cytokine release microspheres combined with bone marrow mesenchymal stem cell transplantation

Title: cytokine release microspheres combined with bone marrow mesenchymal stem cell transplantation for treatment of ischemic heart disease Clinical evaluation and MRI studies in vivo tracer experiments Author: Liu Qiong Degree awarded: China Union Medical University Keywords: microspheres;; gelatin;; vascular endothelial growth factor; magnetic resonance imaging;; microspheres;; gelatin;; myocardial infarction; magnetic resonance imaging;; stem cells;; myocardial infarction; magnetic resonance imaging;; mesenchymal Strong magnets stem cells; ; myocardial infarction; magnetic resonance imaging;; mesenchymal stem cells;; superparamagnetic iron oxide Abstract:

 The first part

 Vascular endothelial growth factor release gelatin microspheres preparation and performance evaluation

 Objective: To prepare gelatin micro-vascular endothelial growth factor release the ball and to evaluate its performance. Methods: VEGF release prepared by condensation emulsion gelatin microspheres. Dropping 20% ​​gelatin solution and stir in the olive oil to form emulsion, curing acetone, isopropanol, suspended in a different aperture sieve after sieving. Whizzer, the glutaraldehyde cross-linking, freeze-dried after cleaning, light yellow gelatin microspheres. Cobalt 60 irradiation sterilization. The beads soaked in PBS for 24 hours, full swelling microspheres, the microscope comes with software to measure particle size of microspheres. Take sterile freeze-dried gelatin microspheres 9 mg, were divided into three groups, the 1.5μCi ~ (125) Ⅰ-VEGF soluble in 30μl PBS, the average dropping to 3 groups of gelatin microspheres, 4 ℃ overnight. PBS centrifugal washing 2 times. The microspheres resuspended in 2ml PBS, the Γ-counter test in the total radioactivity. After re-suspension of microspheres at 37 ℃ constant temperature shaking, the whole volume replacement every 24 h dissolution fluid. Γ-counter to detect the point of microspheres remaining radioactivity, drawing release curve. 42 Kunming mice were randomly divided into 14 groups of three. Freeze-dried powder obtained gelatin microspheres 126 mg, were divided into 42 parts, will 21μCi ~ (125) Ⅰ-VEGF dissolved in 420μl PBS, the average dropping in each gelatin microspheres, 4 ℃ overnight. The beads were resuspended each in 100μl PBS, was injected into the left hind limb subcutaneous tissue in mice. Each set of mice were sacrificed 24 h, clipping the left lower limb, Γ-counter to detect radioactivity remaining in the organization. The average radioactivity in each draw ~ (125) Ⅰ-VEGF release profile.

 Results: The swelling of gelatin microspheres after the average particle size (202.0 ± 44.7) μm. Microspheres in vitro release performance test experiments, the gelatin microspheres release the first four days faster VEGF, in the first 96 hours, released from microspheres in the dissolution fluid accumulated in the accounts of VEGF VEGF microspheres http://www.chinamagnets.biz/Neodymium/Ball-Neodymium-Magnets.php in the initial total content of 48.73%. After the release rate gradually slowed down in the first 144 hours microspheres ~ (125) Ⅰ-VEGF remaining amount of the initial amount of 46.98%. Since then the remaining of VEGF microspheres reached a plateau. Microspheres in vivo sustained release performance test experiments, microspheres ~ (125) Ⅰ-VEGF accounts for the remaining percentage of the initial total content showed a gradual downward trend. In the first 96 hours, mice hind legs ~ (125) Ⅰ-VEGF remaining amount of 51.9%, in the first 144 hours in mice hindlimb ~ (125) Ⅰ-VEGF remaining amount of 41.89%, in 288 hours, ~ (125) Ⅰ-VEGF remaining amount of 9.98%.

 Conclusion: The gelatin microspheres containing VEGF package can be achieved after the slow release of VEGF. Vascular endothelial growth factor release microspheres for the treatment of ischemic heart disease in rats

 Objective: To investigate vascular endothelial growth factor (vascular endothelial growth factor, VEGF) release gelatin microspheres can induce angiogenesis in ischemic myocardium.

