EGF-bFGF-壳聚糖缓释微球诱导骨髓基质细胞向神经样细胞分化的实验研究

发布时间：2018-07-15 20:52

【摘要】：目的：研究证实,骨髓基质细胞(bone mesenchymal stem cells, BMSCs)在表皮生长因子(epidermal growth factor,EGF)和碱性成纤维生长因子(basic fibroblast growth factor,bFGF)诱导下可向神经样细胞分化,具有促进脊髓损伤(Spinal cord injury, SCI)修复的作用,但由于EGF、bFGF进入体内后迅速扩散、变性或酶解,无法保持其生物活性,难以达到体内持续诱导骨髓基质细胞向神经样细胞分化的作用。运用壳聚糖缓释微球负载细胞因子能够达到抵御人体代谢的作用。本实验的目的是制备壳聚糖缓释微球负载EGF和bFGF,在体外培养条件下应用于BMSCs中,观察：1、EGF-bFGF-壳聚糖缓释微球对BMSCs活性、增殖能力的影响；2、诱导BMSCs分化为神经样细胞的情况,为进一步联合BMSCs、 EGF-bFGF-壳聚糖缓释微球进行体内移植治疗SCI提供理论依据。方法：1.骨髓基质细胞的体外培养及鉴定：采用贴壁筛选法对取自成年S-D大鼠股骨和胫骨的BMSCs进行分离培养,并用细胞免疫荧光方法鉴定BMSCs的干细胞标记物CD44、CD90,骨髓造血干细胞标记物CD45。2. EGF-bFGF-壳聚糖缓释微球的制备及检测采用乳化交联法,制备EGF-bFGF-壳聚糖缓释微球。采用elisa方法进行EGF和bFGF的检测,计算载药率、包装率及微球的缓释率。流式细胞术检测空白壳聚糖缓释微球对BMSCs的细胞毒性。3. EGF-bFGF-壳聚糖缓释微球诱导BMSCs向神经元样细胞分化的观察：准备如下分组：a、对照组,BMSCs+DMEM;b、空白壳聚糖缓释微球组,BMSCs+DMEM+空白壳聚糖缓释微球；c、EGF/bFGF直接给药组,BMSCs+DMEM+bFGF(终浓度lOng/ml)+EGF(终浓度20ng/ml);d、EGF-bFGF-壳聚糖缓释微球组,BMSCs+DMEM+EGF-bFGF-壳聚糖缓释微球细胞(使之终释放浓度与C组相同)；光镜下观察BMSCs细胞学形态的变化,MTT比色法检测BMSCs的生长增殖情况,进一步利用免疫荧光检测神经前体细胞标记物Nestin、神经元特异性烯醇化酶NSE和星形胶质细胞标记物GFAP染色情况。结果：1、原代培养的骨髓基质细胞10-14d时细胞融合成片,细胞形态多呈长梭形。经4-5次传代,细胞纯度较高,主要以呈细长梭形的BMSCs为主。第5代体外培养BMSCs用细胞免疫荧光方法进行鉴定,观察到细胞呈CD44和CD90阳性表达,CD45呈阴性表达。2、乳化交联法进行缓释微球的制备,经ELISA法检测得知：壳聚糖终浓度为2mg/ml时,平均载药量最高；而缓释微球的包装率随着TPP浓度的升高逐渐升高,当其浓度为0.45mg/ml时,包封率达到峰值。EGF和bFGF在PH7.4和PH5.8的溶剂中,24h内均出现了一个约40-60%的突释过程,随后开始缓慢释放。3、采用流式细胞结合Annexin V-FITC/PI双染法检测细胞的凋亡情况,正常对照组和壳聚糖缓释微球处理组24h后细胞凋亡率无显著性差异(P0.05)。MTT法检测BMSCs的生长增殖情况,四组BMSCs细胞存活率的比较结果：与对照组相比,空白壳聚糖缓释微球组的细胞增殖率没有统计学差异(P0.05),EGF/bFGF组和EGF-bFGF-壳聚糖缓释微球组其细胞存活率在相同时间点均较对照组和空白壳聚糖缓释微球组显著升高(P0.05)。Annexin V-FITC/PI双染法检测结果表明空白壳聚糖缓释微球无生物学毒性。MTT法检测结果表明空白壳聚糖缓释微球作为载体对BMSCs的存活无显著影响,EGF/bFGF组和EGF-bFGF-壳聚糖缓释微球组可促进BMSCs的生长增殖。4、四组BMSCs诱导培养5天后,空白壳聚糖缓释微球组和对照组均未发生变化,仍然维持BMSCs的典型形态。EGF/bFGF直接给药组和EGF-bFGF-壳聚糖缓释微球组部分细胞呈现神经细胞样的改变。高内涵免疫荧光检测：EGF/bFGF组与EGF-bFGF-壳聚糖缓释微球组：Nestin、NSE、GFAP荧光染色均有阳性表达。共聚焦免疫荧光鉴定：EGF/bFGF组：(36.51±2.53)%的细胞表达神经前体细胞标记物Nestin, (17.66±3.44)%的细胞表达神经元细胞标志物NSE,(27.39±3.03)%的细胞表达星形胶质细胞标记物GFAP; EGF-bFGF-壳聚糖缓释微球组：(30.87±3.53)%的细胞表达神经前体细胞标记物Nestin,(10.57-2.24)%的细胞表达神经元细胞标志物NSE,(21.31±4.69)%的细胞表达星形胶质细胞标记物GFAP,结论：1、采用贴壁筛选法对大鼠BMSCs进行培养是一种简单易行的方法,可迅速获得大量纯度较高的BMSCs.2、乳化交联法制备EGF-bFGF-壳聚糖缓释微球,工艺简单,包封率高,载药量大,在中性PH值条件下有良好的缓释性能,对BMSCs无生物学毒性,可以促进BMSCs的生长增殖。3. EGF-bFGF-壳聚糖缓释微球能诱导骨髓基质细胞分化为神经样细胞。4.EGF-bFGF-壳聚糖缓释微球诱导BMSCs分化为神经样细胞的效率较直接EGF/bFGF低,可能与EGF-bFGF-壳聚糖缓释微球释放细胞因子的初始浓度低有关。EGF-bFGF-壳聚糖缓释微球诱导BMSCs分化的最佳浓度有待进一步检测。
