-RGD3
-1), low (the 50.0 μ gkg of D-rLj-RGD3
-1) dosage group, E-positive control drug Edaravone (1.5 mgkg
-1) group and F-eptifibatide (100 μ gkg
-1) group.Each organizes rat through cerebral ischemia 2 h, recover ischemic region blood flow and pour into after 10 min from sublingual vein administration again, and sham operated rats and model group waits capacity normal saline.
The jamprey-ell gene recombinant protein rLj-RGD3 of rLj-RGD3 used for obtaining according to above-mentioned patented method.
2. model preparation: 10 % chloral hydrate (0.35 mgkg
-1) after intraperitoneal injection of anesthesia, rat dorsal position is fixed on operating-table, preserved skin, along cervical region medisection skin, blunt separation subcutaneous tissue.With reference to bolt line blocked method (MCAO) in the intraluminal middle cerebral artery occlusion in rats of Zea Longal, with arteries and veins in line bolt method blocking-up brain, cause middle cerebral artery supply area blood flow to interrupt, focal cerebral ischemia.After ischemia 2 h, bolt line is slowly extracted out, recovered ischemic region blood flow and pour into again 24 h.Sham operated rats is only anaesthetized separation, exposure and ligation blood vessel, does not import bolt line.
Rat is poured into after 24 h through cerebral ischemia 2 h again, with 10 % chloral hydrate (0.35 mgkg
-1) intraperitoneal injection of anesthesia, from abdominal aortic blood, prepare serum to be measured; Quick broken end is got brain and is carried out TTC dyeing and HE dyeing.
3. observation index
3.1 TTC dyeing: after getting brain and with the residual blood of normal saline flushing, excision olfactory bulb, cerebellum and low brain stem, interval 2 mm are crown to be cut into 5 continuous brain sheets (the first cutter is the utmost point and optic chiasma line midpoint before brain; The second cutter is in optic chiasma place; The 3rd cutter is in infundibular stalk position; Four blade is between infundibular stalk and leaf tail core), be placed in 2 %TTC buffer solution, 37 ℃ of lucifuges, water-bath 20 min, during every 7～8 min, stir and once make brain sheet even dyeing.TTC can be dyed redness by mitochondrion catalase reduction ，Hui Jiang normal cerebral tissue, and ischemic infarction district is because being white in color without this reaction.
Result is as shown in Figure 1: A group in Fig. 1: cerebral tissue has no infarct, and full brain is redness; B group: cerebral infarction volume is maximum; Each administration group of C-D is compared infarct size with model group all have remarkable reduction.
3.2 cerebral infarction volume ratios: brain section is after TTC dyeing, fixing in 10 % formaldehyde.Every brain sheet tow sides are taken pictures, utilize Image Pro-Plus photoshop 6.0 computed in software to go out Infarction volume, and calculate cerebral infarction volume ratio.Computing formula is as follows: Infarction volume ratio=[(each sheet positive and negative Infarction volume sum/2) * sheet thick/(each sheet positive and negative gross area sum/2) * sheet is thick] * 100%.Result is as shown in Figure 2: C, D and E, F group significantly reduce (p<0.05) to the impact of cerebral infarction volume and model group, and high dose (100 μ gkg<sup TranNum="85">-1</sup>) effect and positive drug Edaravone 1.5 mgkg of rLj-RGD3<sup TranNum="86">-1</sup>effect is (p>0.05) quite, low dose group (50 μ gkg<sup TranNum="87">-1</sup>) effect and the similar medicine eptifibatide 100 μ gkg of rLj-RGD3<sup TranNum="88">-1</sup>effect is (p>0.05) quite, shows that rLj-RGD3 is all stronger than above-mentioned two kinds of positive drug effects.
