摘要
目的探讨由细菌脂多糖（lipopolysaccharide，LPS）诱导的活性小胶质细胞（MG）对少突胶质细胞（OL）前体的毒性作用及选择性iNOS抑制剂1400W对毒性作用的阻断效果。方法取2日龄SD大鼠脑内MG和OL前体共培养，分为共培养对照组，共培养LPS组以及共培养LPS＋1400W组。对共培养细胞经LPS100ng／ml诱导后48h，分别应用硝酸还原比色法检测一氧化氮（NO）含量，免疫细胞染色法检测过氧亚硝酸盐（ONOO^-）含量，Westernblot法检测诱生型一氧化氮合酶（Inducible Nitric Oxide Synthase，iNOS）蛋白合成量，Hochest33342／PI荧光染色观察细胞凋亡形态学改变，以及流式细胞仪检测细胞凋亡率。结果与共培养对照组比LPS可诱导培养细胞内NO含量[（82．27±3．41）μmol／L vs．（167．86±9．87）μmol／L，t=8．593，P〈0．01]、ONOO^-含量[（6．14±1．27）×10^7／L vs．（34．38±7．75）×10^7／L，t=5．892，P〈0．01]以及iNOS蛋白合成量相对值[（0．18±0．027）VS．（0．79±0．068），t=9．26，P〈0．01]明显增高，OL前体的凋亡率亦显著增加[（6．73±1．39）％vs．（24．77±2．05）％，t=12．619，P〈0．01]。应用1400W10μmol／L则可显著抑制因LPS诱导而增高的NO含量[（69．55±5．07）μmol／L，t=8．896，P〈0．01]、ONOO^-含量[（10．33±3．47）×10^7／L，t=14．96，P〈0．01]以及iNOS蛋白的合成量（0．35±0．042，t=5．506，P〈0．01），并显著降低了OL前体的细胞凋亡率[（11．8±2．06）％，t=7．715，P〈0．01]。结论NO、iNOS以及ONOO^-等物质在LPS诱导OL前体的死亡通路中扮演了重要角色，1400W通过选择性抑制iNOS，减少了NO以及ONOO^-的生成，从而有效阻断了由LPS诱导活性MG对OL前体的毒性作用，提高了OL前体的存活率。
Objective To explore the toxicity of LPS-induced activated microglia to preoligodendrocytes （preOLs） and the effect of 1400W, a selective inhibitor of inducible nitric oxide synthetase （iNOS）, on the blockage of the toxicity. Methods Co-cultured microglia and preOLs obtained from two-day-old Sprague-Dawley （SD） rats were divided into three groups： co-culture control group, co-culture LPS group and co-culture LPS plus 1400W group. After cultured cells were induced by LPS （ 100 ng/ml） for 48 hours, the concentration of nitric oxide （NO） was measured by nitric acid-deoxidize- colorimetry, the level of peroxynitrite （ ONOO^- ） was determined by immunocytochemistry, and the synthetic level of iNOS was detected by Western blotting, respectively. The morphologic observation of apoptotic preOLs stained with Hoechst 33342/PI and the apoptotic rate of preOLs detected by flow cytometry were processed simultaneously. Data were analyzed with SPSS 11.0 software. Results Compared to co-culture control group, there was significant increase in levels of NO [ （ 82. 27 ± 3.41 ） μmol/L vs. （ 167.86 ±9. 87） μmol/L, t = 8. 593, P 〈 0. 01 ], ONOO^ - [ （6. 14 ± 1.27） × 10^7/L vs. （34. 38 ± 7.75 ） × 10^7/L, t =5. 892, P〈0. 01 ], and iNOS [ （0. 18 ±0. 027） vs. （0. 79 ±0. 068） ,t =9. 26,P 〈0. 01 ] induced by LPS in co-cuhure LPS group, and with a higher apoptotie rate of preOLs [ （ 6. 73 ± 1.39 ） % vs. （ 24. 77± 2.05）%,t=12.619,P〈0. 01]. However, all levels of NO [（69.55 ±5.07） μmol/L,t =8.896,P〈 0.01], ONOO^- [ （10. 33 ±3.47） ×10^7/L, t =14.96, P〈0.01]and iNOS （0.35 ±0.042,t =5.506,P〈 0. 01 ） decreased significantly with the use of 1400W at a dose of 10μmol/L in co-culture LPS plus 1400W group, and the apoptotie rate of preOLs [（11.8 ±2.06）%,t =7.715,P 〈0.01] was also reduced evidently. Conclusions NO, ONOO^- and iNOS, etc. play important roles in the death pathway of preOLs induced by LPS. 1400W can block effectively the toxicity
出处
《中华儿科杂志》
CAS
CSCD
北大核心
2009年第7期537-543,共7页
Chinese Journal of Pediatrics
基金
国家自然科学基金资助项目（30672246）
