CN110151791B - Application of endothelial progenitor cells in transplantation treatment of rat coronary artery microembolism - Google Patents
Application of endothelial progenitor cells in transplantation treatment of rat coronary artery microembolism Download PDFInfo
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Abstract
The present invention relates to the treatment of coronary artery microembolism. The invention discloses a coronary artery microembolism (CME) which is a common and important clinical event in acute coronary syndrome and coronary artery interventional therapy, and discovers that low-dose EPC transplantation can increase the generation of endothelial growth factor VEGF and capillaries, inhibit fibroblast growth factor bFGF, relieve myocardial tissue pathological changes and myocardial tissue collagen fiber hyperplasia caused by CME, and provide a new strategy for CME treatment.
Description
Technical Field
The invention relates to the treatment of coronary artery micro-embolism, in particular to the application of endothelial progenitor cells in the transplantation treatment of coronary artery micro-embolism.
Background
Rupture of atherosclerotic coronary plaque leads to platelet activation, aggregation and thrombus formation, while complete occlusion of the epicardial larger coronary blood vessels is an important pathogenesis of acute myocardial infarction (ami), whereas the falling of ruptured atherosclerotic plaque fragments into distal small vessels may directly or secondarily cause distal vessel occlusion, i.e., Coronary Microembolism (CME).
Micro-thrombi are often found in the coronary microcirculation (mostly 10-100 μm in inner diameter) of patients who die from ischemic heart disease. This coronary microthrombus is characterized by platelet aggregation, fibrinogen and atherosclerotic material (including cholesterol crystals), with the perivascular involvement of microthrombus often being accompanied by myocardial microtrauma and inflammatory responses. Echocardiography tests show that the wall motion of the ventricle on cme animal models caused by color microspheres with the diameter of 25 mu m is weakened, and myocardial micro-infarction and the like can be seen on heart pathological specimens. Cme, the prognosis of the patient is poor, and its recent and long-term complications include heart failure, fatal arrhythmia, and micro-myocardial infarction. The causes may be associated with plaque rupture, an inflammatory response of the microvasculature in the acute phase, and cardiac remodeling in the chronic phase.
There is currently no effective treatment for coronary artery microembolism. The invention provides an effective way for treating coronary artery microembolism through a large number of experiments.
Disclosure of Invention
The invention provides an application of effective amount of endothelial progenitor cells in preparing a medicament for treating coronary artery micro-embolism by transplantation.
Preferably, the effective amount of endothelial progenitor cells is a low dose.
Preferably, wherein the low dose is 2 x 10 when treating coronary artery microembolism disease in rats5And endothelial progenitor cells.
Further, the medicine can remarkably reduce the content of cTNI and vWF in serum.
Further, the medicine can remarkably increase the expression of VEGF and F8 in myocardial tissues and reduce the expression of bFGF in the myocardial tissues.
Furthermore, the drug can obviously increase the expression of microRNA-21, obviously reduce the expression of microRNA-19a, increase the expression of microRNA-214 and obviously increase the expression of microRNA-486-3 p.
The invention also provides a medicine for treating coronary artery micro-embolism by transplantation, which is characterized by comprising the following components in part by weight: the medicament comprises an effective amount of endothelial progenitor cells suitable for transplantation treatment of coronary artery microembolism disease.
Preferably, wherein the effective amount of endothelial progenitor cells is a low dose.
Preferably, wherein the low dose is 2 x 10 when treating coronary artery microembolism disease in rats5And endothelial progenitor cells.
The invention also provides application of an effective amount of endothelial progenitor cells in preparation of a reagent for changing microRNA expression in individuals suffering from coronary artery microembolism diseases, wherein the changed microRNA expression specifically means that the microRNA-21 expression is remarkably increased, the microRNA-19a expression is remarkably reduced, the microRNA-214 expression is increased or the microRNA-486-3p expression is remarkably increased.
The invention has the following positive effects:
1. the invention proves the obvious curative effect of the transplantation of the endothelial progenitor cells, especially the endothelial progenitor cells with low dose on the coronary artery micro-embolism diseases, and provides a new idea for the treatment of the coronary artery micro-embolism diseases.
