CN101229241B - Application of Chinese traditional medicine compounds on preparing medicines promoting mesenchymal stem cells surviving in the body and cardioblast differentiation - Google Patents
Application of Chinese traditional medicine compounds on preparing medicines promoting mesenchymal stem cells surviving in the body and cardioblast differentiation Download PDFInfo
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Abstract
The invention discloses the application of a traditional Chinese medicine combination in the preparation of medicine which enhances the in-vivo survival and cardio blast differentiation of mesenchymal stem cells. The invention also relates to the application of the traditional Chinese medicine compound to the preparation of medicine which cures the cardiovascular disease with autologous mesenchymal stem cells.
Description
Technical field
The present invention relates to a kind of new purposes of Chinese medicine composition, particularly, relate to a kind of Chinese medicine composition and promote the application of bone marrow interstital stem cell in body survival and the medicine that becomes cardiac muscle to break up in preparation.
Background technology
Cardiovascular disease seizes 1,200 ten thousand people's life every year, near 1/4 of the total death of world population, becomes the No.1 formidable enemy of human health.For myocardial infarction or heart failure, traditional treatment comprises that medicine, intervention and surgical operation all can not make the myocardial cell regeneration of disappearance, because the cardiac muscle disappearance causes irreversible Myocardial Remodeling process, eventually to heart failure and death.In recent years; Stem cell regenerating medical science has obtained breakthrough in treating cardiovascular disease; This technology cell cardiomyoplasty art (cellular cardiomyoplasty) that is otherwise known as; Promptly, perhaps mobilize peripheral circulation hemocytoblast or bone marrow stem cell to move to the reparation that myocardium damaged part is realized injury of myocardium through transplanting stem cell or myocardial cell.Transplanting stem cell enforcement cell cardiomyoplasty art is to improve hematodinamics and the disorderly a kind of feasible method of neuro humor that myocardial infarction causes.Various zooperies in early stage show that stem cell has the ability of repairing myocardial cell; Can improve perfusion and cardiac function [Schuster MD, Kocher AA, the Seki T of infarcted region; Martens TP; Xiang G, Homma S, et al.Myocardial neovascularization bybone marrow angioblasts results in cardiomyocyte regeneration.Am JPhysiol Heart Circ Physiol 2004; 287:H525-532. (Schuster MD, KocherAA, Seki T, Martens TP, Xiang G, Homma S etc., the angiogenesis that the bone marrow angioblast causes cause myocardial cell regeneration. U.S.'s physiology magazine heart and circulation fascicle 2004; 287:H525-532.)].
Although stem cell is used to the clinical research of myocardial repair because that the influence of ischemia/reperfusion and inflammatory factor etc. causes at the donorcells of ischemia heart is dead, so the development of cell cardiomyoplasty art cell survival rate is low to be hindered because of implanting.Research shows that a large amount of cells are dead after being implanted into impaired heart, and wherein most cells run off only remaining 15% survival [Muller-Ehmsen J after 12 weeks in transplanting in back 24 hours; Whittaker P; Kloner RA, Dow JS, Sakoda T; Long TI, et al.Survival and development of neonatal rat cardiomyocytestransplanted into adult myocardium.J Mol Cell Cardiol 2002; [34:107-116. PMID:11851351] (Muller-Ehmsen J, Whittaker P, Kloner RA; Dow JS, Sakoda T, Long TI; Deng. the myocardial cell of neonate rat is implanted into existence and the growth behind the adult rat cardiac muscle. molecular cell cardiology magazine, 2002; 34:107-116.)].
Acute myocardial infarction can cause serious regional myocardial ischemia, inflammatory reaction, oxidative stress and apoptosis, and this will obviously reduce the survival rate of transplanted cells.Therefore, the local stem cell of transplanting of protection reduces or avoids its death significant to clinical practice.Existing at present several kinds can be improved the method for implanting cell survival rate: (1) heat shock treatment can improve the toleration of transplanted cells to ischemia/reperfusion injury in the body; Survival rate [the Suzuki K behind the heart is implanted in raising; Smolenski RT; Jayakumar J; MurtuzaB, Brand NJ, Yacoub MH.Heat shock treatment enhances graft cellsurvival in skeletal myoblast transplantation to the heart.Circulation 2000; [102:III216-221. PMID:11082390]) (Suzuki K, Smolenski RT, Jayakumar J, Murtuza B, Brand NJ, Yacoub MH. heat shock treatment strengthen the survival of bone marrow myoblast transplantation behind the heart. circulation .2000; 102:III216-221.)]; (2) bone marrow interstital stem cell of Akt modification can further improve infarcted hearts function [Mangi AA; Noiseux N; Kong D, He H, Rezvani M; Ingwall JS, etal.Mesenchymal stem cells modified with Akt prevent remodeling andrestore performance of infarcted hearts.Nat Med 2003; [9:1195-1201. PMID:12910262] (Mangi AA, Noiseux N, Kong D, He H, Rezvani M, IngwallJS etc., the mescenchymal stem cell that Akt modifies stop infarcted hearts reconstruct and recover cardiac function. natural medical science, 2003; 9:1195-1201.)]; (3) make heme oxygenase-1 overexpression at ischemia myocardial injection plasmid vector; Reduce the quantity of monocyte infiltration, expression [Tang YL, the Tang Y of downward modulation inflammatory factor; Zhang C; Qian KP, Shen LP, Phillips I.Improvedgraft mesenchymal stem cell survival in ischemic heart with ahypoxia-regulated Heme Oxygenase-1 vector.J Am Coll Cardiol 2005; [46:1339-1350. PMID:1619885]) (Tang Y; Zhang C; Qian KP, Shen LP, the mesenchymal stem cell transplantation of Phillips I. heme oxygenase-1 overexpression existence after the Ischemic Heart improves. Journal of the American College of Cardiology 2005; 46:1339-1350.)].But; Above method all is based upon on the basis of donorcells level; And the key of the destiny of decision transplanted cells in heart is the regional myocardial microenvironment of infarction, and the intervention of therefore carrying out to the infarcted myocardium microenvironment possibly more effectively promote the survival and the performance biological effect of transplanted cells.。
The present invention is the improvement invention of on the basis of No. 01131203.3 Chinese patent and No. 200410048292.2 patent application, carrying out, and quotes in full the content of this two patent documents record at this.The invention provides a kind of Chinese medicine composition and promote the new application of bone marrow interstital stem cell in body survival and the medicine that becomes cardiac muscle to break up in preparation; Improve the quality of local microenvironment through the intervention treatment, and then effectively promote to implant the survival and the biological effect of cell.
