甲基强的松龙与促红细胞生成素联合应用对原代星形胶质细胞作用的研究

发布时间：2018-04-06 22:36

本文选题：星形胶质细胞　切入点：原代培养　出处：《苏州大学》2009年硕士论文

【摘要】： 第一部分原代星形胶质细胞模型制作及鉴定目的通过新生鼠大脑制作原代星形胶质细胞,并进行鉴定和星形胶质细胞纯度测定方法采用出生三天内的SD大鼠大脑制成细胞悬浊液后,按1.5×107计数接种于已用0.01%多聚赖氨酸包被好的底面积为75cm2培养瓶中,放置于培养箱中(5%CO2,95%O2,37OC)培养,采用含15%的胎牛血清的DMEM培养液培养7d,通过摇床和逐渐传代纯化星形胶质细胞,观察细胞的变化,通过免疫荧光染色鉴定星形胶质细胞。结果通过摇床与高血清培养可获得高纯度星形胶质细胞,并随逐渐传代更加纯化,形态也逐渐变化,培养的细胞GFAP染色呈阳性,显示正常星形胶质细胞胞体呈多角形,胞膜光滑,边界清楚,突触长且多相互连接,GFAP阳性细胞占总细胞数比例在95%以上。结论通过新生鼠大脑制作原代星形胶质细胞,高血清培养基和摇床后培养可获得高纯度的星形胶质细胞。第二部分原代星形胶质细胞缺营养损伤模型制作目的制作理想的原代星形胶质细胞的损伤模型,观察形态变化方法采用出生三天内的SD大鼠大脑制成细胞悬浊液后,采用含15%的胎牛血清的DMEM培养液培养,通过摇床和逐渐传代纯化星形胶质细胞。采用PBS缓冲液代替培养基模拟缺营养模型,缺营养3h后恢复营养,观察细胞缺营养前后形态变化。结果缺营养3h后部分细胞胞体变小,突触变短,细胞间相互连接减少或消失,个别细胞呈圆形,复营养后大多数细胞胞体变大,突出变长并相互交错,基本恢复缺营前形态,个别细胞死亡成圆形。结论高血清培养和摇床后可获得高纯度的星形胶质细胞,原代星形胶质细胞可耐受一定的缺营养损伤,恢复营养后可基本恢复至损伤前的形态第三部分甲基强的松龙与促红细胞生成素联合应用对原代星形胶质细胞作用的研究目的研究甲基强的松龙(MPSS)与促红细胞生成素(EPO)单独以及联合应用对体外培养的原代星形胶质细胞的作用。方法采用出生3d内的SD大鼠大脑制成细胞悬浊液,采用含15%的胎牛血清的DMEM培养液培养,通过摇床和逐渐传代纯化星形胶质细胞,按1.5×107计数接种于已用0.01%多聚赖氨酸包被好的底面积为75cm2培养瓶中,放置于培养箱中(5%CO2,95%O2,37OC)培养,通过摇床和逐渐传代纯化星形胶质细胞,实验分为正常组、正常+ MPSS组、正常+EPO组、正常+ MPSS+EPO组、损伤组、损伤+ MPSS组、损伤+EPO组、损伤+ MPSS+EPO组。通过PBS缓冲液代替培养基模拟缺营养模型3小时后即刻分别加入甲基强的松龙(10umol/l)、促红细胞生成素(10u/l)、甲基强的松龙(10umol/l)与促红细胞生成素(10u/l),然后分别继续培养三天,免疫荧光技术鉴定星形胶质细胞;MTT法检测细胞的增殖活性和凋亡; PCR技术测定细胞GFAP RNA表达的变化。结果星形胶质细胞摇床后传至第三代,经鉴定星形胶质细胞纯度达95%以上;缺营养3h后部分细胞胞体变小,突触变短,细胞间相互连接减少或消失,个别细胞呈圆形,复营养后大多数细胞胞体变大,突出变长并相互交错,基本恢复缺营前形态,个别细胞死亡成圆形;正常组和损伤组分别加入MPSS、EPO、MPSS和EPO三天后,损伤组细胞活性与GFAP表达均明显升高,并且联合应用MPSS与EPO与单独应用MPSS、EPO相比,细胞活性与GFAP表达明显升高,但是正常组没有明显变化。结论适当剂量的MPSS与EPO联合应用对缺营养损伤的星形胶质细胞有显著的保护作用,并且与单独应用MPSS、EPO相比有显著性作用,而MPSS、EPO单独应用,MPSS与EPO联合应用对正常的星形胶质细胞均无显著性保护作用。
[Abstract]:The first part of the primary astrocyte model and identification
Objective to make the primary astrocytes from the brain of newborn rats and to identify and determine the purity of astrocytes.
Using the method of birth within three days of the SD rat brain into cell suspension, according to a 1.5 x 107 count with 0.01% seeded on polylysine coated bottom area good for the development of 75cm2 bottle, placed in the incubator (5%CO2,95%O2,37OC) by culture, containing 15% fetal bovine serum DMEM medium 7d, by shaking table and gradually purified astrocytes, observe the changes of the cells and identification of astrocytes by immunofluorescence staining.
Results by shaking table and high serum medium can obtain high purity astrocytes, and gradually with the passage more purified form also changes gradually, the cultured cells with GFAP positive staining, normal astrocytes cells were polygonal, membrane smooth, clear boundary, synaptic long and mutually connected, GFAP positive cells the total cell number ratio above 95%.
Conclusion primary astrocytes are produced by the brain of newborn rats, and high purity astrocytes can be obtained by high serum medium and rocking bed.
The second part of the primary astrocyte deficiency injury model
Objective to make an ideal damage model of primary astrocytes and observe the morphological changes
Using the method of birth within three days of the SD rat brain into cell suspension, using fetal bovine serum containing 15% DMEM medium, by shaking and gradually purified astrocytes. Using PBS buffer instead of the medium simulation model the lack of nutrition, lack of nutrition 3H recovery after nutrition, morphological changes before and after the lack of nutrition cells were observed.
