KR100573881B1 - Pharmaceutical Composition for Recovering Ischemia - Google Patents

Abstract

Phospholipase C-δ (phospholipase C-δ, PLC-δ) as an active ingredient, and relates to a therapeutic agent for ischemic symptoms comprising a pharmaceutically acceptable carrier. The ischemic treatment agent of the present invention can prevent a symptom of causing an ischemic state due to deposition of excess calcium in a cell, and thus may be widely used for the prevention and treatment of ischemia.

Phospholipase C-δ, excessive deposition of calcium, ischemia

Description

Pharmaceutical Composition for Recovering Ischemia             

1 is an electrophoresis picture showing the degree of expression of PLC in each site.

Figure 2 is a graph showing the expression level of PLC-δ1 with time after occlusion.

Figure 3 is an electrophoresis picture showing the expression of PLC over time in cultured cardiomyocytes in ischemia.

4 is a graph showing the effect of protease inhibitors on the expression of PLC-δ.

5 is a genetic map of the expression vector pcDNA3.1-HA (PLC-δ1).

6 is a graph showing the degree of calcium deposited on the sample using the fluorescence of fluorochrome.

The present invention relates to a treatment for ischemic symptoms. More specifically, the present invention relates to a therapeutic agent for ischemic disease comprising phospholipase C-δ (PLC-δ) as an active ingredient and a pharmaceutically acceptable carrier.

Destruction of calcium homeostasis in cardiac cells leads to various physiological changes, including loss of consciousness and cardiac muscle ischemia accompanied by mitochondrial dysfunction, and reduction of contractility associated with electrical ischemia, which is initially associated with ischemic acidosis. Of the contractile proteins induced by and stimulated by magnesium and inorganic phosphates are notable, and these symptoms are the same not only in vivo but also in cultured heart muscle (Tanaka M., et. al., Circ.Res., 75: 426-433, 1994; Long X., et al., J. Clin. Invest., 99: 2635-2643, 1997; Chen SJ, et al., Mol. Biochem., 178: 141-149, 1998; Bialik S., et al., Circ.Res., 85: 403-414, 1999).

It is known that the main cause of this ischemia is the excessive accumulation of calcium ions in the heart muscle, and this increase in calcium is caused by intracellular intracellular activities such as phospholipase C (PLC), protein kinase, protease, and nuclease. It is known to change levels (Bolli R. and Marban E., Physiol. Rev., 79: 609-634, 1999).

In particular, phospholipase C hydrolyzes membrane phospholipids and phosphatidylinositol 4,5-bisphosphate (PIP ₂ ) to diacylglycerol (DAG) and inositol 1,4,5-tri It produces phosphate (inositol 1,4,5-triphosphate, IP ₃ ), which has been reported to mediate intracellular calcium transport with protein kinase C (PKC). SG and Bae YS, J. Biol. Chem., 272: 15045-15048, 1997). To date, 11 PLC isozymes have been reported, which are largely classified into four groups: PLC-β, PLC-γ, PLC-δ, and PLC-ε. Among them, PLC-β, PLC-γ and PLC-ε are activated by G-protein coupled receptor, receptor tyrosine kinase and Ras pathway, PLC-δ is GTP binding It is known to be activated by proteins. In particular, PLC-δ1 is known to be activated by the α ₁ -adrenergic pathway to which transglutaminase II (G _h ), a type of GTP binding protein, functions (see Rhee SG, Annu). Rev. Biochem., 70: 281-312, 2001; Hwang KC and Gray CD, J. Biol. Chem., 270: 27058-27062, 1995). Electrical pathways have been reported to play an important role in signaling systems that regulate calcium homeostasis and regulate physiological control processes such as blood pressure control and neurotransmitter release (Murthy SNP, Proc. Natl). Acad. Sci., USA., 96: 11815-11819, 1999).

