siRNA下调AEG-1表达对神经母细胞瘤细胞MMP-9的表达及侵袭能力的影响

目的 观察下调Astrocyte elevated gene-1(AEG-1)基因对神经母细胞瘤细胞中基质金属蛋白酶-9(MMP-9)表达以及细胞侵袭能力的影响.方法 根据RNA干扰原理,设计合成AEG-1 siRNA.通过荧光定量RT-PCR和Western blotting观察神经母细胞瘤细胞中AEG-1基因mRNA和蛋白下调情况;采用Western blotting观察下调AEG-1表达对神经母细胞瘤细胞中MMP-9蛋白表达的影响;通过侵袭实验(Transwell法)观察下调AEG-1表达对神经母细胞瘤细胞侵袭能力的影响.结论 采用AEG-1 siRNA转染神经母细胞瘤细胞,下调细胞内AEG-1的mRNA和蛋白表达(P＜0.05)后MMP-9蛋白表达抑制率分别为63.1%和58.9%.此外下调AEG-1表达使细胞侵袭能力明显减弱(P＜0.05).结论 AEG-1基因下调使神经母细胞瘤细胞中MMP-9的蛋白表达明显减少、活性明显减弱,同时使细胞的侵袭能力降低.由此可见AEG-1在肿瘤细胞的侵袭转移中可能扮演重要角色,将会成为神经母细胞瘤基因治疗的新靶点.

Abstract：

Objective To investigate the effect of astrocyte elevated gene-1 (AEG-1)siRNA induced inhibition of invasion in neuroblastoma cells.Methods A small interference RNA(siRNA) targeting to AEG-1 mRNA(AEG-1 siRNA) was constructed and transfected into neuroblastoma cells with Lipofectamine 2000.A non-specific siRNA(control siRNA) and non-treatment were used as negative control group and blank group.The expression of AEG-1 mRNA was detected by RT-PCR.The proteins of AEG-1 and MMP-9 were detected by Western blotting.Cell invasion after AEG-1 knockdown was observed by Transwell assay.Results Compared with the control group,the expression of AEG-1 mRNA and protein were significantly decreased in the cells transfected with AEG-1 siRNAs(P＜0.05).AEG-1 knockdown by siRNA markedly decreased the expression of MMP-9 protein by 63.1% in neuroblastoma cells and the ability of cell invasion (P＜0.05).Conclusions The expression of AEG-1 mRNA is down-regulated by AEG-1 siRNA in neuroblastoma cells.Moreover,knockdown of AEG-1 expression in human neuroblastoma cells significantly inhibits the expression of MMP-9 protein and cell invasion.Therefore,AEG-1 may play a key role for MMP-9 activity and cell invasion,making it a potential target gene for gene-therapy in neuroblastoma.     

儿童肾母细胞瘤HMGB1、RAGE及VEGF的表达及意义

目的 探讨高迁移率族蛋白-1(HMGB1)、晚期糖基化终末产物受体(RAGE)和血管内皮生长因子(VEGF)在小儿肾母细胞瘤组织中的表达及其在肿瘤血管浸润、淋巴结转移中的意义.方法 收集郑州大学第一附属医院2003年1月至2010年1月小儿外科手术切除经病理证实为肾母细胞瘤的石蜡标本50例、15例同期相应瘤旁组织和7例正常肾组织标本.应用免疫组织化学SP染色辅以计算机图像分析系统分析结果的方法,研究HMGB1、RAGE及VEGF在各组标本中的表达及意义.结果 HMGB1、RAGE在肾母细胞瘤组织中的表达(144.46±13.55、138.18±12.26)与两者在瘤旁组织和正常肾组织中表达(107.47±5.27、103.91±4.29;100.98±4.82、100.82±3.32)相比差异均有统计学意义(P＜0.05),HMGB1在瘤旁与正常肾之间表达差异有统计学意义(P＜0.05).VEGF在瘤体组与瘤旁组中(147.57±13.77,140.28±7.85)和在正常肾组织组中的表达(106.38±1.92)相比差异显著(P＜0.05).三者在临床分期Ⅲ～Ⅳ期和预后不良组织型组及淋巴结转移组的肾母细胞瘤组织中的表达(148.69±12.17、147.73±6.71、163.14±7.50;157.88±4.44、155.29±3.97、169.17±4.42;152.11±7.36、151.56±5.46、163.83±7.20)显著高于临床分期Ⅰ～Ⅱ期和预后良好组织型及无淋巴结转移组(P＜0.05)(139.23±5.83、133.30±11.23、140.85±8.87;139.52±5.22、135.39±9.71、143.94±10.39;138.61±5.59、133.00±8.75、141.18±8.95).相关分析显示肾母细胞瘤组织中HMGB1与RAGE、VEGF的表达均成正相关(r=0.424,P=0.002;r=0.453,P=0.001),RAGE与VEGF之间无明显相关(r=0.237,P=0.101).结论 HMGB1、RAGE和VEGF的高表达与肾母细胞瘤的临床分期、病理类型、淋巴结转移有关,参与了肿瘤血管浸润及淋巴结转移,HMGB1/RAGE可能是促进肾母细胞瘤转移的一条信号通路,给我们靶向治疗肾母细胞瘤提供了新的思路.

