本文選題：色素上皮　切入點(diǎn)：眼　出處：《河北北方學(xué)院》2011年碩士論文

【摘要】：) 細(xì)胞微囊化是近年發(fā)展起來的一種有效避免免疫排斥反應(yīng)從而提高移植細(xì)胞的存活率的方法，人視網(wǎng)膜色素上皮(Human RetinalPigment Epithelial Cell, hRPE)細(xì)胞具有酪氨酸羥化酶活性且能分泌多巴胺，有可能在帕金森病的細(xì)胞移植治療中作為供體細(xì)胞發(fā)揮巨大潛能。本實(shí)驗(yàn)探索了微膠囊制備的條件，制備了hRPE細(xì)胞微膠囊，而且在微囊化過程后加入了一些細(xì)胞因子或細(xì)胞保護(hù)性物質(zhì)以期對微囊化所造成的hRPE細(xì)胞功能的損傷進(jìn)行改善。 我們利用自制的靜電微囊發(fā)生器制備hRPE細(xì)胞海藻酸鈉-殼聚糖-海藻酸鈉(Alginate-Chitosan-Alginate, ACA)微膠囊，制備過程中影響所成微囊質(zhì)量及穩(wěn)定性的因素包括電壓、注射泵推進(jìn)速度、電極距離、針頭內(nèi)徑、海藻酸鈉溶液濃度、殼聚糖溶液濃度、成膜反應(yīng)時(shí)間等物理因素和化學(xué)因素，我們通過改變其他參數(shù)進(jìn)行單因素試驗(yàn)研究，考察各影響因素對微囊的作用。倒置光學(xué)顯微鏡觀察微囊形態(tài)，用計(jì)數(shù)板測量微膠囊粒徑大小，并用膨脹度間接表征微膠囊的穩(wěn)定性。本次實(shí)驗(yàn)最終確定制備細(xì)胞微膠囊的條件參數(shù)為電極距離為30mm，針頭內(nèi)徑為0.292mm，，電場電壓為7kv，推進(jìn)速度為20mL·h~(-1)，海藻酸鈉及殼聚糖溶液濃度均為15g·L~(-1)，成膜反應(yīng)時(shí)間為15min。 由于在微囊化的過程中，不可避免對細(xì)胞生長會造成一定的傷害，而微囊化后細(xì)胞環(huán)境的變化也會對細(xì)胞的生長產(chǎn)生影響。為了改善細(xì)胞的生長狀態(tài)，我們在制備了細(xì)胞微膠囊的基礎(chǔ)上初步觀察上皮細(xì)胞培養(yǎng)液中添加果糖、成纖維細(xì)胞生長因子、牛磺酸后對人視網(wǎng)膜色素上皮（hRPE）細(xì)胞海藻酸鈉-殼聚糖-海藻酸鈉（ACA）微囊化后的細(xì)胞總數(shù)、存活率及活細(xì)胞數(shù)的影響。hRPE細(xì)胞微囊化后將其分為四組，一組作為空白對照組只加入上皮細(xì)胞培養(yǎng)液、另外三組在上皮細(xì)胞培養(yǎng)液中分別加入果糖、成纖維細(xì)胞生長因子、牛磺酸后培養(yǎng)7d，在第0d、1d、3d、7d測定微膠囊內(nèi)細(xì)胞的細(xì)胞總數(shù)、存活率及活細(xì)胞數(shù)。細(xì)胞計(jì)數(shù)板進(jìn)行常規(guī)計(jì)數(shù),臺盼藍(lán)染色法檢測囊內(nèi)細(xì)胞存活率。 制備出的hRPE細(xì)胞的ACA微膠囊直徑為450μm±13μm。四組細(xì)胞微囊化后的hRPE細(xì)胞至培養(yǎng)7d,細(xì)胞存活率最低為(75±16.8)%,但各組間沒有明顯差異(P0.05);而空白組、果糖組、�；撬峤M及成纖維細(xì)胞生長因子組微膠囊內(nèi)細(xì)胞總數(shù)分別為(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,牛磺酸組、成纖維細(xì)胞生長因子組比空白對照組明顯增多(P0.05),有統(tǒng)計(jì)學(xué)意義;各組活細(xì)胞數(shù)為細(xì)胞總數(shù)×細(xì)胞存活率,有統(tǒng)計(jì)學(xué)意義。說明培養(yǎng)液中加入細(xì)胞生長因子、牛磺酸后在7d之內(nèi)可促進(jìn)海藻酸鈉-殼聚糖-海藻藻酸鈉(ACA)微膠囊內(nèi)的hRPE細(xì)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.

【學(xué)位授予單位】：河北北方學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R774.1

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