Because all PLC isoenzymes contain C2 domains that react with calcium ions, and among PLC isoenzymes, PLC-δ1 has a three-dimensional structure that is most sensitive to intracellular calcium ions, the study of PLC-δ1 It is expected to provide important clues in the study of receptor-mediated and calcium channel activation of related PLC isoenzymes (Rebecchi MJ, et al., Physiol. Rev., 80: 1291-1335, 2000). To date, the primary hydrolysates of PIP ₂ , DAG, and IP ₃ by PLC in the ischemic heart muscle caused by excessive calcium deposition are important for intracellular signaling and activation of PKC through intracellular calcium transport. Only studies that play a role have been reported (Hansen CA, et al., J. Mol. Cell. Cardiol., 27: 471-484, 1995; Schnabel P., et al., J. Mol Cell.Cardiol., 28: 2419-2427, 1996).

Increased intracellular calcium ions activate protein kinases and calcium activating proteolytic enzymes. These activated proteases degrade proteins that regulate the level of intracellular calcium, reducing their reactivity to calcium ion concentrations. In addition, excess calcium ions are deposited in cells that have decreased reactivity to calcium ions, and excessively deposited calcium ions change the physiological activity of the tissue. In particular, in the case of cardiac muscle, as described above, excessive deposition of calcium ions may lead to cardiac ischemia, which may result in death of the individual, thereby preventing the level of calcium ions from being increased within a certain level. Efforts have been made to develop ways to do this, but no results have been reported.

Therefore, there is a constant need to develop a method for treating ischemia by regulating the level of calcium ions in the cell not to be raised above a certain level.

Therefore, the present inventors have made intensive studies to develop a method for treating cardiac ischemia by regulating the level of calcium ions in the cell not to be increased above a certain level. Excessive deposition of calcium ions was reduced, confirming that it is possible to treat ischemic symptoms, and completed the present invention.

After all, the main object of the present invention is to provide a treatment for ischemic symptoms comprising PLC-δ as an active ingredient.

The agent for treating ischemic symptoms of the present invention is phospholipase C-δ (PLC-δ) as an active ingredient, and includes a pharmaceutically acceptable carrier, wherein the phospholipase C-δ used is Although not particularly limited thereto, it is preferable to use phospholipase C-δ1 (PL-δ1, PLC-δ1).

The inventors note that the ischemic state of cardiac muscle is mainly caused by excessive deposition of calcium, and that all PLC isoenzymes have a C2 domain capable of reacting with calcium, and thus, how PLC isozymes affect calcium metabolism. I wanted to see. First, as a result of measuring the expression patterns of various PLC isoenzymes in the ischemic heart, only PLC-δ1 (SEQ ID NO: 1), belonging to the PLC-δ family, is expressed over time in the ischemic heart muscle. It was found that the level decreased.

As a result of analyzing the cause of the decrease of the expression level of PLC-δ1 over time in the ischemic heart muscle, it was confirmed that PLC-δ1 is degraded by calcium-activated protease, It was found that -δ1 was directly affected. Increased intracellular calcium ions activate protein kinases and calcium-activated proteolytic enzymes. These activated proteases degrade proteins that regulate the levels of intracellular calcium, resulting in decreased reactivity to calcium ion concentrations. In light of this, we expect that PLC-δ1 will play a role in regulating intracellular calcium levels and predict that intracellular calcium levels can be controlled by increasing intracellular PLC-δ1 levels. It was.

In order to prove the above prediction, the present inventors construct an expression vector pcDNA3.1-HA (PLC-δ) capable of expressing PLC-δ1 in eukaryotic cells, and introduce it into cardiomyocytes to artificially induce an ischemic state. After the calcium concentration was measured. As a result, it was confirmed that the intracellular calcium concentration did not increase even in the ischemic state in the cardiomyocytes into which the electric expression vector was introduced.

