| BackgroundEndothelial progenitor cells (EPCs) were firstly found by Pro. Asahara, and were defined as precursors of endothelial cells which positive expressed CD34, CD133, VEGFR2. EPCs derived from the bone marrow, existed in the systemic circulation. It can proliferate, migrate, and differentiate into mature endothelial cells and participate in angiogenesis, besides it is capable of binding in the blood vessels and stimulating the proliferation of neighboring endothelial cells. Many EPCs agonists such as granulocyte-colony stimulating factor, vascular endothelial growth factor (VEGF) and statins, can mobilize EPCs in bone marrow. However adverse reactions, such as increased vascular permeability and high ratio of restenosis and liver damage, limit their use in clinical.Prepared Rehmannia glutinosa (PRG), belongs to the family of Scrophulariaceae, is a widely used traditional Chinese medicinal herb. It has been used to treat hypodynamia caused by many kinds of diseases. It has been effective and safe, but the involved mechanism has not been verified. Recently, Rehmannia glutinosa extract (RGE) has been used in modern medicine studies. RGE can stimulate the proliferation and differentiation of hematopoietic stem cells in bone marrow and increase the DNA content of bone marrow. So we supposed that Rehmannia glutinosa extract (RGE) could activate EPCs.Objectives Grasp the method of RGE systemic administration. Investigate the changed effect of RGE on EPCs in bone marrow and circulation blood of normal rats along with the differ in concentration and duration of RGE. Screen the optimal concentration and duration for RGE, and provide basis for the subsequent experiments. Explore the separation, induced cultivation, identification and functional measurements for EPCs in vitro.Method1. Preparations of Rehmannia glutinosa extract (RGE)Rude polyoses was extracted form Rehmannia glutinosa powder by water dissolving and alcohol extracting repeatedly. And then RGE was extracted and purified in Sevag way. The concentration, purification and extraction ratio were tested.2. Animal group80male Wistar rats were random divided into four groups:in the control group (n=20) rats were oral-treated with normal saline, in the RGE-L group (n=20) rats were oral-treated with RGE at0.38g·kg-1·day-1, in the RGE-M group (n=20) rats were oral-treated with RGE at0.75g·kg-1·day-1, in the RGE-H group rats were oral-treated with RGE at1.5g·kg-1·day-1.5rats in each group were sacrificed at4,8,12,16weeks after systemic delivery.3Isolation and cultivation of EPCs10ml peripheral blood was obtained from rats by aspiration of the heart. Bone-marrow cells were obtained by flushing the cavity of femurs, tibias, and humerus with growth medium EBM-2. Peripheral blood and bone marrow mononuclear cells were isolated by Ficoll density-gradient centrifugation.10million isolated cells were resuspended in growth medium EBM-2and plated in25-cm2culture flasks. After48h, non-adherent cells were discarded and growth medium was changed every2days.4. Identification of EPCsDirect fluorescent staining was used to detect dual binding of FITC-UEA-land Dil-acLDL and the nuclei were stained with DAPI. Cells double stained for Dil-Ac-LDL and FITC-UEA-1were considered EPCs. Immunocytochemistry followed standard protocols.5. Determination of EPC numberFluorescence-activated cell sorting (FACS) was used to determine the EPC population in blood and bone marrow of rats. Briefly, fresh anticoagulation blood or bone-marrow PBS suspension (200μl) was incubated with the monoclonal antibodies: anti-VEGFR2, anti-CD133and anti-CD34-PerCP-Cy5.5for20min at room temperature, then with2ml Lysing solution for10min and washed with PBS twice by centrifugation. The cells were resuspended with200μl PBS, then incubated with the secondary antibodies:goat polyclonal rabbit IgG-FITC and goat polyclonal mouse IgG-F(ab)2fragment PE for30min at room temperature. Cells were washed with PBS and resuspended in400μl PBS.6. function test of EPCs(1) MTT assay was used to evaluate EPC proliferation.(2) EPC migration was evaluated by use of a Transwell chamber.