 Methods: VEGF release prepared by condensation emulsion gelatin microspheres, and 1,1 '- bis octadecane 3 ,3,3', 3'-tetramethyl - indole - carboxy cyanine - perchloric acid salt (DiI) fluorescent tags. Chinese mini-pigs 18, according to randomization tables animals were divided into VEGF release microspheres transplantation group, no-load microspheres transplantation group and control group. Open-chest left anterior descending coronary artery ligation preparation of miniature swine myocardial infarction model. Myocardial infarction model building underwent MRI 14 d after the second thoracotomy, exposing the left ventricular anterior wall near the apex, VEGF release microspheres in animals transplanted myocardial infarct border zone injections of VEGF release package containing microspheres, each animals injected with 3 points, each point of injection volume of 3 mg (containing VEGF 3μg); in the empty microsphere group injected the same amount of myocardial infarct border zone is not wrapped release of VEGF microspheres, the animals injected with three points each. Control group was injected with the same amount of VEGF in phosphate buffer. Microspheres after transplantation 24 h (baseline) and 21 d (the end), film-enhanced MRI acquisition MRI and animal heart function parameters and measure infarct size. Endpoint test animals were sacrificed after the end of histology, immunohistochemistry assay microspheres transplant myocardial ischemic fibrosis, capillary density and microsphere degradation.

 Results of miniature pigs after left anterior descending artery ligation on day 14, delayed enhancement magnetic resonance imaging showed left ventricular anterior wall, apex and interventricular septum were delayed enhancement stove. In the microspheres after transplantation and transplantation after 24h and 21d, each group of myocardial infarct size and cardiac function parameters were not statistically different. Histological results suggest that transplanted microspheres 3 weeks after myocardial ischemia, VEGF release microspheres microspheres transplantation group graft fibrosis in myocardial tissue at the point slightly below the no-load ball transplantation group and control group. VEGF release microspheres injected microspheres transplantation myocardial capillary density is higher than the point of no-load microspheres transplantation group and control group (9.8 ± 1.5/HPF vs 4.1 ± 0.9 and 3.7 ± 1.1/HPF, P <0.05). Transplanted gelatin microspheres 3 weeks after myocardial ischemia, interstitial degradation of gelatin microspheres visible debris.

 Conclusion vascular endothelial growth factor release gelatin microspheres can induce neovascularization of ischemic myocardium area. Cytokine release microspheres to enhance bone marrow mesenchymal stem cell transplantation for myocardial infarction studies

 Purpose of this study was to observe vascular endothelial growth factor (vascular endothelial growth factor, VEGF) release gelatin microspheres and autologous bone marrow mesenchymal stem cells (mesenchymal stem cells, MSCs) transplantation in the pig model of myocardial infarction infarct border zone 3 weeks after the MSCs survival situation, combined with magnetic resonance imaging (magneticresonance imaging, MRI), to clarify and improve the local microenvironment of autologous MSCs transplantation on outcome after myocardial ischemia effects.

 Preparation of an emulsion condensation method VEGF release gelatin microspheres. Chinese mini-pigs 18. Extraction and preparation of autologous iliac bone marrow MSCs, the injection of paramagnetic iron oxide particles (superparamagnetic iron oxide particles, SPIO), 4 ', 6 - two amidine-2 - phenylindole (4' ,6-Diamidino-2 -phenylindole dihydrochloride, DAPI) labeled cells and mark rate detected. Table according to randomized animals were divided into control group, MSCs transplantation group (MSCs group) and MSCs-VEGF release microspheres transplantation group (MSCs-VEGF group). Open-chest left anterior descending coronary artery ligation preparation of miniature swine model of myocardial infarction. Modeling after 14 days, magnetic resonance delayed enhancement imaging (delatedenhancement MRI, DE-MRI) detection of infarction underwent re-exploration area, look into the outer membrane attentive group of animals at myocardial infarction, MSCs injected MSCs 3 surrounding area points ( 3 × 10 ~ 7 / points) in MSCs-VEGF release microspheres injected each animal group three points, each point of injection of MSCs 3 × 10 ~ 7 and VEGF release gelatin microspheres 3mg (including the recombinant human VEGF165 3μg) , myocardial infarction in control animals injected around the area corresponding parts of the medium DMEM. Microspheres after transplantation 24 h (baseline) and 21 d (the end), film-enhanced MRI acquisition MRI and animal heart function parameters including ejection fraction, left ventricular end-diastolic volume, left ventricular end-systolic volume, increased left ventricular anterior wall thickness ratio, and measure infarct size. Endpoint test animals were sacrificed after the end of histology, immunohistochemistry assay microspheres transplant myocardial ischemic fibrosis, capillary density and stem cell survival.