Therefore, the ischemic symptom treatment agent of the present invention can prevent the symptom of causing an ischemic state due to the deposition of excess calcium in the cell, and thus may be widely used for the prevention and treatment of ischemia.

On the other hand, the ischemic symptoms therapeutic agent containing PLC-δ of the present invention as an active ingredient and a pharmaceutically acceptable carrier can be administered in an injection form. The injectable compositions are preferably aqueous isotonic solutions or suspensions, and the compositions mentioned are sterile and / or adjuvants (e.g., preservatives, stabilizers, wetting or emulsifier solution promoters, salts and / or buffers for controlling osmotic pressure). It contains. In addition, they may contain other therapeutically valuable substances.

Effective amount

In the present invention, the dose of the active ingredient PLC-δ may be administered once daily to 0.1 to 1 mg / kg body weight, depending on the age, sex, symptoms, administration method or prevention of the patient. Dosage levels for patients with specific symptoms may vary by those skilled in the art depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion, severity of disease, and the like.

Acute Toxicity Test of PLC-δ

6 to 7-week-old non-rodent beagle dogs (beagle) determining the LD ₅₀ values, and subcutaneously injected PLC-δ of the present invention, to measure the number of deaths of beagles over the 7 days after administration targeting bar, hayeotneun LD ₅₀ The value was about 580 mg / kg. Therefore, it was found that the ischemic symptom treatment agent containing PLC-δ of the present invention as an active ingredient in the range of the effective amount indicated above was a sufficiently safe drug.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 : Change of PLC isoenzyme expression in ischemic heart muscle

In order to observe the expression of PLC isoenzyme in ischemic heart muscle, myocardial infarction was induced by surgically occluding the left artery from the rat to the coronary artery. That is, after 200 days of age, 30 g of male Sprague-Dawley rats were anesthetized with 10 mg / kg of ketamine and 5 mg / kg of xylazine, and then the third and fourth ribs were removed. The amputation opened the chest and the heart was pulled through the intercostal space. The left coronary artery was then occluded 2-3 mm from its origin using a 5-0 prolen suture (ETHICON, UK) and the heart was repositioned to induce myocardial infarction. As a control, rats which did not occlude the coronary artery were used.

The heart of the experimental group and the control group were removed, and the heart of the experimental group was separated into the border and wound areas, and then each was homogenization buffer (homogenization buffer, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 10 µg / ml leupetin, Homogenized in 10 μg / ml aprotinin, and 4 μg / ml of calpine inhibitors I and II, 10 mM Tris-HCl buffer, pH 7.4), and centrifuged at 4 ° C., 100,000 × g for 1 hour to obtain supernatant. . KCl was added to the supernatant to 2M, stirred at 4 ° C. for 2 hours, centrifuged at 35,000 × g for 30 minutes to give a supernatant, and dialyzed overnight in 4L homogenization buffer, followed by the same conditions. Centrifugation gave a supernatant containing 80 mg of protein. The supernatant obtained above was applied to a Heparin-Sepharose CL-6B column equilibrated with 20 mM HEPES-NaOH (pH 7.0) containing 1 mM EGTA and 0.1 mM DDT, and eluted with an equilibration buffer containing 1.2 M NaCl. , An active fraction showing PLC activity was obtained. At this time, PLC activity measurement was performed by measuring the hydrolytic activity using [ ³ H] -PIP ₂ and [ ³ H] -PI as a substrate. Hydrolytic activity using [ ³ H] -PIP ₂ as substrate was measured for lipid colloid (12 μM [ ³ H] -PIP ₂ , 12,000 cpm), 50 mM HEPES-NaOH (pH 7.0), 0.1% sodium deoxycholate, 120 mM KCl 5 μl of the sample was mixed in a 95 μl reaction mixture containing 10 mM NaCl, 2 mM MgCl ₂ , 2 mM EGTA, and 1.4 mM CaCl ₂ , and reacted at 30 ° C. for 10 minutes, followed by [ ³ H] levels. Hydrolysis activity using [ ³ H] -PI as a substrate was performed at 150 μM [ ³ H] -PI (20,000 cpm), 50 mM HEPES-NaOH (pH 7.0), 3 mM CaCl ₂ , 2 mM EGTA, and 0.1 50 μl of the sample was mixed with 150 μl reaction mixture solution containing% sodium dioxycholate, reacted at 30 ° C. for 10 minutes, and then measured by measuring the level of [ ³ H] in the reaction solution.

