本文選題：視網(wǎng)膜神經(jīng)節(jié)細(xì)胞 + 巨噬細(xì)胞�。� 參考：《第四軍醫(yī)大學(xué)》2010年碩士論文

【摘要】： 研究背景: 哺乳動(dòng)物的視神經(jīng)和視網(wǎng)膜是腦的衍生物,屬于中樞神經(jīng)系統(tǒng)(central nervous system,CNS)。視神經(jīng)纖維由視網(wǎng)膜神經(jīng)節(jié)細(xì)胞(retinal ganglion cells,RGCs)的軸突構(gòu)成。在視神經(jīng)損傷后,RGCs發(fā)生不可逆行性退變與、凋亡,,是導(dǎo)致視功能不可逆性損害的主要原因,也是在臨床中青光眼、視網(wǎng)膜缺血性疾病等眼科疾病的主要損傷細(xì)胞。由此常造成嚴(yán)重的視力損害,臨床上的治療十分棘手。近年來視神經(jīng)的保護(hù)和再生成為困擾醫(yī)學(xué)界的世界性難題,對(duì)視神經(jīng)再生各方面的研究也是眾說紛紜。周圍神經(jīng)系統(tǒng)(peripheral nerveous system,PNS)和CNS損傷后都將發(fā)生一系列形態(tài)學(xué)變化:損傷遠(yuǎn)端軸突發(fā)生潰變,近端軸突少量潰變后長(zhǎng)出神經(jīng)芽突,同時(shí)受損區(qū)域周圍膠質(zhì)細(xì)胞增生。但是CNS和PNS不同,軸突發(fā)芽后不繼續(xù)生長(zhǎng)并且很快夭折。發(fā)生這種變化的一個(gè)主要原因是CNS和PNS的微環(huán)境不同,它們的髓鞘細(xì)胞類型不同,PNS的髓鞘是雪旺細(xì)胞(Schwann cell),而CNS是少突膠質(zhì)細(xì)胞(oligodendroglia)。雪旺細(xì)胞可產(chǎn)生多種神經(jīng)營(yíng)養(yǎng)因子,能促進(jìn)PNS軸突再生,少突膠質(zhì)細(xì)胞則產(chǎn)生神經(jīng)生長(zhǎng)抑制因子,不利于CNS軸突的再生。 近年來,有學(xué)者發(fā)現(xiàn)在晶狀體損傷后,可以在視神經(jīng)損傷后促進(jìn)視網(wǎng)膜神經(jīng)節(jié)細(xì)胞的軸突再生。而晶狀體損傷后可以引起眼內(nèi)巨噬細(xì)胞的浸潤(rùn),這提示巨噬細(xì)胞可能在視神經(jīng)損傷后RGC存活和軸突再生中扮演重要角色。 因此,本實(shí)驗(yàn)通過分別對(duì)新生大鼠視網(wǎng)膜神經(jīng)節(jié)細(xì)胞及成年大鼠腹腔巨噬細(xì)胞進(jìn)行原代培養(yǎng),利用Transwell小室建立兩種細(xì)胞的共培養(yǎng)模型;觀察巨噬細(xì)胞對(duì)RGC存活時(shí)間及軸突再生的影響。 目的 分別對(duì)成年SD大鼠腹腔巨噬細(xì)胞和新生SD大鼠視網(wǎng)膜神經(jīng)節(jié)細(xì)胞進(jìn)行的原代培養(yǎng)、純化及鑒定;建立巨噬細(xì)胞/RGC的共培養(yǎng)模型,觀察巨噬細(xì)胞對(duì)RGC存活時(shí)間及軸突再生長(zhǎng)度的影響。 方法 1.大鼠腹膜腔注射DMEM培養(yǎng)液約6 mL,按摩腹腔10分鐘后,用生理鹽水10 mL灌洗腹膜腔。抽取腹膜腔細(xì)胞懸液,離心2次(1000r·min-1,5 min)。加DMEM培養(yǎng)液混懸細(xì)胞,37℃培養(yǎng)2 h后,D-Hanks液洗除漂浮的淋巴細(xì)胞,加入培養(yǎng)液繼續(xù)培養(yǎng)。兔抗大鼠CD68單克隆抗體免疫細(xì)胞化學(xué)進(jìn)行鑒定。 2.取出生1d SD仔鼠,斷頭處死后,在無菌條件下取眼球,解剖顯微鏡下去除眼前段,鈍性分離視網(wǎng)膜,加入0.125%胰蛋白酶消化,37℃條件下放置20 min后,500 r·min-1離心3分鐘,漂洗2次,去除上清液。加入培養(yǎng)液(含2% B27,15μg/mL BRDU的DMEM),鈍頭吸管吹打成單細(xì)胞懸液,行細(xì)胞計(jì)數(shù),接種于24孔板中,置于培養(yǎng)箱中培養(yǎng)。兔抗大鼠GAP-43單克隆抗體免疫細(xì)胞化學(xué)進(jìn)行鑒定。 3.采用transwell小室建立大鼠腹膜腔巨噬細(xì)胞和RGCs的共培養(yǎng)模型,不用巨噬細(xì)胞共培養(yǎng)的RGCs作為對(duì)照組,用胎盤藍(lán)染色計(jì)數(shù)及相差顯微鏡觀察RGCs存活的時(shí)間,并測(cè)量培養(yǎng)1 d、3 d和5 d時(shí)RGCs突起的平均長(zhǎng)度,進(jìn)行組間比較。 結(jié)果 成功獲取并培養(yǎng)了大鼠巨噬細(xì)胞和RGCs,建立了兩種細(xì)胞的共培養(yǎng)系統(tǒng)。1 d、3 d、5 d和7 d,共培養(yǎng)組平均RGCs活細(xì)胞數(shù)分別為(35.50±2.92)、(28.20±3.36)、(18.70±3.95)和(8.80±1.55),與對(duì)照組(36.20±2.35)、(27.10±2.96)、(15.80±3.04)和(8.40±2.01)比較,兩組之間均無統(tǒng)計(jì)學(xué)顯著性差異(P0.01)。但是,培養(yǎng)1 d、3 d和5 d,共培養(yǎng)組RGCs突起的平均長(zhǎng)度分別為(19.79±3.98)μm、(68.30±4.07)μm和(95.51±6.51)μm,明顯較長(zhǎng)于對(duì)照組[(15.28±1.28)μm、(58.18±4.22)μm和(82.61±3.75)μm] (P0.01)。 結(jié)論 巨噬細(xì)胞可明顯促進(jìn)共培養(yǎng)的RGCs軸突的生長(zhǎng),但對(duì)其存活時(shí)間沒有顯著影響。
[Abstract]:Background of Study :


