CN101361745B - Use of SIP in preparing medicine for preventing mesenchymal stem cells apoptosis - Google Patents
Use of SIP in preparing medicine for preventing mesenchymal stem cells apoptosis Download PDFInfo
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Abstract
The invention discloses the application of S1P in preparing a medicament for inhibiting mesenchymal stem cells from apoptosis, in preparing a medicament for curing ischemic heart disease and a medicament for curing limb ischemia. The invention can significantly improve the survival rate of mesenchymal stem cells in clinical transplantation.
Description
Technical field
The invention belongs to biomedicine field, relate in particular to a kind of new purposes of S1P.
Background technology
(English name is 1-phosphoric acid sphingol: sphingosine 1-phosphate, be called for short S1P) be the sphingomyelins metabolite with biologic activity in serum source, has the important physical function, both can be used as that the second message,second messenger plays a role in the cell, and can combine with the special receptor of cell surface again and bring into play important biological function.1-phosphoric acid Chemistry of Sphingosine structural formula is:
Its molecular formula is: C
18H
38NO
5P, its molecular weight is: 379.47.200480037861.1) and " antilymphocyte antibody of combination S 1P receptor agonist/modulator and immunosuppressant is induced " (application number: some purposes that disclose S1P 200680003305.1) etc. at present, patent application " sphingosine-1-phosphate (S1P) receptor stimulating agent is used for the treatment of the application of brain degenerative diseases " (application number:.
For a long time, S1P is considered to the sphingolipid metabolite that forms in the eukaryotic cell membrane Umklapp process always.Yet, found that before 10 years it has regulated (Zhang since the cell growth, H etc. (1991) J.CellBiol.114,155-167), S1P has been in the news and has participated in many different biological processes, such as, the cell growth, differentiation, survival, blood vessel takes place and cell migration (Goetzl, E.J etc. (1998) FASEBJ.12,1589-1598.Spiegel, S. etc. (2000) Biochim.Biophys.Acta 1484,107-116.Pyne, S. etc. (2000) Biochem.J.349,385-402.Hla, T etc., (2001) Science294,1875-1878).Recently find, S1P is by influencing the adjusting (Jolly that the transportation of T cell function and lymphocyte has participated in immunologic function equally, P. etc. (2002) Mol.Immunol.38,1239-1245.Mandala, S etc. (2002) Science 296,346-349.Brinkmann, V etc. and (2002) J.Biol.Chem.277,21453-21457).In addition, Aventis Pharma GmbH is at " purposes of S1P " (application number: disclose S1P and function fragment thereof 200480015055.4) in effects such as adjusting, prevention and eliminate pains.Playing the part of multiple player especially at cardiovascular system S1P, comprise myocardial cell (P.Robert etc., J.Mol.Cell.Cardiol.33 (2001) 1589-1606), cardiac fibroblast (Lucia Cavallini etc. by adjusting, Arch Biochem Biophys, Oct 1999; 370 (2): .Am J Physiol Heart Circ Physiol such as 156-62.Lee K, Jun2007; 292:H2698-H2711), blood vessel endothelium and vascular smooth muscle cell (Waeber C etc., Vascular sphingosine-1-phosphate S1P1 and S1P3 receptors.Drug NewsPerspect 2004,17:365-382.Tamama K etc., Oka jima F:Sphingosine1-phosphate signaling in atherosclerosis and vascular biology.Curr OpinLipidol 2002,13:489-495.Panetti TS:Differential effects ofsphingosine 1-phosphate and lysophosphatidic acid on endothelial cells.Biochim Biophys Acta 2002,1582:190-196) at the hypertrophy of interior cell, propagation, the generation and the development of multiple physiology of cardiovascular system and pathophysiological processes regulated and control in survival and biological characteristics such as migration and then participation.