Abstract：

Objective This study aim to assess the expressions of high mobility group box chromosomal proteinl ( HMGB1) and receptor foradvanced glycation end products (RAGE) and vascular endothelial growth factor (VEGF) in Wilms'tumor and their clinical significance both in the tumor blood vessel infiltrates and in the lymph node metastasis. Methods Fifty cases of Wilms'tumor samples, which had been confirmed by pathology, were collected from The First Affiliated Hospitals of Zhengzhou University from january 2003 to january 2010. And 15 cases were taken from the adjacent kidney tissues at the same time. Other 7 cases were normal kidney tissues. The expression of HMGB1, RAGE and VEGF were detected by the Immunohistochemical SP staining in each group of specimens. The expression intensity was analyzed by computer image processing and their significance in each group of specimens were studied. Results The expressions of HMGB1 and RAGE in the Wilms'tumor tissues (144. 46 ± 13. 55、138.18 ±12. 26) were compared with those in the adjacent kidney tissues and in the normal kidney tissues( 107. 47 ± 5. 27、103. 91 ± 4. 29; 100. 98 ± 4. 82、 100. 82 ± 3. 32). The expressions of HMGB1 in the adjacent kidney tissues and in the normal kidney showed significant differences. There was a significant differences between the Wilms' tumor group and the adjacent kidney tissues or in the normal kidney tissues ( F ＜ 0.05 ). The expressions of VEGF proteins in Wilms'tumor tissues and in the adjacent kidney tissuess(147. 57 ± 13. 77,140. 28 ± 7. 85) comoared to those in the normal kidnev tissues(106. 38 ± 1. 92)were remarkably different(P＜0. 05). The expressions of HMGB1 ,RAGE and VEGF proteins in Wilms' tumor tissues of stage Ⅲ-Ⅳ and high risk histopathology and lymph node metastasis group (148. 69 ± 12.17、147. 73 ± 6. 71、163.14 ± 7. 50; 157. 88 ± 4. 44、155. 29 ± 3. 97、169. 17±4.42;152. 11 ± 7. 36、151. 56 ± 5. 46、163. 83 ± 7. 20)were significantly higher than those of stage Ⅰ-Ⅱ and low risk histopathology and no lymph node metastasis group(P＜0. 05) (139. 23 ± 5. 83、133. 30 ± 11. 23、140. 85 ± 8. 87; 139. 52 ± 5. 22、135. 39 ± 9. 71、143. 94 ± 10. 39; 138. 61 ± 5. 59、133. 00 ±8. 75、141.18 ± 8. 95). There was a positive correlation between the expressions of HMGB1 with RAGE and VEGF(r = 0. 424, P = 0. 002; r = 0. 453, P = 0. 001), but the correlation between the RAGE and VEGF is not clear (r = 0. 237, P = 0. 101). Conclusions There was a high expression of HMGB1 ,RAGE and VEGF in the Wilms'tumor tissues and they are partly related to the clinical stages and pathological type and lymph node metastasis of Wilms'tumor. They perhaps participated in the tumor blood vessel infiltration and the lymph node metastasis and the pathway of HMGB1/RAGE is perhaps the main mechanism correlated with the metastasis of Wilms'tumor.     

叉头样转录因子3及转化生长因子β在小儿肾母细胞瘤中的表达和意义

目的 探讨叉头样转录因子3(FOXP3)及转化生长因子β(TGF-β)mRNA在小儿肾母细胞瘤中的表达和临床意义.方法 采用RT-PCR方法检测30例肾母细胞瘤组织、30例瘤旁组织和3例正常肾组织中FOXP3及TGF-βmRNA的表达情况,观察两者表达与临床分期的关系.结果 与瘤旁组织和正常组织比较,FOXP3及TGF-βmRNA在肿瘤组织中表达均上升,为0.706±0.107、0.667±0.121,差异具有统计学意义(P＜0.001);二者mRNA在Ⅰ期肿瘤中的表达强度为0.535±0.086、0.564±0.050均低于Ⅱ期的0.741±0.074、0.704±0.067和Ⅲ期的0.843±0.053、0.734±0.085,差异具有统计学意义(P＜0.001);FOXP3和TGF-βmRNA表达强度呈明显正相关(r=0.749,P＜0.001).结论 小儿肾母细胞瘤组织中,FOXP3及TGF-βmRNA表达增高,可能与肾母细胞瘤的发展和转移有关.

Abstract：

Objective To investigate the expression levels and the clinical significance of FOXP3and TGF-β in nephroblastoma. Methods The expression levels of FOXP3 and TGF-β were evaluated using RT-RCR in 30 nephroblastoma,30 adjacent tissues and 3 normal tissues. Results The expression levels of FOXP3 and TGF-β in nephroblastoma tissues were higher than adjacent and normal tissues(P＜0. 001). Compared to the stage Ⅱ and Ⅲ the difference of the expression levels of FOXP3and TGF-β in stage Ⅰ were significant in nephroblastoma tissues(P＜0. 001). There was a positive correlation between FOXP3 and TGF-β. Conclusions The expression levels of FOXP3 and TGF-β were higher in tumor tisssues, suggesting that it may be related to metastasis and progression of the nephroblastoma.     