(3) Capillary-like tube formation was analyzed by use of Matrigel Matrix.7. Statistical analysisData are expressed as mean±SD and were assessed by one-sample Kolmogorov-Smirnov test to check for normal distribution. Differences between2groups were assessed by unpaired t-test and among multiple groups by ANOVA followed by post-hoc two-tailed Newman-Keuls test. Data analysis involved use of SPSS11.5(SPSS Inc., Chicago, IL). Statistical significance was set at P<0.05.Results1. General situation of ratsThe rats (6weeks old, body weight at160-180g) were fed a regular rat chow and housed in normal night-day conditions under standard temperature and humidity. The rats in each group were normally health and no natural mortality during the test.2. The number of EPCs was evaluated by FACS The FASC showed that the number of EPCs increased with RGE systemic delivery both in blood and in bone marrow, and this tendency intensified with the time went by. In peripheral blood, at the end of8weeks, the EPCs number in RGE-M and RGE-H groups was statistically increased compared to control group (P<0.01-0.05); at the end of12and16weeks, the EPCs number in all the three RGE groups was increased significantly (P<0.01), however the ratios from12to16weeks (8.53%,1.57%,1.79%) were much lower than that from8to12weeks (10.43%,12.84%,16.50%). In bone marrow, at the end of8weeks, the EPCs number in RGE-M and RGE-H groups was increased compared with control group (P<0.05); at the end of12and16weeks, the EPCs number in all the three RGE groups was increased significantly (P<0.01), however the ratios were positive from8to12weeks (6.95%、12.10%.17.14%) and the ratios decreased (RGE-H3.34%) or negative (RGE-L RGE-M groups) from8to12weeks.3. The functional test of EPCs in peripheral bloodMononuclear cells were isolated from peripheral blood, induced cultured by EVM-2for48h, the nonadherented cells were centrifuged and co-cultured for another48h, and then discarded the cells still not adherent culture for the third48h. These cells were digested by trypsin and counted for use.(1) Proliferation testMTT showed the proliferation of EPCs from peripheral blood. At the end of16weeks, the proliferation of EPCs increased in RGE-M and RGE-H group compared with control group (P<0.05) and the same group at the end of4weeks (P<0.05). The other concentration at other time points had no statistical different.(2) Migration testMigration of EPCs in peripheral blood was evaluated by use of Transwell chamber. At the end of12weeks, it increased in RGE-M and RGE-H group compared to control group and the same group at the end of4weeks (P<0.05), however these changes seemed shyly. And as from12to16week, it had no further growth.(3) Tube-formation test Tube-formation capacity of peripheral blood EPCs was evaluated by Matrigel Matrix. In RGE-H group, at the end of8weeks it had increased compared to the end of4weeks (P<0.05), and at the end of12weeks it was more than control group (P<0.05). At the end of16weeks, the tube-formation capacity of EPCs in all the RGE groups significantly increased compared with control group and the same group at the end of4weeks as well.4. The functional test of EPCs in bone marrowMononuclear cells were isolated from bone marrow PBS solution, induced cultured by EVM-2for48h, the nonadherented cells were centrifuged and co-cultured for another48h, then transfer of culture and discarded the cells still not adherent. After culture for6days, cells were digested by trypsin and counted for use.(1) Proliferation testMTT showed the proliferation of EPCs from bone marrow. At the end of12weeks, the proliferation of EPCs increased in RGE-M and RGE-H group compared with control group (P<0.05) and the same group at the end of4and8weeks (P<0.05). At the end of16weeks, it increased further more in all RGE groups than control group.(2) Migration testMigration of EPCs in bone marrow was evaluated by use of Transwell chamber. At the end of12and16weeks, it increased in all the RGE groups compared to control group and the same group at the end of4,8weeks (P<0.05), however the extent didn’t significant.(3) Tube-formation testTube-formation capacity of bone marrow EPCs was evaluated by Matrigel Matrix. At the end of12weeks, the tube-formation capacity of EPCs increased in RGE-M and RGE-H group compared with control group (P<0.05). At the end of16weeks, it significantly increased in all the RGE groups compared with control group and the same group at the end of4weeks (P<0.01-0.05). The RGE-H group was more significant than other groups (P<0.01). Conclusion1. Under physiological condition, rats systemic delivery with three different concentration of RGE, and lead to the number of EPCs in peripheral blood and bone marrow increased. This increase was much more significant in bone marrow than peripheral blood. The RGE-H group had the most extrusive effect among the three concentrations. The increment speed was maximum at about12weeks after oral-feed.2. At the condition of physiology, RGE systemic delivery was able to active