Objective To determine the transfection efficiency of recombinant adenovirus to endothelial progenitor cells(EPCs) and provide the base of lung cancer therapy by transfecting human herpes simplex virusthymidine kinase（HSV-TK） gene to EPCs. Methods Admove recombinant adenovirus 5F35(AD5F35) which transfected with βgalactosidase(AD5F35LacZ) to the 24 well plate cultivated with EPCs and transfect the EPCs. Stain the EPCs with LacZ kit and calculate the transfection efficiency. Results The blue stain cells were cells transfected successfully with AD5F35LacZ under the optical microscope. The transfection efficiencies of adenovirus to EPCs were different under the premise of the different multiplicity of infection(MOI). In a certain range, the transfection efficiencies rise with the MOI rise. When MOI was 400,the proportion of blue stain cell is the highest, which was 98.38%±1.25%. Conclusion Recombinant adenovirus can transfect EPCs successfully. The transfection efficiencies rise with the MOI rise. When the MOI is 400,the transfection efficiency is the highest.
Abstract： Objective To investigate and improve the method of isolation, induction and culture, amplification in vitro of human endothelial progenitor cells (EPCs) in human bone marrow, thus establish a foundation for EPCs to participate in basic research and clinical application. Methods WHuman bone marrow mononuclear cells (hBMMNCs) were isolated by density gradient centrifugation and cultured in the 6plateds coating human fibronections(HFN group), coating gelatinum (coating gelatinum group) and coating nothing (coating nothing group) respectively. After culturing for 4-7d endothelial cell basal medium-2(EBM-2) cell colonyforming units (CFUs) appeared, then select EPCsliked CFUs for cultivation which was named the pick method, CD34+ KDR+ and CD133+ KDR+ double positive cells were detected in flow cytometry, and CD133, CD34, CD31, vWF and KDR expression were detected with cell immunochemical test. Results hBMMNCs were isolated from human bone marrow more effectively with OptiprepTM cell separating medium, and induced and obtained more EPCs, and cultured and amplificated in vitro. Flow cytometer showed CD133+ KDR+ double positive cells reaching up to 70.4%±5.4%, CD34+ KDR+ double positive cells reaching up to 69.1%±8.7%. EPCs grew vigorously in coating HFN group and coating gelatinum group, both HFN and gelatinum promote EPCs adherence and growth, but there were no statistically difference in two groups (Pgt;0.05).Surface mark of adherent cells cultured 7d such as CD133, CD31, vWF and KDR showed positive, and cells cultured 14d such as CD34 showed positive. Conclusion The method of picking CFUs can obtain more EPCs from human bone marrow with success and can amplificate EPCs in vitro, thus introducing another simple and effective method to purify EPCs, further widening range and increasing method to purify EPCs.
Objective To study the short and medium term effect of myocardial contractile force by implantation of endothelial progenitor cells （EPCs） in the myocardial infarction model. Methods Hundred and twenty SD rats were equally and randomly divided into experimental group and control group （60 rats in each group）. Acute myocardial infarction model was created by ligation of LAD. Autologous EPCs were purified from peripheral blood then implanted into the acute myocardial infarct site via topical injection. IMDM were used in control group. Specimens and muscle strip were harvested at 3, 6 weeks, 6, 8 and 12 months after EPCs implantation for contractile force study and to detect the expression of vascular endothelial growth factor（VEGF）, basic fibroblast growth factor （bFGF） and Ⅷ factor by immunohistology and video image digital analysis system. Results The expression of VEGF, bFGF and the microvessel counts in experimental group were much higher than those of control group（P〈 0.01） at 3, 6 weeks and 6 months after transplantation. The contractile force in experimental group was better than that in control group（P〈0.01） at the same time. But from 8 months after implantation, the contractile force and so on were not up in the experimental group. Conclusion EPCs, after being implanted into infarct myocardium, shows the ability of improvement of the contractile performance in infarcted myocardium by means of angiogenesis and vasculogenesis and the medium term results are persistent.