Summary of the invention
The object of the invention provides a kind of Chinese medicine composition and promotes the application of bone marrow interstital stem cell in body survival and the medicine that becomes cardiac muscle to break up in preparation, and said Chinese medicine composition is processed by following bulk drugs:
Radix Ginseng 3-10 Hirudo 3-11 Eupolyphaga Seu Steleophaga 5-10 Olibanum (system) 1-5 Radix Paeoniae Rubra 3-9 Lignum Dalbergiae Odoriferae 1-5
Lignum Santali Albi 1-5 Scorpio 3-9 Periostracum Cicadae 3-12 Scolopendra 1-3 Borneolum Syntheticum 1-7 Semen Ziziphi Spinosae (stir-fry) 3-10;
Preferably, this Chinese medicine composition is processed by following bulk drugs:
Radix Ginseng 6 Hirudo 10 Eupolyphaga Seu Steleophagas, 7 Olibanums (system) 2 Radix Paeoniae Rubra 5 Lignum Dalbergiae Odoriferaes 2
Lignum Santali Albi 2 Scorpios 7 Periostracum Cicadaes 7 Scolopendra 1 Borneolum Syntheticum, 5 Semen Ziziphi Spinosaes (stir-fry) 5;
Or:
Radix Ginseng 10 Hirudo 8 Eupolyphaga Seu Steleophagas, 7 Olibanums (system) 2 Radix Paeoniae Rubra 5 Lignum Dalbergiae Odoriferaes 2
Lignum Santali Albi 2 Scorpios 9 Periostracum Cicadaes 7 Scolopendra 1 Borneolum Syntheticum, 5 Semen Ziziphi Spinosaes (stir-fry) 5;
Or:
Radix Ginseng 6 Hirudo 11 Eupolyphaga Seu Steleophagas, 7 Olibanums (system) 2 Radix Paeoniae Rubra 5 Lignum Dalbergiae Odoriferaes 2
Lignum Santali Albi 2 Scorpios 3 Periostracum Cicadaes 7 Scolopendra 1 Borneolum Syntheticum, 5 Semen Ziziphi Spinosaes (stir-fry) 5;
Or:
Radix Ginseng 5.5 Hirudo 10.375 Eupolyphaga Seu Steleophagas, 6.875 Olibanums (system) 2.25
Radix Paeoniae Rubra 4.75 Lignum Dalbergiae Odoriferaes 2.375 Lignum Santali Albis 2.25 Scorpios 6.875
Periostracum Cicadae 6.875 Scolopendra 1.375 Borneolum Syntheticums, 1.375 Semen Ziziphi Spinosaes (stir-fry) 4.625;
More preferably, the active component of above-mentioned Chinese medicine composition is made up of following ingredients:
The a mean diameter is less than Scorpio, Hirudo, Scolopendra, Eupolyphaga Seu Steleophaga, Periostracum Cicadae and the Olibanum (processed) medicated powder of 100 μ m;
B Borneolum Syntheticum medicated powder;
The volatile oil that c is extracted by Lignum Dalbergiae Odoriferae and Lignum Santali Albi;
The alcohol-extracted extract of the alcohol extract of d Radix Ginseng after after concentrating with ethanol extraction;
The water extracted immersing paste that is condensed into behind the water extract after the Lignum Dalbergiae Odoriferae behind the e extraction composition c and water extract, Radix Paeoniae Rubra and the Semen Ziziphi Spinosae (parched) decocte with water of Lignum Santali Albi medicinal residues and the water extract filtration of the medicine residues of Radix Ginseng after the extraction ingredient d, the mixing.
The invention also discloses that to contain above-mentioned Chinese medicine composition be capsule, tablet, pill, oral liquid, soft capsule or drop pill as the pharmaceutical preparation of active component.
Another object of the present invention provides above-mentioned Chinese medicine composition and is preparing with the application in the medicine of ABM interstital stem cell treatment cardiovascular disease, and preferably, this cardiovascular disease is a myocardial infarction, is preferably acute myocardial infarction especially.
In the Chinese medicine composition of the present invention, as the latin name of the crude drug of active component and processing method thereof from " Chinese medicine voluminous dictionary " (in July, 1977, front page, Shanghai science tech publishing house) and " Chinese pharmacopoeia (version in 2005, Chemical Industry Press).
Chinese medicine composition of the present invention can also be by the preparation process of routine; For example; The preparation technology of Fan Biting " pharmacy of Chinese materia medica " (Shanghai Science Press 1997 December the 1st edition) record; Process the acceptable any conventional dosage form of pharmaceutics, for example capsule, tablet, pill, oral liquid, soft capsule, drop pill etc.
In the preparation of the present invention, can also contain the conventional adjuvant of optional formulation art, for example filler, disintegrating agent, binding agent, fluidizer, antioxidant, correctives, sweeting agent, suspending agent etc.Said adjuvant comprises for example starch, sucrose, lactose, dextrin, pregelatinized Starch; Acceptable other adjuvant of crospolyvinylpyrrolidone etc. or pharmacy of Chinese materia medica (adjuvant of each dosage form record among the Fan Biting " pharmacy of Chinese materia medica ", Shanghai Science Press December in 1997 the 1st edition).
Preparation of the present invention is preferably processed through following method for preparing: five kinds of Chinese medicine such as the Hirudo of said ratio, Scorpio, Periostracum Cicadae, Eupolyphaga Seu Steleophaga, Scolopendra cleaned, and oven drying at low temperature, subsequent use; Lignum Santali Albi, Lignum Dalbergiae Odoriferae extract volatile oil, and medicinal residues and aqueous solution are subsequent use; Radix Ginseng extracts secondary with 70% alcohol heating reflux, and 3 hours for the first time, 2 hours for the second time, merge extractive liquid, reclaimed ethanol to there not being the alcohol flavor; The medicinal residues of medicine residues of Radix Ginseng and Lignum Santali Albi, Lignum Dalbergiae Odoriferae merge with aqueous solution, add Radix Paeoniae Rubra, Semen Ziziphi Spinosae (stir-fry), the decocte with water secondary; 3 hours for the first time, 2 hours for the second time, collecting decoction; Filter, filtrating is concentrated into the clear paste that relative density is 1.20-1.25 (60 ℃), adds above-mentioned panaxynol extract; Mixing, cold drying is ground into fine powder; The five tastes such as Olibanum (system) and Hirudo are ground into fine powder altogether; The Borneolum Syntheticum porphyrize, with above-mentioned fine powder facing-up, mixing sprays into volatile oil respectively, and mixing incapsulates, and processes 1000, promptly gets.