2. The invention further proves the mechanism of the endothelial progenitor cells for treating the coronary artery micro-embolism diseases, including the influence on the expression levels of VEGF, bFGF and F8 in the myocardial tissues and the influence on the expression of microRNA-21, microRNA-19a, microRNA-214 and microRNA-486-3p in the cardiac tissues, and provides a basis for deeply explaining the pathogenesis of the coronary artery micro-embolism.
Drawings
FIG. 1 is a morphological observation of EPC cells.
FIG. 2 shows the ratio of the EPC surface markers CD34-FITC and VEGFR2-PE measured by flow.
FIG. 3 shows the results of echocardiographic examination of rats in each group. Note: sham denotes the Sham group; CME denotes coronary microemboli group; CME + EPC (low) represents coronary microembolism & EPC transplantation group (low dose group); CME + EPC (high) represents coronary microembolism & EPC transplantation group (high dose group). Before indicates preoperative, 1d indicates postoperative 1d, 7d indicates postoperative 7d, and 28d indicates postoperative 28 d.
Fig. 4 is a standard curve.
FIG. 5 shows ELISA assay of rat serum from each group. Note: sham denotes the Sham group; CME denotes coronary microemboli group; CME + EPC (low) represents coronary microembolism & EPC transplantation group (low dose group); CME + EPC (high) represents coronary microembolism & EPC transplantation group (high dose group). Before indicates preoperative, after indicates postoperative 28 d.
FIG. 6 shows the VEGF immunohistochemical detection of rat heart tissues in each group. Note: sham denotes the Sham group; CME denotes coronary microemboli group; CME + EPC (low) represents coronary microembolism & EPC transplantation group (low dose group); CME + EPC (high) represents coronary microembolism & EPC transplantation group (high dose group).
FIG. 7 shows the observation of bFGF immunohistochemical staining of myocardial tissues of rats in each group. Note: sham denotes the Sham group; CME denotes coronary microemboli group; CME + EPC (low) represents coronary microembolism & EPC transplantation group (low dose group); CME + EPC (high) represents coronary microembolism & EPC transplantation group (high dose group).
FIG. 8 shows the F8 immunohistochemical staining of rat myocardial tissues in each group. Note: sham denotes the Sham group; CME denotes coronary microemboli group; CME + EPC (low) represents coronary microembolism & EPC transplantation group (low dose group); CME + EPC (high) represents coronary microembolism & EPC transplantation group (high dose group).
FIG. 9 shows HE staining observation of myocardial tissues of rats in each group. Note: sham denotes the Sham group; CME denotes coronary microemboli group; CME + EPC (low) represents coronary microembolism & EPC transplantation group (low dose group); CME + EPC (high) represents coronary microembolism & EPC transplantation group (high dose group).
FIG. 10 shows the macroson staining of myocardial tissue of rats in each group. Note: sham denotes the Sham group; CME denotes coronary microemboli group; CME + EPC (low) represents coronary microembolism & EPC transplantation group (low dose group); CME + EPC (high) represents coronary microembolism & EPC transplantation group (high dose group).
FIG. 11 shows the detection of microRNA in rat heart tissue of each group. Note: sham denotes the Sham group; CME denotes coronary microemboli group; CME + EPC (low) represents coronary microembolism & EPC transplantation group (low dose group); CME + EPC (high) represents coronary microembolism & EPC transplantation group (high dose group).
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
Example 1: isolation culture identification of EPC
1. Extraction of EPCs
1) SD rats with the mass of 200-250 g and the age of 8-9 weeks are weighed, 5% chloral hydrate is used for abdominal anesthesia (0.6mL/100g), and then the hairs of the legs of the rats are removed by an electric shaver. Fixing the prone position of the rat, unhairing, disinfecting with iodophor alcohol, and spreading a towel. Both legs of the SD rat were removed with scissors. All the operation operations are completed by the same person, and the aim is to reduce unnecessary errors possibly brought in the operation process.
2) The rough flesh and bone separation of the two legs was performed in a 50mL centrifuge tube containing 75% absolute ethanol, and then the centrifuge tube was placed in a transfer chamber and UV-sterilized for 10 min.