不同转移潜能人神经母细胞瘤细胞系趋化因子受体CXCR4的表达及对瘤细胞趋化作用的影响

目的 以自建的人神经母细胞瘤转移瘤(QDDQ-NM1)及原位瘤(QDDQ-NM2)细胞系的细胞为研究对象,探讨CXCR4在不同转移潜能人神经母细胞瘤细胞系细胞中的表达情况及其对瘤细胞趋化作用的影响.方法 采用RT-PCR检测两神经母细胞瘤细胞系细胞CXCR4 mRNA的表达;流式细胞仪直接免疫荧光法检测两细胞系细胞膜表面CXCR4蛋白的表达;通过趋化和趋化抑制实验观察CXCR4特异性配体CXCL12对QDDQ-NM1和QDDQ-NM2细胞系瘤细胞的趋化作用.结果 RT-PCR显示QDDQ-NM1细胞系CXCR4mRNA的相对含量为0.57±0.08,QDDQ-NM2细胞系为0.29±0.05,两者比较差异有统计学意义(P=0.005＜0.05),流式细胞仪直接免疫荧光法检测两细胞系细胞膜表面CXCR4蛋白表达的阳性率分别为(64.59±1.57)%和(36.72±0.57)%,两者的差异有统计学意义(P=0.00＜0.05),CXCR4特异性配体CXCL12可在一定范围内呈浓度依赖性的趋化QDDQ-NM1和QDDQ-NM2细胞系细胞的迁移,以100 ng/ml的效果较为明显,两细胞系迁移到聚碳酸酯膜下的细胞数差异有统计学意义(P＜0.05).CXCR4特异性拮抗剂AMD3100能有效抑制这种趋化作用(P＜0.05).结论 QDDQ-NM1细胞系的细胞功能性高表达趋化因子受体CXCR4,可能与人神经母细胞瘤QDDQ-NM1细胞系细胞的体外高转移潜能有关.

Abstract：

Objective To study the expression of functional chemokine receptor CXCR4 and its effects on the metastatic potential of human neuroblastoma.Methods Two human neuroblastoma cell lines were used in this study,including QDDQ-NM1 with high metastatic potential and QDDQ-NM2 with low metastatic potential.The expression of CXCR4 was explored at mRNA level using RT-PCR,and the protein level by flow cytometry.Chemotaxis assay was also performed to study the migratory response of QDDQ-NM1 and QDDQ-NM2 to CXCR4 ligand CXCL12.Results The mRNA of CXCR4 was higher in QDDQ-NM1 group than that in QDDQ-NM2 group (0.57±0.08 vs 0.29±0.05,P =0.005).The expression of CXCR4 on QDDQ-NM1 group was also higher than that in QDDQ-NM2 group [(64.59 ±1.57) % vs (36.72 ± 0.57) %,P＜0.05] The QDDQ-NM1 cells exhibited stronger migratory response to CXCL12 in a concentration dependent manner (P＜0.05),and the response peaked to CXCL12 at 100 ng/ml.AMD3100,a specific CXCR4 antagonist,could reverse the tumor's migratory response to CXCL12.Conclusions The expression of CXCR4 is associated with the metastatic potential of human neuroblastoma.     

