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
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摘要
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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(P>0.05);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(P<0.05),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。
Cell microencapsulation is a kind of effective method which avoidsimmune rejection so as to improve the survival rate of transplanted cells.Human Retinal Pigment Epithelial Cell (hRPE) has a huge potential in celltransplantation about Parkinson disease because of its Tyrosine hydroxyl ofenzyme activity and the dopamine. This research explores the optimizatingcondition for the microcapsule preparation and tries to prepare themicrocapsule with hRPE cells. We expect that the microcapsuled hRPE cellinjury during the preparation can be reduced by some cytokines and cellprotective substances.
Prepared Alginate-Chitosan-Alginate (ACA) microcapsule with hRPEcells by using our self-made high voltage electrostatic system, we canobserve the effects of every factor to microcapsule based on the otherfactors unchanged. These factors include physical and chemical factorssuch as woltage, injection rate, electrode distance, needle inner diameterand sodium alginate and chitosan solution concentration, film reaction timeand so on. Microcapsule morphology was observed by inverted opticalmicroscope, its diameter done by cell counter and its stability testifiedindirectly. The best factors are electrode distance with 30mm, needle innerdiameter with 0.292mm, electric voltage with 7kv, injection rate with20mL·h~(-1)，sodium alginate and chitosan solution concentration with 15g·Land film reaction time with 15minutes.
The cell growth could be harmed inevitably by microencapsulationand changing environments. In order to improve the cell’s growth condition,we observed initially the effects of fructose , fibroblast growth supplement (FGS) and taurine on the total number, the survival rate and the survivalnumber of Human Retinal Pigment Epithelial（hRPE）Cell which areencapulsed on the basis of preparing the cell microcapsule.
Microcapsule with hRPE cells were inoculated into four kinds ofculture mediums respectively which contained nothing, fructose, FGS andtaurine. Then observe the total number, the survival rate and the survivalnumber of the microencapsulated hRPE cell on the 0, 1, 3, 7 days. The cellnumber is counted by cell counter. The survival rate of the cells was testtedby trypan blue staining.
The diameter of ACA microcapsule of hRPE is 450μ±13μm diametercan be gained. After 7days, four groups of hRPE cells microencapsulatedsurivial rate is (75±16.8)% at least. But they have no statistically significantdifferences(P>0.05); the total number of blank group, fructose group,taurine group and fibroblast growth supplement group is（6.1±0.6)×10~4、(6.0±0.5)×10~4、(7.2±0.4）×10~4 and (8.0±0.5）×10~4 , taurine group andfibroblast growth supplement group has significant increased (P<0.05).Four groups’survival number of the cells has significant difference just astheir total number (P<0.05). The total number of cells and the survivalnumber of microencapsulated hRPE cells microencapsulated into culturemediums which contained FGS and taurine can be increased within 7 days.
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(P>0.05);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(P<0.05),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。
Cell microencapsulation is a kind of effective method which avoidsimmune rejection so as to improve the survival rate of transplanted cells.Human Retinal Pigment Epithelial Cell (hRPE) has a huge potential in celltransplantation about Parkinson disease because of its Tyrosine hydroxyl ofenzyme activity and the dopamine. This research explores the optimizatingcondition for the microcapsule preparation and tries to prepare themicrocapsule with hRPE cells. We expect that the microcapsuled hRPE cellinjury during the preparation can be reduced by some cytokines and cellprotective substances.
Prepared Alginate-Chitosan-Alginate (ACA) microcapsule with hRPEcells by using our self-made high voltage electrostatic system, we canobserve the effects of every factor to microcapsule based on the otherfactors unchanged. These factors include physical and chemical factorssuch as woltage, injection rate, electrode distance, needle inner diameterand sodium alginate and chitosan solution concentration, film reaction timeand so on. Microcapsule morphology was observed by inverted opticalmicroscope, its diameter done by cell counter and its stability testifiedindirectly. The best factors are electrode distance with 30mm, needle innerdiameter with 0.292mm, electric voltage with 7kv, injection rate with20mL·h~(-1)，sodium alginate and chitosan solution concentration with 15g·Land film reaction time with 15minutes.
The cell growth could be harmed inevitably by microencapsulationand changing environments. In order to improve the cell’s growth condition,we observed initially the effects of fructose , fibroblast growth supplement (FGS) and taurine on the total number, the survival rate and the survivalnumber of Human Retinal Pigment Epithelial（hRPE）Cell which areencapulsed on the basis of preparing the cell microcapsule.
Microcapsule with hRPE cells were inoculated into four kinds ofculture mediums respectively which contained nothing, fructose, FGS andtaurine. Then observe the total number, the survival rate and the survivalnumber of the microencapsulated hRPE cell on the 0, 1, 3, 7 days. The cellnumber is counted by cell counter. The survival rate of the cells was testtedby trypan blue staining.
The diameter of ACA microcapsule of hRPE is 450μ±13μm diametercan be gained. After 7days, four groups of hRPE cells microencapsulatedsurivial rate is (75±16.8)% at least. But they have no statistically significantdifferences(P>0.05); the total number of blank group, fructose group,taurine group and fibroblast growth supplement group is（6.1±0.6)×10~4、(6.0±0.5)×10~4、(7.2±0.4）×10~4 and (8.0±0.5）×10~4 , taurine group andfibroblast growth supplement group has significant increased (P<0.05).Four groups’survival number of the cells has significant difference just astheir total number (P<0.05). The total number of cells and the survivalnumber of microencapsulated hRPE cells microencapsulated into culturemediums which contained FGS and taurine can be increased within 7 days.
引文
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47. Lim F, Sun AM. Microencapsulated is lets as bioartificial endocrinepancreas [J]. Science. 1980, 210(4472): 908-910.
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