脑梗死继发皮质脊髓束华勒氏变性大鼠模型制备及标准化评估

目的 优化大鼠光化学法脑梗死的实验方法,制备稳定的脑梗死继发脊髓束华勒氏变性大鼠模型并进行标准化评估。方法 通过光化学法建立SD大鼠大脑右侧运动皮层终末动脉永久性缺血性损伤模型(实验组)。第一阶段实验组根据45只SD大鼠质量分为3组:A组,150~250g;B组,250~350g;C组,350~450g。每组再根据5%玫瑰红B剂量随机平均分3个亚组:Ⅰ组,12.5mg/100g体重;Ⅱ组,20mg/100g体重;Ⅲ组,30mg/100g体重。第二阶段实验组在BⅡ组基础上,根据玫瑰红B的浓度分为3组:a组,5%;b组,7.5%;c组,10%;每组再根据注射时间随机平均分3个亚组:1组,注射时间<1min;2组,注射时间1~2min;3组,注射时间>2min。造模后24h扫描MRI,28d扫描扩散峰度成像(Dif fusional kurtosis imaging,DKI)后牺牲大鼠,取脑组织做常规HE、髓鞘LFB及免疫荧光GFAP染色。结果 脑梗死继发脊髓束华勒氏变性大鼠模型制备的最佳参数组合为:SD大鼠体重250~350g、5%玫瑰红B 20mg/100g体重、注射时间≥2min、冷光源探头距离暴露的颅骨大约20mm、照射强度300~320klx、照射时间30min,该参数组合制备模型的24h脑梗死阳性率、28d脑软化灶阳性率及28d存活率均为100%。结论 本研究详细描述了光化学法脑梗死继发脊髓束华勒氏变性大鼠模型建立过程并进行标准化评估,为神经变性疾病研究提供了一种稳定、可靠的动物模型。

Construction and expression of the bicistronic expression vector with RANTES and SDF-1 genes

Objective: To construct bicistronic expression vector with RANTES and SDF-1 genes, the ligands of HIV-1 principal coreceptors, and identify its expression. Methods: RANTES-KDEL was amplified from plasmid pCMV-R-K by PCR and cloned into eukaryotic expression vector pCMV-S/K. Gene transfection into HeLa cells was carried out by lipofectin. Indirect immumofluorescence and radioimmunoprecipitation were used to confirm the expression of RANTES and SDF-1. Results: The construction of pCMV-R-K-S-K was confirmed by enzymatic digestion and sequencing. RANTES and SDF-1 were shown expressed in HeLa cells by indirect immumofluorescence and radioimmunoprecipitation. Conclusion: pCMV-R-K-S-K was constructed and expressed in cell line Hela successfully, which will contribute to further study of gene therapy of AIDS by HIV-1 coreceptors knockout.

GAD67-GFP基因敲入小鼠5-羟色胺与γ-氨基丁酸共存神经元在脑内的分布

目的:采用免疫荧光组织染色方法,以GAD67-GFP基因敲入小鼠为实验动物,系统地观察了小鼠脑干内5-羟色胺(5-HT)与γ-氨基丁酸(GABA)的共存情况,为中枢神经系统内5-HT与GABA共存神经元的分布提供形态学证据。方法:应用免疫荧光组织染色技术,对GAD67-GFP基因敲入小鼠脑干内5-HT与GABA免疫荧光双重染色,观察双重标记神经元在5-HT细胞群(B1-B9)中的分布情况并计数。结果:在所有脑干的5-HT细胞群中,双重标记神经元在中缝大核区域内所占的比率最高,平均为10.0%,其次是中缝苍白核和中缝隐核,分别为1.8%和1.4%。中缝桥核与中缝背核所含的共存神经元比例小,仅0.9%和0.3%。而在其他细胞群即B4,B6和B8-9细胞组,未观察到双重标记神经元。结论:在中枢神经系统内,只有部分延髓中线核团的5-HT能神经元同时含有GABA,而中脑中线核团和脑干的其他非中线5-HT细胞群内未见这种共存神经元的存在。