舒尼替尼及联合顺铂对小儿睾丸卵黄囊瘤荷瘤鼠模型的抗肿瘤作用

目的 探讨舒尼替尼(Sunitinib)、顺铂(CDDP)及两者联合用药对小儿睾丸卵黄囊瘤(TYST)异种移植荷瘤鼠模型的抗肿瘤作用及相关作用机制.方法 肿瘤标本来自本实验室的小儿睾丸卵黄囊瘤裸鼠第17代模型,并接种在雄性裸鼠单侧腹股沟皮下区,成瘤后随机分成4组(n=5):对照组、CDDP组、Sunitinib组和Sunitinib+CDDP组.绘制肿瘤体积和裸鼠体重变化曲线图,计算肿瘤消退率;HE染色观察肿瘤组织形态学变化;免疫组织化学法检测AFP、Ki-67、Glypican-3、CD105在各组肿瘤中的表达:CD105测定微血管密度(MVD),Ki-6表示细胞增殖率(PI);TUNEL法检测肿瘤细胞凋亡率(AI);实时荧光定量PCR(RT-qPCR)验证靶向因子的mRNA表达变化.结果 各治疗组均能显著抑制肿瘤生长,并能消退肿瘤体积.在治疗后肿瘤体积上,除顺铂组与舒尼替尼组间无统计学差异外(41.61±7.61比67.15±5.39,P＞0.05),其余各组间都有统计学差异:对照组与顺铂(651.72±121.16比41.61±7.61,P＜0.05),对照组与舒尼替尼组(651.72±121.16比67.15±5.39,P＜0.05),对照组联合舒尼替尼±顺铂组(651.72±121.16比23.03±2.37,P＜0.05),舒尼替尼组与联合舒尼替尼+顺铂组(67.15±5.39比23.03±2.37,P＜0.05),顺铂组与联合舒尼替尼+顺铂组(41.61±7.61比23.03±2.37,P＜0.05);在裸鼠体重上,相比对照组,除舒尼替尼组无统计学差异外(25.90±0.75比26.66±0.65,P＞0.05),其余各组间差异均有统计学意义:对照组与顺铂组(25.90±0.75比18.90±0.63,P＜0.05),对照组与联合舒尼替尼+顺铂组(25.90±0.75比18.26±1.20,P＜0.05);AFP、Glypican-3在各治疗组阳性表达面积(Pixels)均少于对照组(AFP:对照组与顺铂组,1.26×106土1.48×105比5.54×105±8.14×104,P＜0.05;对照组与舒尼替尼组,1.26×106±1.48×105比7.09×105±6.64×104,P＜0.05;对照组与联合舒尼替尼+顺铂组,1.26×106±1.48×105比3.62×105±4.83×104,P＜0.05.Glypican-3:对照组与顺铂组,9.68×105±7.63×104比4.04×105±5.04×104,P＜0.05;对照组与舒尼替尼组,9.68×105±7.63×104比4.59×105±2.32×104,P＜0.05;对照组与联合舒尼替尼+顺铂组,9.68×105±7.63×104比1.89×105±2.55×104,P＜0.05).两者在顺铂组与舒尼替尼组的比较中差异无统计学意义(P＞0.05);PI和AI在各治疗组中相比对照组,差异都具有统计学意义(PI和AI:对照组与顺铂组,58.97土1.38比42.36±1.28和1.69±0.20比54.62±2.49,P＜0.01;对照组与舒尼替尼组,58.97±1.38比43.48±1.00和1.69±0.20比47.32±2.00,P＜0.01;对照组与联合舒尼替尼+顺铂组,58.97±1.38比33.34±1.19和1.69±0.20比63.41土2.23,P＜0.01).顺铂组相比联合舒尼替尼+顺铂组,PI:P=0.001,AI:P=0.002;舒尼替尼组相比联合舒尼替尼+顺铂组,PI和AI:P＜0.001;顺铂组相比舒尼替尼组,PI.P=0.597,AI:P=0.059;RT-qPCR证实M-CSFR、PDGFR-β、RET、VEGFR-2的mRNA表达受到抑制.结论 Sunitinib能显著抑制小儿睾丸卵黄囊瘤的生长,并消退肿瘤体积:主要通过直接抑制肿瘤细胞的生长和肿瘤血管的新生,从而诱导肿瘤细胞凋亡,同时伴有直接细胞毒作用引起坏死;Sunitinib相比CDDP具有更轻的毒副作用,且联合CDDP具有增强抗肿瘤作用.

Abstract：

Objective To study the antitumor effects of Sunitinib or Sunitinib combind with cis - diamminedichloroplatinum (CDDP) on an athymic mouse human testicular yolk sac tumor xenograft model. Methods The athymic mouse human testicular yolk sac tumor xenograft model was established by subcutaneous injection of 17th passage pediatric testicular yolk sac tumor cells in the unilateral inguinal region of the male nude mice. The mice of control group didn't receive any treatment. The tumor bearing mice were treated with either CDDP, or Sunitinib group, or Sunitinib combined with CDDP. The tumor-bearing nude mice were divided into 4 groups (5 in each) according the treatment they underwent. The tumor volumes and mice weight were measured to calculate the regression rate of tumor. The tumor was collected for H&E staining and immunohistochemical staining of AFP, Ki-67,Glypican-3 and CD105. Microvessel density (MVD) was measured by analyzing the CD105 expression. The tumors' proliferation index (PI) was studied by analyzing Ki-67 expression. The apoptosis of the tumor was quantitated using TUNEL staining. The mRNA expressions of cytokines were determined by quantitative real-time PCR (RT-qPCR). Results The tumor volumes were significantly decreased after chemotherapy. No difference of tumor volume was found between CDDP group and Sunitinib group (41.61 ± 7. 61 vs. 67. 15 ± 5. 39, P＞0. 05). Significant differences of tumor volumes were found between CDDP group and CDDP+ Sunitinib group (41.61 ± 7. 61 vs. 23. 03 ± 2. 37, P＜0. 05), and between Sunitinib group and CDDP+ Sunitinib group (67. 15 ± 5. 39 vs. 23.03 ± 2. 37, P＜0. 05). Of the body weight of tumor-bearing mice, no difference was found between controls and Sunitinib group (25. 90 ± 0. 75 vs. 26. 66 ± 0. 65, P＞0. 05). And significant differences of the body weight were noted between controls and CDDP group (25.90 ± 0. 75 vs. 18. 90 ± 0. 63, P＜0. 05),controls and CDDP+ Sunitinib group (25. 90 ± 0. 75 vs. 18. 26 ± 1.20,P＜0. 05). The positive areas (pixels) of AFP in the mice with chemotherapy were less than those of control mice (Control vs. CDDP: 1.26 × 106±1.48 × 105 vs. 5. 54 × 105 ± 8. 14 × 104 , P＜0. 05. Control vs. Sunitinib: 1.26 × 106 ± 1.48 × 105 vs. 7. 09 × 105 ± 6. 64 × 104, P＜0. 05. Control vs. CDDP + Sunitinib: 1.26 × 106 ± 1.48 × 105 vs. 3. 62 × 105 ± 4. 83 × 104, P＜0. 05). The positive areas (pixels) of Glypican-3 in the mice with chemotherapy were less than those of control mice (Control vs. CDDP: 9. 68 × 105 ± 7. 63 × 104 vs. 4. 04 × 105 ± 5. 04 × 104 , P＜0. 05. Control vs. Sunitinib: 9. 68 × 105 ± 7. 63 × 104 vs. 4. 59 × 105 ± 2. 32 × 104 , P＜0. 05. Control vs. CDDP + Sunitinib: 9. 68 × 105 ± 7. 63 × 104 vs. 1.89 × 105 ± 2. 55 × 104, P＜0. 05). However, there were no statistical differences of the positive areas (pixels) of AFP and Glypican-3 between CDDP and Sunitinib groups (P＞0. 05). The PI was significantly decreased after chemotherapy (Control vs. CDDP: 58. 97 ± 1.38 vs. 42. 36 ± 1.28, P＜ 0. 01. Control vs.Sunitinib: 58. 97 ± 1.38 vs. 43. 48 ± 1.00, P＜0. 01. Control vs. CDDP+ Sunitinib: 58. 97 ± 1.38 vs.33. 34 ± 1.19, P＜0. 01 ). The apoptosis index (AI) was also significantly increased after chemotherapy (Control vs. CDDP: 1.69 ± 0. 20 vs. 54. 62 ± 2. 49, P＜0. 01. Control vs. Sunitinib: 1.69 ± 0. 20vs. 47. 32 ± 2. 00, P＜0. 01. Control vs. CDDP + Sunitinib: 1.69 ± 0. 20 vs. 63. 41 ± 2. 23, P＜0. 01). Significantly differences of PI and AI were found between CDDP and CDDP + Sunitinib groups (P＜0. 01 ), and Sunitinib and CDDP + Sunitinib (P＜0. 01 ). RT-qPCR study confirmed that the mRNA expressions of M-CSFR, PDGFR-β, RET and VEGFR-2 were decreased in the mice underwent chemotherapy. Conclusions Sunitinib is effective to suppress the pediatric testicular yolk sac tumor growth, and reduce the tumor volume. Sunitinib can inhibit the angiogenesis in tumor, induce apoptosis of tumor cells, and kill the tumor cells directly. Sunitinib is less toxic than CDDP, and synergistic with the antitumor effect of CDDP.     

