目的:探讨炎症因子转化生长因子(transforming growth factor-α,TNF-α)对SD大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成脂分化的影响及相关的分子机制.方法:将生长良好的第3代(P3)SD大鼠BMSCs分为对照组(Con组)、成脂诱导组(Ad组)和10 ng/mL TNF-α干预成脂诱导组(Ad+TNF-α组),每组各接种5块6孔板,培养14 d后,收集各组细胞的RNA和蛋白质.于第14天时间点处进行油红O染色和异丙醇萃取,酶标仪490 nm处检测吸光度(absorbance,A)值,观察各组成脂分化的程度.qRT-PCR检测各组基因Wnt10b、前体脂肪细胞因子-1 (preadipocyte factor-1,Pref-1)、CCAAT区增强子结合蛋白-α(CCAAT/enhancer-binding protein-α,C/EBP-α)、过氧化物酶体增殖物激活受体-γ(peroxisome proliferator-activated receptor-y,PPAR-γ)、脂肪酸结合蛋白-4(fatty acid binding protein-4,FABP-4)的mRNA表达水平.Western blot检测第14天3组基因β联蛋白(β-catenin)、PPAR-y、C/EBP-α、FABP-4的蛋白表达水平.结果:TNF-α可以明显抑制SD大鼠BMSCs的成脂分化.在Ad+TNF-α组,油红O染色明显低于Ad组,异丙醇萃取后A值检测也明显低于Ad组(0.360±0.035 vs.0.770±0.025,P=0.000).qRT-PCR结果显示,在Ad+TNF-α组,Wnt10b、Pref-1的mRNA水平(17.050±2.706,4.135±0.280)明显高于Con组(P=0.019,P=0.003)和Ad组(0.575±0.065,0.514±0.060) (P=0.020,P=0.006),而C/EBP-α、PPAR-y、FABP-4的mRNA水平(0.200±0.012,0.768±0.030,0.883±0.048)明显低于Ad组(2.965±0.455,2.330±0.211,3.847±0.171)(P=0.019,P=0.000,P=0.001).Western blot结果显示,诱导14 d后,Ad+TNF-α组的C/EBP-α、PPAR-γ、FABP-4蛋白表达水平(0.586±0.013,0.356±0.008,0.118±0.002)明显低于Ad组(1.082±0.018,0.840±0.112,1.094±0.038)(均P=0.000).在Ad+TNF-α组,磷酸化β-catenin(P-β-catenin)的蛋白水平(0.648±0.005)明显低于Ad组(3.376±0.211)(P=0.000).结论:TNF-α抑制SD大鼠BMSCs成脂过程分化相的作用可能与Wnt/β-catenin信号通路活性的增加和Pref-1的高表达有关,由此BMSCs的成脂分化停留在了前体脂肪细胞阶段.

目的:研究星形胶质细胞CTX TNA2在氧糖剥夺(oxygen-glucose deprivation,OGD)后N-myc downstream regulated gene 2 (NDRG2)和其磷酸化分子p-NDRG2 (Thr-348)的表达变化,明确NDRG2的磷酸化修饰是否对星形胶质细胞存活产生影响.方法:将CTX TNA2细胞进行3 h OGD处理,复氧复糖后于0h,2h,6h,12 h,24 h,通过Western Blot检测NDRG2和p-NDRG2(Thr-348)水平的变化.再通过感染慢病毒或给予Akt改变p-NDRG2(Thr-348),研究不同水平的p-NDRG2(Thr-348)对CTX TNA2细胞的存活是否产生影响.结果:(1)CTXTNA2细胞在OGD后2 h NDRG2(Thr-348)磷酸化水平达到峰值,以后逐渐下降,24 h后会恢复到OGD后0h的水平;(2)抑制p-NDRG2(Thr-348),降低CTX TNA2细胞活力(P＜0.05),增加乳酸脱氢酶(lactate dehydrogenase,LDH)释放(P＜0.05);(3)促进p-NDRG2(Thr-348),提高CTX TNA2细胞活力(P＜0.05),减少LDH释放(P＜0.05);结论:星形胶质细胞CTX TNA2细胞在OGD后NDRG2磷酸化水平发生明显变化.促进NDRG2磷酸化,有助于细胞存活;抑制NDRG2磷酸化,加重细胞凋亡.

