高压静电法制备人视网膜色素上皮细胞微胶囊及微囊化后对细胞进行保护的研究探索

发布时间：2018-04-07 15:01

本文选题：色素上皮　切入点：眼　出处：《河北北方学院》2011年硕士论文

【摘要】：) 细胞微囊化是近年发展起来的一种有效避免免疫排斥反应从而提高移植细胞的存活率的方法，人视网膜色素上皮(Human RetinalPigment Epithelial Cell, hRPE)细胞具有酪氨酸羟化酶活性且能分泌多巴胺，有可能在帕金森病的细胞移植治疗中作为供体细胞发挥巨大潜能。本实验探索了微胶囊制备的条件，制备了hRPE细胞微胶囊，而且在微囊化过程后加入了一些细胞因子或细胞保护性物质以期对微囊化所造成的hRPE细胞功能的损伤进行改善。我们利用自制的静电微囊发生器制备hRPE细胞海藻酸钠-壳聚糖-海藻酸钠(Alginate-Chitosan-Alginate, ACA)微胶囊，制备过程中影响所成微囊质量及稳定性的因素包括电压、注射泵推进速度、电极距离、针头内径、海藻酸钠溶液浓度、壳聚糖溶液浓度、成膜反应时间等物理因素和化学因素，我们通过改变其他参数进行单因素试验研究，考察各影响因素对微囊的作用。倒置光学显微镜观察微囊形态，用计数板测量微胶囊粒径大小，并用膨胀度间接表征微胶囊的稳定性。本次实验最终确定制备细胞微胶囊的条件参数为电极距离为30mm，针头内径为0.292mm，，电场电压为7kv，推进速度为20mL·h~(-1)，海藻酸钠及壳聚糖溶液浓度均为15g·L~(-1)，成膜反应时间为15min。由于在微囊化的过程中，不可避免对细胞生长会造成一定的伤害，而微囊化后细胞环境的变化也会对细胞的生长产生影响。为了改善细胞的生长状态，我们在制备了细胞微胶囊的基础上初步观察上皮细胞培养液中添加果糖、成纤维细胞生长因子、牛磺酸后对人视网膜色素上皮（hRPE）细胞海藻酸钠-壳聚糖-海藻酸钠（ACA）微囊化后的细胞总数、存活率及活细胞数的影响。hRPE细胞微囊化后将其分为四组，一组作为空白对照组只加入上皮细胞培养液、另外三组在上皮细胞培养液中分别加入果糖、成纤维细胞生长因子、牛磺酸后培养7d，在第0d、1d、3d、7d测定微胶囊内细胞的细胞总数、存活率及活细胞数。细胞计数板进行常规计数,台盼蓝染色法检测囊内细胞存活率。制备出的hRPE细胞的ACA微胶囊直径为450μm±13μm。四组细胞微囊化后的hRPE细胞至培养7d,细胞存活率最低为(75±16.8)%,但各组间没有明显差异(P0.05);而空白组、果糖组、牛磺酸组及成纤维细胞生长因子组微胶囊内细胞总数分别为(6.1±0.6)×10~4、(6.0±0.5)×10~4、(7.2±0.4)×10~4和(8.0±0.5)×10~4,牛磺酸组、成纤维细胞生长因子组比空白对照组明显增多(P0.05),有统计学意义;各组活细胞数为细胞总数×细胞存活率,有统计学意义。说明培养液中加入细胞生长因子、牛磺酸后在7d之内可促进海藻酸钠-壳聚糖-海藻藻酸钠(ACA)微胶囊内的hRPE细胞的增殖。
[Abstract]:)
Cell microencapsulation is a recently developed to avoid immune rejection so as to improve the survival rate of transplanted cells, retinal pigment epithelium (Human RetinalPigment Epithelial Cell, hRPE) cells with tyrosine hydroxylase activity and can secrete dopamine, there may be cell transplantation in the treatment of Parkinson disease as donor cells play a huge potential. The experiment explored the microcapsule preparation conditions, hRPE cell microcapsules were prepared, and the function of hRPE cells of some cytokines or cell protective substances in order to cause the damage of the microcapsule was improved by adding microencapsulated process.
We use self-made microcapsule electrostatic generator for preparation of hRPE cell alginate chitosan alginate (Alginate-Chitosan-Alginate, ACA) micro capsule, preparation process, influence factors and the stability of the microcapsule quality including voltage, speed, injection pump needle electrode distance, diameter, concentration of sodium alginate solution, the concentration of chitosan solution, membrane reaction time and other physical and chemical factors, we performed single factor experiment by changing other parameters, the influence of various factors on the morphology of microcapsules. Microcapsules were observed by inverted optical microscope, counting plate measurement of microcapsule particle size, and the stability of the expansive degree indirectly characterize microcapsules. The experimental parameters were finally determined conditions preparation of microcapsules for cell electrode distance is 30mm, the needle diameter is 0.292mm, voltage is 7kv, promote the speed of 20mL h~ (-1) and sodium alginate. The concentration of chitosan solution is 15g. L~ (-1), and the time of film forming reaction is 15min.
Because in the process of microencapsulation on cell growth, inevitably will cause some harm, and the change of cell environment after microencapsulation can affect cell growth. In order to improve the growth state of cells, we prepared the basic cell microcapsules on the preliminary observation of epithelial cells in the culture medium for adding fructose, into fibroblast growth factor on human retinal pigment epithelium (hRPE) cells after taurine alginate chitosan alginate (ACA) the total number of cells after microencapsulation, the survival rate and the number of live cells the effects of.HRPE cells after microencapsulation will be divided into four groups, one group as control group with epithelial cell culture medium in addition, the three groups in the epithelial cells of fructose were added in liquid, fibroblast growth factor, 7d culture after taurine in 0d, 1D, 3D, 7d determination of total number of cells within the cell micro capsule, and the survival rate of living cells The cell count board was counted and the cell survival rate was detected by trypan blue staining.
The prepared hRPE cells ACA microcapsule diameter of hRPE cells to culture 7d 450 m + 13 M. four cells after microencapsulation, the cell survival rate was lowest (75 + 16.8)%, but there is no significant difference between the groups (P0.05); and the control group, taurine group and fructose group. Fibroblast growth factor group of micro capsule total cells respectively (6.1 + 0.6) * 10~4 (6 + 0.5) * 10~4 (7.2 + 0.4) * 10~4 and (8 + 0.5) * 10~4, taurine group, fibroblast growth factor group than in control group significantly increased (P0.05). Each group was statistically significant; the number of live cells for cell number x cell survival rate was statistically significant. The cell growth factor added to the culture medium, taurine can promote the alginate chitosan alginate sodium alginate within 7d (ACA) proliferation in microcapsules of hRPE cells.

【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R774.1

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