本文選題：海藻酸鈉 + 凝膠��； 參考：《華中科技大學(xué)》2015年博士論文

【摘要】：第一部分海藻酸鈉凝膠的制備及對成血管因子的封裝目的：設(shè)計(jì)并合成具有一定生物活性的海藻酸鈉凝膠,觀察其顯微結(jié)構(gòu);并封裝成血管生長因子VEGF和bFGF,測定并比較海藻酸鈉對這兩種生長因子的釋放規(guī)律。方法：購買天然的高分子量及低分子量海藻酸鈉大分子,通過高碘酸鈉氧化修飾法部分氧化海藻酸鈉分子；氧化型的高分子量及低分子量海藻酸鈉溶解后,按照1：1等體積混合后,在二價(jià)鈣離子的作用下發(fā)生交聯(lián)反應(yīng)形成凝膠,掃描電鏡下觀察其顯微結(jié)構(gòu)；同時(shí)加入VEGF及bFGF一起形成凝膠,通過酶聯(lián)免疫吸附實(shí)驗(yàn)測定兩種因子的釋放規(guī)律。結(jié)果：成功合成了海藻酸鈉凝膠,并完成了凝膠對VEGF及bFGF的封裝。掃描電鏡下顯示凝膠內(nèi)部呈多孔網(wǎng)狀結(jié)構(gòu),孔隙直徑在10 μ m左右；ELISA結(jié)果顯示：短時(shí)程內(nèi),兩種生長因子都平穩(wěn)緩慢釋放,在24h時(shí)釋放率達(dá)到20%左右；長時(shí)程內(nèi),兩種生長因子釋放速度也較平穩(wěn),第七天時(shí)釋放速度明顯加快,釋放率達(dá)到85%左右。采用配對資料的t檢驗(yàn)發(fā)現(xiàn),海藻酸鈉凝膠對兩種生長因子的釋放率沒有統(tǒng)計(jì)學(xué)差異。結(jié)論：海藻酸鈉能夠形成凝膠,而且能封裝生長因子VEGF和bFGF;封裝后,能較平穩(wěn)釋放生長因子,在一周左右有一個(gè)釋放高峰；海藻酸鈉能保證兩種生長因子的協(xié)同釋放。第二部分內(nèi)皮祖細(xì)胞的分離培養(yǎng)鑒定及與海藻酸鈉凝膠的負(fù)荷目的：分離培養(yǎng)大鼠骨髓來源內(nèi)皮祖細(xì)胞,觀察內(nèi)皮祖細(xì)胞的生物特點(diǎn),并鑒定其表面標(biāo)志物；用海藻酸鈉凝膠封裝VEGF及bFGF,同時(shí)負(fù)荷內(nèi)皮祖細(xì)胞共培養(yǎng),觀察海藻酸鈉凝膠對內(nèi)皮祖細(xì)胞生長的影響。方法：分離SD大鼠骨髓,采用密度梯度離心法獲得單個(gè)核細(xì)胞；用EGM培養(yǎng)基誘導(dǎo)培養(yǎng),根據(jù)差速貼壁原理,分別獲得早、晚期內(nèi)皮祖細(xì)胞；倒置顯微鏡下觀察兩種內(nèi)皮祖細(xì)胞的原代生長規(guī)律；晚期內(nèi)皮組細(xì)胞經(jīng)傳代培養(yǎng)后采用流式細(xì)胞術(shù)檢測其表面標(biāo)記CD34及VEGFR2的陽性率,免疫熒光法觀察這兩種表面分子的染色情況。使用封裝有VEGF及bFGF的海藻酸鈉凝膠負(fù)荷內(nèi)皮祖細(xì)胞,使用細(xì)胞計(jì)數(shù)法觀察內(nèi)皮祖細(xì)胞單獨(dú)培養(yǎng)及與凝膠共培養(yǎng)時(shí)的生長速度。結(jié)果：我們成功分離培養(yǎng)了大鼠骨髓來源的內(nèi)皮祖細(xì)胞：早期內(nèi)皮組細(xì)胞在種植兩天后貼壁,四天左右開始出現(xiàn)“集落”,一周時(shí)觀察到典型的“內(nèi)皮祖細(xì)胞集落”,其特點(diǎn)是中央為大量圓球形細(xì)胞,周圍梭形細(xì)胞呈放射狀排列；晚期內(nèi)皮祖細(xì)胞在第五日二次貼壁后呈圓球形,之后大量擴(kuò)增,兩周時(shí)達(dá)到80%融合,呈典型的“鋪路石”樣外觀。免疫熒光檢測顯示內(nèi)皮祖細(xì)胞VEGFR染色較強(qiáng),而CD34分子染色較弱；流式細(xì)胞術(shù)檢測顯示VEGFR2和CD34雙陽性細(xì)胞比例為(1.50±0.04)%;VEGFR2單陽性細(xì)胞比例為(18.50±0.12)%；CD34單陽性細(xì)胞比例為(2.41±0.08)%。細(xì)胞生長曲線測試顯示與單純內(nèi)皮祖細(xì)胞培養(yǎng)相比,海藻酸鈉凝膠共培養(yǎng)使細(xì)胞增殖更平穩(wěn),增殖期延長。結(jié)論：密度梯度離心法結(jié)合差速貼壁原理能夠獲得穩(wěn)定傳代的大鼠骨髓來源內(nèi)皮祖細(xì)胞,為后續(xù)的組織工程研究提供“種子細(xì)胞”；海藻酸鈉凝膠與內(nèi)皮祖細(xì)胞有良好的生物相容性,與VEGF及bFGF一起可以促進(jìn)內(nèi)皮祖細(xì)胞的生長和增殖。第三部分封裝成血管因子VEGF及bFGF的海藻酸鈉凝膠負(fù)荷內(nèi)皮祖細(xì)胞在大鼠皮膚創(chuàng)面模型中促血管生成的效果的觀察目的：觀察封裝成血管因VEGF及bFGF的海藻酸鈉凝膠,負(fù)荷大鼠內(nèi)皮祖細(xì)胞在大鼠皮膚創(chuàng)面模型中促血管生成的效果,并探討其可能的機(jī)制。