Perhaps, preparation of the present invention is preferably processed through following method for preparing:
A) weight ratio of crude drug is: Radix Ginseng 3-10 part, Hirudo 3-11 part, Eupolyphaga Seu Steleophaga 5-10 part, Olibanum (processed) 1-5 part, Radix Paeoniae Rubra 3-9 part, Lignum Dalbergiae Odoriferae 1-5 part, Lignum Santali Albi 1-5 part, Scorpio 3-9 part, Periostracum Cicadae 3-12 part, Scolopendra 1-3 part, Borneolum Syntheticum 1-7 part, Semen Ziziphi Spinosae (parched) 3-10 part;
B) pulverizing medicinal materials technology:
Scorpio, Hirudo, Scolopendra, Eupolyphaga Seu Steleophaga and five kinds of worm medicines of Periostracum Cicadae are prepared burden by prescription through the Olibanum (processed) that cleans, washes after handling the back and cleaning the process of preparing Chinese medicine, pulverized by pulverizer, the medicated powder fineness reaches more than 80 orders; Medicated powder behind the coarse powder carries out micronizing through various superfine communication techniques, makes the medicated powder mean diameter less than 100 μ m; Medical material to be pulverized is prepared burden after the cleaning, drying sterilization;
C) extract concentrated and drying process:
Reuse water extraction behind Lignum Dalbergiae Odoriferae and the Lignum Santali Albi elder generation extracting in water volatile oil, Radix Paeoniae Rubra and Semen Ziziphi Spinosae decocte with water, after the water extract filters, extractum to be condensed into; Radix Ginseng with ethanol extraction after, reuse water extraction, alcohol extract are condensed into alcohol-extracted extract after reclaiming ethanol, the water extract filter with all water extract mixings after be condensed into the water extracted immersing paste;
D) preparation process:
In Fluidbedgranulatingdrier, add the superfine powder flour, again the step c) gained is extracted extractum and spray into granulation; The granule of processing through granulate, is added the Borneolum Syntheticum fine powder, spray into the volatile oil that extracts by Lignum Dalbergiae Odoriferae and Lignum Santali Albi, by the capsule filler filling, process capsule behind the mixing.
Perhaps, preparation of the present invention is preferably processed through following method for preparing:
A) weight ratio of crude drug is: Radix Ginseng 3-10 part, Hirudo 3-11 part, Eupolyphaga Seu Steleophaga 5-10 part, Olibanum (system) 1-5 part, Radix Paeoniae Rubra 3-9 part, Lignum Dalbergiae Odoriferae 1-5 part, Lignum Santali Albi 1-5 part, Scorpio 3-9 part, Periostracum Cicadae 3-12 part, Scolopendra 1-3 part, Borneolum Syntheticum 1-7 part, Semen Ziziphi Spinosae (parched) 3-10 part;
B) pulverizing medicinal materials technology:
Scorpio, Hirudo, Scolopendra, Eupolyphaga Seu Steleophaga and five kinds of worm medicines of Periostracum Cicadae are prepared burden by prescription through the Olibanum (processed) that cleans, washes after handling the back and cleaning the process of preparing Chinese medicine, pulverized by pulverizer, the medicated powder fineness reaches more than 80 orders; Medicated powder behind the coarse powder carries out micronizing through various superfine communication techniques, makes the medicated powder mean diameter less than 100 μ m; Medical material to be pulverized is prepared burden after the cleaning, drying sterilization;
C) extract concentrated and drying process:
Reuse water extraction behind Lignum Dalbergiae Odoriferae and the Lignum Santali Albi elder generation extracting in water volatile oil, Radix Paeoniae Rubra and Semen Ziziphi Spinosae decocte with water, after the water extract filters, extractum to be condensed into; Radix Ginseng with ethanol extraction after, reuse water extraction, alcohol extract are condensed into alcohol-extracted extract after reclaiming ethanol, the water extract filter with all water extract mixings after be condensed into the water extracted immersing paste, extractum directly is spray dried to spray powder;
D) preparation process:
The superfine powder flour is added in the Fluidbedgranulatingdrier with step c) gained spray drying powder, sprays solvent again and process granule; The granule of processing through granulate, is added the Borneolum Syntheticum fine powder, spray into the volatile oil that extracts by Lignum Dalbergiae Odoriferae and Lignum Santali Albi, by the capsule filler filling, process capsule behind the mixing.
The consumption of of the present invention group of thing by active component crude drug gross weight, is each 0.8-3 gram, and take 2-4 every day, is preferably each 1.11-2.22 gram, takes every day three times.
The present invention provides a large amount of experimental study data to show, uses drug group intervention of the present invention that utilization ABM interstital stem cell is carried out cell cardiomyoplasty art and plays facilitation.Microarray is surveyed the gene expression section of infraction back heart and is found that single use low dosage drug group of the present invention can make gene expression produce actively and change; Comprise up regulation to antiinflammatory, anti-apoptosis, fibrosis gene; Therefore; Think and use the local microenvironment after drug group intervention of the present invention can improve acute myocardial infarction, thereby significantly improve the survival and the differentiation capability of the bone marrow interstital stem cell of implantation.And then; Experimental data provided by the invention also proves; Use the local interior environment after drug group intervention of the present invention can effectively improve acute myocardial infarction; Utilization ABM interstital stem cell is carried out cell cardiomyoplasty art play facilitation, thus significant to the clinical practice of the transplanting of bone marrow interstital stem cell.
Description of drawings
Haematoxylin-Yihong (HE) dyeing of four groups of laboratory animal infarcted region of Fig. 1 microscopically and Ma Song (Masson ' s) trichrome stain.Picture shows; First group (matched group), second group (the pharmaceutical intervention treatment of the present invention of single use low dosage), the 3rd group (the single bone marrow interstital stem cell that carries out is transplanted therapeutic intervention) all exist serious fibrosis and inflammatory cell infiltration, infarcted region not to have the myocardial cell survival basically; Yet the 4th group of (using bone marrow interstital stem cell to transplant with pharmaceutical intervention of the present invention simultaneously treats) infarcted region fibrosis and inflammatory cell infiltration are lighter.Picture A amplification is 400 *, picture B amplification is 40 *.
Fig. 2 implants the survival ability of the bone marrow interstital stem cell of infarcted hearts.Figure (A) shows that the 3rd group (the single bone marrow interstital stem cell that carries out is transplanted therapeutic intervention) almost do not observe the transplanted cells of 4 ', 6 diamidinos-2-benzene indole hydrochloride (DAPI) labelling; The 4th group (using bone marrow interstital stem cell to transplant and pharmaceutical intervention of the present invention treatment simultaneously) can be observed the transplanted cells of more DAPI labelling; Figure (B) shows that there is remarkable significant difference in the 3rd group (the single bone marrow interstital stem cell that carries out is transplanted therapeutic intervention) with the cell survival ability of the 4th group (using bone marrow interstital stem cell to transplant and pharmaceutical intervention of the present invention treatment simultaneously).
*P＜0.0001, the amplification of figure (A) is 400 *.
The bone marrow interstital stem cell that Fig. 3 implants is divided into myocardial cell and blood vessel structure.Figure (A) and figure (B) show the cellular expression α-muscle rhabdomyosarcoma filamentous actin of part DAPI labelling, cardiac troponin T; Figure (C) shows that the cell of part DAPI labelling is participated in angiogenesis, expresses vascular smooth muscle actin and the specific Factor IX of vascular endothelial cell.Figure (D) shows; The 3rd group (the single bone marrow interstital stem cell that carries out is transplanted therapeutic intervention) becomes the statistical comparisons of myocardium differentiation ratio with the 4th group of (using bone marrow interstital stem cell to transplant and pharmaceutical intervention of the present invention treatment simultaneously) cell of DAPI labelling in heart, there were significant differences for the ability of two groups of implantation cell differentiation cardioblasts.