the EPCs in peripheral blood and bone marrow. This activation in peripheral blood EPCs was not so obvious as that in bone marrow EPCs. And in bone marrow, the activation of EPCs often appeared12weeks after oral-feed. BackgroundMyocardial infarction (MI) occurs with the deprivation of coronary blood and is usually caused by stenosis or occlusion of the coronary artery. The culminating event is necrosis of myocardial tissue and dysfunction of the left ventricle. Therefore, recanalization and establishment of collateral circulation are primary measures for saving ischemic myocardium, improving the prognosis of myocardial infarction and reducing mortality. Many studies proved that cell transplantation and therapeutic angiogenesis is a most potential and significant method for the treatment of end-stage coronary artery disease. Recent studies showed that endothelial progenitor cells (EPCs) which were differentiated from bone marrow-derived stem cells can be activated after myocardial infarction, and mobilization, migration, homing to the site of injury and participated in angiogenesis, so that protect the infarcted myocardium. The modern medical studies about Prepared Rehmannia Glutinosa showed that itcould effectively promote the proliferation of blood cells and bone marrow, it could also be used for the treatment of ischemic cardiovascular and cerebrovascular diseases. Obsolete research methods, relatively single mechanism, superficial studies about mechanism, however superficial limits the promotion of Rehmannia in clinical research. And so far no research connected the mechanism of Rehmannia Glutinosa Extra (RGE) with the action of endothelial progenitor cells yet.Objectives1. Establish a rat model of myocardial infarction, and investigate the protective effect of Chinese medicine Prepared Rehmannia Glutinosa on infarcted rat myocardium in acute and chronic phase.2. Observed the change of endothelial progenitor cells in rat peripheral blood and bone marrow after myocardial infarction, including number and function. Explore the therapeutic effect of endothelial progenitor cells in the acute and chronic phase of myocardial infarction. Discuss the role of RGE on endothelial progenitor cells’ therapeutic effect.Methods1. Animal groupA total of120male Wistar rats (8weeks old; body weight180-200g) were randomized to2groups (n=60each) for treatment:high-dose RGE (1.5g·kg-1·day-1orally) for8weeks, then left anterior descending coronary artery ligation (n=28), mock surgery (n=16) or no treatment (n=16), then RGE orally for4weeks; or normal saline (NS) as the above protocol. Then rats were sacrificed,4～7each on the3rd day and the end of1,2,4weeks, recording as day3, week1, week2and week4respectively.2. The establishment of a rat myocardial infarction model.Method1:The rats were narcotized by2.5%napental (30mg/kg), and fixed the rat on the operating table in supine position, cut off its chest hair. Horizontal cut the chest shin about1.5～2cm at the3to4left intercostal sternal where the apex beat most obvious and blunt dissection of the subcutaneous tissue, pectoralis major, serratus anterior muscle. Blunt softened3to4intercostal shun intercostal muscle texture, seen the beating apex, fast through the myocardium0.5cm above the apex with the absorbable suture, and closed ligation. Light squeeze together serratus anterior muscle, the pectoralis major, chest skin and discharge pleural gas before suture the skin incision. Subcutaneous injection of penicillin80,000IU/kg of conventional anti-infective.Method2:The rats were narcotized by ethylether, and fixed the rat on the operating table in supine position, cut off its neck and chest hair. Longitudinal cut open the rat neck skin about1.5～2cm, separate subcutaneous tissue and muscle, blunt separation trachea, cut open tracheal half cycle between trachea ring. Do endotracheal intubations, connection breathing machine, adjust the frequency of breathing, breathing ratio and tidal volume. Longitudinal cut open chest skin about4cm at the left edge of the sternal3mm place, blunt separation subcutaneous tissue, and the pectoralis major, former saw muscle. Cut open the intercostal muscle on the third intercostals space to exposure pleura. Open the frame, punctured pleura at the breathe out phase, exposing the heart. Absorpted suture ligation at the anterior descending coronary artery in place2mm below the common border of the pulmonary artery cone and the left atrium. Layered suture muscle and skin. Remove the endotracheal intubation after rats were awake, and clearing the airway blood clot and