视神经节细胞的体外培养与纯化

进行大鼠视网膜神经节细胞体外培养及纯化的方法学研究。取出生72h内的SD大鼠乳鼠视网膜制成细胞悬液,放于已包被多聚鸟氨酸和层粘连蛋白(laminin,LN)的培养皿中,加20%EMDM血清培养液、于37℃、5%二氧化碳孵箱中培养,同时分别在混合培养的视网膜神经细胞中加入5’-溴-2’-脱氧脲苷(Brdu)和羊抗鼠IgG、小鼠抗大鼠Thy1.1抗体,于4小时,24小时,48小时,72小时观测细胞存活情况;并采用MTT法测定不同方法培养的视网膜细胞在不同时间的存活率,绘制细胞生长曲线。通过对不同方法培养的细胞观察可见,视网膜混合培养细胞和加入Brdu培养的细胞,于3～4小时开始贴壁,48小时开始伸出粗细长短不等的突起,72小时后突起增多,并伸长;加入羊抗鼠IgG、小鼠抗大鼠Thy1.1抗体的细胞,其呈多边形,大多数细胞质折光强,72小时后很少见到活细胞;混合培养细胞数量在48小时后增加约70%,加入Brdu后,混合培养的细胞数量在48小时无明显减少,纯化的视网膜神经细胞在48小时后明显减少。本实验通过用两种方法培养视网膜神经细胞发现,Brdu可抑制视网膜非神经细胞的增长,RGCs纯度为60-70%,采用羊抗鼠IGg和小鼠抗大鼠Thy1.1抗体可起到纯化RGCs的目的,RGC纯度≥90%。

To set up the means to cultivate and purify the retina ganglion ces of the big rat externally. Take out the retinae of the SD rat in 72 hours after birth and execute it cell suspension, then set it in the cultivated utensil covered with poly - 1 - ornithine and laminin （LN） . Add 20% EMDM cultivated liquid and cultivate it in an incubator filled with 5% carbon dioxide.At the sarnetime, add 5 - Bromo- 2＇ - Deoxyuridine in mixed cultivated cells and observe the vegetal circumstances of cells and purify the retina ganglion cells with the goat anti - mouse IgG and the mouse anti - rat Thyl. 1 antibody then observe the vegetal circumstances of cells respectively.Observe the vegetal circumstances of cells respectively at the 4th, 24th, 48th and 72th hour time. And use the MTr method to mensurate the livability of the mixed retina cells and the RGCs during different externally cultivating time, and protract the vegetal curve of the cells. We can find the cells cultured with two way, that retina nerve ceils and the cells cultured with Brdu began to stick to the cliff 3--4 hours after cultivated. After 48 hours the cells began to protrude protuberances which differ in thickness and length and they have manifolded and extended in 72 hours. Purified RGCs have already sticked to the cliff 3 hours after the cultivation. The cells appeared circular at this time with relatively dark in the middle part of the cell body and the apparent halation. In 48 hours the cells have protruded protuberances which differ in thickness and length and meanwhile the ceils appeared polygon. The majority d cytoplasm had strong refractivity. There are hardly any ceils alive after three days. The amount of retina nerve cells which were mixed cultivated increased about 70% after 48 hours; after added Brdu, they had no evident increase after 48 hours; purified retina ganglion cells decreased obviously after 48 hours. The experiment has proved that brdu is able to restrain the increase of retina nerve cells, and the purity of ganglion cells ranges from 60--70%. The goat anti- mouse IgG and the mouse anti - rat Thyl. 1 antibody are able to purify ganglion cells, and the purity of ganglion cells is no less than 90%.