*P＜0.0001, figure (A), figure (B) and the amplification of scheming (C) is 400 *.Annotate: MSCs is a mesenchymal stem cells MSCs among the figure, and VWF is a von Wilebrand factor, and SM-actin is the vascular smooth muscle actin, and Overlay representes the colour developing result of first three visual field superposition.
The iuntercellular that Fig. 4 in vivo implants cell connects proteic expression.Figure (A) shows that the cellular expression slit of DAPI labelling connects protein 43 (Cx43).Scheme (B) and show, the slit of the 3rd group (the single bone marrow interstital stem cell that carries out is transplanted therapeutic intervention) and the 4th group (using bone marrow interstital stem cell to transplant and pharmaceutical intervention of the present invention treatment simultaneously) is connected the protein 43 expression and has significant difference.
*P＜0.0001, the amplification of figure (A) is 400 *.Annotate: Overlay representes the colour developing result of first three visual field superposition among the figure.
Fig. 5 transplants the capillary density of 6 all back infarcted region and infarction surrounding zone.After transplanting for 6 weeks, the capillary density of second group (single use low dosage of the present invention pharmaceutical intervention treatment), the 3rd group (the single bone marrow interstital stem cell that carries out is transplanted therapeutic intervention) and matched group infarcted region and infarction surrounding zone do not have significant difference (
*P＞0.05,
*But all be lower than the 4th group (using bone marrow interstital stem cell to transplant with pharmaceutical intervention of the present invention simultaneously treats) and (be respectively P＞0.05),
#P＜0.0001,
##P＜0.0001).
Fig. 6 detects the damaged area of heart muscle perfusion after transplanting all backs and transplanting for six weeks with single photon emission computed tomography (SPECT).Figure (A) shows every group SPECT representative of graphics.Figure (B) transplant a week initial SPECT result in back show between four groups the filling defect area do not have significant difference (
*P=0.984).After transplanting for six weeks; The 4th group of (using bone marrow interstital stem cell to transplant and pharmaceutical intervention of the present invention treatment simultaneously) filling defect area significantly is reduced to 22.1 ± 9.3%; (the n=7 that compares with matched group, second group (the pharmaceutical intervention treatment of the present invention of single use low dosage), the 3rd group (the single bone marrow interstital stem cell that carries out is transplanted therapeutic intervention) that there were significant differences
#P＜0.0001).
The anti-apoptotic effect of Fig. 7 medicine of the present invention.(A) deoxidation uridine triphosphate (dUTP) the breach end-labelling (TUNEL) of pig heart infarction myocardium resistive connection hop protein antibody of periphery and deoxynucleotidyl transferase mediation detects the apoptotic cell of dna break in the karyon.Investigating terminal point, the 2nd group (the pharmaceutical intervention treatment of the present invention of single use low dosage) and the 4th group of (using bone marrow interstital stem cell to transplant and pharmaceutical intervention of the present invention treatment simultaneously) infarction periphery have less apoptosis karyon (red arrow).Apoptotic index statistics between 4 groups of amplification: * 20. (B).Compare with matched group, the obviously minimizing of the 2nd group of (single use low dosage pharmaceutical intervention of the present invention is treated) apoptosis cardiac muscle (
*P＜0.0001).And the 4th group of (using bone marrow interstital stem cell to transplant and pharmaceutical intervention of the present invention treatment simultaneously) apoptotic index obviously be less than the 2nd group (single use low dosage pharmaceutical intervention of the present invention is treated) (
#P＜0.0001).But with matched group relatively, the 3rd group of (the single bone marrow interstital stem cell that carries out is transplanted therapeutic intervention) apoptotic index do not have significant difference (
*P=0.289).
Fig. 8 tests the myocardium oxidative stress horizontal detection of terminal point infarction periphery.(A) the 2nd group (single use low dosage of the present invention pharmaceutical intervention treatment) and the 4th group of (use bone marrow interstital stem cell to transplant simultaneously and pharmaceutical intervention of the present invention is treated) superoxide dismutase (SOD) vigor than matched group significantly raise (
*P＜0.05,
#P＜0.05).But the 3rd group (single carry out bone marrow interstital stem cell transplant therapeutic intervention) and matched group relatively do not have significant difference (
*P=0.449).(B) the 2nd group (single use low dosage of the present invention pharmaceutical intervention treatment) and the 4th group of (use bone marrow interstital stem cell to transplant simultaneously and pharmaceutical intervention of the present invention is treated) malonaldehyde (MDA) content than matched group significantly reduce (
*P＜0.05,
#P＜0.05).There is not significant difference (P=0.195) between the 3rd group (the single bone marrow interstital stem cell that carries out is transplanted therapeutic intervention) and the matched group.
The specific embodiment
Embodiment 1: the preparation of medicine of the present invention
A) the crude drug prescription is:
Radix Ginseng 55 gram Hirudos 103.75 gram Eupolyphaga Seu Steleophagas 68.75 gram Olibanums (system) 22.5 grams
Radix Paeoniae Rubra 47.5 gram Lignum Dalbergiae Odoriferaes 23.75 gram Lignum Santali Albis 22.5 gram Scorpios 68.75 grams
Periostracum Cicadae 68.75 gram Scolopendras 13.75 gram Borneolum Syntheticums 13.75 gram Semen Ziziphi Spinosaes (stir-fry) 46.25 grams;
B) pulverizing medicinal materials technology:
Scorpio, Hirudo, Scolopendra, Eupolyphaga Seu Steleophaga and five kinds of worm medicines of Periostracum Cicadae are prepared burden by prescription through the Olibanum (processed) that cleans, washes after handling the back and cleaning the process of preparing Chinese medicine, pulverized by pulverizer, the medicated powder fineness reaches more than 80 orders; Medicated powder behind the coarse powder carries out micronizing through various superfine communication techniques, makes the medicated powder mean diameter less than 30-40 μ m; Medical material to be pulverized is prepared burden after the cleaning, drying sterilization;
C) extract concentrated and drying process:
Reuse water extraction behind Lignum Dalbergiae Odoriferae and the Lignum Santali Albi elder generation extracting in water volatile oil, Radix Paeoniae Rubra and Semen Ziziphi Spinosae add suitable quantity of water and decoct secondary, and each 3 hours, merge decocting liquid, after the water extract filters, extractum to be condensed into; Radix Ginseng is with an amount of 70% ethanol extraction secondary; Each 3 hours, merge extractive liquid, reclaimed ethanol to there not being the alcohol flavor; The reuse water extraction; It is 0.9～1.1 (60 ℃) alcohol-extracted extract that alcohol extract is condensed into relative density, is concentrated into the clear paste that relative density is 0.9～1.1 (60 ℃) behind the filtration of water extract and above-mentioned all water extract mixings, subsequent use;
D) preparation process:
In Fluidbedgranulatingdrier, add the superfine powder flour, again the step c) gained is extracted extractum and spray into granulation; The granule of processing through granulate, is added the Borneolum Syntheticum fine powder, spray into the volatile oil that extracts by Lignum Dalbergiae Odoriferae and Lignum Santali Albi, by the capsule filler filling, process 1000 capsules behind the mixing.
