本文選題：星形膠質(zhì)細(xì)胞 + 細(xì)胞培養(yǎng)技術(shù)　； 參考：《蘇州大學(xué)》2011年碩士論文

【摘要】：第一部分大鼠星形膠質(zhì)細(xì)胞的培養(yǎng)及鑒定 目的采用新生大鼠大腦皮質(zhì)制作原代星形膠質(zhì)細(xì)胞,傳代培養(yǎng),并進(jìn)行星形膠質(zhì)細(xì)胞鑒定和細(xì)胞純度測定 方法使用出生后3天內(nèi)的SD乳鼠(圖1)大腦皮質(zhì)制成細(xì)胞懸液后,按10~7~10~9 /L個細(xì)胞接種于已用0.01%多聚左旋賴氨酸包被好的底面積為75 cm~2培養(yǎng)瓶中,放置于培養(yǎng)箱(圖2)中(5% CO_2,37℃)培養(yǎng),采用含15%的胎牛血清的DMEM/F12培養(yǎng)基培養(yǎng)7-10 d,通過差速貼壁和不同速度的搖床(圖3)及逐漸傳代純化星形膠質(zhì)細(xì)胞,觀察細(xì)胞形態(tài)的變化,通過GFAP免疫熒光染色鑒定星形膠質(zhì)細(xì)胞,通過對比GFAP陽性細(xì)胞與DAPI染色細(xì)胞核測定細(xì)胞純度。 結(jié)果使用差速貼壁法與搖床技術(shù)相結(jié)合可獲得高純度星形膠質(zhì)細(xì)胞,并傳代純化,細(xì)胞的形態(tài)逐漸發(fā)生變化,培養(yǎng)的細(xì)胞GFAP染色呈陽性,顯示正常星形膠質(zhì)細(xì)胞胞體多呈三角形,胞膜光滑,邊界清晰,胞突較長(圖19、20),熒光染色顯示GFAP陽性細(xì)胞占總細(xì)胞數(shù)比例在95%以上(圖11、12)。 結(jié)論通過新生乳鼠大腦皮質(zhì)制作原代星形膠質(zhì)細(xì)胞,使用差速貼壁法與搖床技術(shù)培養(yǎng)后可獲得高純度的星形膠質(zhì)細(xì)胞。 第二部分大鼠星形膠質(zhì)細(xì)胞缺營養(yǎng)損傷模型制作 目的制作理想的大鼠星形膠質(zhì)細(xì)胞的損傷模型,觀察形態(tài)變化,檢測細(xì)胞損傷前后統(tǒng)計學(xué)差異性 方法純化后的星形膠質(zhì)細(xì)胞,采用PBS緩沖液代替培養(yǎng)基模擬脊髓缺血損傷模型(缺營養(yǎng)),缺營養(yǎng)3小時后更換正常培養(yǎng)基恢復(fù)營養(yǎng)。⑴、倒置顯微鏡(圖4)觀察細(xì)胞缺營養(yǎng)前后形態(tài)變化;⑵、MTT(圖5)檢測星形膠質(zhì)細(xì)胞增殖活性;⑶、PCR(圖6)檢測星形膠質(zhì)細(xì)胞AQP4 mRNA表達(dá)。 結(jié)果⑴、缺營養(yǎng)3小時后部分細(xì)胞胞體變小,胞突變短,個別細(xì)胞脫落,呈圓形�；謴�(fù)營養(yǎng)后細(xì)胞胞體變大,胞突變長并相互交錯,基本恢復(fù)缺營前形態(tài),仍然可見少數(shù)死亡細(xì)胞漂浮在貼壁細(xì)胞層面上;⑵、MTT示:損傷后星形膠質(zhì)細(xì)胞增殖活性降低,恢復(fù)培養(yǎng)基后細(xì)胞增殖活性升高,差異有統(tǒng)計學(xué)意義;⑶、PCR檢測示:星形膠質(zhì)細(xì)胞損傷后AQP4表達(dá)降低,恢復(fù)培養(yǎng)基后AQP4表達(dá)升高,差異有顯著統(tǒng)計學(xué)意義。 結(jié)論通過上述方法模擬脊髓缺血損傷,原代星形膠質(zhì)細(xì)胞缺營養(yǎng)后可耐受一定的損傷,恢復(fù)營養(yǎng)后可基本恢復(fù)至損傷前的形態(tài),MTT、PCR檢測星形膠質(zhì)細(xì)胞損傷前后差異有顯著統(tǒng)計學(xué)意義。 第三部分EPO對星形膠質(zhì)細(xì)胞的AQP4 mRNA表達(dá)的作用 目的研究EPO (Erythropoietin)對體外培養(yǎng)原代星形膠質(zhì)細(xì)胞缺營養(yǎng)3 h后aquaporin4 (AQP4) mRNA表達(dá)的作用。 