二氧化碳对小鼠神经母细胞瘤细胞跨间皮细胞迁移影响的实验研究

目的 本实验研究不同气体条件对神经母细胞瘤细胞跨间皮细胞迁移的影响,探讨CO2气腹对腹腔肿瘤细胞迁移影响的潜在机制.方法 经0.125%胰酶预处理后,通过腹腔灌洗分离培养C57/BL6小鼠腹膜间皮细胞.间皮细胞纯化通过细胞形态学及免疫组化方法(小鼠间皮细胞角蛋白特异性抗体AE1/AE3)鉴定.MTT实验确定细胞活力.使用Transwell系统培养连续间皮细胞层,分别暴露于100%CO2及5%CO2 2 h,经荧光染色后的小鼠神经母细胞瘤细胞(Neuro2a)加入至Transwell系统上室,细胞迁移数目经多功能检测仪进行测定.结果 MTT实验提示,间皮细胞于5%CO2及100%CO2暴露后4 h、6 h、10 h及24 h后两组细胞活力分别为0.82±0.28比0.62±0.22、0.87士0.24比0.62±0.28、0.88±0.69比0.69±0.81及1.05±0.86比0.97±0.25,差别无统计学意义.迁移实验表明,间皮细胞于100%CO2中暴露后,细胞屏障功能下降,Neuro2a迁移数目增加258.56±71.08比77.05±27.89(P＜0.05).结论 100%CO2利于神经母细胞瘤细胞跨间皮细胞迁移.因此,小鼠模型中发现的CO2气腹后神经母细胞瘤转移增加可能与间皮细胞屏障削弱有关.

Abstract：

Objective This study was to assess the effect of CO2 pneumoperitoneum on transepithelial neuroblastoma cell migration. Methods Purification of primary murine peritoneal mesothelial cells was achieved by sequential, peritoneal lavage after 0. 125% trypsin pretreatment. Purity of the mesothelial cell culture was confirmed by cell morphology and immunohistochemical staining for specific cytokeratine (AE1/AE3). In all experiments vitality of the cells was confirmed by MTT assay. Monolayers of mesothelial cells were established on transwell systems. Following incubation with 100% CO2 or 5% CO2 for 2 h, fluorescent stained Neuro2a (neuroblastoma) cells were added to the upper chamber and their migration to the lower chamber was measured by multi - detection reader. Results The conversion of MTT was nearly same 4h, 6 h, 10 h and 24 h after 5% CO2 or 100% CO2 exposition,0. 82 ± 0. 28 VS 0. 62 ± 0. 22,0. 87 ± 0. 24 VS 0. 62 ± 0. 28, 0. 88 ± 0. 69 VS 0. 69 ± 0. 81 and 1. 05 ± 0. 86 VS 0. 97 ± 0. 25 respectively. Migration studies showed that the barrier function of the mesothelial monolayer decreased. A significantly increased migration of neuroblastoma cells was found after 100% CO2 exposure 258. 56 ± 71. 08 VS 77. 05 ± 27. 89 (P＜0. 05). Conclusions 100% CO2 facilitated transepithelial neuroblastoma cell migration. Thus, the increased neuroblastoma metastasis observed after CO2 pneumoperitoneum in mice might be related to an impaired mesothelial barrier function.     