Amount of drug of the present invention by active component crude drug gross weight, for each 2-4 grain, is taken three times every day.
Experimental example medicine of the present invention is in the facilitation of utilization bone marrow interstital stem cell
Materials and methods
The Chinese miniature pig at 10 monthly ages of laboratory animal, body weight (30kg ± 5kg), provide by China Agricultural University's Experimental Animal Center.All laboratory animals all receive humanity and treat, and meet " management of laboratory animal and the guide for use " of NIH's promulgation.And all experimental programs have all obtained the approval of management of laboratory animal committee of the Chinese Academy of Medical Sciences and laboratory animal Ethics Committee of the outer cardiovascular diseases hospital of China Concord Medical Science University's mound.
The separation of Medulla Sus domestica interstital stem cell and cultivation
Intramuscular injection of ketamine and diazepam are anaesthetized pig, and two kinds of drug doses are respectively 25mg/kg and 1mg/kg.Left crista iliaca place preserved skin, shop to pig under the sterile working are single, extract about 50ml bone marrow with the syringe that contains 12,500 units heparin.All laboratory animals are all intramuscular injection buprenorphine 0.3mg pain relievings before sending back between raising.
The separation of bone marrow interstital stem cell and cultivation are according to former reported method somewhat modified.Briefly, the bone marrow of extraction adds Silicon stone colloidal suspension liquid (Percoll separating medium, 1.077g/ml, Sigma company) with 1 times of phosphate buffer (PBS) dilution, and under 4 ℃ of conditions, 800g isolated mononuclearcell in centrifugal 30 minutes.The cell precipitation thing is with PBS flushing 2 times; Back density with 5 * 10/cm is laid on the normal cultured base and [contains LG DMEM (Gibco company); 10% hyclone (Gibco), 100U/ml penicillin and streptomycin] on, the moist incubator that place 37 ℃, contains 5% carbon dioxide is cultivated.After three days, remove hematopoietic cell, fibroblast and other non-adherent cell through changing culture medium.The adherent further multiplication by culture of purification bone marrow interstital stem cell that retains.Per three days replacing one subcultures in the experimentation.Cultivated the cell clone of attached cell formation homogeneous through 10 days.After attached cell reaches 80% fusion, add 0.25% pancreatin-0.02%EDTA liquid (Sigma) it is suspended again, go down to posterity with 1: 3 ratio and further cultivate.
Myocardial infarction model, the preparation of transplanted cells and the facilitation of drug group of the present invention
28 Chinese miniature pigs are equally divided into 4 groups: first group is matched group; N=7, second group of (single use low dosage drug group therapeutic intervention of the present invention; N=7), the 3rd group (the single bone marrow interstital stem cell that carries out is transplanted therapeutic intervention; N=7) and the 4th group (use bone marrow interstital stem cell to transplant and drug group therapeutic intervention of the present invention simultaneously, n=7).
After cell reaches 80% fusion; It is separated from culture bottle, in the DMEM that contains 10% hyclone (GIBCO) culture medium, it is suspended again, with 4 '; 6 diamidinos-2-benzene indole hydrochloride (DAPI) (50 μ g/ml, Sigma) labelling 30 minutes under 37 ℃ of conditions.Cell washed in PBS liquid remove unconjugated DAPI 6 times, each laboratory animal is chosen 3 * 10 cells and is placed warm DMEM to preserve after several minutes to transplant then.Labeling process is extremely important, need guarantee that all transplanted cells nuclear is painted.
Intramuscular injection of ketamine and diazepam are anaesthetized pig, and two kinds of drug doses are respectively 25mg/kg and 1mg/kg, and tracheal intubation connects mechanical respirator and carries out artificial ventilation, keeps anesthesia through intravascular injection ketamine and diazepam.Open breast along the breastbone median line, separate LADCA (LAD) and prop up, and use the plastic casing-pipe ligation, guarantee the formation at ischemia position to first diagonal angle.Intravenous injection lignocaine 2mg/kg before the coronary ligation, the back continues intravenous administration and finishes until operation, and lasting dosage is 0.5mg/min.Block LADCA (LAD) and formed myocardial infarction/re-perfusion model in 90 minutes.
Poured into again back 30 minutes, and injected ABM interstital stem cell (3 * 10 cells) suspension 500 μ l in the infarcted region and the surrounding zone thereof of each laboratory animal.Control animals is injected isopyknic DMEM liquid.
After transplanting end, close the thoracic cavity, place a 18F mediastinum conduit simultaneously to rebuild negative pressure and drain remained blood and irrigating solution in the thoracic cavity.After this, stop using anesthetis, pull out tracheal intubation at the appropriate time and cicatrize a wound.When not having gas permeation or residual blood, pull out the chest conduit.All laboratory animal postoperatives are all accepted antibacterial therapy, intramuscular injection cephazolin 1.0, every day twice, continuous three days; The pain relieving of the buprenorphine of intramuscular injection simultaneously, every day twice, each 0.3mg, logotype three days.
Dosage according to the past experimentation is confirmed carries out therapeutic intervention from implementing first three sky of bone marrow interstital stem cell transplanting to transplanting back four days nursing medicines of the present invention, and using dosage is 0.05gkgd.Nuclear magnetic resonance image (MRI)
Cell transplantation one week the back with six weeks after adopt cine MR I and Contrast-enhanced MRI collection laboratory animal cardiac functional parameter respectively.Adopt clinical use the 1.5T MRI scan machine of being furnished with RF receiving coil (Siemens, Germany (Siemens, Germany)) carry out NMR-imaging.Intramuscular injection of ketamine and diazepam are anaesthetized laboratory animal, and dosage is respectively 25mg/kg and 1mg/kg.MRI adopts wireless ecg-gating spin echo.Cine MR I and the every 4mm scanning of corresponding Contrast-enhanced MRI one deck begin from mitral value level, altogether the 6-8 layer.Measure laterally with the arrowhead view to confirm the correct plane of minor axis, the images of per 60 ° of definite major axis.Unite and use total focus stable state fast gradient echo (TrueFisp) pulse train and sensitivity coding (TSENSE) parallel imaging technology to obtain cine MR I image.Typical case's imaging parameters is following: circulation time (TR)=41.7ms, echo time (TE)=1.39ms, bandwidth (BW)=965Hz/PX; Flip angle (FA)=48 °, image matrix=109 * 192, spatial resolution=3.2mm * 2.0mm; Bed thickness (SL)=6.0mm, parallel factor=3.Adopt the echo-wave imaging element of TSENSE imaging technique to obtain the flow process perfusion image and one four Room image (the TR=6.0ms first time of 3-4 minor axis position; TE=1.22ms, FA=30 °, spatial resolution=2.8mm * 2.8mm; SL=10.0mm; Parallel factor=2, each heartbeat 4-5 opens image, all aspects are once saturated prepulse).The image of about 60 cardiac cycles is obtained in first round scanning.0.1mmol Gd-DTPA (Schering Corp (Schering)) is washed (flow velocity 4mL/s) with 20mL 0.9%NaCl.Perfusion posterior vein injection 0.1mmol/kg magnevist solution, 5 minutes posterior vein injection 0.2mmol/kg diethylene-triamine pentaacetic acid gadoliniums (Gd-DTPA) then directly carry out the Contrast-enhanced MRI photography.Use inversion recovery (PSIR) flashing sequence to accomplish the T1 weighting, utilization PSIR Technique on T 1 is adjusted.The typical image parameter is following: TR=700ms, TE=4.8ms, BW=130kHz, flat resolution=1.8 * 1.3mm, image array=156 * 256, SL=8mm.Take all cine MR I and Contrast-enhanced MRI image 6 weeks once more after transplanting stem cell.Make the corresponding of the short-axis slice of this time taking and baseline shooting for the first time according to anatomy location.In addition, for normal healthy controls is provided, 5 sham operated rats animals adopt identical MRI experimental design to study.
