红细胞生成素（EPO）与甲泼尼龙（MPSS）联合应用对损伤星形胶质细胞影响的研究

发布时间：2018-05-11 02:44

本文选题：星形胶质细胞 + 细胞培养技术　；参考：《苏州大学》2011年硕士论文

【摘要】：第一部分大鼠星形胶质细胞的培养及鉴定目的采用新生大鼠大脑皮质制作原代星形胶质细胞,传代培养,并进行星形胶质细胞鉴定和细胞纯度测定方法使用出生后3天内的SD乳鼠(图1)大脑皮质制成细胞悬液后,按10~7~10~9 /L个细胞接种于已用0.01%多聚左旋赖氨酸包被好的底面积为75 cm~2培养瓶中,放置于培养箱(图2)中(5% CO_2,37℃)培养,采用含15%的胎牛血清的DMEM/F12培养基培养7-10 d,通过差速贴壁和不同速度的摇床(图3)及逐渐传代纯化星形胶质细胞,观察细胞形态的变化,通过GFAP免疫荧光染色鉴定星形胶质细胞,通过对比GFAP阳性细胞与DAPI染色细胞核测定细胞纯度。结果使用差速贴壁法与摇床技术相结合可获得高纯度星形胶质细胞,并传代纯化,细胞的形态逐渐发生变化,培养的细胞GFAP染色呈阳性,显示正常星形胶质细胞胞体多呈三角形,胞膜光滑,边界清晰,胞突较长(图19、20),荧光染色显示GFAP阳性细胞占总细胞数比例在95%以上(图11、12)。结论通过新生乳鼠大脑皮质制作原代星形胶质细胞,使用差速贴壁法与摇床技术培养后可获得高纯度的星形胶质细胞。第二部分大鼠星形胶质细胞缺营养损伤模型制作目的制作理想的大鼠星形胶质细胞的损伤模型,观察形态变化,检测细胞损伤前后统计学差异性方法纯化后的星形胶质细胞,采用PBS缓冲液代替培养基模拟脊髓缺血损伤模型(缺营养),缺营养3小时后更换正常培养基恢复营养。⑴、倒置显微镜(图4)观察细胞缺营养前后形态变化;⑵、MTT(图5)检测星形胶质细胞增殖活性;⑶、PCR(图6)检测星形胶质细胞AQP4 mRNA表达。结果⑴、缺营养3小时后部分细胞胞体变小,胞突变短,个别细胞脱落,呈圆形。恢复营养后细胞胞体变大,胞突变长并相互交错,基本恢复缺营前形态,仍然可见少数死亡细胞漂浮在贴壁细胞层面上;⑵、MTT示:损伤后星形胶质细胞增殖活性降低,恢复培养基后细胞增殖活性升高,差异有统计学意义;⑶、PCR检测示:星形胶质细胞损伤后AQP4表达降低,恢复培养基后AQP4表达升高,差异有显著统计学意义。结论通过上述方法模拟脊髓缺血损伤,原代星形胶质细胞缺营养后可耐受一定的损伤,恢复营养后可基本恢复至损伤前的形态,MTT、PCR检测星形胶质细胞损伤前后差异有显著统计学意义。第三部分EPO对星形胶质细胞的AQP4 mRNA表达的作用目的研究EPO (Erythropoietin)对体外培养原代星形胶质细胞缺营养3 h后aquaporin4 (AQP4) mRNA表达的作用。方法采用出生三天内的SD大鼠大脑皮层制成单细胞悬液后,应用含15%胎牛血清的DMEM/F12培养,通过差速贴壁、摇床和传代纯化星形胶质细胞,GFAP免疫荧光鉴定细胞,通过PBS缓冲液代替培养基模拟缺营养模型3小时后即刻应用促EPO(10 u/l)然后按照加入药物0.5 h、1 h、1.5 h、2 h、3 h、6 h、12 h、18 h收集细胞。PCR技术测定细胞AQP4 mRNA表达的变化。结果星形胶质细胞缺营养三小时AQP4表达降低;EPO作用0.5 h后细胞AQP4 mRNA表达较正常生理盐水对照组明显升高(图14、15)。结论EPO作用0.5 h后对损伤的星形胶质细胞AQP4 mRNA表达有显著作用。第四部分EPO与MPSS联合应用对体外培养星形胶质细胞AQP4 mRNA表达的研究目的EPO与MPSS(Methylprednisolone)联合应用对体外培养星形胶质细胞的作用,并探讨其对脊髓缺血水肿损伤的作用机制。方法体外培养的星形胶质细胞传至第三代,免疫荧光显微镜鉴定细胞种类,在PBS液中作用3 h后,建立星形胶质细胞缺营养损伤的实验模型;给予DMEM/F12培养基加15%胎牛血清恢复营养,然后按生理盐水对照组、MPSS (10 ug/ml)组、EPO(10 u/ml)组、MPSS (10 ug/ml)和EPO (10 ug/ml)组、EPO (5 ug/ml)和MPSS (5 ug/ml)组的剂量干预,干预后分别于0.5 h、1 h、1.5 h、2 h收集细胞;观察:⑴、光镜观察不同干预组合对星形胶质细胞形态影响;⑵、应用四甲基偶氮唑蓝(MTT)比色法测定细胞活性;⑶、使用RT-PCR检测星形胶质细胞AQP4 mRNA表达水平的变化。结果⑴、缺营养可抑制星形胶质细胞增殖,恢复培养基后细胞形态均有恢复;⑵、单独应用EPO对细胞增殖恢复无统计学意义,单独MPSS对损伤细胞增殖恢复有统计学意义;⑶、EPO和MPSS联合应用对细胞增殖恢复有明显统计学意义;⑷、生理盐水对照组对细胞增殖无明显作用;⑸、半量EPO和半量MPSS联合应用对细胞增殖恢复有统计学意义;⑹、单独EPO和单独MPSS均促进损伤细胞AQP4 mRNA表达增加;⑺、联合应用EPO和MPSS对细胞AQP4 mRNA表达有明显影响作用;⑻、半量EPO和半量MPSS联合应用对细胞AQP4 mRNA表达亦有明显影响作用; 结论半量EPO+半量MPSS联合应用的对星形胶质细胞细胞增殖、AQP4 mRNA表达增加作用优于单独大剂量应用MPSS或EPO。