方法：取SD大鼠24只,隨機(jī)分為A、B、C、D四組,設(shè)計(jì)大鼠背部創(chuàng)面模型；A組大鼠背部創(chuàng)面移植單純的海藻酸鈉凝膠,B組大鼠背部創(chuàng)面移植封裝有VEGF及bFGF的海藻酸鈉凝膠,C組大鼠背部創(chuàng)面移植負(fù)荷有內(nèi)皮祖細(xì)胞的海藻酸鈉凝膠,D組大鼠背部創(chuàng)面移植封裝有VEGF及bFGF、同時(shí)負(fù)荷內(nèi)皮祖細(xì)胞的海藻酸鈉凝膠。連續(xù)大體觀比較創(chuàng)面愈合的基本情況；術(shù)后7天時(shí),活體成像顯微鏡下觀察各組大鼠創(chuàng)面血管密度及血液供應(yīng)的基本情況；處死大鼠并分離創(chuàng)面組織,HE染色觀察創(chuàng)面的組織變化情況；免疫熒光法檢測各組創(chuàng)面Ang-1及VEGFR抗原的表達(dá)情況；實(shí)時(shí)熒光定量PCR檢測創(chuàng)面組織中PECAM1、 VE-cadherin、Flk-1 mRNA的轉(zhuǎn)錄水平。結(jié)果：大體觀見D組大鼠創(chuàng)面造模后4天傷口已明顯干燥、結(jié)痂,在術(shù)后一周時(shí)形成了一層痂皮保護(hù)層,顯示創(chuàng)面愈合較其他組快；活體成像顯微鏡結(jié)果示A、B、C、D四組大鼠創(chuàng)面血管平均計(jì)數(shù)依次分別為(條)：4.0±0.8；12.5±1.3；14.0±0.8；27.8±2.5。A組的血管數(shù)與B、C、D組對比有統(tǒng)計(jì)學(xué)差異(P0.05)；D組的血管數(shù)與B、C組對比有統(tǒng)計(jì)學(xué)差異(P0.05)。A、B、C、 D四組大鼠創(chuàng)面的平均血流速度依次分別為(μm/秒)：41.60±1.76；53.45±1.67；55.03±1.50；64.88±2.12。A的血流速度與B、C、D組均有統(tǒng)計(jì)學(xué)差異(P0.05)；B、C組與D組對比均有統(tǒng)計(jì)學(xué)差異(P0.05)。免疫熒光法顯示D組熒光強(qiáng)度最高。實(shí)時(shí)熒光定量PCR結(jié)果顯示A、B、C、D四組大鼠創(chuàng)面組織中PECAM的mRNA相對表達(dá)量依次為0.198±0.021；0.393±0.027；0.409±0.019；0.805±0.017。A組與B、C、D組對比差異有統(tǒng)計(jì)學(xué)意義；B、C組與D組對比差異有統(tǒng)計(jì)學(xué)意義(P0.05)。四組的VE-cadherin mRN A相對表達(dá)量依次為0.479±0.008；0.507±0.007；0.494±0.005；0.871±0.023。D組與A、B、C組對比差異有統(tǒng)計(jì)學(xué)意義；A、B、C三組之間相互對比差異無統(tǒng)計(jì)學(xué)意義(P0.05)。四組的Flk-1 mRNA相對表達(dá)量依次為0.186±0.017；0.406±0.010；0.404±0.008；0.690±0.020。A組與B、C、D組對比差異有統(tǒng)計(jì)學(xué)意義；B、C組與D組對比差異有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論：封裝VEGF及bFGF的海藻酸鈉凝膠,同時(shí)負(fù)荷內(nèi)皮祖細(xì)胞用于大鼠背部創(chuàng)面的損傷修復(fù),在宏觀上可以促進(jìn)創(chuàng)面的結(jié)痂及炎癥反應(yīng)的消退,增加創(chuàng)面血管密度及改善血供；微觀上可以增加創(chuàng)面組織中相關(guān)血管營養(yǎng)因子比如PECAM、 VE-cadherin及Flk-1的(?) RNA的表達(dá),而這些因子與血管的營養(yǎng)和再生有密切聯(lián)系。以上結(jié)果提示該種方法可能在創(chuàng)面修復(fù)過程中有促進(jìn)血管新生、改善創(chuàng)面血供的作用,在今后關(guān)于創(chuàng)面修復(fù)的組織工程學(xué)研究方面具有良好的應(yīng)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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R965

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