Single photon emission computed tomography (SPECT)
For measuring the damaged area of heart muscle perfusion, with six all backs cardiac muscle is carried out single photon emission computed tomography after one week at cell transplantation.Quiet notes 99m technetium-methoxy isobutyl isonitrile 296MBq (8mCi) carried out myocardium tomography with gammacamera after 45-60 minute.Employing low energy consumption double detector gamma camera (Fa Ruikemu, General Corporation (Varicum, GE)) is furnished with the fine-resolution collimator of 20% energy window, and set is in arriving 140KeV γ peak value.Every hardwood is gathered 40s, 32 images, and acquisition matrix 64 * 64 is thrown illumination range from right front oblique 45 ° ~ left back oblique 45 °, totally 180 °.SPECT rebuilds and adopts the low filtering of Bart wet this (Butterworth), cut-off frequency 0.45, and type is 5, through the heart axle is adjusted, rebuilds the view data of heart minor axis, vertical long axis, three axial planes of horizontal long axis.Adopt scintigraphic buphthalmos technique computes filling defect area.
Histologic analysis
Become cardiac muscle and blood vessel differentiation potentiality for detecting the bone marrow interstital stem cell of transplanting, myocardium frozen tissue section is carried out fluorescence immunoassay, with 5 μ m thickness serial section.The antibody that detects comprises: the specific Factor IX of vascular endothelial cell (VWF 1: 50; DAKO), α-vascular smooth muscle actin (SM-actin, 1: 50, DAKO), α-muscle rhabdomyosarcoma filamentous actin (1: 50; DAKO), cardiac troponin T (cTn-T; 1: 50, Sigma), the slit connect protein 43 (1: 50, Sigma).Section is hatched with the sheep anti mouse or the rabbit igg of rhodamine or marked by fluorescein isothiocyanate after the PBS rinsing.At last, under laser scanning co-focusing microscope, observe and take pictures.
Measure the survival and the differentiation capability of the bone marrow interstital stem cell of transplanting in the body, at the bottom of the apex of the heart to the heart, be cut into 8, the thick frozen section of every picked at random 5 5-μ m with left ventricle is cross-section.Under fluorescence microscope, the cell that DAPI and cTn-T are positive is observed and is counted in 5 visuals field of every frozen section picked at random.The cardiac-like muscle cell that the cell that cTn-T is positive is considered to break up.The iuntercellular stain density that the slit connects protein 43 is measured in 5 sections of picked at random at the infarction position, and the utilization image analysis system is analyzed.
Measure the capillary density of infarcted region and infarction surrounding zone; The tissue preparation method is Weidner N; Semple JP; Welch WR, Folkman J.Tumor angiogenesis and metastasis-correlation in invasive breast carcinoma.N Engl J Med 1991; [324:1-8. PMID:1701519] (Weidner N, Semple JP, Welch WR, tumor vessel takes place and the transfer dependency in the Folkman J. invasive breast carcinoma. new Glan medical journal, 1991; 324:1-8.) put down in writing.With VWF antibody (1: 200, DAKO) section is dyeed.Choose 5 and 8 sections from the infarcted region of each experimental animal respectively with the infraction neighboring area, analyze the blood vessel of counting positive staining by a research worker that has neither part nor lot in the cell processing.5 high power fields of picked at random are counted from every section of choosing, and the result expresses with the blood capillary number under each high power field.
Deoxidation uridine triphosphate (dUTP) the breach end-labelling (TUNEL) of deoxynucleotidyl transferase mediation detects apoptosis
We are with TUNEL analytic process (Luo Shi; Germany) detect apoptosis situation in the cardiac muscular tissue; When the experiment terminal point; Obtain cardiac muscular tissue's section of all animal infarction neighboring areas, paraffin section dewaxes with trypsinization, hatches 60 minutes in 37 ℃ of wet tanks with terminal deoxynucleotidyl transferase (TdT) and fluorescein-labeled dUTP.Hatched 30 minutes with the conjugated alkali phosphatase specific antibody of resorcinolphthalin. then, TUNEL dyes with 3, and 3-diaminobenzidine (DAB) colour developing contains the pulsating karyon of dna break and dyed blueness.In order to detect the ratio of apoptosis karyon in the section; Tissue was redyed (1: 100 with myocardium specific desmin (Desmin) monoclonal antibody; DAKO); Tissue slice is observed under 400 power microscopes, and the minimum myocardial cell of under 8 high power fields, counting more than 100, apoptotic index (apoptotic index) account for the percentage ratio of myocardial cell sum in the visual field for the myocardial cell of apoptosis.
Activities of antioxidant enzymes and MDA
In order to detect the oxidative stress level of infarcted myocardium; We obtain infarction periphery cardiac muscular tissue when the experiment terminal point; Superoxide dismutase detects (company is built up in Nanjing) with xanthine oxidation method, and MDA is represented (company is built up in Nanjing) with the myocardium malonaldehyde level that the thiobarbituricacid method detects.
Statistical analysis
Continuous variable is represented with mean ± standard deviation, with X 2 test (x
2) group difference of the 3rd group and the 4th group differentiation rate is analyzed.Data are carried out homogeneity test of variance and normal distribution assessment; Then carry out variance analysis; Confirm the difference that each group existed in each analysis phase (transplant one week of back and detect for baseline, transplanting six weeks of back be end point determination), with transplant one week the back data serve as reference to transplant six all after data analyze.With least significant difference method (LSD) check a plurality of parameters between two groups are compared.Proofread and correct with Ba Fuluoni (Bonferroni) method, when P＜0.05, the difference significance.All data all use SPSS13.0. to carry out statistical analysis.