3) The sterilized centrifuge tube was taken to a clean bench, two legs were taken out, the femur and tibia were separated with sterilized forceps and small scissors, and the both ends of the femur and tibia were cut with rongeur.
4) The bone marrow in femur and tibia was washed out with sterile 1 XPBS, and the 1 XPBS containing bone marrow was added dropwise to an equal volume of the separation medium (4.5mL of Percoll mixed with 0.5mL of 8.5% NaCl solution, 0.2mL of the mixture was discarded, and 3.8mL of 0.85% NaCl solution was added) and centrifuged at 2000rpm/min for 20 min.
5) Sucking the middle layer cloud substance of the centrifuged liquid with a suction tube, adding 1 XPBS with the same amount, mixing uniformly, and centrifuging at 2000rpm/min for 20 min.
6) The supernatant was discarded, 4mL of 1 XPBS was added thereto and the mixture was mixed well, followed by centrifugation at 2000rpm for 20 min.
7) The supernatant was discarded, 1ml of LEGM-2 medium was added to the suspension, the suspension was added to a sterile flask with a pipette, and 3ml of LEGM-2 medium was added to the flask and cultured in a cell culture incubator.
8) The extracted cells can be used for identification and subsequent experiments after being continuously purified.
2. EPC cell identification
1) Continuously purifying the cells extracted in step 1, selecting 0.25% pancreatin without EDTA to digest the cells for 5min, observing the digestion effect under an inverted microscope, and stopping digestion by using a complete culture medium containing 5% fetal calf serum after the cells contract and become round;
2) lightly blowing and beating by using a suction pipe to fully suspend adherent cells and blow away cell clusters;
3) centrifuging, washing cells for 2 times by PBS, resuspending the cells, taking 100 mu L of the cell suspension, respectively placing the cell suspension in a tube A and a tube B of a flow tube, respectively adding an anti-CD 34-FITC flow antibody and an anti-VEGFR 2-PE flow antibody in the tube A, respectively adding an isotype control antibody in the tube B, and incubating for 30min at normal temperature in a dark place;
4) centrifuging, discarding supernatant, adding PBS, mixing in flow tube, centrifuging at 2000rpm for 5min, repeating once, and washing away unbound antibody. And resuspend the cells with 500. mu.L PBS, store at 4 ℃ and wait for machine detection.
3. Analysis of results
(1) EPC cell identification
FIG. 1 shows microscopic EPC cell morphology. As can be seen from the figure, the cells under the microscope have blood vessel-like structures, and the cells shrink and become round, and need to be further identified by a subsequent flow.
FIG. 2 shows the ratio of the EPC cell surface markers CD34-FITC and VEGFR2-PE measured by flow. From the figure, it is found that the single-label positive ratio of CD34-FITC is 99.95%, the single-label positive ratio of VEGFR2-PE is 99.71%, and the double-label positive ratio of CD34-FITC and VEGFR2-PE is 98.96%. The result shows that CD34-FITC and VEGFR2-PE are positively expressed, and the cells obtained by separation culture are EPC cells which can be used for subsequent experiments.
Example 2: rat coronary artery microembolism model establishment
This example establishes a rat coronary artery microembolism model:
1. experiment grouping
1) False operation group (Sham)
2) Coronary artery micro-embolism set (CME)
3) Coronary Microembolism (CME) & EPC transplant group (Low dose group)
4) Coronary Microembolism (CME) & EPC transplant group (high dose group)
2. Animal modeling
Half an hour before the operation of ultraviolet irradiation operation, anesthetizing a rat by 10 percent chloral according to the dose of 3 mu L/g, collecting 0.5mL of blood from the tail vein after the rat is numb, and forming blood clots in a 37 ℃ incubator. Performing electric coagulation hemostasis on the tail of a rat, shaving hair on the chest, inserting a trachea, connecting a breathing machine, wherein the breathing ratio is 1:2, the tidal volume is 18, and the correct intubation is shown when the breathing rhythm of the rat is consistent with that of the breathing machine. Then, the blood clots are fully ground by a homogenizer and then pass through a 300-mesh screen, and the large blood clots are filtered out to obtain the micro thrombus for later use. The iodophor sterilizes the chest of the rat, the skin, the muscle and the ribs are cut off by scissors in sequence near the left side of the chest, the heart is exposed, and the micro thrombus injected into the left ventricle by an insulin injection needle of 300 mu L is found. Squeezing air out of chest, closing chest, suturing muscle and skin layer by layer, sterilizing wound with iodophor, and injecting penicillin subcutaneously to prevent wound infection. After the mouse feels consciousness, the mouse is separated from the breathing machine to breathe autonomously. Sham operated groups were injected with 0.3mL PBS into the left ventricle, and the EPC low dose groups were dissolved in 2X 10. mu.L microthrombus suspension5EPCs of (2X 10) dissolved in 300. mu.L of microthrombus suspension6The EPCs of (1). After the operation, the rats in each group were normally kept.
