封装VEGF及bFGF的海藻酸钠凝胶负荷内皮祖细胞修复大鼠皮肤创面的实验研究

发布时间：2018-07-02 07:26

本文选题：海藻酸钠 + 凝胶　；参考：《华中科技大学》2015年博士论文

【摘要】：第一部分海藻酸钠凝胶的制备及对成血管因子的封装目的：设计并合成具有一定生物活性的海藻酸钠凝胶,观察其显微结构;并封装成血管生长因子VEGF和bFGF,测定并比较海藻酸钠对这两种生长因子的释放规律。方法：购买天然的高分子量及低分子量海藻酸钠大分子,通过高碘酸钠氧化修饰法部分氧化海藻酸钠分子；氧化型的高分子量及低分子量海藻酸钠溶解后,按照1：1等体积混合后,在二价钙离子的作用下发生交联反应形成凝胶,扫描电镜下观察其显微结构；同时加入VEGF及bFGF一起形成凝胶,通过酶联免疫吸附实验测定两种因子的释放规律。结果：成功合成了海藻酸钠凝胶,并完成了凝胶对VEGF及bFGF的封装。扫描电镜下显示凝胶内部呈多孔网状结构,孔隙直径在10 μ m左右；ELISA结果显示：短时程内,两种生长因子都平稳缓慢释放,在24h时释放率达到20%左右；长时程内,两种生长因子释放速度也较平稳,第七天时释放速度明显加快,释放率达到85%左右。采用配对资料的t检验发现,海藻酸钠凝胶对两种生长因子的释放率没有统计学差异。结论：海藻酸钠能够形成凝胶,而且能封装生长因子VEGF和bFGF;封装后,能较平稳释放生长因子,在一周左右有一个释放高峰；海藻酸钠能保证两种生长因子的协同释放。第二部分内皮祖细胞的分离培养鉴定及与海藻酸钠凝胶的负荷目的：分离培养大鼠骨髓来源内皮祖细胞,观察内皮祖细胞的生物特点,并鉴定其表面标志物；用海藻酸钠凝胶封装VEGF及bFGF,同时负荷内皮祖细胞共培养,观察海藻酸钠凝胶对内皮祖细胞生长的影响。方法：分离SD大鼠骨髓,采用密度梯度离心法获得单个核细胞；用EGM培养基诱导培养,根据差速贴壁原理,分别获得早、晚期内皮祖细胞；倒置显微镜下观察两种内皮祖细胞的原代生长规律；晚期内皮组细胞经传代培养后采用流式细胞术检测其表面标记CD34及VEGFR2的阳性率,免疫荧光法观察这两种表面分子的染色情况。使用封装有VEGF及bFGF的海藻酸钠凝胶负荷内皮祖细胞,使用细胞计数法观察内皮祖细胞单独培养及与凝胶共培养时的生长速度。结果：我们成功分离培养了大鼠骨髓来源的内皮祖细胞：早期内皮组细胞在种植两天后贴壁,四天左右开始出现“集落”,一周时观察到典型的“内皮祖细胞集落”,其特点是中央为大量圆球形细胞,周围梭形细胞呈放射状排列；晚期内皮祖细胞在第五日二次贴壁后呈圆球形,之后大量扩增,两周时达到80%融合,呈典型的“铺路石”样外观。免疫荧光检测显示内皮祖细胞VEGFR染色较强,而CD34分子染色较弱；流式细胞术检测显示VEGFR2和CD34双阳性细胞比例为(1.50±0.04)%;VEGFR2单阳性细胞比例为(18.50±0.12)%；CD34单阳性细胞比例为(2.41±0.08)%。细胞生长曲线测试显示与单纯内皮祖细胞培养相比,海藻酸钠凝胶共培养使细胞增殖更平稳,增殖期延长。结论：密度梯度离心法结合差速贴壁原理能够获得稳定传代的大鼠骨髓来源内皮祖细胞,为后续的组织工程研究提供“种子细胞”；海藻酸钠凝胶与内皮祖细胞有良好的生物相容性,与VEGF及bFGF一起可以促进内皮祖细胞的生长和增殖。第三部分封装成血管因子VEGF及bFGF的海藻酸钠凝胶负荷内皮祖细胞在大鼠皮肤创面模型中促血管生成的效果的观察目的：观察封装成血管因VEGF及bFGF的海藻酸钠凝胶,负荷大鼠内皮祖细胞在大鼠皮肤创面模型中促血管生成的效果,并探讨其可能的机制。方法：取SD大鼠24只,随机分为A、B、C、D四组,设计大鼠背部创面模型；A组大鼠背部创面移植单纯的海藻酸钠凝胶,B组大鼠背部创面移植封装有VEGF及bFGF的海藻酸钠凝胶,C组大鼠背部创面移植负荷有内皮祖细胞的海藻酸钠凝胶,D组大鼠背部创面移植封装有VEGF及bFGF、同时负荷内皮祖细胞的海藻酸钠凝胶。连续大体观比较创面愈合的基本情况；术后7天时,活体成像显微镜下观察各组大鼠创面血管密度及血液供应的基本情况；处死大鼠并分离创面组织,HE染色观察创面的组织变化情况；免疫荧光法检测各组创面Ang-1及VEGFR抗原的表达情况；实时荧光定量PCR检测创面组织中PECAM1、 VE-cadherin、Flk-1 mRNA的转录水平。结果：大体观见D组大鼠创面造模后4天伤口已明显干燥、结痂,在术后一周时形成了一层痂皮保护层,显示创面愈合较其他组快；活体成像显微镜结果示A、B、C、D四组大鼠创面血管平均计数依次分别为(条)：4.0±0.8；12.5±1.3；14.0±0.8；27.8±2.5。A组的血管数与B、C、D组对比有统计学差异(P0.05)；D组的血管数与B、C组对比有统计学差异(P0.05)。A、B、C、 D四组大鼠创面的平均血流速度依次分别为(μm/秒)：41.60±1.76；53.45±1.67；55.03±1.50；64.88±2.12。A的血流速度与B、C、D组均有统计学差异(P0.05)；B、C组与D组对比均有统计学差异(P0.05)。免疫荧光法显示D组荧光强度最高。实时荧光定量PCR结果显示A、B、C、D四组大鼠创面组织中PECAM的mRNA相对表达量依次为0.198±0.021；0.393±0.027；0.409±0.019；0.805±0.017。A组与B、C、D组对比差异有统计学意义；B、C组与D组对比差异有统计学意义(P0.05)。四组的VE-cadherin mRN A相对表达量依次为0.479±0.008；0.507±0.007；0.494±0.005；0.871±0.023。D组与A、B、C组对比差异有统计学意义；A、B、C三组之间相互对比差异无统计学意义(P0.05)。四组的Flk-1 mRNA相对表达量依次为0.186±0.017；0.406±0.010；0.404±0.008；0.690±0.020。A组与B、C、D组对比差异有统计学意义；B、C组与D组对比差异有统计学意义(P0.05)。结论：封装VEGF及bFGF的海藻酸钠凝胶,同时负荷内皮祖细胞用于大鼠背部创面的损伤修复,在宏观上可以促进创面的结痂及炎症反应的消退,增加创面血管密度及改善血供；微观上可以增加创面组织中相关血管营养因子比如PECAM、 VE-cadherin及Flk-1的(?) RNA的表达,而这些因子与血管的营养和再生有密切联系。以上结果提示该种方法可能在创面修复过程中有促进血管新生、改善创面血供的作用,在今后关于创面修复的组织工程学研究方面具有良好的应用前景。
[Abstract]:The first part of the preparation of sodium alginate gel and the encapsulation of vascular factors: design and synthesize a certain bioactive sodium alginate gel, observe its microstructure, and encapsulate the vascular growth factor VEGF and bFGF, determine and compare the release of these two growth factors by sodium alginate. Methods: purchase natural high Molecular weight and low molecular weight alginate macromolecules are partially oxidized by sodium periodate to oxidize sodium alginate molecules; after the oxidation of high molecular weight and low molecular weight sodium alginate is dissolved, the gel is formed by crosslinking reaction under the action of two valence calcium ions, and the microjunction is observed under the scanning electron microscope under the action of two valence calcium ions. The gel was formed together with VEGF and bFGF, and the release regularity of two factors was determined by enzyme linked immunosorbent assay. Results: the sodium alginate gel was successfully synthesized and the encapsulation of VEGF and bFGF was completed by the gel. The porous network structure in the gel was shown under