JP2005068122A - Application of midkine for prevention and treatment of myocardiopathy - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To elucidate a mechanism of midkine (MK) for myocardium and effect on myocardiopathy for aiming at the application of the MK for the prevention and treatment of the myocardiopathy. <P>SOLUTION: A myocardium ischemia reperfusion model is prepared by using a wild type mouse for analyzing the appearance pattern of the MK. Also, a similar model is prepared by using an MK-deleted mouse for elucidating the role of the MK in a living body by investigating the similar model comparatively with the case of the wild type mouse. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

Detailed Description of the Invention

  The present invention relates to the prevention and treatment of myocardial injury.

  Midkine (MK) is a heparin-binding growth factor discovered as a gene product that is transiently expressed during the differentiation induction process of embryonic tumor cells by retinoic acid. (Kadomatsu, K. et al .: Biochem. Biophys. Res. Commun., 151: 1312-1318, 1990). MK maintains a high degree of protein conservation between species, and human and mouse MKs have 87% homology in amino acid sequences. MK has various biological activities such as survival and differentiation of nerve cells, tissue repair, and fibrinolytic system activation ability, and its relevance to pathological conditions is also attracting attention. In particular, expression of MK increases in many human cancers, and normal cells become cancerous by forced expression by introduction of MK cDNA (Kadomatsu, K. et al .: Br. J. Cancer, 75: 354-359, 1997). Therefore, it is considered to be deeply involved in the development and development of human cancer. In addition, in knockout mice lacking the MK gene, the formation of neointima is remarkably reduced in the carotid artery injury model, and therefore MK is considered to be involved in the occurrence of vascular lesions (Horiba, M. et al .: J. Clin. Invest. , 105: 489-495, 2000). Furthermore, MK also has an apoptosis inhibitory effect by enhancing the expression of Bcl-2 (Qi, M. et al .: J. Biochem., 127: 269-277, 2000). By applying these MK biological activities, it is considered that there is a therapeutic effect on several cytotoxic diseases.

  Myocardial disorders such as ischemic heart disease and dilated cardiomyopathy are fatal diseases, and even if they can maintain their lives, there is a high possibility of falling into heart failure, and the quality of life is significantly limited. In particular, there is currently no curative treatment for severe heart failure, and heart transplantation is the only effective means, but the donor shortage continues worldwide. In recent years, attention has been focused on apoptosis in the process of myocardial injury (Ginard, BN. Et al. J. Clin. Invest., 111: 1457-1459, 2003), and MK is expected to lead to prevention of myocardial injury. Is done.

Problems to be solved by the invention

  It is an object of the present invention to clarify the mechanism of MK to the myocardium and to elucidate the effect on the myocardial disorder with the main purpose of preventing and applying myocardial damage to treatment.

Means for solving the problem

  As a model of myocardial injury, a wild-type mouse myocardial ischemia / reperfusion model was prepared, and the MK expression pattern in the myocardium after reperfusion was examined by Western blotting, and the localization of MK was examined in detail by immunohistochemical staining. Furthermore, in order to examine the influence of MK in the living body, the same model was created even in MK-deficient (MKKO) mice, and the degree of lesion was compared with that of wild-type mice. As an analysis of the direct action of MK on myocardial injury, MK was added to cultured cardiomyocytes in which apoptosis was induced, and suppression of cell death was examined.

  Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. FIG. 1 is Western blotting in which the MK expression pattern after myocardial ischemia reperfusion was examined. FIG. 2 demonstrates MK expression in the myocardium by immunohistochemical staining. FIG. 3 is a comparative study of the degree of myocardial injury in wild-type and MKKO mice.

    A method for creating a mouse myocardial ischemia / reperfusion model will be described. After abdominal anesthesia with pentobarbital, intubate intubation and ventilator management. After the left parathoracic incision is made to expose the heart, the left coronary artery is ligated under a stereomicroscope and rereleased 1 hour later. After closing the chest, confirm the appearance of spontaneous breathing, extubate, wait for recovery, and return to normal breeding.