3. Echocardiography detection
Before and after the operation for 1 and 7 days, the change of the heart function of the rat is detected, and the specific steps are as follows:
1) anesthetizing a rat;
2) carrying out intraperitoneal injection anesthesia on rats by using 3% sodium pentobarbital according to the dose of 0.25 mL/rat;
3) after anesthesia, supplementing ketamine according to the dosage of 75-100 mg/kg (ip) and continuing anesthesia;
4) then, injecting atropine 20-30 mu g/kg into the abdominal cavity to reduce airway secretion;
5) fixing the rat on an operating table after anesthesia, shearing off the left chest coat, removing the residual coat with 5% sodium sulfide, and cleaning with absorbent cotton and a 75% alcohol cotton ball;
6) placing the rat on an operation table, and performing machine detection;
7) left Ventricular Ejection Fraction (LVEF), left ventricular end systolic diameter (lvdd), Left Ventricular End Diastolic Diameter (LVEDD), and short axis shortening rate (LVFS) were collected over 3 consecutive cardiac cycles.
4. ELISA detection
The experimental principle is as follows:
coating a cardiac troponin I (TNNI3) antibody (von Willebrand factor (vWF) antibody) in a 96-well microplate to prepare a solid phase carrier, adding a standard or a specimen into each well, wherein the cardiac troponin I (TNNI3) antibody (von Willebrand factor (vWF)) is bound to the antibody bound to the solid phase carrier, then adding a biotinylated cardiac troponin I (TNNI3) antibody (von Willebrand factor (vWF) antibody), washing the unbound biotinylated antibody, adding HRP-labeled avidin, washing thoroughly again, and adding a TMB substrate for color development. TMB is converted to blue by the catalysis of peroxidase and to the final yellow by the action of an acid. The shade of the color was positively correlated with the cardiac troponin I (TNNI3) antibody (von willebrand factor (vWF)) in the sample. The absorbance (OD value) was measured at a wavelength of 450nm with a microplate reader, and the sample concentration was calculated.
And (3) ELISA detection:
1) preparing a standard substance, a reagent and a sample before an experiment;
2) adding 100 mu L of sample (standard substance and sample), and incubating for 1h at 37 ℃;
3) removing by suction, adding 100 mu L of detection solution A, and incubating for 1h at 37 ℃;
4) washing the plate for 3 times;
5) adding 100 μ L of detection solution B, and incubating at 37 deg.C for 30 min;
6) washing the plate for 5 times;
7) adding TMB substrate 90 μ L, and incubating at 37 deg.C for 10-20 min;
8) add stop solution 50. mu.L and read immediately at 450 nm.
5. Analysis of results
FIG. 3 shows the results of echocardiographic examination of rats in each group. The results show that the LVEDD, LVESD, LVEF and LVFS indexes of rats in each group before the operation are not obviously different, and the LVEDD after the operation is not obviously different in each group. LVESD was significantly increased, with a slight decrease in LVEF and LVFS, compared to the sham group. After EPC transplantation, LVESD is remarkably reduced, LVEF and LVFS are slightly increased, and the effect of a low-dose group is obvious.
FIG. 4 is a standard curve, and FIG. 5 is ELISA assay of rat sera from each group. The result shows that the serum cTNI and vWF of rats in each group before the operation have no obvious difference, and the serum cTNI and the vWF after the operation have obvious difference in each group. The CME group rats had increased serum cTNI and vWF levels compared to the sham group. After EPC transplantation, the content of cTNI and vWF in rat serum is reduced, wherein the effect of a low-dose group is obvious.