the scanning electron microscope, the diameter of the pore was about 10 mu m, and the result of ELISA was obvious. In short time, the two growth factors were released slowly and slowly, the release rate reached about 20% at 24h; the release rate of two growth factors was also more stable in the long period, and the release rate was accelerated obviously at seventh days, and the release rate was about 85%. The release rate of the two growth factors by the paired data of the alginate gel and the release rate of the alginate gel on the two growth factors. There is no statistical difference. Conclusion: sodium alginate can form gels, and can encapsulate growth factors VEGF and bFGF. After encapsulation, the growth factor can be released smoothly, and there is a release peak in a week or so; sodium alginate can guarantee the synergistic release of two growth factors. Isolation and identification of second partial progenitor cells and alginic acid The purpose of sodium gel load: to isolate and culture the endothelial progenitor cells from rat bone marrow, to observe the biological characteristics of endothelial progenitor cells and to identify the surface markers; to encapsulate VEGF and bFGF with sodium alginate gel, and to co culture the endothelial progenitor cells, and to observe the effect of sodium alginate gel on the growth of inner skin progenitor cells. Methods: SD rats were separated. Mononuclear cells were obtained by density gradient centrifugation. The early and late endothelial progenitor cells were obtained by EGM medium, and the primary growth of two endothelial progenitor cells was observed under the inverted microscope. The surface markers were detected by flow cytometry in the late endothelial cells after transmission. The positive rates of CD34 and VEGFR2 were recorded. The staining of these two surface molecules was observed by immunofluorescence. The endothelial progenitor cells were loaded with sodium alginate gel encapsulated with VEGF and bFGF. The growth rate of endothelial progenitor cells was observed by the cell count method, and the growth rate of the endothelial progenitor cells was observed individually and in co culture with the gel. Endothelial progenitor cells: the early endothelial cells were adhered to the wall for two days and began to appear "colonies" around four days, and a typical "endothelial progenitor cell colony" was observed at one week, characterized by a large number of round cells in the central area and radially arranged around the spindle cells, and the late endothelial progenitor cells were attached to the wall at two times on fifth days. Round ball, then expanded in large quantities and reached 80% fusion at two weeks, showing a typical "paving stone" appearance. Immunofluorescence test showed that the VEGFR staining of endothelial progenitor cells was stronger, but CD34 molecule staining was weak; flow cytometry showed that the ratio of VEGFR2 and CD34 double positive cells was (1.50 + 0.04)%, and the proportion of VEGFR2 single positive cells was (18.50 + 0.12)%. The ratio of CD34 single positive cells was (2.41 + 0.08)%. The cell growth curve test showed that compared with the pure endothelial progenitor cell culture, the alginate gel co culture made the cell proliferation more stable and the proliferation period prolonged. Conclusion: the density gradient centrifugation combined with differential adherence principle can obtain the endothelial progenitor cells of the stable generation of rat bone marrow. Subsequent tissue engineering studies provide "seed cells"; sodium alginate gels have good biocompatibility with endothelial progenitor cells, and together with VEGF and bFGF can promote the growth and proliferation of endothelial progenitor cells. Third part of the alginate gel negative endothelial progenitor cells encapsulated into vascular factor VEGF and bFGF in the rat skin wound model Objective: To observe