    The expression of MK by Western blotting will be described. For wild-type mice, the heart of the reperfusion model is taken out over time, such as 6 hours, 12 hours, and 24 hours after myocardial ischemia reperfusion, and protein is extracted. After SDS gel electrophoresis (SDS-PAGE) for the same amount of protein as each, Western blotting using anti-MK antibody was performed, and the time-dependent change in MK protein expression was evaluated by quantification with a densitometer To do.

    The evaluation of MK expression by immunohistochemical staining will be described. Wild-type mice 24 hours after myocardial ischemia / reperfusion are fixed with 4% paraformaldehyde, and then the heart is removed. After embedding in paraffin, 5 μm-thick sections are prepared. The enzyme antibody method is performed using an anti-MK antibody as a primary antibody and an HRP-labeled IgG antibody as a secondary antibody, and then the color of the MK protein is evaluated by color development with a chromogenic substrate (AEC).

    A method for evaluating myocardial injury will be described. After 24 hours after the creation of a myocardial ischemia / reperfusion model, the chest was collected again and blood was collected. The left coronary artery was ligated, and Evans Blue was injected into the beating heart chamber. Then, the normal site where the coronary blood flow is maintained is stained blue, and the other sites are identified as ischemic risk areas. After cardiac arrest, the heart is removed and cut into approximately 1 mm thick sections and stained again with TTC. Of the risk area, a portion stained red with TTC is a region where the myocardial survival is maintained, and a complete infarct area remains white. By measuring the areas of these colors and comparing the ratios, the degree of myocardial injury can be quantified. Furthermore, myocardial damage is evaluated from various aspects by measuring serum CPK.

EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to these Examples.

(Example 1) The pattern of MK expression after myocardial ischemia reperfusion was analyzed by Western blotting. As shown in FIG. 1, only a slight MK expression was observed in normal myocardium, but gradually increased from 12 hours after the operation, and a peak of expression was observed at 24 hours. Relatively strong expression persisted after 48 hours.

(Example 2) The expression range of MK was evaluated by immunohistochemical staining for specimens 24 hours after myocardial ischemia reperfusion. As shown in FIG. 2a, MK was strongly expressed around a normal site around the infarction (a site stained red). The boundary of the expression region is clear, and when it is strongly expanded, expression restricted mainly to the myocardial cell membrane was observed (FIG. 2b). When combined with the findings in (Example 1), MK was strongly expressed in the normal myocardial region around the infarct lesion after myocardial ischemia reperfusion, suggesting that it is involved in the repair of myocardial tissue.

(Example 3) The degree of myocardial injury 24 hours after myocardial ischemia-reperfusion was compared between wild-type mice and MKKO mice, and the effect of MK in the living body was examined. As shown in FIG. 3, the myocardial infarction area that remained white without staining with Evans Blue or TTC was significantly larger in the MKO mice than in the wild type, and in the blood showing the degree of damage. CPK was also significantly higher in MKKO mice. Furthermore, the mortality rate within 24 hours after surgery was also significantly higher in MKKO mice. From these facts, it was shown that myocardial damage worsens when MK is deficient in myocardial ischemia reperfusion.

The invention's effect

  As described above with reference to the drawings, MK is expressed around the impaired myocardium, indicating that the progression of the disorder is suppressed. If it summarizes as a conceptual diagram, it will be guessed that MK is acting like FIG. From these facts, it is considered that administration of MK to the impaired myocardium leads to prevention of heart failure.

  It is a figure of Western Blotting which shows the pattern of MK expression after myocardial ischemia reperfusion concerning one Example of this invention.   It is a figure of the immunohistochemical staining which showed the expression range of MK in connection with one Example of this invention.   It is the figure which compared the grade of the myocardial injury 24 hours after ischemia reperfusion concerning one Example of this invention between a wild-type-MKKO mouse | mouth.   It is the conceptual diagram put together from the Example of this invention.

Claims (1)

Prevention of myocardial damage by midkine.