The previously obtained active fraction was applied to a TSKgel heparin-5PW HPLC column (7.5X75 mm) equilibrated with 20 mM HEPES-NaOH (pH 7.0) containing 1 mM EGTA and 0.1 mM DDT, washed with 15 ml of equilibration buffer, , 40 mL of an equilibration buffer containing NaCl having a concentration of 0 to 0.64 M and 10 mL of an equilibration buffer containing NaCl having a concentration of 0.64 M to 1M were eluted to obtain an active fraction showing PLC activity. The activity fractions obtained previously were analyzed by immunoblot method using antibodies of PLC-δ1, PLC-γ1, PLC-β1 and PLC-β3, and the expression levels of PLC-δ1, PLC-γ1, PLC-β1 and PLC-β3 were analyzed. Was measured (see FIG. 1). Figure 1 is an electrophoresis picture showing the degree of expression of PLC at each site, N represents the control, B represents the border, S represents the wound. As shown in FIG. 1, PLC-δ1, PLC-β1, PLC-β3 and PLC-γ1 were expressed at a constant level in the control group, but PLC-β1, PLC-β3, and PLC-γ1 were normally expressed in the experimental group, but PLC The expression of -δ1 was found to be significantly reduced.

Therefore, in order to determine the expression level of PLC-δ1 according to the infarction time, the boundary area and the wound area of the experimental group immediately after the occlusion procedure, 2 hours after the occlusion procedure, 1 day after the occlusion procedure, and 3 days after the occlusion procedure, PLC-δ1 was obtained from the control group, respectively, and the expression level thereof was measured (see FIG. 2). Figure 2 is a graph showing the expression level of PLC-δ1 over time after the occlusion procedure, (□) represents the control group, (경계) represents the border site, (■) represents the wound site. As shown in FIG. 2, the level of PLC-δ1 expression in the border and wound area suddenly decreased at the time of day 1 after the occlusion procedure, and no further decrease was observed even after a lapse of time.

Example 2 Change in the Expression Pattern of PLC in Ischemia

In order to analyze the result of Example 1, it was confirmed that the cardiomyocytes were cultured to show the same result even when artificial ischemia was induced.

First, ventricular cells were obtained from the heart of 1 to 2 day old Sprague-Dawley rats, suspended in a culture buffer solution (α-MEM, Gibco-BRL, USA) containing 0.1 mM BrdU, and then cultured in a culture dish. The cells were inoculated at a concentration of 10 ⁵ cells / ml and then cultured in a cell incubator at 37 ° C. and 5% CO ₂ . Then, a modified crab buffer (137 mM NaCl, 3.8 mM KCl, 0.49) containing 1 mM 2-dioxyglucose, 20 mM sodium lactate, 12 mM KCl, 1 mM sodium dithionite, and 0.2% FBS pH 6.5 Ischemic state was induced by replacing the culture with mM MgCl ₂ , 0.9 mM CaCl ₂ , 4 mM Hepes) and then incubating for several hours at 37 ° C. Subsequently, after 1 day, 2 days, and 3 days after the ischemic induction, sample cells were obtained, and the expression levels of PLC-δ1, PLC-β1, and PLC-γ1 were measured in each sample (see FIG. 3). ). Figure 3 is an electrophoresis picture showing the expression of PLC over time in cultured cardiomyocytes in ischemia. As shown in FIG. 3, the expression levels of PLC-β1 and PLC-γ1 did not change with time, but PLC-δ1 decreased with time.

