目的  检测胆固醇对ob/ob肥胖小鼠神经干细胞（NSCs）增殖和分化能力的影响，探讨肥胖引起中枢神经系统功能障碍的可能机制。    方法  选取4月龄ob/ob和野生型（WT）小鼠各6只，运用细胞增殖抗原（Ki67）和双皮质素（DCX）免疫荧光染色检测ob/ob小鼠侧脑室室管膜下区（SVZ）神经发生水平；分离培养18只4月龄ob/ob和WT小鼠SVZ的 NSCs，运用BrdU掺入实验和β-Ⅲ-微管蛋白（Tuj1）免疫荧光染色检测NSCs的自我更新和分化能力。运用基质辅助激光解飞行时间质谱（MALDI-MS）分别检测3只ob/ob和WT小鼠脑组织的脂质分布，并分析胆固醇（ST）含量和胆固醇合成相关基因的表达变化。体外培养15只WT新生鼠（P0）SVZ 的NSCs，并通过电穿孔法转染胆固醇合成限速酶3-羟基-3-甲基-戊二酰辅酶 A还原酶（Hmgcr）的小干扰RNA（siRNA），验证敲减效率，并通过BrdU掺入实验和Tuj1免疫荧光染色检测Hmgcr基因敲减对NSCs的影响。   结果  与WT小鼠相比，ob/ob 小鼠SVZ部位 Ki67+和DCX+ 细胞数量均显著下降（P<0.05）。体外实验结果显示，ob/ob小鼠NSCs的BrdU阳性率（P<0.05）和Tuj1阳性率（P<0.05）较WT小鼠NSCs显著下降。MALDI-MS结果显示，在ob/ob小鼠SVZ部位，ST（26∶0）和ST（27∶3）分布较WT小鼠明显减少；同样，试剂盒检测结果显示，与WT小鼠相比，ob/ob小鼠SVZ组织及NSCs的胆固醇含量显著降低（P<0.05）； Real-time PCR结果显示，ob/ob小鼠SVZ组织和NSCs的胆固醇合成相关基因Hmgcr、羊毛甾醇 14α-脱甲基酶（Cyp51）、3-β-羟基类固醇-Δ8,Δ7-异构酶（Ebp）和角鲨烯合酶（Fdft1）的mRNA表达水平均较WT小鼠显著下降（P<0.05），提示ob/ob小鼠SVZ的胆固醇合成障碍。Real-time PCR结果显示，Hmgcr-siRNA的干扰效率为55.27%±8.768%（P<0.05），Western blotting结果显示，Hmgcr-siRNA的干扰效率为32.69%±8.056%（P<0.05）；BrdU掺入实验结果显示，Hmgcr-siRNA组的BrdU阳性率较对照（control）组显著下降（P<0.05）；1% FBS诱导分化后，Hmgcr-siRNA组Tuj1阳性率较control组也显著下降（P<0.05），提示胆固醇合成受损抑制神经干细胞的自我更新和分化能力。   结论  ob/ob小鼠脑内胆固醇水平下降可影响SVZ部位 NSCs增殖分化能力。 

Objective  To investigate the effect of cholesterol on the proliferation and differentiation of neural stem cells (NSCs) in ob/ob obese mice, and to explore the possible mechanism of central nervous systym dysfunction caused by obesity.     Methods  Selected 6 4-month-old ob/ob and wild type (WT) mice, and cell proliferation antigen (Ki67) and doublecortin (DCX) immunofluorescenct staining were used to detect ob/ob mice lateral ventricle subventricular zone (SVZ) neurogenesis level. Cultured SVZ NSCs isolated from 18 4-month-old ob/ob and WT mice, and BrdU incorporation experiment and β-Ⅲ-tubulin (Tuj1) immunofluorescent staining were employed to detect the self-renewal and differentiation ability of NSCs. Matrix-assisted laser desorption/ionization time of flight mass spectrometry（MALDI- MS）was used to detect the lipid distribution in 4-month-old ob/ob and WT mice brain tissues, and measure the changes of cholesterol(ST) content and the expression genes related to cholesterol synthesis. Cultured 15 WT postnatal day 0(P0) mouse SVZ NSCs in vitro and electrotransfected with the small interfering RNA(siRNA) sequence of cholesterol synthesis rate-limiting enzyme 3-hydroxy-3-methyl-glutaryl coenzyme A reductase（Hmgcr）verified the knockdown efficiency, to detecte the effect of Hmgcr gene knockdown on NSCs by BrdU incorporation experiment and Tuj1 immunofluorescent staining.     Results  Compared with the WT mice, the number of Ki67+ and DCX+ cells in the SVZ tissue of ob/ob mice decreased significantly (P<0.05); The result  of in vitro experiments showed that the BrdU positive rate (P<0.05) and Tuj1 positive rate (P<0.05) of ob/ob mice NSCs were also significantly lower than that of WT mice NSCs. MALDI-MS test result  showed that the lipid distribution of ST (26∶0) and ST (27∶3) reduced significantly on SVZ of ob/ob mice. Meanwhile, Kit assay result  showed that the cholesterol content of ob/ob mice SVZ tissue and NSCs both reduced significantly (P<0.05). In addition, the Real-time PCR result  showed that the mRNA expression levels of cholesterol synthesis-related genes Hmgcr, Lanosterol 14-alpha demethylase（Cyp51）, 3-beta-hydroxysteroid-Δ8, Δ7-isomerase（Ebp）and squalene synthase（Fdft1）in the SVZ tissue and NSCs of ob/ob mice were both significantly lower than those in WT mice (P<0.05), suggesting that ob/ob mice SVZ cholesterol synthesis disordered. Real-time PCR result  showed that the interference efficiency of siRNA was 55.27%±8.768% (P<0.05), and the result  of Western blotting showed that the interference efficiency of siRNA was 32.69%±8.056%(P<0.05). The result  of BrdU incorporation experiment indicated that the positive rate of BrdU in the Hmgcr-siRNA group was significantly lower than that in the control group (P<0.05). After differentiation induced with 1% FBS, the positive rate of Tuj1 in the Hmgcr-siRNA group also decreased significantly compared with the control group (P<0.05), suggesting that impaired cholesterol synthesis inhibited the self-renewal and differentiation ability of neural stem cells.      Conclusion  Decreased cholesterol levels in the ob/ob mice brain can affect the proliferation and differentiation of SVZ NSCs.