Results some cells lack of nutrition after 3H were smaller, shorter synaptic connections among cells, reduce or disappear, the individual cells were round, larger cell body nutrition after the most complex, long and prominent interlaced, recovered before the lack of form, individual cell death round. Conclusion high serum culture and shaking to obtain high purity astrocytes, primary astrocytes can tolerate a certain lack of nutritional damage, the restoration of nutrition can be restored to normal form
The study of the effect of the third part of methylprednisolone and erythropoietin on primary astrocytes
Objective to study the role of methylprednisolone (MPSS) and erythropoietin (EPO) alone and in combination with the primary astrocytes cultured in vitro.
Methods 3D was born SD in the rat brain cell suspension was made, containing 15% fetal bovine serum DMEM culture medium, by shaking table and gradually purified astrocytes, according to a 1.5 x 107 count with 0.01% seeded on polylysine coated bottom area good for 75cm2 culture bottle placed in the incubator (5%CO2,95%O2,37OC) cultured by shaking table and gradually purified astrocytes were divided into normal group, normal + MPSS group, +EPO normal group, normal + MPSS+EPO group, injury group, injury injury + MPSS group, +EPO group, MPSS+EPO group. The injury + PBS buffer instead of the medium the simulation model of 3 hours immediately after the lack of nutrition were added methylprednisolone (10umol/l), erythropoietin (10u/l), methylprednisolone (10umol/l) and erythropoietin (10u/l), and then to continue training for three days, immunofluorescence identification of star Glial cells; MTT assay was used to detect cell proliferation and apoptosis, and PCR technique was used to determine the changes in the expression of GFAP RNA.
The results of astrocytes after shaking to the third generation, after identification of astrocytes reached a purity of more than 95%; the lack of nutrition 3h after some cells become smaller, shorter synaptic connections among cells, reduce or disappear, the individual cells were round, larger cell body nutrition after the most complex, long and prominent interlaced the lack of basic recovery, Yingqian morphology, individual cell death round; the normal group and the injury group were added to MPSS, EPO, MPSS and EPO after three days, the expression was significantly increased in injury group and GFAP cell activity, and the combined application of MPSS and EPO with MPSS alone, compared to EPO, the expression of cell activity and GFAP increased significantly, but the normal group did not change significantly.
Conclusion the appropriate dose of MPSS combined with EPO has a significant protective effect on damaged astrocytes, and MPSS and EPO used alone, have significant effect, compared with MPSS, EPO alone, the combination of MPSS and EPO had no significant protective effect on normal astrocytes.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R651.2;R329

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