CAJ | 학술논문

目的：旨在获得高纯度的星形胶质细胞,并对不同培养时期的细胞进行鉴定,为进一步研究奠定基础。方法常规分离新生 SD 大鼠大脑皮质并制备单细胞悬液,采用差速黏附加摇床震荡法对所获得的细胞进行纯化。倒置相差显微镜及 HE 染色观察细胞形态,GFAP 免疫荧光对获得细胞进行鉴定。结果原代培养的皮质细胞从3 d 开始迅速增殖,9~12 d 即可铺满瓶底,此时细胞呈明显的分层生长,星形胶质细胞位于底层,形态多样,阳性率为(67.2±7.1)%；经过1次传代后,GFAP 阳性率有所提升(84.0±6.0)%,但不能完全去除小胶质细胞及少突胶质细胞,细胞仍呈分层生长；至第3次传代后,获得大量几乎为单一种类的星形胶质细胞,其胞体较大、突起较短粗,一般2~3个,胞核圆形或椭圆形,常偏于一侧,GFAP 染色呈强阳性,阳性率可达(97.6±2.4)%。结论通过差速黏附与摇床震荡相结合的方法,获得了高纯度且生长状态良好的星形胶质细胞。
목적：지재획득고순도적성형효질세포,병대불동배양시기적세포진행감정,위진일보연구전정기출。방법상규분리신생 SD 대서대뇌피질병제비단세포현액,채용차속점부가요상진탕법대소획득적세포진행순화。도치상차현미경급 HE 염색관찰세포형태,GFAP 면역형광대획득세포진행감정。결과원대배양적피질세포종3 d 개시신속증식,9~12 d 즉가포만병저,차시세포정명현적분층생장,성형효질세포위우저층,형태다양,양성솔위(67.2±7.1)%；경과1차전대후,GFAP 양성솔유소제승(84.0±6.0)%,단불능완전거제소효질세포급소돌효질세포,세포잉정분층생장；지제3차전대후,획득대량궤호위단일충류적성형효질세포,기포체교대、돌기교단조,일반2~3개,포핵원형혹타원형,상편우일측,GFAP 염색정강양성,양성솔가체(97.6±2.4)%。결론통과차속점부여요상진탕상결합적방법,획득료고순도차생장상태량호적성형효질세포。
Objective To obtain highly purified astrocytes and identify the cells in each stage to support further studies.Methods The cerebral cortex of a neonatal SD rat was isolated and prepared into single cell suspension.The obtained cells were purified by differential adherence and shook at a constant temperature.By inverted phase contrast microscopy and HE staining,cell morphology was observed.The immunofluorescence staining with anti-mouse GFAP was used to identify the cells.Results The primary cortical cells developed rapidly at 3 d after culture and covered the flasks at 9-12 d.At this time,the cells showed stratification and the astrocytes lay at the lower layer.GFAP positive rate was only about (67.2 ±7.1)%.After the first passage,GFAP positive rate increased obviously (84.0±6.0)%. However, oligodendrocytes and microglias could not be removed completely,and the cells also showed stratification.Through 3 times of passages,we obtained many single species of astrocytes showing satellite shape with 2 or 3 processes,big cell body and round or oval-shaped nuclei leaned to one side.Immunofluorescence staining showed that nearly all of the cells were strong positive and the positive rate reached as high as (97.6 ± 2.4 )%.Conclusion Through differential adherence and shaking at a constant temperature,more astrocytes of high purity and in good state can be obtained.