The M ﹠ M of myocardial infarction raises year by year, is having a strong impact on people's quality of life.The scheming infarction causes the generation of myocardial ischemia, and myocardial ischemia causes myocardial cell because of deficient the carrying out property death of nutrition, forms scar tissue then, and cardiac muscle takes place excessively to reinvent, finally cause heart failure (.RevInvest Clin such as JL Aceves, Mar2005; 57 (2): 156-62).In recent years, the cellular transplantation therapy myocardial infarction has obtained development faster.The mescenchymal stem cell of derived from bone marrow (mesenchymal stem cells, hereinafter to be referred as MSCs) can be used as seed cell and be transplanted to ischemic myocardium, itself and by being differentiated to form myocardial cell, approach such as paracrine action improve ischemic myocardium blood effectively and supply, part recover and improved cardiac function (.Ann.Thorac.Surg. such as Kimikazu Hamano, Apr 2002; 73:1210-1215.JuliaFeygin Deng .Am J Physiol Heart Circ Physiol, Sep 2007; 293:H1772-H1780).At present, the MSCs cell transplantation has become safe and effective method (the Haider H.Expert Rev Cardiovasc Ther.2006 of domestic and international clinical treatment ischemic heart desease; 4 (4): 557-568.Monolayered mesenchymal stem cells repair scarred myocardium aftermyocardial infarction.Y Miyahara etc., Apr 2006; 12 (4): 459-65).Yet, discover that the MSCs that is implanted into the heart infarction district has only part to survive and to play a role, a considerable amount of stem cell a large amount of apoptosis (YONG-JIAN GENG etc., Acad.Sci., Dec2003 in ischemic tissue are arranged; 1010:687～697).Therefore, how effectively to reduce the MSCs apoptosis rate that is implanted into ischemic tissue, improve its survival rate, thereby reach best cellular transplantation therapy effect, be the difficult problem that present cellular replacement therapy ischemic heart desease and other ischemic disease of limb need to be resolved hurrily, and directly have influence on the curative effect of cell transplantation.
By retrieval, do not find that relevant S1P suppresses the report of apoptosis of mesenchymal stem cell effect under the Hypoxia and ischemia environment.
Summary of the invention
Shortcomings such as the survival rate of seed cell-mesenchymal stem cells MSCs that The present invention be directed to present clinical cellular transplantation therapy ischemic cardiomyopathy is low, therapeutic effect difference provide a kind of medicine that can improve the mesenchymal stem cells MSCs survival and then strengthen its curative effect.
First purpose of the present invention is the application of S1P on preparation inhibition apoptosis of mesenchymal stem cell medicine.
Second purpose of the present invention is the application of S1P on preparation treatment ischemic heart medicine.
The 3rd purpose of the present invention is the application of S1P on preparation treatment ischemic disease of limb medicine.
Described S1P is meant S1P or contains the pharmaceutical composition of S1P.Described S1P (sphingosine1-phosphate is called for short S1P), Chinese is a 1-phosphoric acid sphingol, its chemical structural formula is:
Molecular formula is: C
18H
38NO
5The P molecular weight is: 379.47
Above-mentioned apoptosis of mesenchymal stem cell mainly is to be caused by the hypoxic-ischemic environment after transplanting, and described hypoxic-ischemic environment comprises that scarce (low) oxygen lacks (low) blood, lacks (low) oxygen intact (low) blood, lacks (low) blood intact (low) oxygen etc.
S1P of the present invention can obtain by buying, and is dissolved as desired concn (0.1～50%) with methanol or BSA etc. during use and gets final product.
The occupation mode of S1P among the present invention: (1), before transplanting, mesenchymal stem cells MSCs is hatched 20～120min in concentration is the S1P of 0.1～50uM; (2), before Bone Marrow Mesenchymal Stem Cells Transplantation, give the oral S1P of patient or contain the medicine of S1P, as high density lipoprotein (HDL) and low density lipoprotein, LDL (LDL); (3), before Bone Marrow Mesenchymal Stem Cells Transplantation, give patient's intravenous injection S1P or contain the medicine of S1P, improve the S1P content in the serum, as high density lipoprotein (HDL) and low density lipoprotein, LDL (LDL) etc.
The advantage that the present invention has: (1) the present invention is effective, can significantly improve the survival rate (survival rate can improve 50%) of the mesenchymal stem cells MSCs that clinical transplantation is used for the treatment of; (2) applied range of the present invention not only can be used for transplanting mescenchymal stem cell treatment ischemic heart desease, also can be used for ischemic disease of limb etc.
Description of drawings
Fig. 1. the nuclear fluorescence microscope photo of different disposal mesenchymal stem cells MSCs.