Example 3: detection of Effect of EPC transplantation on treating rat coronary artery microembolism
1. Grouping experiments: the same as in example 2.
2. Animal modeling: the same as in example 2.
3 immunohistochemical detection
3.1 pretreatment
Paraffin section: dewaxing and hydrating by a conventional method, soaking the slices in xylene for 5min, replacing the xylene, and soaking for 5 min; soaking in anhydrous ethanol for 5 min; soaking in 95% ethanol for 5 min; soaking in 85% ethanol for 5 min; soaking in 70% ethanol for 5min, and soaking in distilled water for 3min × 3 times.
3.2 Experimental procedures
1) Slicing, baking at 37 deg.C overnight;
2) xylene for 1, 2 and 3min respectively, ethanol for 100%, 95%, 90%, 80%, 70%, 50% for 2min respectively, and distilled water for 1 min;
3) TBS washing for 5min for 3 times;
4) repairing antigen (boiling with high fire, and turning to low fire for 20min), and naturally cooling;
5) TBS washing for 5min for 3 times;
6) placing the slices in 3% H2O2 solution, and incubating at room temperature for 10min to block endogenous peroxidase;
7) TBS washing for 3 times, each for 5min, spin-drying, and blocking with 5% BSA for 20 min;
8) BSA solution was removed and 50 μ L of diluted primary anti-coverage tissue (VEGF 1: 200, bFGF 1: 100, F81: 150) overnight at 4 ℃;
9) TBS washing for 5min for 3 times;
10) removing PBS solution, adding 50-100 μ L of corresponding species of secondary antibody to each slice, and incubating at room temperature for 50 min;
11) TBS washing for 5min for 3 times;
12) TBS solution is removed, 50-100 mu L of fresh DAB solution is added into each section to prepare DAB solution, the color development is controlled by a microscope, and the DAB solution is washed by distilled water;
13) staining with hematoxylin for 25s, washing with tap water for 3-5min, and returning blue;
14) washing with distilled water for 1min, soaking in 50%, 70%, 80%, 90%, 100% for 1min, soaking in xylene for 1, 2min, air drying, adding neutral gum, and sealing.
4. HE detection
4.1 pretreatment
Paraffin section: dewaxing and hydrating by a conventional method, soaking the slices in xylene for 5min, replacing the xylene, and soaking for 5 min; soaking in anhydrous ethanol for 5 min; soaking in 95% ethanol for 5 min; soaking in 85% ethanol for 5 min; soaking in 70% ethanol for 5min, and soaking in distilled water for 3min × 3 times.
4.2 Experimental procedures
1) Hematoxylin staining: the slices with distilled water are placed into hematoxylin water solution for dyeing for 5min, and color separation is carried out in 1% hydrochloric acid ethanol for 1 s. Washing with running water, and returning blue with natural water for 20 min.
2) Eosin staining: adding eosin staining solution for dyeing for 1min, and washing with running water.
3) Transparency and mounting: the slides were placed in xylene for 3min × 2 times and mounted with neutral gum.
4) Image acquisition and analysis: and taking a picture through a microscope, and collecting and analyzing relevant parts of the sample.
5Masson assay
5.1 pretreatment
Paraffin section: dewaxing and hydrating by a conventional method, soaking the slices in xylene for 5min, replacing the xylene, and soaking for 5 min; soaking in anhydrous ethanol for 5 min; soaking in 95% ethanol for 5 min; soaking in 85% ethanol for 5 min; soaking in 70% ethanol for 5min, and soaking in distilled water for 3min × 3 times.
5.2 Experimental procedures
1) Staining with hematoxylin for 5min, and washing with distilled water;
2) differentiation with 0.1% HCl, bluing after washing with tap water;
3) dyeing with ponceau acid reddish staining solution for 5min, and slightly washing with distilled water;
4) treating with 1% phosphomolybdic acid aqueous solution for 50 s;
5) spin-drying, without washing, fixing green and dyeing for 5s, washing with tap water, and drying in an electric heating air-supporting drying oven;
6) after drying, adding neutral gum on the slices, and covering a glass slide;
7) and (5) observing through a microscope and taking a picture.