Example 3 Effect of Protease on PLC-δ

As shown in Example 2, in the ischemic state, since the level of PLC-δ decreased with time, it was intended to determine whether such a decrease in PLC-δ was due to proteolytic enzymes. That is, in the cultured cardiomyocytes inducing ischemic state in the same manner as in Example 2, inhibitors of calpastatin and caspase, which are inhibitors of calpain, a calcium-activated protease zVAD-fmk was added at the beginning of culture at concentrations of 100 mM and 10 μM, respectively, and the expression level of PLC-δ was measured by immunoblot method. At this time, the control group was used as a control group not treated with a protease inhibitor on the cardiomyocytes obtained from normal rats (see Figure 4). Figure 4 is a graph showing the effect of protease inhibitors on the expression of PLC-δ, lane 1 is a control, lane 2 is a cardiomyocyte that induced ischemic state, lane 3 is an ischemic state Cardiac cells were treated with calpastatin, and lane 4 shows ischemia-induced cardiomyocytes with calpastatin and zVAD-fmk. As shown in Figure 4, the protease inhibitors were found to inhibit the degradation of PLC-δ1 in ischemic cardiomyocytes.

Example 4 Effect of PLC-δ1 on Calcium Ion Excess Deposition in Ischemic Cells

To investigate the effect of PLC-δ on calcium ion overdeposition in ischemic cells, calcium deposition was measured in ischemic cardiomyocytes overexpressed with PLC-δ1.

First, an expression vector pcDNA3.1 capable of expressing PLC-δ1 in eukaryotic cells by inserting cDNA (SEQ ID NO: 1) encoding PLC-δ1 into a commercially available pcDNA3.1-HA vector (Invitrogen, USA). -HA (PLC-δ1) was constructed. Ie, the pIBI20 vector known to contain the PLC-δ1 gene (Junliang Pan et al., J. Biol. Chem., 273 (16): 10058-10067, 1998; John A. et al., Proc. Natl Acad.Sci., USA, 95 (2): 638-645, 1998) was digested with HindIII and inserted into the HindIII locus of the pcDNA3.1-HA vector to express the expression vector pcDNA3.1-HA (PLC-δ1). Was constructed (see FIG. 5). 5 is a genetic map of the expression vector pcDNA3.1-HA (PLC-δ1).

Myocardial cells cultured in Example 2 (control), ischemic heart-induced cardiomyocytes (Experimental Group 1), the ischemic state is induced by the method of Example 2, and the expression vector pcDNA3.1-HA (PLC- cardiomyocytes transduced with δ1, overexpressed PLC-δ1 (experimental group 2) and ischemic state induced by the method of Example 2, and treated with 100 mM calpastatin-treated cardiomyocytes (Example 3) Experiment group 3) was prepared, and the control group and the experimental group, which were prepared before, were incubated for 3 days, and then the degree of calcium deposition in each control group and the experimental group was measured (see FIG. 6).

At this time, cardiomyocytes overexpressing PLC-δ1 were constructed as follows. Cardiomyocyte cells cultured by the method of Example 2 were washed with serum-free DMEM (Sigma Chem. Co., USA) medium and diluted with the same medium as LIPOFECTAMIN PLUS (5 μg / ml, Sigma Chem. Co., USA). ) Was mixed with 5 μg of the expression vector pcDNA3.1-HA (PLC-δ1) and added to electro washed cardiomyocytes. Then, the cells were incubated for 12 hours in a 37 ° C. CO ₂ cell incubator, and the culture medium was replaced with DMEM medium containing 10% (v / v) fetal calf serum (FBS), followed by another 48 hours.

