本文選題：嗅鞘細(xì)胞 + 脊髓損傷　； 參考：《寧夏醫(yī)科大學(xué)》2016年碩士論文

【摘要】：目的1.從SD新生鼠嗅黏膜取材原代培養(yǎng)提純鑒定嗅鞘細(xì)胞。2.探討嗅鞘細(xì)胞在體外模擬的脊髓損傷缺氧內(nèi)環(huán)境中是否誘導(dǎo)自噬,并檢測自噬對嗅鞘細(xì)胞增殖能力的影響。3.驗(yàn)證嗅鞘細(xì)胞移植對大鼠半橫斷損傷的治療作用。4.探討SPIO體外標(biāo)記嗅鞘細(xì)胞的合適方案,以及體外成像特點(diǎn)。方法1.用酶消化法和改良的Nash差速貼壁法分離提純嗅鞘細(xì)胞,采用CCK-8法繪制生長曲線,選出增殖較好的細(xì)胞代次;利用免疫熒光法鑒定細(xì)胞屬性和純度。2.采用氧濃度為2%作為低氧條件(低氧處理組),培養(yǎng)SD新生鼠嗅黏膜來源的OECs,以正常條件下培養(yǎng)的OECs作為對照組,采用自噬抑制劑3-MA處理后的OECs繼續(xù)在低氧條件下培養(yǎng)作為3-MA低氧處理組,在相差顯微鏡下觀察細(xì)胞形態(tài);對照組、4 h低氧處理組、8 h低氧處理組,Western blotting法檢測各組OECs中HIF-1α(Hypoxia inducible factor-1,低氧誘導(dǎo)因子-1α)、自噬標(biāo)志蛋白LC3Ⅰ/Ⅱ和自噬標(biāo)志基因Beclin-1的蛋白表達(dá)水平;對照組、自噬抑制劑3-MA處理的OECs然后在正常條件下培養(yǎng)作為3-MA處理組、低氧組、3-MA低氧處理組,采用CCK-8法檢測各組OECs的增殖能力。3.建立SD大鼠脊髓左側(cè)半橫斷損傷模型,實(shí)驗(yàn)組傷后立即注射嗅鞘細(xì)胞,對照組只注射完全培養(yǎng)基,單純損傷組不注射。于1、2、4、6、8周對各組實(shí)驗(yàn)鼠進(jìn)行大鼠后肢運(yùn)動功能(BBB)評分。明確嗅鞘細(xì)胞移植對脊髓半橫斷損傷大鼠的治療效果。4.用多聚左旋賴氨酸包被過的SPIO納米粒子標(biāo)記OECs,普魯士藍(lán)染色和透射電鏡下觀察標(biāo)記結(jié)果,對標(biāo)記后的嗅鞘細(xì)胞進(jìn)行細(xì)胞活性和增殖能力的檢測并實(shí)現(xiàn)體外MRI成像。結(jié)果1.經(jīng)原代分離、提純培養(yǎng)的SD新生鼠嗅黏膜來源嗅鞘細(xì)胞大多數(shù)呈梭形,典型的雙極、三極細(xì)胞形態(tài)。激光共聚焦顯微鏡下GFAP和P75雙染陽性的為嗅鞘細(xì)胞。根據(jù)計(jì)算公式其純度可達(dá)87%。2.與對照組比較,低氧組嗅鞘細(xì)胞中HIF-1α表達(dá)水平增高(P0.05),LC3Ⅰ向LC3Ⅱ的轉(zhuǎn)化明顯,Beclin-1表達(dá)水平增高(P0.05);自噬抑制組中嗅鞘細(xì)胞增殖能力降低(P0.05)。3.實(shí)驗(yàn)組與對照組和單純損傷組比較,P0.05,具有統(tǒng)計(jì)學(xué)差異;對照組與單純損傷組在各個時(shí)間點(diǎn)比較P0.05,無統(tǒng)計(jì)學(xué)差異;隨著時(shí)間的推移各組BBB評分也在升高。4.成功應(yīng)用SPIO標(biāo)記嗅鞘細(xì)胞且不影響其生物學(xué)特性,MRI體外成像顯示1×106數(shù)量級細(xì)胞在軸位和矢狀位的T2WI和SWI序列上信號可較好顯示。結(jié)論1.低氧誘導(dǎo)的自噬對大鼠嗅鞘細(xì)胞的增殖能力具有促進(jìn)作用。2.嗅鞘細(xì)胞移植對脊髓半橫斷損傷有治療作用。3.成功應(yīng)用SPIO標(biāo)記嗅鞘細(xì)胞且不影響細(xì)胞基本生物學(xué)特性和增殖能力,并實(shí)現(xiàn)MRI體外成像,為以后嗅鞘細(xì)胞移植入脊髓的示蹤問題奠定了基礎(chǔ)。
[Abstract]:Objective 1. Olfactory ensheathing cells. 2. 2 were isolated from olfactory mucosa of newborn SD rats. To investigate whether olfactory ensheathing cells induce autophagy in anoxic environment of spinal cord injury in vitro and to detect the effect of autophagy on the proliferation of olfactory ensheathing cells. To verify the therapeutic effect of olfactory ensheathing cell transplantation on hemitranverse injury in rats. 4. 4. Objective: to investigate the appropriate scheme of SPIO labeling olfactory ensheathing cells in vitro and the imaging characteristics in vitro. Method 1. Olfactory ensheathing cells were isolated and purified by enzyme digestion method and modified Nash differential adherence method. The growth curve was drawn by CCK-8 method and the cells with better proliferation were selected. The cell properties and purity were identified by immunofluorescence method. OECs derived from olfactory mucosa of newborn SD rats were cultured under hypoxic conditions (oxygen concentration of 2%) and OECs cultured in normal condition as control group. OECs treated with autophagy inhibitor 3-MA continued to be cultured in hypoxic condition as 3-MA hypoxia group, and the morphology of cells was observed under phase contrast microscope. Western blotting assay was used to detect the expression of HIF-1 偽 -Hypoxia inducible factor-1, hypoxia inducible factor-1 偽, autophagy marker LC3 鈪，

本文編號：1903570