6 fluorescent quantitative PCR
6.1 Total RNA extraction (gun head and centrifuge tube are sterilized by moist heat, no RNase)
1) The homogenizer was taken, 1mL Trizol Reagent was added, and the mixture was pre-cooled on ice.
2) 100mg of tissue was taken and added to the homogenizer.
3) Grind well until no visible tissue mass.
4) The supernatant was centrifuged at 12000rpm for 10 min.
5) Adding 250 μ L of chloroform, inverting the centrifuge tube for 15s, mixing well, and standing for 3 min.
6) Centrifuge at 12000rpm for 10min at 4 ℃.
7) The supernatant was transferred to a new centrifuge tube, 0.8 times the volume of isopropanol was added, and the mixture was mixed by inversion.
8) Standing at-20 deg.C for 15 min.
9) Centrifuging at 12000rpm at 4 deg.C for 10min to obtain white precipitate as RNA.
10) The liquid was removed by suction and 1.5mL of 75% ethanol was added to wash the precipitate.
11) Centrifuge at 12000rpm for 5min at 4 ℃.
12) The liquid was aspirated off and the centrifuge tube was placed on a clean bench and blown for 3 min.
13) Add 15. mu.L of RNase free water to dissolve the RNA.
14) Incubate at 55 ℃ for 5 min.
15) RNA concentration and purity was checked using UV 1800: after the blank of the instrument is zeroed, 2.5 mu L of RNA solution to be detected is placed on a detection base, a sample arm is put down, and light absorption value detection is started by using software on a computer.
16) The RNA having an excessively high concentration is diluted at an appropriate ratio so that the final concentration is about 200 ng/. mu.L.
6.2 reverse transcription (gun head and PCR both sterilized by moist heat, No RNase)
1) A PCR tube was taken and a solution containing 2. mu.g of RNA was added.
2) Add 1. mu.L RT-PCR neck ring primer.
3) Make up to 12 μ L with nuclease-free deionized water.
4) Preserving the temperature for 5min at 65 ℃ on a PCR instrument, and quickly cooling on ice.
5) mu.L of 5 XBuffer, 2. mu.L of 10mM dNTPs, 1. mu.L of RNA inhibitor and 1. mu.L of reverse transcriptase are added in this order and mixed by pipetting.
6) Keeping the temperature of the PCR sample at 42 ℃ for 60min, and keeping the temperature of the PCR sample at 80 ℃ for 5min after the completion of the reaction to inactivate the reverse transcriptase.
6.3 quantitative PCR
1) 0.2mL of PCR tube was used to prepare the following reaction system, and 3 tubes were prepared for each reverse transcription product.
2) PCR amplification
6.4 primer sequences
TABLE 3.6.4.1 primer sequence Listing
7. Analysis of results
7.1 immunohistochemical assay results
FIG. 6 shows the observation of VEGF immunohistochemical staining of rat myocardial tissues in each group. As a result, VEGF expression in the CME myocardial tissues was significantly reduced compared with that in the sham-operated group. Compared with the CME group, the myocardial tissue VEGF expression of the low-dose EPC transplantation group is obviously increased. The VEGF expression of the myocardial tissues of the high-dose EPC transplantation group is not obviously changed.
FIG. 7 shows the observation of bFGF immunohistochemical staining of myocardial tissues of rats in each group. As a result, the expression of bFGF in the myocardial tissue of the CME group is obviously increased compared with that of the sham operation group. Compared with the CME group, the low-dose EPC transplantation group has obviously reduced myocardial tissue FGF expression. The expression of bFGF in myocardial tissue of a high-dose EPC transplantation group is slightly reduced.
FIG. 8 shows the F8 immunohistochemical staining of rat myocardial tissues in each group. As a result, the CME myocardial tissue F8 expression was significantly reduced compared to the sham-operated group. Compared with the CME group, the low-dose EPC transplantation group has obviously increased myocardial tissue F8 expression. There was no significant change in F8 expression in high-dose EPC-transplanted myocardial tissues.