the effect of promoting angiogenesis: To observe the effect of VEGF and bFGF sodium alginate gel on the angiogenesis of rat inner skin progenitor cells in rat skin wound model, and to explore its possible mechanism. Methods: 24 rats of SD were randomly divided into groups of A, B, C, and D, and the model of the back wound of the rat was designed; A The back wound of group rats was transplanted with simple sodium alginate gel, and the back wound graft in group B was encapsulated with sodium alginate gel with VEGF and bFGF. The transplantation load on the back wound of group C rats was loaded with alginate gel of endothelial progenitor cells, and the back wound of group D was encapsulated with VEGF and bFGF, and the alginate gel loaded with endothelial progenitor cells. The basic situation of wound healing was compared continuously. On the 7 day after operation, the blood vessel density and blood supply were observed under the living microscope microscope. The rats were killed and the wound tissue was separated. The tissue change of the wound was observed by HE staining. The expression of Ang-1 and VEGFR antigen in the wound surface was detected by immunofluorescence. Results: the transcriptional level of PECAM1, VE-cadherin, Flk-1 mRNA in wound tissue was detected by real time fluorescence quantitative PCR. Results: the wounds were obviously dry and scab at 4 days after the formation of the wounds in group D, and a layer of crust skin protective layer was formed at one week after the operation, indicating that the wound healing was faster than the other groups; the results of living imaging microscopy showed A, B, C, D. The average count of blood vessels in the four groups was:4.0 + 0.8, 12.5 + 1.3 and 14 + 0.8, and the number of blood vessels in group 27.8 + 2.5.A was significantly different from that of B, C and D group (P0.05). The number of blood vessels in D group was statistically different from B and C group (P0.05).A, and the average blood flow velocity of the wounds of four groups of rats was respectively (micron seconds) respectively The blood flow velocity of 60 + 1.76, 53.45 + 1.67, 55.03 + 1.50, 64.88 + 2.12.A and B, C, D were statistically different (P0.05), B, C group and D group were statistically different (P0.05). The fluorescence intensity of the D group was the highest. The times were 0.198 + 0.021, 0.393 + 0.027 and 0.409 + 0.019, and 0.805 + 0.017.A groups were statistically significant compared with B, C, and D groups, and B, C group and D group were statistically significant (P0.05). The VE-cadherin mRN A relative expression in four groups was 0.479 + 0.008, 0.507 + 0.007, 0.494 + 0.005. There was no statistically significant difference between the three groups of A, B and C (P0.05). The relative expression of Flk-1 mRNA in the four groups was 0.186 + 0.017, 0.406 + 0.010 and 0.404 + 0.008; 0.690 + 0.020.A group was statistically significant compared with B, C and D groups; B, the difference between the C group and the group was statistically significant. Conclusion: encapsulation: Encapsulation GF and bFGF sodium alginate gel, at the same time loading endothelial progenitor cells, can be used to repair the injury of the rat's back wound. On the macro level, it can promote the scab and inflammatory reaction of the wound, increase the blood vessel density and improve the blood supply. In the microcosmic, it can increase the related blood tube nutrient factors such as PECAM, VE-cadherin and Flk-1 in the wound tissue. (?) expression of RNA, which are closely related to the nutrition and regeneration of blood vessels. These results suggest that this method may promote angiogenesis and improve blood supply of the wound in the process of wound repair, and has a good prospect in the future of tissue engineering research on wound repair.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R965

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