Objective To investigate the effect of simvastatin on inducing endothel ial progenitor cells (EPCs) homing and promoting bone defect repair, and to explore the mechanism of local implanting simvastatin in promoting bone formation. Methods Simvastatin (50 mg) compounded with polylactic acid (PLA, 200 mg) or only PLA (200 mg) was dissolved in acetone (1 mL) to prepare implanted materials (Simvastatin-PLA material, PLA material). EPCs were harvested from bone marrow of 2 male rabbits and cultured with M199; after identified by immunohistochemistry, the cell suspension of EPCs at the 3rd generation (2 × 106 cells/mL) was prepared and transplanted into 12 female rabbits through auricular veins(2 mL). After 3 days, the models of cranial defect with 15 cm diameter were made in the 12 female rabbits. And the defects were repaired with Simvastatin-PLA materials (experimental group, n=6) and PLA materials (control group, n=6), respectively. The bone repair was observed after 8 weeks of operation by gross appearance, X-ray film, and histology; gelatin-ink perfusion and HE staining were used to show the new vessels formation in the defect. Fluorescence in situ hybridization (FISH) was performed to show the EPCs homing at the defect site. Results All experimental animals of 2 groups survived to the end of the experiment. After 8 weeks in experimental group, new bone formation was observed in the bone defect by gross and histology, and an irregular, hyperdense shadow by X-ray film; no similar changes were observed in control group. FISH showed that the male EPC containing Y chromosome was found in the wall of new vessels in the defect of experimental group, while no male EPC containing Y chromosome was found in control group. The percentage of new bone formation in defect area was 91.63% ± 4.07% in experimental group and 59.45% ± 5.43% in control group, showing significant difference (P lt; 0.05). Conclusion Simvastatin can promote bone defect repair, and its mechanism is probably associated with inducing EPCs homing and enhancing vasculogenesis.
Objective To observe the adhesion and prol iferation of late endothel ial progenitor cells (EPCs) planted on nanoporous PLLA scaffold in vitro and to provide a new approach that optimizes tissue engineered material. Methods Male and female New Zealand rabbits (weight 2.5-3.0 kg) were used. Isolated late EPCs from rabbit peri pheral blood were cultured. Electrostatic spinning technique was adopted to prepare misal igned nanofibers, al igned nanofibers and super-al igned nanofibers, and low temperature plasma technique was appl ied to prepare misal igned membrane, al igned membrane and super-al igned membrane. After being divided into group A (cells only), B (misal igned membrane), C (normal membrane), D (al igned membrane) and E (super-al igned membrane), the primary late EPCs (1 × 105/mL) werecultured on scaffolds and MTT method was used to detect cell prol iferation abil ity at 3, 5, 7, 9, 11, 13, 15 and 17 days afterculture. After being divided into group A (misal igned membrane), B (normal membrane), C (al igned membrane) and D (superal igned membrane), precipitation method was appl ied to detect cell adhesion rate at 4, 12 and 24 hours after compound culture, and the morphologic changes of cells were observed at 4, 24 and 72 hours after compound culture. Results Fiber diameters in nanofibrous PLLA scaffolds were 300-400 nm, with a porosity rate of above 90%. At 3, 5, 7, 9, 11, 13, 15 and 17 days after culture, A value of each group was increased with time and the cells in each group grew well, showing there was no significant difference between group A and group B at each time point (P gt; 0.05 ); during the period of 7-15 days after culture, the difference between groups C, D and E and groups A and B was significant (P lt; 0.05). At 4 hours after compound culture, the adhesion rate of group A was superior to that of groups B, C and D (P lt; 0.05); at 12 and 24 hours after compound culture, the adhesion rate of groups B, C and D was remarkably higher than that of group A (P lt; 0.05); significant difference was noted in each group between the time point of 4 hours and the time point of 12 and 24 hours after compound culture (P lt; 0.05), but no significant difference between 12 hours and 24 hours was detected (P gt; 0.05). Morphology observation demonstrated that cells grew well on the scaffolds, the cells in groups A and B grew sporadically and disorderly, while the cells in groups C and D attached and al igned along fiber and prol iferated, with an excretion of ECM. Group D was better at maintaining cell morphology. Conclusion Al igned and superal igned nanofibers of PLLA scaffold can promote the adhesion and prol iferation of seed cells on the scaffold and maintain good cell morphology, which is an appropriate candidate scaffold material for blood vessel tissue engineering. Late EPCs is an ideal cell source for blood vessel tissue engineering.