Objective To explore the clinical features and NEMO gene mutation in a boy with ectodermal dysplasia and immunodeficiency (EDA-ID).Methods According to the patient's clinical presentation and family history,the integrity of the IL-12/IFN-γ axis was detected by Q-RT-PCR after ruling out the common primary immunodeficiency disease (PID),and then NEMO gene was sequenced.Results The patient showed recurrent fever,pale skin,sparse hair,mild frontal bossing,and fungus and BCG infections.The X-ray of the head revealed no obvious tooth germ.The expression of IL-12B mRNA in the whole blood cells was reduced more after stimulation with BCG plus IFN-γ than BCG alone (P ＜ 0.05).NEMO gene sequencing analysis found 1241T ＞ A substitution mutation in exon 10,predicting valine-to-aspartic acid missense mutation at the 414th amino acid site (p.V414D).The mother and one of her sisters had heterozygous mutations at the same site.Cornclusion Male children with BCG infections and recurrent fever should be considered to have mutation of NEMO gene,especially when there are some ectodermal dysplasia phenotypes such as pale and dry skin without sweat,sparse hair and no teeth.Matrilineal family history of incontinentia pigmenti is helpful to diagnosis.EDA-ID can be diagnosed early by combining the integrity test of IL-12/IFN-γ axis and genetic analysis.

Objective To investigate the effect of dopamine on lipopolysaccharide (LPS)-induced inflammatory response in mouse peritoneal macrophages (MPMs).Methods MPMs were isolated after injection of thioglycolate broth into the peritoneal cavity.MPMs from wild type C57 mice were distributed into control group,LPS group,dopamine pretreatment group,dopamine D1-like and and D2-like receptor antagonist groups (D1 and D2 antagonist groups).In the latter two groups,MPMs were pretreated with Dl-like and D2-like receptor antagonist respectively for 30 min,and then stimulated with dopamine and LPS.MPMs from wild type (TLR4 +/+) and TLR4 knock-out (TLR4-/-) mice were only divided into LPS group and dopamine pretreatment group.In LPS group,MPMs were stimulated with 1 μg/ml LPS for 6 h.In dopamine pretreatment group,MPMs were pretreated with 10-4 mol/L dopamine for 2 h,and then stimulated with 1μg/ml LPS for 6 h.Expressions of TLR4 and pro-IL-1βwere detected by Western blot and tumor necrosis factor-α (TNF-α) in cell culture supernatant by ELISA method.Results (1) Expressions of TLR4,pro-IL-1β and TNF-α in control group were (0.56 ± 0.07),(0.65 ± 0.11) and (1,770.6 ±448.8) pg/ml;in LPS group were (1.12 ± 0.15),(1.24 ± 0.20) and (15,569.5 ± 822.7) pg/ml;in dopamine pretreatment group were (0.28 ± 0.11),(0.22 ± 0.08) and (7,800.7 ±862.6)pg/ml;in D1 antagonist group were (0.25 ±0.12),(0.18 ±0.09) and (7,065.0 ± 1016.8)pg/ml;in D2 antagonist group were (0.80 ±0.09),(0.44 ±0.08) and (14,299.6 ± 1430.9)pg/ml.The three indicators in LPS group were increased compared to control group and dopamine pretreatment group (P ＜ 0.05),and in D2 antagonist group were increased compared to dopamine pretreatment group (P ＜ 0.05).There were no obvious differences between D1 antagonist group and dopamine pretreatment group(P ＞0.05).(2) After LPS stimulation,expressions of pro-IL-1 β and TNF-α in TLR4+/+ mice were (0.94 ±0.17) and (15,109.0 ± 1,903.4)pg/ml,and were (0.08 ±0.04) and (5,063.6 ± 512.8) pg/ml in TLR4-/-mice (P ＜0.05).After dopamine pretreatment,expressions of proIL-1β and TNF-α were (0.45 ±0.12) and (6,383.9 ± 1,287.3) pg/ml in TLR4+/+ mice,and were (0.05 ± 0.02) and (4,863.0 ± 824.7) pg/ml in TLR4-/-mice.Expressions of pro-IL-1β and TNF-o were higher in TLR4 +/+ mice than in TLR4-/-mice after LPS stimulation (P ＜ 0.05).Expressions of pro-IL-1β and TNF-αin TLR4 +/+ mice were reduced after dopamine pretreatment compared to LPS stimulation (P ＜ 0.05),while were similar in TLR4-/-mice (P ＞ 0.05).Conclusion Dopamine can inhibit LPS-induced inflammatory response in MPMs,and the inhibition is strongly related to dopamine D2-1ike receptor and TLR4.

