This study was reviewed and approved by the Animal Ethics Committee of Tianjin First Central Hospital. Adult Wistar rats, 8 weeks of age (weighing 250-280 g), were purchased from the experimental animal center of the Military Academy of Medical Sciences, China. All surgical procedures and care administered to the animals were approved by the Animal Care Committee and performed according to institutional guidelines.
Isolation and culturing of BM-EPCs
Wistar rats were sacrificed by decapitation and soaked in 75% ethanol twice.
Mononuclear cells were isolated from rat BM, especially femur and tibia of the rats, by density gradient centrifugation using Histopaque 1083(Sigma, Stlouis, USA). Isolated cells were resuspended in M199 medium(Gibco, Carlsbad, USA, involved 10% FBS, 20% double antibody, 20 ng/ml VEGF (Sigma), and 1 ng/ml bFGF (Sigma)) and cultured in fibronectin-coated dishes and maintained at a 37℃+5% CO2 incubator. After 4 days in culture, non-adherent cells were removed and fresh medium was added. After the 7 days of culture, BM-EPC cells were identified by a combination of specific surface marker expression.
a. Identification of BM-EPCs by endothelial marker
Afer 7 days of incubation, the adherent cells were digested by 0.25% trypsin and counted 2×106 cells. Aliquotes containing 1×106 cells/100μL into each tubes, were added primary antibody at an appropriate dilution and incubate for 2 h at room temperature. These endothelial markers include CD133 (Proteintech Group, Inc, WuHan, China), CD34 (Proteintech Group, Inc, WuHan, China), VE-cadherin (Bioss, Beijing, China), and KDR (Proteintech Group, Inc, WuHan, China). Then add diluted secondary antibody to the cells and incubate for 1 h at room temperature. The surface markers on BM- EPCs was analyzed by the flow cytometry (BD bioscience, USA), data was analyzed using BD CellQuest™ Pro software Version 5.1.
Identification of BM-EPCs by immunochemistry
The fibronectin-adherent BM- Derived EPCs were identified by incubation with 10μg/ml of fluorescently labeled acetylated-low density lipoprotein (Dil-ac-LDL; Molecular Probes, Eugene, OR, USA) for 4h and 10μg/ml of fluorescently FITC labeled Ulex Europaeus Agglutinin 1 (UEA-1; Sigma-Aldrich, MS, USA) for 1h at room temperature, then co-incubation with DAPI for 15 min, Images were captured by inverted fluorescence microscopy (Olympus IX71, Olympus Optical Co. Ltd, Tokyo, Japan), Double positive cells can be considered BM-EPCs.
The BM-EPCs transwell to SDF-1α assey
Resuspend the cells to 5×104/100μL and coated into 24-well Millipore Transwell chambers. 100μL suspension were coated in upper chambers, meanwhile 600μL SDF-1α (R&D Systems, Inc, USA)solution was injected into the lower chambers with the concentration of 1ng/ml, 10 ng/ml and 100 ng/ml, respectively. Incubated the Millipore Transwell chambers at 37℃ for 45-60 min. Discarded the solution of chambers, washed with 1×PBS and fixed in 4% paraformaldehyde with 600μL for 30 min. Stained with 600μL 0.1% crystal violet for 20 min. Finally, Images were captured by fluorescence microscopy (Leica Microsystems, Wetzlar, Germany).
Fabrication of BM-EPC cell sheets and thickness assey
1.5×106 BM-EPCs were seeded into Nunc UpCell Surface dishes (Thermo Scientific, USA) coated with vitronectin. Under temperature-responsive culture, incubated for 37℃and 7 days firstly, then reducing the temperature to 25℃ for 30 min, the cells spontaneously detached as contiguous cell sheets and were harvested from the dishes.
To observe detached EPCs sheets thickness, one sheet were fixed with 20 mL -20℃ ice-cold methanol solution under 4℃for10 min,3ml primary Anti-Collagen I antibody (1:300,Bioss,Beijing Biosynthesis Biotechnology, Beijing, China) were added to the dishes. After incubation for 5 min at 37℃, 3ml secondary antibody Alexa Flour 488 (1:500, Proteintech Group, Inc, WuHan, China) were injected, incubate at 37℃in the dark for 1 h. Cell nuclei were visualized with DAPI after incubation with secondary antibodies. Images were obtained using fluorescence microscope.
BM-EPC cell sheets proliferation assay.
