本文選題：星形膠質(zhì)細(xì)胞　切入點(diǎn)：原代培養(yǎng)　出處：《蘇州大學(xué)》2009年碩士論文

【摘要】： 第一部分原代星形膠質(zhì)細(xì)胞模型制作及鑒定 目的通過新生鼠大腦制作原代星形膠質(zhì)細(xì)胞,并進(jìn)行鑒定和星形膠質(zhì)細(xì)胞純度測(cè)定 方法采用出生三天內(nèi)的SD大鼠大腦制成細(xì)胞懸濁液后,按1.5×107計(jì)數(shù)接種于已用0.01%多聚賴氨酸包被好的底面積為75cm2培養(yǎng)瓶中,放置于培養(yǎng)箱中(5%CO2,95%O2,37OC)培養(yǎng),采用含15%的胎牛血清的DMEM培養(yǎng)液培養(yǎng)7d,通過搖床和逐漸傳代純化星形膠質(zhì)細(xì)胞,觀察細(xì)胞的變化,通過免疫熒光染色鑒定星形膠質(zhì)細(xì)胞。 結(jié)果通過搖床與高血清培養(yǎng)可獲得高純度星形膠質(zhì)細(xì)胞,并隨逐漸傳代更加純化,形態(tài)也逐漸變化,培養(yǎng)的細(xì)胞GFAP染色呈陽(yáng)性,顯示正常星形膠質(zhì)細(xì)胞胞體呈多角形,胞膜光滑,邊界清楚,突觸長(zhǎng)且多相互連接,GFAP陽(yáng)性細(xì)胞占總細(xì)胞數(shù)比例在95%以上。 結(jié)論通過新生鼠大腦制作原代星形膠質(zhì)細(xì)胞,高血清培養(yǎng)基和搖床后培養(yǎng)可獲得高純度的星形膠質(zhì)細(xì)胞。 第二部分原代星形膠質(zhì)細(xì)胞缺營(yíng)養(yǎng)損傷模型制作 目的制作理想的原代星形膠質(zhì)細(xì)胞的損傷模型,觀察形態(tài)變化 方法采用出生三天內(nèi)的SD大鼠大腦制成細(xì)胞懸濁液后,采用含15%的胎牛血清的DMEM培養(yǎng)液培養(yǎng),通過搖床和逐漸傳代純化星形膠質(zhì)細(xì)胞。采用PBS緩沖液代替培養(yǎng)基模擬缺營(yíng)養(yǎng)模型,缺營(yíng)養(yǎng)3h后恢復(fù)營(yíng)養(yǎng),觀察細(xì)胞缺營(yíng)養(yǎng)前后形態(tài)變化。 結(jié)果缺營(yíng)養(yǎng)3h后部分細(xì)胞胞體變小,突觸變短,細(xì)胞間相互連接減少或消失,個(gè)別細(xì)胞呈圓形,復(fù)營(yíng)養(yǎng)后大多數(shù)細(xì)胞胞體變大,突出變長(zhǎng)并相互交錯(cuò),基本恢復(fù)缺營(yíng)前形態(tài),個(gè)別細(xì)胞死亡成圓形。結(jié)論高血清培養(yǎng)和搖床后可獲得高純度的星形膠質(zhì)細(xì)胞,原代星形膠質(zhì)細(xì)胞可耐受一定的缺營(yíng)養(yǎng)損傷,恢復(fù)營(yíng)養(yǎng)后可基本恢復(fù)至損傷前的形態(tài) 第三部分甲基強(qiáng)的松龍與促紅細(xì)胞生成素聯(lián)合應(yīng)用對(duì)原代星形膠質(zhì)細(xì)胞作用的研究 目的研究甲基強(qiáng)的松龍(MPSS)與促紅細(xì)胞生成素(EPO)單獨(dú)以及聯(lián)合應(yīng)用對(duì)體外培養(yǎng)的原代星形膠質(zhì)細(xì)胞的作用。 方法采用出生3d內(nèi)的SD大鼠大腦制成細(xì)胞懸濁液,采用含15%的胎牛血清的DMEM培養(yǎng)液培養(yǎng),通過搖床和逐漸傳代純化星形膠質(zhì)細(xì)胞,按1.5×107計(jì)數(shù)接種于已用0.01%多聚賴氨酸包被好的底面積為75cm2培養(yǎng)瓶中,放置于培養(yǎng)箱中(5%CO2,95%O2,37OC)培養(yǎng),通過搖床和逐漸傳代純化星形膠質(zhì)細(xì)胞,實(shí)驗(yàn)分為正常組、正常+ MPSS組、正常+EPO組、正常+ MPSS+EPO組、損傷組、損傷+ MPSS組、損傷+EPO組、損傷+ MPSS+EPO組。通過PBS緩沖液代替培養(yǎng)基模擬缺營(yíng)養(yǎng)模型3小時(shí)后即刻分別加入甲基強(qiáng)的松龍(10umol/l)、促紅細(xì)胞生成素(10u/l)、甲基強(qiáng)的松龍(10umol/l)與促紅細(xì)胞生成素(10u/l),然后分別繼續(xù)培養(yǎng)三天,免疫熒光技術(shù)鑒定星形膠質(zhì)細(xì)胞;MTT法檢測(cè)細(xì)胞的增殖活性和凋亡; PCR技術(shù)測(cè)定細(xì)胞GFAP RNA表達(dá)的變化。 結(jié)果星形膠質(zhì)細(xì)胞搖床后傳至第三代,經(jīng)鑒定星形膠質(zhì)細(xì)胞純度達(dá)95%以上;缺營(yíng)養(yǎng)3h后部分細(xì)胞胞體變小,突觸變短,細(xì)胞間相互連接減少或消失,個(gè)別細(xì)胞呈圓形,復(fù)營(yíng)養(yǎng)后大多數(shù)細(xì)胞胞體變大,突出變長(zhǎng)并相互交錯(cuò),基本恢復(fù)缺營(yíng)前形態(tài),個(gè)別細(xì)胞死亡成圓形;正常組和損傷組分別加入MPSS、EPO、MPSS和EPO三天后,損傷組細(xì)胞活性與GFAP表達(dá)均明顯升高,并且聯(lián)合應(yīng)用MPSS與EPO與單獨(dú)應(yīng)用MPSS、EPO相比,細(xì)胞活性與GFAP表達(dá)明顯升高,但是正常組沒有明顯變化。 結(jié)論適當(dāng)劑量的MPSS與EPO聯(lián)合應(yīng)用對(duì)缺營(yíng)養(yǎng)損傷的星形膠質(zhì)細(xì)胞有顯著的保護(hù)作用,并且與單獨(dú)應(yīng)用MPSS、EPO相比有顯著性作用,而MPSS、EPO單獨(dú)應(yīng)用,MPSS與EPO聯(lián)合應(yīng)用對(duì)正常的星形膠質(zhì)細(xì)胞均無(wú)顯著性保護(hù)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.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R651.2;R329

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本文編號(hào)：1719243