 The results of DAPI labeled MSCs in 100%. Myocardial infarction 14 days after model building, miniature swine myocardial infarct size of 33.6% ± 8.9%. Cell transplantation after 24 h, three groups myocardial infarct size and heart function parameters were not statistically different. 3 weeks after cell transplantation, MSCs and MSCs-VEGF group left ventricular anterior wall thickening rate was higher (-5.23 ± 3.82 and -1.52 ± 4.81 compared with -15.53 ± 3.42, P <0.05), MSCs-VEGF left ventricular end-systolic volume is less than the control group (31.20 ± 3.56 vs 37.05 ± 6.26, P <0.05). MSCs-VEGF cells of myocardial capillary density is higher than the injection site MSCs group and control group (18.18 ± 6.6 compared with 10 ± 3.58 and 12.81 ± 4.26/HPF; P <0:05). MSCs-VEGF injection of sustained-release microsphere group point of the survival of MSCs transplanted stem cells than MSCs group (383.07 ± 96.33 / high power field compared with 285.17 ± 73.97 / high power field, P <0.0001), while the apoptosis rate was lower than MSCs MSCs group 5.24 ± 5.44% vs 10.24 ± 5.04%, P <0.05].

 Conclusions VEGF release gelatin microspheres can improve the transplanted MSCs in ischemic myocardium area three weeks the survival rate. Survival of transplanted stem cells micro-environment is an important factor affecting the outcome. Iron oxide magnetic resonance nanoparticles in vivo tracer labeled stem cell transplantation and reliability evaluation of quantitative analysis

 Objective To observe the transplanted pig model of myocardial infarction infarct border zone and normal myocardial areas paramagnetic iron oxide particles labeled autologous bone marrow mesenchymal stem cells (mesenchymal stem cells, MSCs) formed by the low signal area signal changes, combined with histological examination results, evaluation of MRI for stem cell transplantation in myocardial ischemia monitoring the evolution of the value after the number.

 Method of Chinese miniature pig 6, iliac bone marrow extracted and prepared autologous bone marrow mesenchymal stem cells by injection with paramagnetic iron oxide particles (superparamagnetic iron oxide particles, SPIO) and 4 ', 6 - amidine-2 - phenylindole indole (4 ',6-Diamidino-2-phenylindole dihydrochloride, DAPI) labeled cells and mark rate detected. Open-chest left anterior descending coronary artery ligation preparation of miniature swine model of myocardial infarction. Modeling after 14 d, the line re-exploration, outer membrane attentive look directly at the border zone of myocardial infarction each animal and the injection of normal cardiac area labeled MSCs 2 points, each point of injection of MSCs 3 × 10 ~ 7. Both were injected at various points as the control medium two points. 24 h after cell transplantation, and 21 d, fast gradient echo sequence (FGRE sequence) T_2 ~ * detect stem cell transplantation point as low signal area and signal strength area. T_2 ~ * signal to reduce the area of ​​measurement achieved by the FGRE area method, the signal to reduce the level of a normal cardiac area and the area T_2 ~ * value of the difference with normal myocardial area T_2 ~ * values ​​expressed as a percentage. Histological examination of myocardial cell morphology, scar formation, capillary density and stem cell survival. MSCs injection points at different time points with low signal area and T_2 ~ * signal intensity compared using repeated measures analysis of variance and paired t test. Stem cell injection point and control point comparison of capillary density, myocardial infarct border zone and normal MSCs injection site area was used to compare the signal attenuation data into a group t test.

 The results of DAPI and SPIO labeled MSCs rate of 100%. Cell transplantation after 24 h, SPIO labeled MSCs injection point in the MRI showed a clear boundary oval T_2 ~ * low signal area, MSCs injected after 24 h, myocardial infarct border zone and normal zone MSCs injection point T_2 ~ * low signal area The signal intensity [(67 ± 5.48)% vs (61.92 ± 7.76)% t = 1.65 P = 0.1158)] and area [(0.56 ± 0.24) cm ~ 2 vs (0.52 ± 0.25) cm ~ 2, t = 0.39, P = 0.7044)] were not statistically different. 3 weeks after transplantation, T_2 ~ * low signal area compared with normal myocardial areas extent than before the fall, the surrounding area infarction reduced to (40.12 ± 5.93)% (t = 9.53, P <0.0001); reduced to normal myocardial area ( 46.92 ± 6.25)% (t = 11.03, P <0.0001). Infarct border zone and a low signal area MSCs injection point to reduce the extent of T_2 ~ * is greater than the normal myocardial zone between the two groups MSCs injection point and the magnitude of the signal intensity decreased with significant difference (26.88 ± 7.27 vs 15 ± 4.51, F = 20.08, P = 0.0003), infarct border zone MSCs injected at this time point T_2 ~ * low signal area signal contrast is lower than normal cardiac area MSCs injection point (t =- 2.48, P = 0.0234). Meanwhile, T_2 ~ * low signal area should also lower than before the small, low-signal area but the extent of reduction in myocardial infarct border zone and normal zone was no significant difference (F = 1.15, P = 0.2982). Normal myocardial tissue in the area point of MSCs injected fluorescent nuclei density is higher than the marginal zone infarction MSCs injection point (106 ± 25/HPF vs143 ± 31/HPF, t =- 2.47, P = 0.0293). Infarct marginal zone MSCs injection site tissue capillary density is higher than the control point area (13.4 ± 4.0/HPF vs 9.4 ± 3.1/HPF, t = 2.49, P = 0.0229).