secretion.The rats in mock groups underwent mock surgery with a silk suture across the coronary artery without ligation.3. Mortality analysis:Reserved survival record and draw the survival curve.4. Electrocardiography and echocardiography analysisBefore and after surgery, rats underwent electrocardiography (ECG) by use of a Micromaxx P04224system and ultrasonic cardiography (UCG) by a high-frequency duplex ultrasonic cardiogram system and a transducer. Rats underwent ultrasonic cardiography at day3, weeks1,2and4before sacrificed. The transducer for ultrasonic cardiography was placed at the left thoraces between the3rd and4th ribs to obtain B-mode tracings of the heart from just below the level of the papillary muscles of the mitral valve. We obtained left-ventricular end-diastolic diameters (LVD-d) and end-systolic diameters (LVD-s) with M-mode tracings between the anterior and posterior walls. The time of end-diastole and end-systole was defined as time of maximum and minimum diameter of the left ventricle, respectively, in one heart cycle. Following the American Society of Echocardiology leading-edge method, we obtained3images, on average, in each view, which were averaged over three consecutive cycles. The system calculated the left-ventricular end-diastolic volume (LV-d), left-ventricular end-systolic volume (LV-s), mass of the left ventricle (LV-mass), left-ventricular fractional shortening (LVFS) and left-ventricular ejection fraction (LVEF)5. Serological markers detectionELISA was used to measure cardiac troponin T (Tn-T) and brain natriuretic peptide (BNP) concentration in serum for left ventricular function evaluation, by use of a BNP kit (Rat-45, Abcam, USA) and Tn-T kit (TSZ ELISA, USA). Briefly, standards and diluted serum of rats were added into the pre-coated96-well plates and incubated for30min in37℃. After a washing with PBS, the horseradish peroxidase-conjugated anti-body was added for30min incubation at37℃. After a washing by PBS, the tetramethylbenzibine substrate was added. After reaching the desired color density, the reaction was terminated by stop solution. OD450was determined by use of an ELISA plate reader (Varioskan Flash, Thermo Fisher, Germany). Each samples repeated in3wells.6. Determination of EPC number in peripheral blood and bone marrowFluorescence-activated cell sorting (FACS) was used to determine the EPC population in blood and bone marrow of rats. Briefly, fresh anticoagulation blood or bone-marrow PBS suspension (200μl) was incubated with the monoclonal antibodies: anti-VEGFR2(Abeam, USA,1mg/ml,1:100), anti-CD133(Abeam, USA,0.5mg/ml,1:100) and anti-CD34-PerCP-Cy5.5(Santa Cruz Biotechnology, Santa Cruz, CA;0.2mg/ml,1:10) for20min at room temperature, then with2ml Lysing solution (BD, USA) for10min and washed with PBS twice by centrifugation. The cells were resuspended with200μl PBS, then incubated with the secondary antibodies:goat polyclonal rabbit IgG-FITC (Abeam, USA,2mg/ml,1:80) and goat polyclonal mouse IgG-F(ab)2fragment PE (Abeam, USA,0.5mg/ml,1:40) for30min at room temperature. Cells were washed with PBS and resuspended in400μl PBS. Flow cytometry involved use of a FACS calibur flow cytometer and Cell-Quest software (BD Biosciences, USA). Each analysis included at least10,000cells.7. Function detection of EPCs from bone marrow.(4) MTT assay was used to evaluate EPC proliferation. (5) EPC migration was evaluated by use of a Transwell chamber.(6) Capillary-like tube formation was analyzed by use of Matrigel Matrix.8. Histological analysisMyocardial tissues (approximately2mm thick) in the left ventricle of rats were removed and fixed in4%pre-cooled paraformaldehyde for72h, then embedded in paraffin, and sectioned into slices5μm thick. Haematoxylin eosin (HE) and Poley’s stain was used to observe the form of the myocardium and assess the ischemic myocardial area. Images were visualized under an optical microscope at×200magnification.9. Apoptosis of myocardium detection.Myocardial tissue sections underwent the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) using an in situ detection kit (Roche, Germany) following the manufacturer’s instructions. The TUNEL apoptotic index was determined by calculating the ratio of TUNEL-positive cells to total myocardial cells.10. Immunohistochemical analysisImmunohistochemical staining involved standard techniques as described. Briefly, endogenous peroxidase activity was inhibited by incubation with3%H2O2. Sections were blocked with5%calf serum in PBS and incubated overnight at4℃with the monoclonal