The results suggest that transplantation of low-dose EPC can increase the production of endothelial growth factor VEGF and capillary vessel, and inhibit fibroblast growth factor bFGF.
7.2HE assay results
FIG. 9 shows HE staining observation of myocardial tissues of rats in each group. As a result, the myocardial cells in the sham-operated group were well-arranged, and there was no edema in the interstitium. CME group myocardial cell arrangement disorder, interstitial edema, enlargement, partial infarction focus, HE expression of deep staining, inflammatory cell infiltration. The low-dose EPC transplanted group has the advantages of regular arrangement of myocardial cells, no interstitial edema, basic disappearance of focus and no obvious inflammatory cell infiltration. The myocardial pathological state of the high-dose EPC transplantation group is not obviously improved compared with that of the model group. The results suggest that low dose EPC transplantation can alleviate CME-induced myocardial tissue pathology.
7.3Masson assay results
FIG. 10 shows the macroson staining of myocardial tissue of rats in each group. The collagen fibers are blue, and the cardiomyocytes are red. As a result, the myocardial cells of the sham-operated group were aligned and no collagen fibers were evident. CME group myocardial cells are disorderly arranged, and collagen fibers are obviously proliferated. The myocardial cells of the low-dose EPC transplantation group are arranged more regularly, and compared with the CME group, the collagen fiber hyperplasia condition is obviously improved. The arrangement of the myocardial cells of the high-dose EPC transplantation group is still relatively disordered, and the collagen proliferation condition is relatively serious. The results suggest that low-dose EPC transplantation can alleviate CME-induced collagen fibril proliferation in myocardial tissue.
7.4PCR assay results
FIG. 11 shows the detection of microRNA in rat heart tissue of each group. The result shows that compared with a sham operation group, the expression of the microRNA-21 in the heart tissue of the rat in the CME group is obviously reduced, the expression of the microRNA-19a is obviously increased, the expression of the microRNA-214 is reduced, and the expression of the microRNA-486-3p is obviously reduced. After the EPC is transplanted, the expression of microRNA-21 of rat heart tissue in a low-dose group is obviously increased, the expression of microRNA-19a is obviously reduced, the expression of microRNA-214 is increased, and the expression of microRNA-486-3p is obviously increased; the expression of microRNA-21 of the heart tissue of the rat in the high-dose group has no obvious change, the expression of microRNA-19a is reduced, the expression of microRNA-214 has no obvious change, and the expression of microRNA-486-3p is increased. The result indicates that the EPC low-dose group has obvious improvement on the microRNA expression change caused by rat CME injury.
While embodiments of the invention have been disclosed above, it is not intended to be limited to the uses set forth in the specification and examples. It can be applied to all kinds of fields suitable for the present invention. Additional modifications will readily occur to those skilled in the art. It is therefore intended that the invention not be limited to the exact details and illustrations described and illustrated herein, but fall within the scope of the appended claims and equivalents thereof.
Claims (5)
1. Use of effective amount of endothelial progenitor cells in preparation of medicine for treating rat coronary artery micro-embolism disease by transplantation, whereinThe effective amount of the endothelial progenitor cells is low dose, and the low dose is 2X 105And endothelial progenitor cells.
2. The use according to claim 1, wherein the medicament is capable of significantly reducing serum cTNI and vWF levels.
3. The use according to any one of claims 1-2, wherein the medicament is capable of significantly increasing the expression of VEGF and F8 in myocardial tissue and decreasing the expression of bFGF in myocardial tissue.
4. The use according to any one of claims 1 to 2, wherein the medicament is capable of significantly increasing the expression of microRNA-21, significantly decreasing the expression of microRNA-19a, increasing the expression of microRNA-214, and significantly increasing the expression of microRNA-486-3 p.
5. The application of an effective amount of endothelial progenitor cells in preparing a reagent for changing microRNA expression in a rat suffering from coronary artery microembolism disease specifically means that the microRNA-21 expression is remarkably increased, the microRNA-19a expression is remarkably reduced, the microRNA-214 expression is increased or the microRNA-486-3p expression is remarkably increased.
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