In addition, the deposition degree of calcium was measured by the following method. Each cardiomyocytes obtained from the control group and each experimental group were inoculated on a cover slip of glass material at 5 μg / cm ² , and α- containing 0.1 μM BrdU and 10% (v / v) fetal calf serum (FBS). After culturing for one day in MEM medium, 0.265 g / l CaCl ₂ , 0.214 g / l MgCl ₂ , 0.2 g / l KCl, 8.0 g / l NaCl, 1.0 g / l glucose, 0.05 g / l Washed with a modified Tyrode's solution containing NaH ₂ PO ₄ and 1.0 g / L of NaHCO ₃ . Then, 5 μM acetoxymethyl ester of Fura-2 kit (Fura-2 AM, Molecular probes, Eugen, Germany) for calcium ion measurement was added and left in the dark for 20 minutes at room temperature, followed by argon laser confocal microscopy ( confocal microscopy, Leica, Solms, Germany) was used to measure the fluorescence intensity of the fluorochromes emitted. The fluorescence of the electrofluorochromes was emitted by an argon laser 488 mm line, and the emitted fluorescence was measured through a 510-560 mm bandpass filter.

6 is a graph showing the degree of calcium deposited on the sample using the fluorescence of fluorochrome. As shown in Figure 6, compared to the calcium deposited on the ischemic cell induced, the cells overexpressing PLC-δ1 is the same low calcium deposition as the control and calpastatin-treated cells not induced ischemic state The degree was shown.

According to the results of Examples 1 to 4, ischemic state is induced when excessive calcium ions are deposited in cardiac muscle cells, and PLC-δ1 is rapidly degraded by calcium activating protease in ischemic state. In addition, overexpression of PLC-δ1 in cardiac muscle cells in the ischemic state showed that at least calcium excessive deposition was suppressed. Using PLC-δ1 prevents ischemia caused by excessive deposition of intracellular calcium. It was judged to be.

As described in detail and demonstrated above, it provides a therapeutic agent for ischemic symptoms comprising phospholipase C-δ as an active ingredient and a pharmaceutically acceptable carrier. The ischemic treatment agent of the present invention can prevent a symptom of causing an ischemic state due to deposition of excess calcium in a cell, and thus may be widely used for the prevention and treatment of ischemia.