antibodies:anti-VEGFR2(Abcam, USA,1mg/ml,1:100), anti-CD133(Abcam, USA,0.5mg/ml,1:50) and anti-CXCR4(Abcam, USA,1mg/ml,1:100). After a washing with PBS, sections were incubated with secondary antibody at37℃for30min. Immunohistochemical staining was visualized by use of a diaminobenzidine kit (Zhongshan Goldenbridge Biotechnology, Beijing). Samples were counter stained with hematoxylin for nuclei.11. Real-time quantitative polymerase chain reaction (RT-PCR)Tissue samples were frozen with the use of liquid nitrogen. Total RNA was extracted by use of TRIZOL reagent (Invitrogen, USA), quantified by spectrophotometry and reverse transcribed by use of the M-MLV Reverse Transcriptase System (Osaka, Japan) with oligo-dT primers. The mRNA expression of VEGFR2, CD133, and CXCR4in myocardium was examined by real-time RT-PCR with SYBR Green Real-time PCR Master Mix (TOYOBO, Life Science Department, Japan) and an MYIQTM Single Color Real-Time PCR Detection System (Bio-Rad, Germany). The mRNA sequences were obtained from Gene-bank (NCBI, Bethesda, MD; Tablel). Actin level was an internal control. Experiments were performed in triplicate, and data were analyzed by the2-△△CT method.12. Statistical analysisData are expressed as mean±SD and were assessed by one-sample Kolmogorov-Smirnov test to check for normal distribution. Differences between2groups were assessed by unpaired t-test and among multiple groups by ANOVA followed by post-hoc two-tailed Newman-Keuls test. Data analysis involved use of SPSS11.5(SPSS Inc., Chicago, IL). Statistical significance was set at P<0.05Results1. General situation of ratsThe rats (6weeks old, body weight at160-180g) were fed a regular rat chow and housed in normal night-day conditions under standard temperature and humidity. The rats in each group were normally health and no natural mortality during the test.2. Electrocardiography and echocardiography analysisAfter surgery induction, the ECG revealed elevated ST segment and pathologic waveforms, the UCG revealed changes in left ventricular wall mobility, blood flow at the mitral valve and the increased LV-d, LV-s, LV-mass, the decreased LVEF, LVFS, the reversed E/A ratio (P<0.01-0.05). These revealed the successful establishment of the MI model. After MI, the function of left ventricular was reflected by LVEF of UCG. In acute stage (day3to week1), the MI groups with both treatment showed almost no difference in LVEF, while as to the chronic stage (week2and week4), the recovery of LVEF was greater with RGE than NS (P<0.05). These revealed that RGE systemic delivery protected the function of left ventricular in chronic stage after MI. 3. Serological markers detectionThe significantly up-regulated serum levels of Tn-T and BNP in NS-MI and GRE-MI groups also showed the successful establishment of mouse MI model (P<0.01). After MI, the high level of Tn-T decreased in RGE group (from week1, P<0.01～0.05) earlier than that in NS group (from week4, P<0.01). As for BNP, the level increased from day3to week4with NS (P<0.01～0.05), while had no changes with RGE treatment after MI. These revealed that RGE systemic delivery decreased myocardial damage and protected them from further inflammatory reaction after MI.4. Determination of EPC number in peripheral blood and bone marrowFACS was used to analyze the quantity of EPCs marked by CD34, VEGFR2and CD133in blood and bone marrow. After MI, the quantity of EPCs in peripheral blood increased (P<0.01) and it decreased in bone marrow (P<0.05) with both RGE and NS. These suggested that the EPCs in bone marrow were mobilized to peripheral blood as the injury of myocardium. In chronic stage after MI, the increase of EPCs in peripheral blood was more significant with RGE compared to NS (P<0.01), and the decrease of EPCs in bone was not so much with RGE as it with NS. These suggested that in chronic stage after MI, RGE was able to increase EPCs mobilizing to ischemic myocardium and maintain the quantity of EPCs stored in bone marrow. With the increased EPC population in both bone marrow and peripheral blood, the total number of EPCs in vivo was much more with RGE than NS.5. Function detection of EPCs from bone marrowBy MTT assay, we tested the proliferation of EPCs in each group. The MI surgery made the proliferated activity of EPCs up-regulated with both GRE and NS (P<0.01). However, it maintained at a high level with RGE compared to it decreased in week2and4with NS (P<0.05). These showed that RGE was able to up-regulated the proliferation of EPCs after MI, especially in chronic stage.At week4after MI, the migration of EPCs was more active with RGE than NS (P<0.05). From week2after MI, EPCs participated in capillary-like tube formation were increased with RGE compared to NS (P<0.05), and a similar increase occurred in normal rats with RGE relative to NS (P<0.05). These suggested REG’s function on motivating EPCs tube-formation capacity after MI as well as in normal physiological status6. Histological analysisHE staining showed that at weekl after MI, myocardial tissue infarction occurred obvious histomorphology changes:infarction area muscle fiber dissolving, fracture, arrangement disorder.Poley’s stain showed the ischemic myocardial zone (stained red) in normal myocardium (stained blue). In chronic stage after MI, the relative ischemic area was lower with RGE than NS (P<0.05).7. Apoptosis of myocardium detectionTUNEL stain and quantitative analysis showed that the apoptotic myocardium was less with RGE than NS in chronic stage after MI (P<0.01～0.05). These showed RGE’s function on improving ischemic myocardium and decreasing myocardial apoptosis.8. Immunohistochemical analysisImmunohistochemistry showed the fluctuant expression of VEGFR2and CD133, which were signals of new-born capillary. After MI, the expression of VEGFR2increased from week1until week4with RGE (P<0.05-0.01), and was much more significant than that with NS (P<0.05～0.01). In chronic stage after MI, the expression of CD133also increased more with RGE than NS (P<0.01). The expression fluctuation at the level of mRNA was almost the same. These revealed that RGE was able to promote the newborn of capillary at the chronic stage of MI.9. Real-time quantitative polymerase chain reaction(RT-PCR)Real-time PCR showed the fluctuant expression at the level of mRNA on VEGFR2and CD133, which were signals of new-born capillary. The expression of VEGFR2increased from week1until week4with RGE (P<0.05～0.01) after MI, and was more significant than that with NS (P<0.05～0.01). In chronic stage of MI, the expression of CD133mRNA also increased with RGE compared to NS (P<0.01). Conclusions1. RGE systermic delivery after myocardial infarction can reduce necrosis and apoptosis of the myocardial ischemic cells, protect the infarction ventricular function through increasing angiogenesis and improving blood supply in infarction region, thus improve the prognosis of myocardial infarction.2. RGE systermic delivery can increase the quantity of EPCs mobilized from bone marrow to peripheral blood after myocardial infarction, and maintain a highly active of EPCs, promote their participated into tube cavity formation. At the same time, RGE could also keep the relative stability number of EPCs in bone marrow.3. Compared with the NS group, the effects of RGE in the treatment of myocardial infarction almost occurred in the chronic stage rather than acute stage. BackgroundIn physiological state, the number of endothelial progenitor cells (EPCs) in peripheral blood and tissue is very low, there are a certain number of EPCs in the bone marrow, however, most of them were under the resting state. Local damage or some EPCs agonist was able to mobilize the EPCs from bone marrow to the peripheral blood, and plante in the damage localization. Stromal cell-derived factor (SDF) played an important role in the process of EPCs’ mobilization and migration.Activated EPCs first migrate to the ischemic tissue for their roles. Stromal-derived factor-1(SDF-1, or CXCL12) is the only known chemokine capable of migration of hematopoietic stem cells (HSCs), as the fluctuations in SDF-1expression controlled the fluctuated steady-state of HSCs and their progenitors in peripheral blood. Among these, the SDF-1α and its receptor4(CXCR4) play a key role in mobilization and migration of EPCs. After MI, SDF-1α/CXCR4interaction plays a crucial role in recruiting EPCs to the ischemic myocardium, the increased CXCR4expression lead to increased EPCs homing to the ischemic zone and participated in therapeutic angiogenesis. These suggest that the SDF-1α/CXCR4cascade is critical for the regulation of EPCs, and it might be an important therapeutic target for cardiovascular diseases especially in MI.In experiment Ⅰ and experiment Ⅱ, we had confirmed that Rehmannia Glutinosa Extract (RGE) could up-regulated the number and function of EPCs in bone marrow and peripheral blood, under the state of pathologic and physiologic caused by myocardial infarction model. In the chronic phase of myocardial infarction, RGE treated the myocardial infarction through activating EPCs to participate in angiogenesis in ischemic myocardium and the surrounding areas as well. We suggested that the