Objective To compare canine decel luarized venous valve stent combining endothel ial progenitor cells (EPC) with native venous valve in terms of venous valve closure mechanism in normal physiological conditions. Methods Thirty-six male hybrid dogs weighing 15-18 kg were used. The left femoral vein with valve from 12 dogs was harvested to prepare decelluarized valved venous stent combined with EPC. The rest 24 dogs were randomly divided into the experimental group and the control group (n=12 per group). In the experimental group, EPC obtained from the bone marrowthrough in vitro ampl ification were cultured, the cells at passage 3 (5 × 106 cells/mL) were seeded on the stent, and the general and HE staining observations were performed before and after the seeding of the cells. In the experimental group, allogenic decelluarized valved venous stent combined with EPC was transplanted to the left femoral vein region, while in the control group, the autogenous vein venous valve was implanted in situ. Color Doppler Ultrasound exam was performed 4 weeks after transplantation to compare the direction and velocity of blood flow in the distal and proximal end of the valve, and the changes of vein diameter in the valve sinus before and after the closure of venous valve when the dogs changed from supine position to reverse trendelenburg position. Results General and HE staining observations before and after cell seeding: the decelluarized valved venous stent maintained its fiber and collagen structure, and the EPC were planted on the decelluarized stent successfully through bioreactor. During the period from the reverse trendelenburg position to the starting point for the closure of the valve, the reverse flow of blood occurred in the experimental group with the velocity of (1.4 ± 0.3) cm/s; while in the control group, there was no reverse flow of blood, but the peak flow rate was decreased from (21.3 ± 2.1) cm/s to (18.2 ± 3.3) cm/s. In the control group, the active period of valve, the starting point for the closure of the valve, and the time between the beginning of closure and the complete closure was (918 ± 46), (712 ± 48), and (154 ± 29) ms, respectively; while in the experimental group, it was (989 ± 53), (785 ± 43), and (223 ± 29) ms, respectively. There was significant difference between two groups (P lt; 0.05).After the complete closure of valve, no reverse flow of blood occurred in two groups. The vein diameter in the valve sinus of the experimental and the control group after the valve closure was increased by 116.8% ± 2.0% and 118.5% ± 2.2%, respectively, when compared with the value before valve closure (P gt; 0.05). Conclusion Canine decelluarized venous valve stent combined with EPC is remarkably different from natural venous valve in terms of the valve closure mechanism in physiological condition. The former rel ies on the reverse flow of blood and the latter is related to the decreased velocity of blood flow and the increased pressure of vein in the venous sinus segment.
Objective To observe the changes in the number and function of bone marrow-derived endothel ial progenitor cells (EPCs) after bone-marrow stimulation, and to investigate the possible mechanism of improving ischemicl imb disease after bone-marrow stimulation through autologue bone-marrow stem cell implantation. Methods Twelvemale Lewis rats, weighing 200-250 g, were classified into the bone marrow stimulation group (n=6) and the control group(n=6). In the stimulation group, the bone marrow of each rat was stimulated by injection of recombinant human granulocytemacrophage colony-stimulatory factor. Mononuclear cells were harvested from bone marrow and cultured in EBM-2 medium. After 7-day culture, EPCs were stained by 1, 1-dioctadecyl-3, 3, 3, 3-tetramethyl indocarbocyanine-labbled acetylated low density l ipoprotein/fluorescein isothiocyanate-ulex europaeus agglutinin 1, and the double positive cells were counted by the fluorescent microscope. The adhesive abil ity of EPCs was determined by counting the number of re-cultured EPCs. The unilateral ischemia hindl imb model was made with 12 Lewis rats. Three days later, EPCs were transplanted into the ischemic tissues. According to different sources of EPCs, the 12 rats were divided into 2 groups: the stimulation group (n=6) and the control group (n=6). At 3 weeks after EPCs transplantation, the quantity of the collateral vascular was observed by digital subtraction angiography (DSA). Results After 7-day culture, the number of EPCs in the stimulation and control groups was (145.2 ± 37.0)/HP and (95.2 ± 39.4)/HP, respectively, and there was significant difference between the two groups (P lt; 0.05). Meanwhile, the number of adhesive EPCs in the stimulation and control groups was (21.8 ± 4.3)/HP and (15.0 ± 5.2)/HP, respectively, and the difference between the two groups was significant (P lt; 0.05). At 3 weeks after the EPCs implantation, the number of the collateral vascular was significantly larger in the stimulation group (4.2 ± 1.2) compared with the control group (2.7 ± 0.8), (P lt; 0.05). Conclusion Bone marrow stimulation increases the number of EPCs and improves the function concurrently, which may be the reason why autologue bone-marrow stem cell implantation improves the curative effect of ischemic l imb diseases after bone-marrow stimulation.