The proliferative capacity of the BM-EPCs sheets was measured using a Cell Counting Kit‑8 (CCK‑8, Dojindo Laboratories, Japan) assay. 5×103/well were seeded into 96‑well plates and maintained at a 37℃atmosphere. At the indicated time points (cultivated after 12h,24h,48h and 72h),10 µl Cell Counting Kit‑8 was added to each well and cells were cultured for an additional 4 h. Finaly, an enzyme immunoassay analyzer was used to measuring the optical density (OD) at an absorbance wavelength of 450 nm. In order to compare the proliferative capacity of the BM-EPCs sheets, EPCs were used as control.
BM-EPC cell sheets tube formation assay
The BM-EPCs and BM-EPC cell sheets (2 x 104 cells) maintained in M199 medium(Sigma-Aldrich, USA) were seeded onto Matrigel(Corning Matrigel Basement Membrane Matrix,Corning,USA)-coated 96-well plates and further cultured in 37℃for 4 h, respectively. To compare the capillary-like tube formation of EPCs sheets and BM-EPCs, Microscope images were captured using inverted phase-contrast microscopy (IX71; Olympus), and the number of network circles for each groups in each image was counted using the Wimasis image analysis program.
Reverse transcription-quantitative PCR (RT-qPCR)
Respectively, total RNA from BM-EPCs and BM-EPCs sheets (1.5×106 cells) was extracted using RNAiso Plus (TaKaRa Biotechnology, Japan) according to the manufacturer's protocol. Total RNA was converted into cDNA using PrimeScriptTM RT Reagent Kit (TaKaRa Biotechnology, Japan), under the following condition: 37°C for 15 min, 85°C for 5 sec and 4°C for 5 min. cDNA was amplified under SYBR Premix Ex TaqTM (TaKaRa Biotechnology, Japan).The sequences of the primers are shown as follows (Table 1) The qPCR thermocycler conditions were as follow：firstly，95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. β-actin were used as internal reference and relative mRNA expression levels were calculated using the 2−ΔCt (fold difference), whereΔCt = (Ct of target genes)-(Ct of endogenous control gene, β-action) in experimental samples.
Western blot analysis of CXCR4/p-CXCR4、PI3K/p-PI3K、AKT/p-AKT、eNOS in BM-EPCs and BM-EPC cell sheets
Respectively, total protein was extracted from BM-EPCs and BM-EPCs sheets(1×106 cells) and quantified using the BCA Protein Assay Kit (TaKaRa, Japan).Proteins lysates were isolated by using 10% SDS-PAGE and then transferred them to PVDF membrane ( Millipore, USA).The membrane were incubated with the antibodies against CXCR4 (1:1000; Proteintech Group, Inc, WuHan, China)、 P-CXCR4 (1:1000; Bioss, Beijing, China.)、PI3K(1:1000;Abcam,USA.ab86714)、P-PI3K(1:1000; Abcam,USA.ab182651)、AKT(1:500; Proteintech Group, Inc, WuHan, China)、P-AKT(1:3000; Proteintech Group, Inc, WuHan, China)、eNOS (1:300; Bioss, Beijing, China) and Tubulin(1:200; Proteintech Group, Inc, WuHan, China). HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) was used as secondary antibodies. The signals were quantified with Image Studio.
Detection the expression of paracrine factors SDF-1α, VEGF and EGF using ELISA
The BM-EPCs and BM-EPC cell sheets (1x106 cells) were incubated in M199 medium which were serum-and growth factor-free under 37℃+5% CO2 atmosphere for 7 d. According to the manufacturer's protocol, the medium was subsequently collected to discover the expression of paracrine factors SDF-1α, VEGF and EGF using ELISA kit. ELISA Kit information is as follows: Rat SDF-1α ELISA kit (Quanzhou Konodi Biotechnology Co. Ltd, China), Rat VEGF ELISA kit (Quanzhou Konodi Biotechnology Co. Ltd, China), Rat EGF ELISA kit (Quanzhou Konodi Biotechnology Co. Ltd, China). The absorbance at 450 nm was measured.
Statistical analyses were conducted using SPSS software version 20.0. Data are expressed as the mean ± standard deviation (SD). Comparisons between two groups were analyzed by a paired samples t-test. Comparisons between groups were analyzed with one-way analysis of variance (ANOVA), following by the least significant difference test (LSD-t). p values of less than 0.05 were considered significantly different.