<110> JANG, Yang Soo          HWANG, Gi Chul <120> Pharmaceutical Composition for Recovering Ischemia <160> 1 <170> KopatentIn 1.6 <210> 1 <211> 2569 <212> DNA <213> phospholipase C <400> 1 atggactcgg gtagggactt cctgaccctg cacgggctcc aggatgaccc ggaccttcag 60 gcccttctga agggcagcca gcttctgaag gtgaagtcca gctcgtggcg tagggaacgc 120 ttctacaagc tacaggagga ctgcaagacc atctggcagg aatctcgaaa ggtcatgagg 180 tccccggagt cgcagctgtt ctccatcgag gacattcagg aggtacggat gggacaccgc 240 acagaaggcc tggagaagtt tgcccgagac atccccgagg atcgatgctt ctccattgtc 300 ttcaaggacc agcgcaacac cctagacctc attgccccat caccagctga cgctcagcac 360 tgggtgcagg gcctgcgcaa gatcatccac cactccggct ccatggacca gcggcagaag 420 ctgcagcact ggattcactc ctgcttgcga aaggctgata aaaacaagga caacaagatg 480 aacttcaagg agctgaagga cttcctgaag gagctcaaca tccaggtgga tgacggctac 540 gccaggaaga tcttcaggga atgtgaccac tcccagacag actcgctgga ggatgaggag 600 attgagacct tctacaagat gctgacccag cgcgcagaga tcgaccgtgc ttttgaggag 660 gccgcaggat ccgcggagac cctgtcggtg gagaggctag tgacgtttct gcagcaccag 720 cagcgggagg aggaggcagg gccagccttg gccctctctc tcattgagcg ctacgagccc 780 agtgagactg ccaaggccca gcggcagatg accaaggatg gcttcctcat gtacttgctg 840 tcggctgacg gtaacgcctt cagcctggca caccgccgtg tctaccagga catggaccag 900 ccactgagtc actacttagt gtcttcttcc cacaacacct acctgctgga agaccagctc 960 acagggccca gcagcacgga ggcctacatc cgggctctgt gcaaaggctg ccggtgcttg 1020 gagctcgact gctgggatgg tcccaaccag gaacccatca tctaccacgg ctacactttt 1080 acctctaaga tacttttctg tgacgtgctc agggccatca gggactatgc cttcaaggca 1140 tccccctacc cagtcatcct gtccctggag aaccactgta gcctggagca acaacgagtc 1200 atggcccgtc acctgagggc tatcctgggg cccatactgt tggaccagcc actggatggg 1260 gttaccacga gcctgccttc acctgagcaa ctgaagggca agatcctgtt gaaagggaag 1320 aagctgggag ggctgctgcc tgctggaggg gagaatgggt ccgaagccac tgacgtgtct 1380 gacgaggtag aggctgctga aatggaggac gaggctgtgc gcagccaagt gcaacacaag 1440 cccaaggagg ataaactaaa gctggtgccg gagctctccg acatgatcat ttactgcaag 1500 agcgtccact ttggtggctt ctccagtcct ggcacctctg ggcaggcatt ctatgagatg 1560 gcttccttct ctgagagccg tgccctccgg ttgctgcaag aatcaggaaa tggttttgtc 1620 cgtcataacg tgagctgtct gagcaggatc tacccggctg ggtggagaac agattcctcc 1680 aactacagtc ctgtggagat gtggaacggg ggctgccaga ttgttgctct gaacttccaa 1740 acccctgggc cagaaatgga tgtttacctt ggctgcttcc aggacaatgg aggctgtggg 1800 tatgtgctga aacccgcctt tctgagagac cccaacacca ccttcaactc acgtgccctg 1860 actcaggggc cctggtggcg tccagagagg ctccgtgtcc ggatcatctc tggacagcag 1920 ctgccaaagg tcaataagaa taagaattct atcgtggacc ccaaggtgat cgtggagatc 1980 catggcgtgg gccgggacac cggtagccgt cagacagcag ttattaccaa taatggtttc 2040 aacccacggt gggacatgga gtttgagttt gaggtaacag tgcctgacct tgccctcgta 2100 cgcttcatgg tagaggatta tgattcctct tctaagaacg actttattgg ccagagtact 2160 atcccctgga acagcctcaa gcaggggtac cgccatgtcc acctcttgtc taagaatgga 2220 gaccagcatc catcagccac actttttgtg aagatctcca tccaggacta agcccggggg 2280 tctgatgcag tccgacctga gtgagtggag ctgtgtccgc acctcggcca aggctgacat 2340 tattgcaccg acctctggcg cggagcctga ggccctgaac tgccttcagt gccaacacat 2400 cgcttgagct tctgcctctc tggaggccca ggagtggtcc gatccccgga gcgctccaaa 2460 attccgttag gaacaacact gcatccctct ccctaccctc tagctgtctt tatggataag 2520 tttttataaa aaaaaaaaat caaaagaaaa aaaaaaaaaa aaaaaaaaa 2569

Claims (3)

A therapeutic agent for cardiac muscle ischemia comprising phospholipase C-δ (PL-δ) as an active ingredient and a pharmaceutically acceptable carrier. The method of claim 1, Phospholipase C-δ, PLC-δ is characterized in that the phospholipase C-δ1 (PLo-δ1, PLC-δ1) Treatment for ischemic symptoms of myocardium. Transducing the expression vector capable of expressing phospholipase C-δ in eukaryotic cells to cardiomyocytes, thereby overexpressing PLC-δ in cardiomyocytes. A method of treating or preventing ischemia of the heart muscle in a mammal, except humans.

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