 Conclusions MRI can be achieved in a certain period of time tracing of transplanted stem cells and transplanted MSCs in the myocardium reflects the number of local trends. Survival of transplanted stem cells affect the micro-environment is an important factor in survival time. Iron oxide particles labeled mesenchymal stem cells ruled out a preliminary study the mechanism of iron particles

 Purpose of this study from the perspective of experimental cytology in mesenchymal stem cell division and proliferation process of stem cell markers in the cytoplasm of superparamagnetic iron oxide particles (superparamagnetic iron oxide, SPIO) labeled rate of evolution in order to identify stem cell division and proliferation of SPIO-labeled rate of change.

 Pig iliac bone marrow extracted method, density gradient centrifugation to obtain mononuclear cell layer, adherent to the time difference between purified bone marrow mesenchymal stem cells (MSCs). The first generation of cells reached 80% confluence, the ratio of 1:3 in six-well plates were inoculated in the two groups. Containing 10% fetal calf serum with high glucose DMEM culture. In the cells reached 60-70% confluence, changed to iron 25 ng / L and poly-lysine 375 pg / L for liquid medium for the cells, and cultured cells 48 h. After the cells reached 80% confluence. The other six-well plates in the 1:3 ratio of cells passaged to a new three six-well plates, in which two six-hole plate placed at the bottom sterile coverslip. One group of cells for 15% FBS-containing DMEM high glucose culture, while the other cells change to sugar containing 5% FBS DMEM culture. Every 24 hours out of the corresponding groups of cells seeded were fixed, stained, testing. The remaining cells were in two different media continued to train passage. The prepared cells seeded Prussian blue staining for iron oxide particles and estimation of cell labeling yield.

 Results: SPIO labeled stem cells after 48 hours, cells seeded Prussian blue staining showed that all cells contain blue-stained particles, located around the nucleus. SPIO-labeled cells in the different conditions of medium and cultured, two groups of SPIO-labeled cells gradually reduce the rate, the cells gradually reduce the amount of iron particles. 15% FBS-containing DMEM high glucose cultured cells group, labeled 72 hours after the cells reached 80% confluence, the first passage, 24 hours after passage, SPIO labeling rate was slightly lower than before (98 ± 4.39)% (P = 0.0409), cultured for 72 hours after the second passage, 24 hours after passage, SPIO-labeled cells, the rate decreased to (88.67 ± 3.93)%, with the first passage compared to a statistically significant difference (P = 0.0004) . Cultured for 72 hours after the third passage, the passage can be seen 24 hours after SPIO-labeled further reduced to (44.5 ± 7.74)%, and the second 24 hours after the passage of significant difference compared (P <0.0001). The fourth 24 hours after passage, SPIO labeled rate (6.17 ± 2.64)%. To contain 5% FBS of sugar DMEM cultured cells group, labeled 96 hours after cells reached 80% confluence, the first passage, passage 24 hours after, SPIO labeling rate compared with the previous slightly lower (97 ± 0.86)% (P = 0.0172), to develop 120 hours after the second passage, 24 hours after passage, SPIO-labeled cells, the rate reduced to (87 ± 4.29)%, with the first passage compared to a statistically significant difference (P = 0.0004). Continued to train 144 hours after the third passage, the passage can be seen 24 hours after SPIO-labeled further reduced to (46.7 ± 7.06)%, and the second 24 hours after the passage of significant difference compared (P <0.0001). The fourth 24 hours after passage, SPIO mark the fourth passage was 24 hours, SPIO labeled rate (5.17 ± 3.06)%. In the two groups 细胞, both in the fourth passages marked Yihou 24 hours, SPIO labeling yield dropped to below 10% in high glucose DMEM group lasted nine days, and in the sugar DMEM group lasted 16 days. In marked the fourth passage after 24 hours, two groups of SPIO-labeled cells was no significant difference [(5.17 ± 3.06)% compared with (6.17 ± 2.64)%, P = 0.558].

 Conclusion SPIO-labeled mesenchymal stem cells to exclude the SPIO of the mechanism of cell division and proliferation. Faster cell division and proliferation, cell clearance rate is also faster SPIO. Degree Year: 2009