方法采用出生三天內(nèi)的SD大鼠大腦皮層制成單細(xì)胞懸液后,應(yīng)用含15%胎牛血清的DMEM/F12培養(yǎng),通過差速貼壁、搖床和傳代純化星形膠質(zhì)細(xì)胞,GFAP免疫熒光鑒定細(xì)胞,通過PBS緩沖液代替培養(yǎng)基模擬缺營養(yǎng)模型3小時后即刻應(yīng)用促EPO(10 u/l)然后按照加入藥物0.5 h、1 h、1.5 h、2 h、3 h、6 h、12 h、18 h收集細(xì)胞。PCR技術(shù)測定細(xì)胞AQP4 mRNA表達(dá)的變化。 結(jié)果星形膠質(zhì)細(xì)胞缺營養(yǎng)三小時AQP4表達(dá)降低;EPO作用0.5 h后細(xì)胞AQP4 mRNA表達(dá)較正常生理鹽水對照組明顯升高(圖14、15)。 結(jié)論EPO作用0.5 h后對損傷的星形膠質(zhì)細(xì)胞AQP4 mRNA表達(dá)有顯著作用。 第四部分EPO與MPSS聯(lián)合應(yīng)用對體外培養(yǎng)星形膠質(zhì)細(xì)胞AQP4 mRNA表達(dá)的研究 目的EPO與MPSS(Methylprednisolone)聯(lián)合應(yīng)用對體外培養(yǎng)星形膠質(zhì)細(xì)胞的作用,并探討其對脊髓缺血水腫損傷的作用機(jī)制。 方法體外培養(yǎng)的星形膠質(zhì)細(xì)胞傳至第三代,免疫熒光顯微鏡鑒定細(xì)胞種類,在PBS液中作用3 h后,建立星形膠質(zhì)細(xì)胞缺營養(yǎng)損傷的實(shí)驗(yàn)?zāi)Ｐ?給予DMEM/F12培養(yǎng)基加15%胎牛血清恢復(fù)營養(yǎng),然后按生理鹽水對照組、MPSS (10 ug/ml)組、EPO(10 u/ml)組、MPSS (10 ug/ml)和EPO (10 ug/ml)組、EPO (5 ug/ml)和MPSS (5 ug/ml)組的劑量干預(yù),干預(yù)后分別于0.5 h、1 h、1.5 h、2 h收集細(xì)胞;觀察:⑴、光鏡觀察不同干預(yù)組合對星形膠質(zhì)細(xì)胞形態(tài)影響;⑵、應(yīng)用四甲基偶氮唑藍(lán)(MTT)比色法測定細(xì)胞活性;⑶、使用RT-PCR檢測星形膠質(zhì)細(xì)胞AQP4 mRNA表達(dá)水平的變化。 結(jié)果⑴、缺營養(yǎng)可抑制星形膠質(zhì)細(xì)胞增殖,恢復(fù)培養(yǎng)基后細(xì)胞形態(tài)均有恢復(fù);⑵、單獨(dú)應(yīng)用EPO對細(xì)胞增殖恢復(fù)無統(tǒng)計學(xué)意義,單獨(dú)MPSS對損傷細(xì)胞增殖恢復(fù)有統(tǒng)計學(xué)意義;⑶、EPO和MPSS聯(lián)合應(yīng)用對細(xì)胞增殖恢復(fù)有明顯統(tǒng)計學(xué)意義;⑷、生理鹽水對照組對細(xì)胞增殖無明顯作用;⑸、半量EPO和半量MPSS聯(lián)合應(yīng)用對細(xì)胞增殖恢復(fù)有統(tǒng)計學(xué)意義;⑹、單獨(dú)EPO和單獨(dú)MPSS均促進(jìn)損傷細(xì)胞AQP4 mRNA表達(dá)增加;⑺、聯(lián)合應(yīng)用EPO和MPSS對細(xì)胞AQP4 mRNA表達(dá)有明顯影響作用;⑻、半量EPO和半量MPSS聯(lián)合應(yīng)用對細(xì)胞AQP4 mRNA表達(dá)亦有明顯影響作用; 結(jié)論半量EPO+半量MPSS聯(lián)合應(yīng)用的對星形膠質(zhì)細(xì)胞細(xì)胞增殖、AQP4 mRNA表達(dá)增加作用優(yōu)于單獨(dú)大劑量應(yīng)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.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R363

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