[Abstract]:Objective : To study the effects of bone marrow stromal cells ( MSCs ) on the differentiation of nerve - like cells under the induction of epidermal growth factor ( EGF ) and basic fibroblast growth factor ( bFGF ) .
Methods : 1 . In vitro culture and identification of bone marrow stromal cells : 1 . In vitro culture and identification of bone marrow stromal cells : 1 . In vitro culture and identification of bone marrow stromal cells : The stem cell marker CD44 , CD90 and bone marrow hematopoietic stem cell marker CD45.2 were identified by cell immunofluorescence method . The preparation and detection of EGF - bFGF - chitosan sustained - release microspheres by emulsion crosslinking method were used to prepare the EGF - bFGF - chitosan sustained - release microspheres . The detection of EGF and bFGF was carried out by the enzyme - linked immunosorbent assay ( ELISA ) , the drug loading rate , the packing ratio and the slow release rate of microspheres were calculated . The cytotoxicity of the blank chitosan sustained - release microspheres was detected by flow cytometry . The effects of EGF - bFGF - chitosan sustained - release microspheres on neuron - like cell differentiation were observed .
c , EGF / bFGF direct drug administration group , bone marrow + DMEM + bFGF ( final concentration lOng / ml ) + EGF ( final concentration 20ng / ml ) ; d , EGF - bFGF - chitosan sustained - release microsphere group , bone marrow + DMEM + EGF - bFGF - chitosan sustained - release microsphere cells ( the final release concentration is the same as group C ) ;
The growth and proliferation of bone marrow stromal cells ( Nestin , NSE ) and astrocytes ( GFAP ) were detected by MTT colorimetric assay .
There was no significant difference between EGF / bFGF group and EGF - bFGF - chitosan sustained - release microspheres ( P0.05 ) . EGF - bFGF - chitosan sustained - release microspheres can induce differentiation of bone marrow stromal cells into neural - like cells .
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R651.2

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