[Abstract]:Part one: culture and identification of rat astrocytes
Objective to make primary astrocytes from neonatal rat cerebral cortex and to carry out the culture and identification of astrocytes and cell purity.
Methods a cell suspension was made from the cerebral cortex of SD rats (Figure 1) 3 days after birth and was inoculated in 10~7~10~9 /L cells to 75 cm~2 culture bottles with 0.01% poly (L-lysine) with a good bottom area, and placed in the incubator (5% CO_2,37 C) (5% CO_2,37 C) and cultured with a DMEM/F12 medium containing 15% fetal bovine serum, and 7-10 D, Astrocytes were observed by differential adhesion and different speed (Figure 3), and the morphological changes of the cells were observed. Astrocytes were identified by GFAP immunofluorescence staining, and cell purity was measured by comparing GFAP positive cells with DAPI stained nuclei.
Results the high purity astrocytes could be obtained by the combination of differential adherence and rocking bed technology. The cells were purified and the morphology of the cells changed gradually. The GFAP staining of the cultured cells was positive. It showed that the normal astrocytes were mostly triangular, smooth, clear and long (Figure 19,20), and the fluorescent staining showed GFAP Yang. The ratio of sex cells to total cells was more than 95% (Figure 11,12).
Conclusion the primary astrocytes are produced by the cerebral cortex of newborn rats. The high purity astrocytes can be obtained by using the differential adherence method and the technique of rocking bed.
The second part is the establishment of rat model of astrocyte lack of nutrition injury.
Objective to make an ideal rat astrocyte injury model, observe morphological changes, and detect the statistical difference before and after cell injury.
Methods the purified astrocytes were replaced by PBS buffer solution to simulate the model of spinal cord ischemia injury (nutrition deficiency). After 3 hours of nutrient deficiency, the normal medium was replaced to restore nutrition. (1) inverted microscope (Figure 4) observed the morphological changes of the cells before and after nutrition; (2) MTT (Figure 5) detected the proliferation activity of astrocytes; (3) PCR (Figure 6) detection The expression of AQP4 mRNA in astrocytes.