Objective To isolate,culture and identify neural tissue committed stem cells(NTCSCs) from bone marrow of SD rats.Methods NTCSCs were isolated and cultured from rat bone marrow by using serum-free DMEM/F12(1︰1) conditional medium,then identified for differentiation by immunohistochemical assay,and determined for transcriptions of Nestin,CXCR4,CD31,βⅢ-Tubulin and GFAP mRNAs by RT-PCR.Results Cell spheres in suspension were isolated and cultured,in which Nestin and CXCR4 as markers of neural stem cells were expressed.βⅢ-Tubulin as neuron marker and GFAP as gliacyte marker were expressed in the cells after differentiation.The transcriptions of Nestin,CXCR4,CD31,βⅢ-Tubulin and GFAP mRNAs were proved in the cells.Conclusion NTCSCs with neurobiological characters were successfully isolated from bone marrow and might be used as cell seeds,which provided a basis for therapy of diseases and repair of wound in central nervous system.

Objective:To evaluate the feasibility of argon laser-induced choroidal neovascularization(CNV)model in the pigmental rabbits in order to establish the foundation for studying the mechanism of CNV and for the development of treatment for CNV.Methods:Experimental eyes in 20 rabbits were received a series of 20 argon green laser(514.5nm) lesions per eye (700 mW,50um,0.1s).Fundus fluorescein angiography(FFA) and indocyanine green angiography (ICGA) were performed,then the rabbits were sacrificed immediately,the eyes were enucleated and processed for histopathologic and immunohistochemistry examination on days 3,7,14,21,30.Results:CNV first appeared on day 7 after photocoagulation,reaching the peak on day 21.The incidence of CNV was 63.0%.Disciform leakage staining appeared in the FFA.ICGA showed that the disciform area was filled with CNV.CNV was ascertained by light microscopy.The immunohistochemistry examination of VEGF was positive in ganglion cell layer and inner nuclear layer of retina from 2w after photocoagulation.Conclusion:The present studies demonstrated that argon laser photocoagulation could be used to produce choroidal neovascularization experimental model in the pigmental rabbits.CNV can be induced rapidly by the method,persists for a long period and at a high successful rate.VEGF plays a role in the formation of CNV.

Objective To evaluate the effect of Celecoxib,a selective cycloxygenase-2(COX-2) inhibitor,inhibits retinal vascular endothelial growth factor(VEGF),COX-2 expression and vascular leakage with argon laser-induced choroidal neovascularization(CNV) model in the pigmental rabbits.Methods 20 healthy colored rabbits were divided into 4 groups: laser group,Celecoxib group,indomethacin group and control group.Each group has 5 rabbits(10 eyes).Celecoxib was administered B.i.d.by oral gavage(50 mg/kg) and indomethacin by oral also(25 mg/d).Experimental eyes in 15 rabbits were received a series of 20 argon green laser(647 nm) lesions per eye(0.7 W,50 μm,0.1 s).Fundus fluorescein angiography(FFA) and indocyanine green angiography(ICGA) were performed,then the rabbits were terminated immediately.The eyes were enucleated and processed for histopathologic and immunohistochemistry examination on the 3rd day,7th day,14th day,21st day,30th day.Results CNV was firstly appeared on day 7 after photocoagulation,reaching the peak on the 21st day.The incidence of CNV was 63.0%.Disciform leakage staining appeared in the FFA.The immunohistochemistry examination of VEGF and COX-2 were positive in retina from the 2nd week after photocoagulation,and they had the same change trend.Conclusion The present studies demonstrated that celecoxib cannot block CNV induced by laser photocoagulation but it can reduce the express of VEGF and incidence of CNV.

眼底新生血管的形成是一个复杂的生物学过程,其中血管内皮生长因子(VEGF)和色素上皮衍生因子(PEDF)在血管的形成中起着重要作用。现就VEGF和PEDF的结构特征、生成和分布、对眼底新生血管的协同作用等作一综述。