發(fā)布時(shí)間：2018-04-03 16:27

  本文選題：視網(wǎng)膜神經(jīng)節(jié)細(xì)胞　切入點(diǎn)：殼聚糖　出處：《華中科技大學(xué)》2012年碩士論文

【摘要】：目的：研究殼聚糖對(duì)體外分層及純化培養(yǎng)的鼠視網(wǎng)膜神經(jīng)節(jié)細(xì)胞生長(zhǎng)的促進(jìn)作用；制備載基因殼聚糖納米粒，研究其特性及其對(duì)質(zhì)粒gp130-pDNA3.1的保護(hù)能力。 方法：取SD乳鼠視網(wǎng)膜，在解剖顯微鏡下將視網(wǎng)膜分成內(nèi)、外兩層，分別將內(nèi)外層及全層視網(wǎng)膜制成細(xì)胞懸液，各自進(jìn)行培養(yǎng)。實(shí)驗(yàn)組加入含10%殼聚糖的完全培養(yǎng)基，對(duì)照組不加殼聚糖。每天觀察細(xì)胞生長(zhǎng)狀態(tài)，MTT法檢測(cè)細(xì)胞存活率。各層細(xì)胞分別用Thy1.1抗體鑒定神經(jīng)節(jié)細(xì)胞，計(jì)算各層細(xì)胞的節(jié)細(xì)胞率。兩步法提純視網(wǎng)膜神經(jīng)節(jié)細(xì)胞。計(jì)算提純率。提純后分別加入含10%血清、10%殼聚糖及鼠神經(jīng)生長(zhǎng)因子（NGF）的培養(yǎng)基，觀察細(xì)胞生長(zhǎng)狀態(tài)。 復(fù)凝集法制備殼聚糖納米粒，測(cè)定其粒徑大小及zata電位；計(jì)算包封率，凝膠阻滯實(shí)驗(yàn)觀察殼聚糖對(duì)質(zhì)粒gp130-pDNA3.1的包裹能力,DNAaseⅠ消化實(shí)驗(yàn)觀察殼聚糖對(duì)質(zhì)粒的保護(hù)作用。 結(jié)果：分層培養(yǎng)細(xì)胞的存活率：各層細(xì)胞第1、2d存活率最高，MTT示：OD外層1.189±0.158，OD內(nèi)層1.084±0.150，OD全層1.381±0.089；3d后隨著培養(yǎng)時(shí)間的延長(zhǎng)，，細(xì)胞存活率逐漸降低（P＜0.05）：外層OD3d0.835±0.120，OD4d0.805±0.116，OD5d0.782±0.107；內(nèi)層：OD3d0.388±0.095，OD4d0.371±0.084，OD5d0.347±0.052；全層：OD3d0.523±0.047，OD4d0.340±0.084，OD5d0.227±0.057；相同時(shí)間點(diǎn)實(shí)驗(yàn)組（殼聚糖組）比對(duì)照組（不含殼聚糖）的存活率高（P＜0.05）。經(jīng)免疫組化鑒定，內(nèi)層細(xì)胞節(jié)細(xì)胞率最高(19.248±0.999)%，其次為全層(4.986±0.635)%，外層節(jié)細(xì)胞率最低（0.748±0.177）%。兩步法提純神經(jīng)節(jié)細(xì)胞：提純率可達(dá)95%以上。純化的神經(jīng)節(jié)細(xì)胞培養(yǎng)48h后測(cè)量最大軸突長(zhǎng)度，殼聚糖組：62.5μm；NGF組：37.5μm；空白對(duì)照組：12.5μm。 復(fù)凝集法制備的載基因殼聚糖納米粒分布均勻，平均直徑約719nm，zata電位約17.2±0.8mV；包封率可達(dá)100%，瓊脂糖凝膠電泳顯示殼聚糖與gp130-pDNA3.1能有效結(jié)合；被殼聚糖包裹的gp130-pDNA3.1不會(huì)被DNAaseⅠ降解。結(jié)論：混合培養(yǎng)的各層細(xì)胞中，內(nèi)層細(xì)胞的節(jié)細(xì)胞率最高。在混合培養(yǎng)的各層細(xì)胞中加入殼聚糖，能促進(jìn)其生長(zhǎng)并提高存活率。加入殼聚糖或NGF后，能提高純化的神經(jīng)節(jié)細(xì)胞的存活率。 復(fù)凝集法制備的載基因殼聚糖納米粒分布均勻，帶正電荷，能夠有效包裹并保護(hù)質(zhì)粒gp130-pDNA3.1。
[Abstract]:Aim: to study the effects of chitosan on the growth of cultured rat retinal ganglion cells in vitro and to study the properties of chitosan nanoparticles and their protective effect on plasmid gp130-pDNA3.1.Methods: the retina of newborn SD rats was divided into inner and outer layers under anatomic microscope. The inner and outer layers of retina and the whole layer of retina were made into cell suspensions and cultured separately.In the experimental group, 10% chitosan was added to the complete medium, while in the control group, the chitosan was not added.Cell survival rate was determined by MTT assay.The ganglion cells were identified by Thy1.1 antibody and the ganglion cell rate was calculated.Retinal ganglion cells were purified by two-step method.Calculate the purification rate.After purification, the cell growth state was observed by adding 10% serum 10% chitosan and rat nerve growth factor (NGF) medium respectively.Chitosan nanoparticles were prepared by complex agglutination method, their particle size and zata potential were measured, the encapsulation efficiency was calculated, and the encapsulation ability of chitosan to plasmid gp130-pDNA3.1 was observed by gel block assay. The protective effect of chitosan on plasmid was observed.Results: the survival rate of the cells in stratified culture: the highest survival rate was observed on the 1st day of each layer. The outer layer of OD was 1.189 鹵0.158 and the inner layer of OD was 1.084 鹵0.150. The whole layer of OD was 1.381 鹵0.089 after 3 days, and with the extension of culture time, the survival rate of the cells in each layer was higher than that in the control group.緇嗚優(yōu)瀛樻椿鐜囬

本文編號(hào)：1705984