effects of RGE on EPCs mobilization, migration, homing and angiogenesis function was through activating SDF-la/CXCR4cascade.Objectives1. Observe the situation of RGE stimulating EPCs in vitro.2. Investigated the mechanism of RGE activating EPCs.Materials and methods1. Cell resourceGet from long bones (humerus, femur, tibia) of male WISTAR rat, isolated the mononuclear cells from bone marrow, induced and cultured primary endothelial progenitor cells.2. Cultivation and identification of EPCsInduced and cultured the bone marrow-derived mononuclear cells by EGM-2medium. After96hours, collected the adherented cells, analyzed these cells by Fluorescence-activated cell sorting (FACS) and Direct fluorescent staining. The EPCs were double stained by DiL-acLDL and FITC-UEA-1, the nuclei were stained with DAPI, and viewed by laser scanning confocal microscope.3. Cell stimulation(1) Concentration gradient screening:the EPCs were cultured with a serum-free medium for24hours, RGE were added to the6-well plates with endothelial progenitor cells at the concentration of10,25,50,100,500and1000μg/ml, the control group EPCs were cultured only with the medium. Collected the cells and supernatant of each well after72hours, waiting to be tested.(2) The optimal concentration screening:the EPCs were cultured with a serum-free medium for24hours, RGE were added to the6-well plates with endothelial progenitor cells at the concentration of10,25,50and100μg/ml, the control group EPCs were cultured only with the medium. In the inhibitor group, EPCs were pre-stimulated with AMD3100(specific inhibitors of the CXCR4,5μg/ml) for one hour and then with RGE (100μg/ml). Each group of cells and supernatant were collected after72hours stimulation, waiting to be tested.(3) The optimal duration screening:the EPCs were cultured with a serum-free medium for24hours, RGE stimulated EPCs at its optimal concentration (50μg/ml for SDF-1α,25μg/ml for CXCR4). The control group EPCs were cultured only with the medium. Collected the cells and supernatant at6,12,24,48,72hours after stimulation respectively, waiting to be tested.(4) Mechanism analysis:the EPCs were cultured with a serum-free medium for24hours, stimulated the EPCs with RGE at the optimal concentration and duration of SDF-1α (50μg/ml,24h) and CXCR4(25μg/ml,48h), in the control group EPCs were cultured only with the medium. In the inhibitor group, EPCs were pre-stimulated with AMD3100(5μg/ml) for one hour and then with RGE (50μg/ml for24h,25μg/ml for48h), remove the RGE-containing medium, plus normal medium for functional test.4. Proliferation function detection5. Western blot analysis(1) Extract the myocardial tissue protein, measure the protein concentration, and exam the protein expression levels of CXCR4, SDF-1α and β-actin by gel electrophoresis, membrane transform, incubating with antibody and exposure etc.(2) Extract the protein from EPCs, exam the protein expression levels of CXCR4, SDF-1α and p-actin as above.6. RT-PCR analysis(1) Extract the myocardial tissue mRNA, reverse transcribed into cDNA, exam the mRNA levels of CXCR4, SDF-1α and β-actin by quantitative real-time polymerase chain reaction.(2) Extract the mRNA from EPCs, exam the mRNA levels of CXCR4, SDF-1α and β-actin as above.7. Statistical analysis Data are expressed as mean±SD and were assessed by one-sample Kolmogorov-Smirnov test to check for normal distribution. Differences between2groups were assessed by unpaired t-test and among multiple groups by ANOVA followed by post-hoc two-tailed Newman-Keuls test. Data analysis involved use of SPSS11.5(SPSS Inc., Chicago, IL). Statistical significance was set at P<0.05.Results1. Myocardial immunohistochemical stainingImmunohistochemical staining was used to detect the expression of CXCR4in myocardium. We can see that, compared with the control group and the sham group, the CXCR4expression increased in ischemic myocardium (P<0.01); for the infarction groups, compared with normal saline (NS), RGE could increase myocardial expression of CXCR4in the chronic phase of MI (2weeks to4weeks)(P<0.01); RGE could also increase myocardial CXCR4expression in the control group and the sham group (P<0.05～0.01).2. The protein expression in myocardial tissueExtract the myocardial tissue protein, test the expression of SDF-1α/CXCR4cascade by western blot. Image Pro-Plus software was used for quantitative analysis and then we found that:Compared with the control group and the sham group, MI group CXCR4expression was increased (P<0.01), SDF-1α also increas... |
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