SOX10在先天性巨结肠相关性小肠结肠炎发病中的作用

目的 探讨SOX10、潘氏细胞发育及分泌防御素-5与先天性巨结肠相关性小肠结肠炎的关系.方法 收集50例先天性巨结肠病变肠管,根据术前是否发生小肠结肠炎分为HAFC组(n=14)和HD组(n=36),并以20例正常结肠标本作对照组.采用免疫组织化学方法观察结肠中防御素-5蛋白质表达、潘氏细胞发育情况以及SOX10蛋白质表达情况.采用实时荧光定量PCR技术检测防御素-5 mRNA及Sox 10 mRNA表达情况.结果 防御素-5在正常肠管中不表达,HAEC组和HD组在肠腺隐窝基底处呈不同程度阳性表达.但前者阳性区域平均光密度值明显增高(0.33±0.039比0.10±0.031,P＜0.05),HAEC组防御素-5mRNA亦呈显著增高趋势(2.72±0.80比0.78±0.21,P＜0.05).对结肠组织同层切片进行潘氏细胞特异性产物溶菌酶免疫组化染色发现,对照组肠管中除1例存在弱阳性外其他均无阳性表达.HD组和HAEC组结肠中同样在隐窝基底处存在溶菌酶阳性细胞,可鉴别为化生的潘氏细胞,但HAEC组在发生率(78.6%)和细胞个数(2.97±0.80)明显高于HD组(27.8%,0.43±0.85)(P＜0.05).SOX10免疫产物主要在结肠神经节细胞膜及胞浆中表达,对照组、HAEC组、HD组阳性区域的平均光密度值递减(0.75±0.041,0.61±0.048,0.35±0.025),差异具有统计学意义(P＜0.05),同时RT-PCR检测显示Sox10 mRNA在各组中的表达与蛋白质水平呈平行结果.结论 SOX10可能通过影响潘氏细胞发育及分泌防御素-5在先天性巨结肠相关性小肠结肠炎的发生发展中起一定作用.

Abstract：

Objective To study the expression of SOX10 and Human α-defensins-5(HD-5 )in Hirschsprung's disease associated enterocolitis(HAEC) and expore the possible relationship between SOX10 and HAEC.Methods Fifty pathological colons of Hirschsprung's disease (HD) were divided into HAEC group (n = 14) and HD group(n = 36) according to the presence of preoperative enterocolitis,Twenty normal colons as control group.The protein and mRNA expression of HD-5 and SOX 10 were measured by immunohistochemical staining and real-time quantitative PCR Results Normal colons did not express HD-5 but positive expression of HD-5 was detected at the base of the crypts of Lieberkuhn in HAEC and HD groups in different degree.The mean optical density of HD-5 immunohistochemical staining (0.33 ± 0.039 vs 0.10 ± 0.031 )and HD-5 mRNA expression (2.72 ± 0.80 vs 0.78 ± 0.21 ) in HAEC group was apparently higher than those in HD group(P＜0.05).The expression of lysozyme,a specific product by Paneth Cell,on sequential sections was negative in control group except one sample.In HAEC group and HD group,positive expression of lysozyme can be seen in the crypts of Lieberkuhn,where the cells can be identified as Metaplastic Paneth cell,but the incidence (78.6% vs 27.8%) and the number of cells 2.97 ± 0.80 vs 0.43 ± 0.85) in HAEC group were obviously higher than those in HD group(2.97 ± 0.80 vs 0.43 ± 0.85) (P＜0.05).SOX10 was mainly located in the plasmalemma and cytoplasm of ganglion cell and its mean optical density in control group (0.75 ±0.041 ),HAEC group (0.61 ± 0.048)and HD group(0.35 ± 0.025) were decrement,the difference between three groups were statistically significant (P＜0.05).Meanwhile,Sox10mRNA detected by real-time quantitative PCR indicated the parallel result.Conclusions SOX10 may be an important factor in the pathogenesis and development of HAEC.     