The optic nerve and retina of a mammal is a derivative of the brain , and belongs to the central nervous system ( CNS ) . The optic nerve fibers are composed of axons of retinal ganglion cells ( RGCs ) . After optic nerve injury , RGCs are non - reversible and apoptosis .


In recent years , some scholars have found that after lens injury , the axonal regeneration of retinal ganglion cells can be promoted after optic nerve injury , and the infiltration of macrophages in the eyes can be caused after lens injury , which suggests that macrophages may play an important role in RGC survival and axonal regeneration after optic nerve injury .


Therefore , in this experiment , the rat retinal ganglion cells and adult rat peritoneal macrophages were cultured in primary culture , the co - culture model of two kinds of cells was established by Transwell chamber , and the effects of macrophages on RGC survival time and axonal regeneration were observed .


Purpose


The primary culture , purification and identification of mouse retinal ganglion cells in adult SD rats were studied . The coculture model of macrophages / RGC was established to observe the effect of macrophages on RGC survival time and axonal regeneration length .


method


1 . The peritoneal cavity of rats was injected with DMEM culture solution about 6 mL . After 10 minutes of massage , peritoneal cavity was irrigated with 10 mL normal saline . The peritoneal cavity cell suspension was extracted and centrifuged twice ( 1000 r 路 min - 1 , 5 min ) . Add DMEM culture medium suspension cells , culture at 37 鈩，

本文編號(hào)：1917602