| Part 1Synthesis of Sodium Alginate Gel and Its’Encapsulation of Growth Factors for AngiogenesisObjective:Design and compound biological sodium alginate gel, and observe the microscopic structure. Use the gel to encapsulate vesscular growth factors such as VEGF and bFGF, detect and make a comparison of the releasing law about the two growth factors.Methods:Prepare natural sodium alginate of high molecular weight(HMW) and low molecular weight(LMW). Oxygenize sodium alginate by sodium periodate, mix the oxidative sodium alginate of HMW and LMW in the ration of 1:1 into solution. By the activation of calcium ion, the sodium alginate gelatinized through cross link reaction, and we observed its microscopic structure under scanning electron microscopy(SEM). Meanwhile, we added VEGF and bFGF to the solution for gelatinization, and tested the releasing law of these two factors.Results:Sodium alginate gel was synthesized and used for encapsulation of VEGF and bFGF successfully. The gel was found to be multiporous network structure, and the pore diameter is about 10 micrometer. With the ELISA method, the releasing law was found to be like follows:In short term, both growth factors were released slowly and smoothly, and the releasing rate reached 20% at 24h. In long term, the releasing procedure was still smoothly but faster, and the releasing rate reached nearly 85% after a week. Regarding the releasing rate of both factors at each point, we used student’s t test to compare the releasing law of VEGF and bFGF, and there was no statistic difference between the two factors.Concluion:Sodium alginate gel could be formed, and encapsulate growth factor of VEGF and bFGF. The factors encapsulated in the gel could be released smoothly and the releasing rates reach a peak point at 7th day. Sodium alginate gel may guarantee the synergetic release of VEGF and bFGF.Part 2Isolation And Cultivation of The Endothelial Progenitor Cells to Co-culture with Sodium Alginate GelObjective:Isolate and culture the endothelial progenitor cells derived from the bone marrow of SD rat, to observe its biological characteristics and identify its surface markers. Co-cultured with sodium alginate gel encapsulating VEGF and bFGF, to observe the influence of co-cultivation to the growth of EPCs.Methods:The bone marrow of SD rats were isolated, and the mononuclear cell were obtained by density gradient centrifugation.. Via differential adhension, early and late EPCs were obtained using endothelial growth medium(EGM). We observed the growth law of these two type EPCs under inverted microscope, detected the positive rate of surface markers of CD34 and VEGFR2 by flow cytometry and immunofluorescence. Comparing the EPCs cultured alone and with sodium alginate gel encapsulating growth factors, we test its influence of co-cultivation by cytometry.Results:We obtained rat bone marrow derived EPCs successfully. The early EPCs appeared to be adherence at day 2, and the EPC-colony appeared at day 4, with a typical "colony-like" shape at day 7, which is composed with round shape cells in the center and spindle-shaped cells around. The late EPCs appeared after a second-time adherence at day 5, amplified rapidly and made a 80% confluence at week 2, with a feature of slabstone-like form. The flow cytometry results showed VEGFR2-positive rate was (18.50±0.12)%, CD34-positive rate was (2.4±0.08)%, and double-poitive rate was (1.50±0.04)%. The immunofluorescence results showed VEGFR2 staining signal was stronger than CD34. We found EPCs could proliferate more steadily and maintain a longer amplification phase when co-cultured with sodium alginate gel encapsulating VEGF and bFGF, compared with cultured alone.Discussion:Rat bone marrow deprived EPCs could be obtained via density gradient centrifugation method with the principle of differential adhension. So this method is useful to supply "seed-cell" for tissue engineering study. Sodium alginate gel is biocompatible to EPCs, and to promote growth and proliferation of EPCs combined with VEGF and bFGF.Part 3Observation of the Angiogenisis Effect of Sodim Alginate Gel Encapsulating VEGF and bFGF Loaded with Endothelial Progenitor Cells on Rat Dorsal Skin WoundObjective:To study the angionenisis effect of sodium alginate gel encapsulating VEGF and bFGF loaded with endothelial progenitor cells on rat dorsal skin wound, and to investigate its possible mechanism.Methods:Twenty four six-weeks Sprague-Dawley rats were randomly divided into four groups(A, B, C, D group). We designed a rat dorsal skin wound model. Sodium alginate gel alone was transplanted onto skin wound of group A, sodium alginate gel encapsulating VEGF and bFGF to group B, sodium alginate gel loaded with EPCs to group C and sodium alginate gel both encapsulating factors and loaded with cells to group D. Observe the serial wound healing process of each group. To measure the vessel number and blood flow speed in the wound area at post-operation 7 in-vivo. After the in-vivo observation, sacrifice all the rats and dissect the skin of wound area. HE staining method was used to observe the general tissue structure, and the expression of antigen Ang-1 or VEGFR in the wound skin were detected by immunofluorescence. At last, we detected the mRNA level of gene PECAM1, VE-cadherin and Flk-1 employing real-time fluorescent quantitation PCR.Results:We found the skin wound of group D was dry and forming a scab without much inflammatory reaction at day 4, coated with a layer of integral scab.The vessels counted in the wound area of each group were 4.0±0.8,12.5±1.3, 14.0±0.8 and 27.8±2.5 from group A to D. There were statistical difference between group A and group B, C and D, likewise between group D and group B and C. (P value<0.05).The blood flow speed measured in the wound area of each group were 41.60± 1.76,53.45±1.67,55.03±1.50 and 64.88±2.12(μm/s) from group A to D.There were statistical difference between group A and group B, C and D, likewise between group D and group B and C.(P value<0.05).Immunofluorescence showed the fluorescence signal of group D was strongest. The PEC AMI mRNA level normalized to GAPDH of each group were 0.198±0.021, 0.393±0.027,0.409±0.019 and 0.805±0.017 from group A to D. There were statistical difference between group A and group B,C and D, likewise between group D and group B and C.(P value<0.05).The VE-cadherin mRNA level normalized to GAPDH of each group were 0.479 ±0.008,0.507±0.007,0.494±0.005 and 0.871±0.023 from group A to D.There were statistical difference between group A and group B, C and D, but no statistical difference among group B,C and D.The Flk-1 mRNA level normalized to GAPDH of each group were 0.186±0.017, 0.406±0.010,0.404±0.008 and 0.690±0.020·from group A to D. There were statistical difference between group A and group B, C and D, likewise between group D and group B and C. (P value<0.05). Conclusions:The utilization of Sodim Alginate Gel Encapsulating VEGF and bFGF Loaded with Endothelial Progenitor Cells on Rat Dorsal Skin Wound could promote the formation of scab and regression of inflammation reaction generally. Its possible molecular mechanism may be involved in new vessel angiogenesis related with growth factors such as PECAM and VE-cadherin and so on. EPCs is also very important in the healing process. The results above suggested that our method was effective in angiogenesis, and could be a potential project in chronic wound healing. |
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