3.3 tectologies change: HE dyes by optical microphotograph Microscopic observation histopathology morphological change and takes pictures, result is as shown in Figure 3: A group cerebral tissue damages phenomenons without other pathomorphology such as edema, hyperemia, cellularity is fine and close, sharpness of border, neuron morphology structural integrity, marshalling, nucleus is without pyconsis; B group rat cerebral tissue cerebral cortex has congested phenomenon, intercellular substance edema, and cell peripheral is loosely organized, neuron arrangement disorder, the swelling of partial nerve unit; C group, D group, E group, F group are compared tissue edema with model group and are alleviated; cellularity is finer and close, a small amount of neuron swelling, and neuronal damage alleviates; illustrate that rLj-RGD3 has good protective effect to the caused brain injury of ischemia-reperfusion, and its protective effect is better than E group and F group.
By experiment, can draw the following conclusions:
Function of nervous system's scoring and infarct size ratio: after ischemia/reperfusion (2 h, 24 h), compare with sham operated rats, model group rat has obvious function of nervous system defect (scoring 8.88,7.50 is learned by function of nervous system); Compare with model group, rLj-RGD3 is dose dependent and reduces nervous function damage (p < 0.01, high and low dose function of nervous system learns and is respectively 4.50,3.25 and 5.88,4.62).Model group infarct size comparison sham operated rats obviously raise (40.56 %) after ischemia/reperfusion 24 h; Compare with model group, rLj-RGD3 is dose dependent and reduces infarct size than (p < 0.01).
Cerebral morphology changes: after rat MCAO/RF ischemia/reperfusion (2 h, 24 h), model group rat cerebral tissue edema is obvious, pyramidal cell arrangement disorder, and cell boundaries is unclear, and rLj-RGD3 can improve histopathology morphological change.
Claims (1)
1. the jamprey-ell gene recombinant protein rLj-RGD3 application in preparing cerebral ischemia reperfusion protection medicine.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103694330A (en) * | 2013-11-25 | 2014-04-02 | 辽宁师范大学 | Preparation method of affinity chromatography purification tag-free genetic recombinant lamprey Lj-RGD3 protein |
| CN104137799A (en) * | 2014-08-08 | 2014-11-12 | 中山大学 | Artificial breeding method for wild lampetra morii |
| CN108187030A (en) * | 2018-01-31 | 2018-06-22 | 辽宁师范大学 | Recombinant peptide rLj-RGD3 is preparing the application in preventing and treating acute myocardial infarction AMI drug |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1760362A (en) * | 2005-07-13 | 2006-04-19 | 辽宁师范大学 | Gene clone and expression of RGD die body protein of oral gland in Japan lamprey possessing function for anti tumour |
| WO2010127159A2 (en) * | 2009-04-30 | 2010-11-04 | Intezyne Technologies, Incorporated | Polymeric micelles for polynucleotide encapsulation |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1760362A (en) * | 2005-07-13 | 2006-04-19 | 辽宁师范大学 | Gene clone and expression of RGD die body protein of oral gland in Japan lamprey possessing function for anti tumour |
| WO2010127159A2 (en) * | 2009-04-30 | 2010-11-04 | Intezyne Technologies, Incorporated | Polymeric micelles for polynucleotide encapsulation |
Non-Patent Citations (1)
| Title |
|---|
| WANG JIHONG ET AL.: ""A novel RGD-toxin protein, Lj-RGD3, from the buccal gland secretion of Lampetra japonica impacts diverse biological activities"", 《BIOCHIMIE》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103694330A (en) * | 2013-11-25 | 2014-04-02 | 辽宁师范大学 | Preparation method of affinity chromatography purification tag-free genetic recombinant lamprey Lj-RGD3 protein |
| CN103694330B (en) * | 2013-11-25 | 2016-01-06 | 辽宁师范大学 | Go the preparation method of the recombinant lamprey Lj-RGD3 albumen of affinitive layer purification label |
| CN104137799A (en) * | 2014-08-08 | 2014-11-12 | 中山大学 | Artificial breeding method for wild lampetra morii |
| CN104137799B (en) * | 2014-08-08 | 2016-03-30 | 中山大学 | The artificial breeding method of wild northeast lamprey |
| CN108187030A (en) * | 2018-01-31 | 2018-06-22 | 辽宁师范大学 | Recombinant peptide rLj-RGD3 is preparing the application in preventing and treating acute myocardial infarction AMI drug |
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