The result
Before all parameters are successfully collected, in the matched group, second group, the 3rd group an animal dead is arranged respectively, the dead animal data are not included statistical analysis in.
Histologic analysis
Cell transplantation is after 6 weeks, the HE demonstration of dyeing, and the serious fibrosis in matched group infarction position also the chronic inflammation cellular infiltration occurs, and myocardial cell is survived seldom, and second group and the 3rd group of situation are like this too.Comparatively speaking, the 4th group of fibrosis is lighter with chronic inflammation cellular infiltration phenomenon, and infarcted region has myocardial cell survive (Fig. 1) simultaneously.
Observe and find that the positive cell of the 4th group of DAPI labelling is obviously more than the 3rd group (308.9 ± 88.2 pairs 73.2 ± 21.3, P＜0.0001) (Fig. 2 A-B).
After transplanting for six weeks; The immunofluorescence analysis of the 3rd group and the 4th group shows that the positive cell of DAPI labelling expresses cardiac muscle and the peculiar albumen of blood capillary; Comprise α-muscle rhabdomyosarcoma filamentous actin, cardiac troponin T, von Wilebrand factor and vascular smooth muscle actin, show that the bone marrow interstital stem cell that part is implanted has been divided into cardiac muscle and blood capillary (Fig. 3 A-C).Particularly the 4th group, the efficient that the bone marrow interstital stem cell of its implantation is divided into myocardial cell is significantly higher than the 3rd group (45.8 ± 5.1% pairs 8.7 ± 2.4%, P＜0.0001) (Fig. 3 D).
In addition, connect through the intercellular that detects between the positive cell of expression study infarcted region DAPI labelling that the slit connects protein 43.The result shows that the expression of the 4th group of slit connection protein 43 is significantly more than the 3rd group (16.1 ± 1.4 pairs 4.7 ± 1.8, P＜0.0001) (Fig. 4 AB).
Capillary density
Measure the capillary density of infarcted region and infarction surrounding zone according to the immunohistochemical staining of VWF antibody; Matched group and second group, the 3rd group relatively, the infarcted region capillary density does not have significant difference (1.8 ± 0.5/HPF to 2.0 ± 0.6 pairs 1.8 ± 0.8, P＞0.05); Yet; Compare with the 3rd group, the capillary density of the 4th group of infarcted region has increased by 105% (3.7 ± 1.0/HPF, P＜0.0001).The capillary density of the 4th group of infarction surrounding zone is 8.9 ± 1.9/HPF, is significantly higher than other three groups (4.9 ± 1.3/HPF, 5.1 ± 0.9,5.2 ± 1.4, P＜0.0001) (Fig. 5).
Nuclear magnetic resonance image and single photon emission computed tomography
Get 36 sections altogether for every group and analyze, counting dyskinesia sections thickens rate with analysis chamber wall.After transplanting a week; Dyskinesia sections is respectively 8.2 ± 3.0,8.3 ± 3.1,8.7 ± 3.9,8.9 ± 3.6 in matched group, second group, the 3rd group, the 4th group 36 sections; Account for 22.8%, 23.1%, 24.2% and 24.7% of each group sum respectively, do not have significant difference (P=0.983) between each group.All dyskinesia sections are used to measure partial locular wall and thicken rate.After transplanting a week; Other parameter, comprise local locular wall thicken rate (P=0.915), left ventricular ejection fraction (LVEF, P=0.996), infarct size (P=0.991), left ventricular end diastolic volume (EDV; P=0.852), left ventricular end-systolic volume (ESV; P=0.990), (LVmass index P=0.791), does not relatively have significant difference to left ventricular mass index between 4 groups.After transplanting for 6 weeks, compare the 4th group cardiac function parameters except that EDV and ESV, all be significantly improved (P＜0.0001) with matched group.Each is organized the change of left chamber function and ventricle geometry and sees table 1.
Table 1 is transplanted after the week and the MRI result of six all back left chamber functions and structure
After baseline representes to transplant a week; After terminal point represented to transplant six weeks; LVEF representes the left ventricular ejection mark; EDV representes left ventricular end diastolic volume; ESV representes left ventricular end-systolic volume; Dyskinesia sections (Dyskinetic segments); Locular wall thickens rate (Wall thickening); Infarct size (Infarct size)); Left ventricular mass index (LV massindes);
*P＞0.05 (comparing between four groups after one week of transplanting);
#P＞0.05 (transplant six week the back matched groups with second group between relatively);
*P＞0.05 (second group, the 3rd group EDD and end-systolic volume and matched group are relatively after six weeks of transplanting);
##P＜0.0001 (transplant between six second group, the 3rd group of all back and matched group and compare)
Fig. 6 has comprised the typical SPECT image of transplanting all backs and being used for detecting the filling defect area after six weeks of transplanting.SPECT result after initial one week of transplanting shows does not have significant difference between four groups (is respectively 50.7 ± 14.5% pairs 52.7 ± 15.5% pairs 51.8 ± 16.5% pairs 49.4 ± 16.0%, P=0.984).After transplanting for 6 weeks; Experiment terminal point SPECT shows that matched group, second group, the 3rd group average filling defect area become 47.8 ± 11.1%, 50.7 ± 12.5%, 47.3 ± 13.2% (n=6 respectively; P=0.899); And the 4th group average filling defect area is 22.1 ± 9.3%, significantly reduces (n=7, P＜0.0001) than first three groups.
Infarcted myocardium peripheral cell apoptosis
Through dyeing together with the DNA end labelling to myocyte's specific mark thing is conjugated protein; Compare drug group of the present invention with matched group, comprise the 2nd group and the 4th group, infarction left ventricle apoptotic cell significantly reduces; (apoptotic index 6.1 ± 1.4; 2.4 ± 0.9 pair 10.1 ± 1.8, P＜0.0001), and the 4th group apoptotic index also significantly is less than the 2nd group (P＜0.0001).Yet the 3rd group apoptotic index and matched group compare there was no significant difference (P=0.289) (Fig. 7)
The assessment of oxidative stress level
At the experiment terminal point, the 2nd group and the 4th group of infarction periphery SOD of heart tissue activity are significantly higher than matched group, (98.7 ± 9.8; 105.1 ± 7.0 pairs of 83.4 ± 8.8U/mg albumen, P＜0.05), show that drug group medication of the present invention can strengthen radical scavenging activity; Yet there was no significant difference (87.4 ± 10.2U/mg albumen between the 3rd group and the matched group; P=0.449). corresponding with it is, the 2nd group and the 4th group of infarcted myocardium MDA content obviously reduce (6.1 ± 0.7,6.0 ± 0.6 pairs of 9.0 ± 0.8nmol/mg albumen than matched group; P＜0.05); Do not have between the primary cellular defect of pointing out drug group of the present invention significantly to alleviate lipid peroxidation and to cause, matched group and the 3rd group significant difference (8.5 ± 0.8nmol/mg albumen, P=0.195) (Fig. 8).