HGF对TGF-β1诱导尿道瘢痕成纤维细胞α-SMA及细胞外基质过度合成的保护作用

目的 探讨HGF对TGF-β1诱导尿道瘢痕成纤维细胞α-SMA及细胞外基质过度合成的保护作用.方法 收集尿道下裂术后瘢痕组织的标本,进行尿道瘢痕成纤维细胞的分离和培养.待细胞生长成单层后,以胰蛋白酶消化传代.取第四代成纤维细胞用于实验,当细胞达到80%融合时,培养液中加入TGF-β1(5ng/ml)及HGF(10～40ng/ml).培养72 h后,用RT-PCR检测各组α-SMA mRNA的变化;ELISA测定细胞Ⅰ、Ⅲ型胶原及纤维结合素的表达.结果 TGF-β1能显著诱导α-SMA m-RNA表达.随HGF的加入,α-SMA m-RNA的表达则明显受到抑制(P＜0.05),且随HGF浓度的升高其阻抑作用呈逐渐增强趋势.对照组、单纯TGF-β1组、加入TGF-β1与10、20、40ng/ml HGF组的Ⅰ型胶原A值分别为0.51±0.04、0.78±0.05、0.71±0.02、0.63±0.03、0.57±0.02,Ⅲ型胶原A值分别为0.12±0.01、0.29±0.02、0.21±0.02、0.14±0.01、0.08±0.01,纤维结合素A值分别为0.24±0.03、0.51±0.02、0.49±0.01、0.38±0.02、0.28土0.01.表明TGF-β1同样能诱导Ⅰ、Ⅲ型胶原及纤维结合素的表达(P＜0.01),而HGF则可以有效地阻抑其表达,其效应呈剂量依赖性(P＜0.05).结论 HGF对TGF-β1诱导的尿道瘢痕成纤维细胞α-SMA及细胞外基质过度合成具有抑制作用.这为临床预防和治疗尿道瘢痕狭窄提供了理论依据.

Abstract：

Objective To examine the inhibitory effects of HGF on TGF-β1 induced a-SMA and extracellular matrix synthesis in cultured fibroblasts derived from urethral scar. Methods Fibroblasts isolated from urethral scar were cultured ex vivo. After the fibroblasts reached confluence, cells were detached using trypsin/ethylenediamine tetra-acetic acid. All experiments were performed using the cells at the fourth passage. At 80% confluence, TGF-β1 (5 ng/ml) and HGF (10-40 ng/ml) were added to the culture medium. After 72 hours co-incubation, the mRNA of a-SMA was studied by RTPCR. The productions of collagen Ⅰ, Ⅲ and fibronectin in supernatants were also examined using ELISA. Results TGF-β1 markedly induced a-SMA mRNA expression in cultured fibroblasts. However, HGF could abrogated TGF-β1-induced a-SMA mRNA expression in a dose-dependent manner (P＜0. 05). The levels of collagen type Ⅰ were 0. 51 ± 0. 04,0. 78 ± 0. 05,0. 71 ± 0. 02,0. 63 ± 0. 03, and 0. 57 ± 0. 02 in control, TGF-β1, TGF-β1 + 10 ng/ml HGF, TGF-β1 + 20 ng/ml HGF, and TGF-β1 +10 ng/ml HGF group, respectively. The levels of collagen type Ⅲ were 0. 12 ± 0. 01,0. 29 ± 0. 02,0. 21 ± 0. 02,0. 14 ± 0. 01, and 0. 08 ± 0. 01 in control, TGF-β1, TGF-β1 + 10 ng/ml HGF, TGF-β1 +20 ng/ml HGF, and TGF-β1 + 10 ng/ml HGF group, respectively. And the levels of fibronectin were 0.24±0.03,0.51 ± 0.02,0.49 ± 0.01,0.38 ± 0.02,0.28 ± 0.01 in control, TGF-β1, TGF-β1 +10 ng/ml HGF, TGF-β1 + 20 ng/ml HGF, and TGF-β1 + 10 ng/ml HGF group, respectively. TGF-β1 significantly stimulated collagen Ⅰ, Ⅲ and fibronectin production in fibroblasts (P＜0. 01 ). However,HGF could reduced abrogated the up-regulation of collagen Ⅰ, Ⅲ and fibronectin induced by TGF-β1 in a dose-dependent manner (P＜0. 05). Conclusions HGF can effectively inhibit TGF-β1 induced aSMA and extracellular matrix synthesis in cultured fibroblasts derived from urethral scar.     

维生素E拮抗邻苯二甲酸二(2-乙基)己酯尿道发育毒性的实验研究

目的 探寻维生素E(Vitamin E,VitE)对邻苯二甲酸二(2-乙基)己酯[Di(2-ethylhexy1)phthlate,DEHP]所致大鼠尿道发育毒性的拮抗作用及其可能机制.方法 GD12(gestation day12)SD孕鼠随机分为4组,每组20只:玉米油对照组、DEHP组(500 mg·kg-1·d-1)、DEHP(500mg·kg-1·d-1)+VitE(200mg·kg-1·d-1)组和VitE组(200mg·kg-1·d-1).各组分别于母鼠孕期12～19d(GD12～19)持续经口灌注给药.各组分别留取10只孕鼠让其正常分娩,出生第一天,即对新生大鼠计数,并在解剖显微镜下测量雄性新生鼠的肛门生殖器距离(anal genital distance,AGD)并称体重;雄性仔鼠70日龄时逐个检查尿道下裂的发生情况.余孕鼠在GD19d行破宫产取仔代鼠,应用逆转录-聚合酶链反应(RT-PCR)的方法检测胎鼠阴茎TGF-β1,TGF-βR3 mRNA的表达水平.DNA末端原位标记染色法(TUNEL法)检测胎鼠阴茎尿道上皮细胞凋亡情况.结果 各组TGF-β1mRNA表达水平分别为:正常组为0.63±0.07、DEHP组为0.96±0.12、DEHP+VitE组为0.65±0.07、VitE组为0.62±0.06,DEHP组表达明显较其他各组增高(P＜0.05).各组TGF-βR3mRNA表达水平分别为:正常组为0.47±0.10、DEHP组为0.75±0.10、DEHP+VitE组为0.49±0.09、VitE组为0.46±0.09,DEHP组表达明显较其他各组增高(P＜0.05).各组胎鼠阴茎凋亡指数分别为:正常组为(30±2.0)%、DEHP组为(8.8土1.1)%、DEHP+VitE组为(28.9±1.6)%、VitE组为(29.6±2.0)%,DEHP组凋亡细胞数较其他各组相比明显减少,差异有统计学意义(P＜0.01).导致大鼠尿道下裂的发生.VitE可降低DEHP上调的胎鼠阴茎TGF-β1,TGF-βR3 mRNA表达水平,恢复胎鼠阴茎尿道上皮细胞的凋亡水平.结论 VitE对DEHP所致尿道发育毒性具有拮抗作用,其机制可能与调控TGF-βs及胎鼠阴茎尿道上皮细胞的凋亡有关.