A wherein: normal control group, B: apoptosis model group, C:0.1 μ M S1P+ apoptosis model group, D:1 μ M S1P+ apoptosis model group, E:10 μ M S1P+ apoptosis model group, F:20 μ M S1P+ apoptosis model group, G:50 μ M S1P+ apoptosis model group.
Fig. 2. different disposal mesenchymal stem cells MSCs Annexin V/PI Flow cytometry result.
A wherein: normal control group, B: apoptosis model group, C:S1P0.1 μ M+ apoptosis model group, D:S1P 1 μ M+ apoptosis model group, E:S1P 10 μ M+ apoptosis model group, F:S1P 20 μ M+ apoptosis model group, G:S1P 50 μ M+ apoptosis model group.
Fig. 3. the column cartogram of different disposal apoptosis of mesenchymal stem cell rate
A wherein: normal control group, B: apoptosis model group, C:S1P0.1 μ M+ apoptosis model group, D:S1P1 μ M+ apoptosis model group, E:S1P10 μ M+ apoptosis model group, F:S1P20 μ M+ apoptosis model group, G:S1P50 μ M+ apoptosis model group.
Fig. 4. different disposal is to the western blot figure of caspase-3 activation fragment influence.
Wherein 1: normal control group, 2: apoptosis model group, 3:S1P (10 μ M)+apoptosis model group, 4:S1P (10 μ M)+apoptosis model group+LY294002 (25uM), 5:S1P (10 μ M)+apoptosis model group+U0126 (20uM).
Fig. 5. for detecting the western blot figure that S1P activates the PI3K/Akt signal path.
Wherein 1: normal control group, 2: apoptosis model group, 3:S1P (10 μ M)+apoptosis model group, 4:S1P (10 μ M)+apoptosis model group+LY294002 (25uM), 5:S1P (10 μ M)+apoptosis model group+U0126 (20uM)
Fig. 6. for detecting the western blot figure that S1P activates the MEK/ERK1/2 signal path.
Wherein 1: normal control group, 2: apoptosis model group, 3:S1P (10 μ M)+apoptosis model group, 4:S1P (10 μ M)+apoptosis model group+LY294002 (25uM), 5:S1P (10 μ M)+apoptosis model group+U0126 (20uM)
Fig. 7. the comparison diagram of survivaling cell number behind the Bone Marrow Mesenchymal Stem Cells Transplantation ischemic myocardium of different disposal.
Wherein vertical coordinate is a survival cells, and abscissa is the natural law of cell transplantation.
The specific embodiment
The present invention is described further below in conjunction with drawings and Examples, but following specific embodiments only illustrates, but not limit protection scope of the present invention by any way.
Embodiment 1, S1P suppress the in vitro tests of apoptosis of mesenchymal stem cell
Carry out as follows
(1), MSCs separates and cultivates
Take the Sprague-Dawley rat, male and female are regardless of, after ketalar (60-80mg/kg) intraperitoneal anesthesia, and 75% alcohol-pickled sterilization 3～5 minutes; Clip rat femur and tibia in super-clean bench, wash medullary cavity 10 times with complete medium (IMDM+15%FBS) (IMDM and FBS are all available from Gibcol biotech firm), inhale repeatedly with micro pipette and blow bone marrow, form dispersive single cell suspension, be inoculated in the culture bottle, put 37 ℃, 5%CO
2Cell culture incubator is cultivated; Cell can grow to more than 80% and to merge after 5 days, with 1:3 (0.25% pancreatin) (pancreatin is available from Sigma-Aldrich company) of going down to posterity; Getting well-grown first generation cell experimentizes.
(2), experiment grouping and processing
A. normal control group: (IMDM+15%FBS) normally cultivates MSCs with complete medium;
B. apoptosis model group: with serum-free (IMDM) and anoxic treatment MSCs (placing the airtight anoxia jar, built-in oxygen consumption agent), 37 ℃, 5%CO
2Hatched 6 hours;
C.S1P processed group: be the S1P (available from U.S. Sigma-Aldrich company) pretreatment MSCs1 hour of 0.1 μ M, 1 μ M, 10 μ M, 20 μ M, 50 μ M with concentration respectively, under the anoxia serum-free condition, continued to hatch 6 hours then.The preparation method of described S1P is that S1P is dissolved in methanol, and then is diluted to desired concn with BSA and gets final product; As 1mg S1P is dissolved in the 263.5ul methanol, reach the liquid storage of 10uM, diluting respectively with 0.4%BSA then is 0.1 and the concentration of 1uM (when using 20uM, during 50uM, the S1P dosage that adds 2 times and 5 times respectively gets final product).