Discuss
This experimental study shows: (1) acute myocardial infarction/inject the ABM interstital stem cell in the cardiac muscle immediately after the perfusion again, and transplanted cells is in vivo survived, differentiation capability is limited, heart after the infarction is not produced significant function assessment and benefits; (2) single in a short time use low dosage medicine of the present invention can not produce significant function assessment benefit to heart after the infarction yet; Yet; On the basis of using drug group of the present invention; Injection of bone marrow interstital stem cell in the cardiac muscle immediately after the perfusion of acute myocardial infarction/again, transplanted cells existence in vivo significantly strengthens with the simple transplantation group of differentiation capability, simultaneously medicine of the present invention unite with stem cell transplantation can also reduce infarct size, promotes angiogenesis, improves myocardial function, the reverse remodeling ventricle.
This tests most important discovery: use in a short time on the basis of low dosage drug group intervention of the present invention; Myocardium immediately interior injection of bone marrow interstital stem cell after acute myocardial infarction reaches and pours into; Implant cell existence and the bone marrow interstital stem cell of the more single implantation of differentiation capability in vivo and significantly strengthen, have remarkable effect to improving cardiac function simultaneously.This shows, the transplanting of stem cell, survival and the differentiation myocardium microenvironment after to acute myocardial infarction has very strong dependency.
This experiment shows; Compare with simple bone marrow interstital stem cell group; Unite survival and the differentiation of using medicine of the present invention and bone marrow interstital stem cell transplantation group to implant cell and significantly improve, in addition, use low dosage drug group of the present invention significantly not improve cardiac function merely.Based on above-mentioned experimental result, we can infer, drug group of the present invention to acute myocardial infarction after the improvement of local microenvironment improved survival rate and the biological activity of implanting cell.Although use low dosage medicine of the present invention itself can not produce tangible curative effect merely, it can significantly improve the survival and the differentiation capability of the bone marrow interstital stem cell of implantation.This experimental result shows, uses low dosage drug group intervention of the present invention that utilization ABM interstital stem cell is carried out cell cardiomyoplasty art in a short time and plays facilitation.The gene expression profile of heart finds that single use low dosage drug group of the present invention can make gene expression produce actively and change after the microarray biochip technology detection infarction, comprises the rise (data not shown) to antiinflammatory, anti-apoptosis, fibrosis gene.Based on above research, we think and use the local microenvironment after low dosage medicine of the present invention can improve acute myocardial infarction in a short time, thereby make the stable survival of bone marrow interstital stem cell and the differentiation of implantation.
In sum; We find to use in a short time the local microenvironment after low dosage medicine of the present invention can effectively improve acute myocardial infarction first; Utilization ABM interstital stem cell is carried out cell cardiomyoplasty art play facilitation, significant to the clinical practice of the transplanting of bone marrow interstital stem cell.
Claims (7)
1. a Chinese medicine composition carries out cell cardiomyoplasty art in preparation and plays the application in the medicine of facilitation to utilization ABM interstital stem cell, it is characterized in that said Chinese medicine composition processed by following bulk drugs:
Radix Ginseng 3-10 Hirudo 3-11 Eupolyphaga Seu Steleophaga 5-10 Olibanum (processed) 1-5 Radix Paeoniae Rubra 3-9 Lignum Dalbergiae Odoriferae 1-5 Lignum Santali Albi 1-5 Scorpio 3-9 Periostracum Cicadae 3-12 Scolopendra 1-3 Borneolum Syntheticum 1-7 Semen Ziziphi Spinosae (parched) 3-10.
2. application as claimed in claim 1 is characterized in that said Chinese medicine composition processed by following bulk drugs:
Radix Ginseng 6 Hirudos 10 Eupolyphaga Seu Steleophagas 7 Olibanum (processed)s 2 Radix Paeoniae Rubra 5 Lignum Dalbergiae Odoriferaes 2 Lignum Santali Albis 2 Scorpios 7 Periostracum Cicadaes 7 Scolopendras 1 Borneolum Syntheticum 5 Semen Ziziphi Spinosae (parched)s 5.
3. application as claimed in claim 1 is characterized in that said Chinese medicine composition processed by following bulk drugs:
Radix Ginseng 10 Hirudos 8 Eupolyphaga Seu Steleophagas 7 Olibanum (processed)s 2 Radix Paeoniae Rubra 5 Lignum Dalbergiae Odoriferaes 2 Lignum Santali Albis 2 Scorpios 9 Periostracum Cicadaes 7 Scolopendras 1 Borneolum Syntheticum 5 Semen Ziziphi Spinosae (parched)s 5.
4. application as claimed in claim 1 is characterized in that said Chinese medicine composition processed by following bulk drugs:
Radix Ginseng 6 Hirudos 11 Eupolyphaga Seu Steleophagas 7 Olibanum (processed)s 2 Radix Paeoniae Rubra 5 Lignum Dalbergiae Odoriferaes 2 Lignum Santali Albis 2 Scorpios 3 Periostracum Cicadaes 7 Scolopendras 1 Borneolum Syntheticum 5 Semen Ziziphi Spinosae (parched)s 5.
5. application as claimed in claim 1 is characterized in that said Chinese medicine composition processed by following bulk drugs:
Radix Ginseng 5.5 Hirudos 10.375 Eupolyphaga Seu Steleophagas 6.875 Olibanum (processed)s 2.25 Radix Paeoniae Rubra 4.75 Lignum Dalbergiae Odoriferaes 2.375 Lignum Santali Albis 2.25 Scorpios 6.875 Periostracum Cicadaes 6.875 Scolopendras 1.375 Borneolum Syntheticums 1.375 Semen Ziziphi Spinosae (parched)s 4.625.
6. like each described application among the claim 1-5, it is characterized in that the active component of said Chinese medicine composition is made up of following ingredients:
The a mean diameter is less than Scorpio, Hirudo, Scolopendra, Eupolyphaga Seu Steleophaga, Periostracum Cicadae and the Olibanum (processed) medicated powder of 100 μ m;
B Borneolum Syntheticum medicated powder;
The volatile oil that c is extracted by Lignum Dalbergiae Odoriferae and Lignum Santali Albi;
The alcohol-extracted extract of the alcohol extract of d Radix Ginseng after after concentrating with ethanol extraction;
The water extracted immersing paste that is condensed into behind the water extract after the Lignum Dalbergiae Odoriferae behind the e extraction composition c and water extract, Radix Paeoniae Rubra and the Semen Ziziphi Spinosae (parched) decocte with water of Lignum Santali Albi medicinal residues and the water extract filtration of the medicine residues of Radix Ginseng after the extraction ingredient d, the mixing.
7. like each described application among the claim 1-5, it is characterized in that this Chinese medicine composition is capsule, tablet, pill or oral liquid as the pharmaceutical preparation of active component.
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