Results (1), after 3 hours of lack of nutrition, the cell body became smaller, the mutation was short, and some cells fell off, and the cell body became round. After the restoration of nutrition, the cell body became larger, the mutation was long and interlaced with each other, and the morphologic changes were basically restored, and a few dead cells were still floating on the surface of the adherent cells. (2) MTT shows the proliferation activity of astrocytes after injury. After the recovery of the medium, the cell proliferation activity increased, and the difference was statistically significant. (3) PCR detection showed that the expression of AQP4 decreased after the astrocytes injury, and the expression of AQP4 increased after the recovery of the medium. The difference was significant statistically significant.
Conclusion this method is used to simulate the injury of spinal cord ischemia. The primary astrocytes can tolerate a certain amount of damage after nutrition. After the recovery of nutrition, it can basically recover to the morphology before injury. MTT and PCR have significant statistical significance before and after the injury of astrocytes.
The third part is the effect of EPO on the expression of AQP4 mRNA in astrocytes.
Objective to study the effect of EPO (Erythropoietin) on the expression of aquaporin4 (AQP4) mRNA after primary culture of 3 h astrocytes in vitro.
Methods after a single cell suspension was made from the cerebral cortex of SD rats born for three days, the DMEM/F12 culture containing 15% fetal bovine serum was used, the astrocytes were purified by differential adherence, rocking bed and passages were purified, and GFAP immunofluorescence was used to identify the cells, and EPO (10 u/l) was immediately used to promote EPO (10 u/l) after 3 hours after the culture medium was replaced by the PBS buffer solution. Then the cell.PCR assay was used to measure the changes of AQP4 expression in the cells according to the drug.PCR 0.5, h, 1 h, 1.5 h, 2 h, 3 h, 6 h, 12 h, 18 h.
Results the expression of AQP4 decreased after three hours of astrocyte deficiency, and the expression of AQP4 mRNA increased significantly after EPO treatment for 0.5 h compared with normal saline control group (14,15).
Conclusion EPO after 0.5 h has a significant effect on the expression of AQP4 mRNA in injured astrocytes.
The fourth part is to study the expression of AQP4 mRNA in astrocytes cultured in combination with EPO and MPSS.
Objective to investigate the effect of EPO combined with MPSS (Methylprednisolone) on astrocytes in vitro and to explore the mechanism of its effects on spinal cord ischemia and edema.
Methods astrocytes cultured in vitro were passed to third generations, and the species of cells were identified by immunofluorescence microscopy. After 3 h in PBS liquid, an experimental model of astrocyte deficiency was established, and DMEM/F12 culture was used to restore nutrition with 15% fetal bovine serum, and then the normal saline control group, MPSS (10 ug/ml) group, EPO (10 u/ml) group, MPSS. (10 ug/ml) and EPO (10 ug/ml) group, EPO (5 ug/ml) and MPSS (5 ug/ml) group dose intervention, the dry prognosis was 0.5 h, 1 h, 1.5 h, 2 h collection of cells; observation: (1), light microscopy observation of the effects of different intervention combinations on astrocyte morphology; (2) use four methyl azazolium (MTT) colorimetry to determine cell activity; 3. Changes in the expression level of AQP4 mRNA in glial cells.
Results (1) the lack of nutrition could inhibit the proliferation of astrocytes and restore the cell morphology after the recovery of the medium. (2) the use of EPO alone has no statistical significance to the proliferation of cell proliferation, and MPSS alone has statistical significance for the recovery of cell proliferation. (3) the combined application of EPO and MPSS has significant statistical significance for the recovery of cell proliferation; 4. The combined application of half amount of EPO and half quantity of MPSS had statistical significance on cell proliferation recovery, and both EPO and single MPSS alone promoted the increase in the expression of AQP4 mRNA in damaged cells, and the combined application of EPO and MPSS had a clear effect on the expression of AQP4 mRNA in cells; The expression of AQP4 mRNA also had a significant effect on the expression of the cells.
Conclusion the combination of half dose EPO+ and half dose MPSS can increase the proliferation of astrocytes, and the increase of AQP4 mRNA expression is better than that of MPSS or EPO. alone.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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