(3), the variation of observation of cell nuclear under the fluorescence microscope
After the MSCs cell is respectively organized in above-mentioned (2) processing, fix 30 minutes with the PBS room temperature that contains 1% glutaraldehyde, to wash 2 times with PBS then, reuse 5 μ g/ml Hoechst 33342 room temperatures are fixed 30 minutes, the nuclear form of observing apoptosis under the fluorescence microscope.
Big and the homogenizing of normal control group (seeing Figure 1A) nucleus as a result, pale blue dyeing; Apoptosis model group (seeing Figure 1B) and 0.1 μ M S1P processed group (seeing Fig. 1 C) karyopyknosis, cracked, form is irregular, and dyeing is dark; 1 μ M, 10 μ M, 20 μ M, 50 μ M S1P processed group (seeing Fig. 1 D-G) nucleus are examined big and homogenizing, pale blue dyeing like the normal control group.Illustrate that S1P can suppress the nucleus generation apoptosis form change of mesenchymal stem cells MSCs, promptly S1P can suppress the generation of MSCs apoptosis.
Test method is as follows:
(1) the experiment grouping is with embodiment 1.
(2) according to Annexin V/PI apoptosis detection kit (Beijing Bao Sai Bioisystech Co., Ltd) explanation, to respectively organize cell with what gained was handled in trypsinization among the embodiment 1, wash 2 times with cold PBS, reuse Annexin V solution incubated at room 30 minutes, then add PI solution and hatched 5 minutes, (the Annexin-V/PI streaming detects early apoptosis and the middle and advanced stage apoptosis that can distinguish cell to up flow type cell instrument detection apoptosis rate then.PS during early apoptosis on the cell membrane translates into the cell membrane skin outward, shows as the Annexin-V positive, but cell membrane is complete, and PI is negative, and wherein Annexin V+/PI-represents early stage apoptosis).
The normal control group is (as Fig. 2 A as a result, Fig. 3 A) early apoptosis rate is 2.03%, apoptosis model group (Fig. 2 B, Fig. 3 B) apoptosis rate is 31.28%, 0.1uM, the early apoptosis rate of each processed group of S1P of 1uM, 10uM, 20uM, 50uM concentration is (as Fig. 2 C-G, Fig. 3 C-G) be respectively 28.29%, 22.91%, 13.12%, 9.97%, 10.86%, the early apoptosis rate that adds each processed group of S1P all is lower than the apoptosis model group.The S1P that variable concentrations is described all can effectively suppress the MSCs apoptosis.
(1) test is handled:
(a) normal control group: cultivate so that complete medium (IMDM+15%FBS) is normal; (b) apoptosis model group: with serum-free (IMDM) and anoxic treatment (placing the airtight anoxia jar, built-in oxygen consumption agent), 37 ℃, 5%CO
2Hatched 6 hours; (c) S1P (available from U.S. Sigma company) processed group:, under the anoxia serum-free condition, continued to hatch 6 hours then with the S1P pretreatment MSCs of 10uM 1 hour; (d) S1P (10 μ M)+apoptosis model+LY294002 (25uM) organizes (LY294002 is available from cellsignal company); (e) S1P (10 μ M)+apoptosis model group+U0126 (20uM) organizes (U0126 is available from cellsignal company).