【摘要】 目的 比较密度梯度离心法及全骨髓培养法分离培养内皮祖细胞的差异。方法 取4周雄性近交系C57BL/6J小鼠骨髓,分别使用密度梯度离心法及全骨髓培养法培养,观察细胞贴壁情况和细胞形态,并行DiIacLDL及FITCUEAI双染、vWF、eNOS及细胞表面标志检测。结果 密度梯度离心法培养细胞可形成典型铺路石样改变及形成血管样结构;而全骨髓培养法贴壁细胞形态多样,较多呈长梭形铺展生长,部分细胞呈类圆形及纺锤形。比较两种方法培养细胞摄取DiIacLDL、结合FITCUEAI双阳性率以及vWF、eNOS及细胞表面标志表达阳性率,差别均有统计学意义(Plt;005)。应用密度梯度离心法,随着培养时间延长,表达CD34、CD133及FLk1细胞逐渐增多(Plt;005)。结论 密度梯度离心法及全骨髓培养法在EGM2MV培养体系下均可培养出内皮祖细胞,但密度梯度离心法较全骨髓培养法培养的内皮祖细胞纯度高。
Objective To observe the effects of Galectin-3 on proliferation of vascular endothelial cells derived from peripheral blood endothelial progenitor cells. Methods The cultured peripheral blood endothelial progenitor cells in vitro were isolated and purified from human peripheral blood, and the cells were differentiated into vascular endothelial cells. Then the cells were cultivated with the galectin-3 of different concentrations, and to observe the proliferation of endothelial cells derived from peripheral blood endothelial progenitor cells. Results The abilities of proliferation of endothelial cells derived from peripheral blood endothelial progenitor cells of 0.1, 1.0, 2.5, 5.0, and 10.0 μg/ml groups were higher than that of 0 μg/ml group, there were not statistic significance of the differences between the 0.1，1.0, 2.5, and 0 μg/ml groups （P＞0.05）. But the abilities of proliferation of 5.0 and 10.0 μg/ml groups were obviously higher than that of 0, 0.1, 1.0, and 2.5 μg/ml groups （P＜0.05）, and the abilities of proliferation of 10.0 μg/ml group was also higher than that of 5.0 μg/ml group （P＜0.05）. Conclusion Galectin-3 can promote the proliferation of endothelial cells derived from peripheral blood endothelial progenitor cell.
Objective To establish the three diamension-model and to observe the contribution of endothelial progenitor cell (EPC) in the angiogenesis and its biological features. MethodsEPC was obtained from the rats’ peripheral blood. Its cultivation and amplification in vitro were observed, and the function of the cultural EPC in vitro was detected. The three diamension-model was established and analyzed. ResultsEPC was obtained from the peripheral blood successfully. The proliferation of the EPC which induced with VEGF(experimental group) was better than that without VEGF （control group） at every different phase (P＜0.01). It was found that EPC grew into collagen-material from up and down in the three diamension-model, and its pullulation and infiltration into the collagen were seen on day 1 after cultivation. With the time flying, there were branch-like constructions which were vertical to the undersurface of collagen and interlaced to net each other. It showed that in experimental group the EPC grew fast, its infiltration and pullulation also were fast, the branch-like construction was thick. But in control group, the EPC grew slowly, infiltration and pullulation were slow, the branch-like construction was tiny and the depth of infiltration into collagen was superficial. The number of new vessels in experimental group was larger than that in the control group at every different phase (P＜0.01). ConclusionRat tail collagen can induce EPC involved in immigration, proliferation and pullulation in angiogenesis. The three-diamension model of EPC can be used to angiogenesis research. VEGF can mobilize and induce EPC to promote the angiogenesis.