(2) test method: each organized the cell non-serum anoxia after 6 hours, and pancreatin (0.25%) digestion, collecting cell extract cell protein, survey protein concentration with the Coomassie brilliant blue method.By sample electrophoresis on the 50 μ g albumen qualities, Western blot (seeing molecular cloning second edition the 18 chapter the 3rd joint 888-898 page or leaf for details) detects caspase-3 activation fragment
The MSC of normal control group (Fig. 4-1) does not almost have Caspase3 to activate segmental generation as a result; And apoptosis model group (Fig. 4-2) has a large amount of Caspase3 activation fragments to produce; The Caspase3 activation fragment of S1P processed group (Fig. 4-3) significantly reduces; After using PI3K and ERK inhibitor LY294002 and U0126 (Fig. 4-4,5) to suppress these two paths, Caspase3 activation fragment increases than S1P processed group again.Illustrate that S1P activates the apoptosis that segmental generation has suppressed MSCs by suppressing Caspase3.
Embodiment 4, inhibitor are tested the anti-apoptosis of mesenchymal stem cell function influence of S1P
(1) test is handled: (a) normal control group: cultivate so that complete medium (IMDM+15%FBS) is normal; (b) apoptosis model group: with serum-free (IMDM) and anoxic treatment (placing the airtight anoxia jar, built-in oxygen consumption agent), 37 ℃, 5%CO
2Hatched 6 hours; (c) S1P (available from U.S. Sigma company) processed group:, under the anoxia serum-free condition, continued to hatch 6 hours then with the S1P pretreatment MSCs of 10uM 1 hour.(d) S1P (10 μ M)+apoptosis model+LY294002 (25uM) group; (e) S1P (10 μ M)+apoptosis model group+U0126 (20uM) group.
(2), above-mentioned respectively organize the cell processing and finish after, the up flow type cell instrument detects PI3K inhibitor LY294002 and/or the ERK1/2 inhibitor U0126 influence to the anti-apoptotic effect of S1P.
Result (as Fig. 5 and Fig. 6) illustrates that LY294002 and U0126 all can obviously suppress the anti-apoptosis of mesenchymal stem cell effect of S1P.PI3K and ERK pathway inhibitor LY294002 and U0126 all can suppress the anti-apoptosis of mesenchymal stem cell effect of S1P separately, also side light the anti-apoptosis of mesenchymal stem cell effect of S1P.
The animal experiment of the short mesenchymal stem cells MSCs survival effect of S1P
(1), test is handled:
A. matched group: cultivate (IMDM contains L-glutaminate and 25mM HEPES) 50ul injection rat heart infarction model heart infarction surrounding zone with complete medium;
B.S1P processed group: cultivate (IMDM) 50ul with the complete medium that contains S1P and suspend 2 * 10
6Mescenchymal stem cell, pretreatment 1h (the S1P final concentration is 10uM).Then inject rat heart infarction model heart infarction surrounding zone together;
(2), test method: the mode of male rat mesenchymal stem cells MSCs injection female rats heart infarction model is adopted in test, 3 groups back 1 hour respectively in injection, 1 day and 1 week back extraction heart tissue DNA pass through to detect the content of sry gene and then detect the mescenchymal stem cell number of surviving with the method for Realtime PCR then.
After 1 hour, the number of the MSC of matched group survival is 490939.3615 to (see figure 7) at cell transplantation as a result, and S1P processed group cell survivaling number is 1051256.348, apparently higher than matched group; Behind the cell transplantation 1 day, the number of the MSC of matched group and the survival of S1P processed group is respectively 331059.9 and 682976.6, and the MSCs number of S1P processed group survival also is significantly higher than matched group; Cell transplantation is after 1 week, and the number of the MSC of S1P processed group survival is 27721.43, is higher than mescenchymal stem cell group 13357.12.Explanation S1P in experimental animals has significantly improved the survival rate of transplanting MSC.
Claims (2)
1.1-the application of phosphoric acid sphingol on preparation inhibition apoptosis of mesenchymal stem cell medicine.
2. according to the described application of claim 1, it is characterized in that described 1-phosphoric acid sphingol is meant 1-phosphoric acid sphingol or contains the compositions of 1-phosphoric acid sphingol.
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| KR102097191B1 (en) * | 2016-11-10 | 2020-04-06 | 울산대학교 산학협력단 | Composition for enforcing the priming effect of stem cell comprising histone deacetylase inhibitor and priming factors |
| CN115961056A (en) * | 2022-12-21 | 2023-04-14 | 武汉光谷中源药业有限公司 | Method for detecting content of umbilical cord source mesenchymal stem cells in human whole blood |
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