Abstract：

Objective To study the therapeutic effects of Vitamin E (VitE) on urethral development toxicity induced by di(2-ethylhexyl) phthalate (DEHP). Methods From gestational day 12 to gestational day 19 (GD 12-19) , the timed-pregnant Sprague-Dawley (SD) rats were fed with the eiwith 20 in each group: DEHP group, DEHP + VitE group, and VitE group. The control rats were fed with corn oil. Ten pregnant rats of each group delivered full-term infant rats. The male infant rats were counted, and the anal genital distance (AGD) and body weight were measured. Hypospadias morbidity was also counted on male rats after 17 postnatal days. In the rest 10 pregnant rats of each group, fetuses were harvested on GD19. Semi-quantitive RT-PCR was used to measure mRNA expression of TGF-β1 and TGF-βR3. TUNEL staining was used to measure cell apoptosis in the fetus' genital tubercles. Results Hypospsdias was observed in male rat after 17 postnatal days. The TGF-β1 mRNA of the control group, DEHP group, DEHP + VitE group, and VitE group is 0. 63 ± 0. 07,0. 96±0. 12, 0. 65 ±0. 07, and 0. 62± 0. 06, respectively. The TGF-βR3 mRNA of the control group,DEHP group, DEHP + VitE group, and VitE group was 0. 47 ± 0. 10, 0. 75 ± 0. 10, 0. 49 ± 0. 09,and 0. 46 ± 0. 09, respectively. The TGF-β1 mRNA and TGF-βR3 mRNA was up-regulated in the fetus of DEHP group (P＜0. 05). Cell apoptosis rate in fetus' genital tubercles on GD19 of the control group, DEHP group, DEHP + VitE, and VitE group was 30% ± 2. 0%, 8. 8% ± 1.1%, 28. 9% ±1.6%, and 29. 6% ± 2. 0%, respectively. Cell apoptosis rate was significantly reduced in the fetus of DEHP group (P＜0. 01). In the GD19 fetus treated with VitE, hypospadias morbidity, the TGF-β1 and TGF-βR3 mRNA expressions were significantly decreased. And cell apoptosis rate was significantly increased. Conclusions Vitamin E ameliorates the urethral development toxicity induced by DEHP via regulating TGFβs expression and cell apoptosis in rat fetus.     

RNA干扰趋化因子受体4基因表达对神经母细胞瘤体外侵袭能力的影响

目的 构建趋化因子受体4(CXCR4)小分子干扰RNA(siRNA)表达载体,研究其对体外神经母细胞瘤侵袭能力的影响.方法 选择CXCR4高表达的神经母细胞瘤SH-SY5Y细胞系,设计合成人CXCR4基因不同靶点的能编码siRNA的3条双链DNA序列,克隆到真核表达载体pSilencerTMneo中构建siRNA表达载体,体外脂质体介导转染SH-SY5Y细胞,用半定量RT-PCR分析CXCR4基因mRNA的变化,用免疫组织化学和Western blot分析CXCR4蛋白表达,Transwell小室检测细胞的侵袭能力.结果 成功构建了CXCR4-siRNA表达载体,转染后半定量RT-PCR检测神经母细胞瘤细胞CXCR4 mRNA丰度分别为siR1转染组0.32±0.09、siR2转染组0.35±0.13和siR3转染组0.33±0.11,相对于对照组0.58±0.13表达下降(P＜0.05);转染后免疫组化检测神经母细胞瘤细胞CXCR4的蛋白表达分别为siR1转染组75.98±4.81、siR2转染组75.52±3.95和siR3转染组76.35±6.51,相对于对照组92.196±3.89表达下降(P＜0.01);转染后Western b1ot检测神经母细胞瘤细胞CXCR4的蛋白表达分别为siR1转染组0.1103±0.0023、siR2转染组0.1203±0.015和siR3转染组0.1308±0.0018,相对于对照组0.4832±0.0012表达下降(P＜0.01);且转染后神经母细胞瘤细胞侵袭能力较对照组53.11±3.72降低(P＜0.05),siR1转染组为25.48±2.81、siR2转染组为30.89±2.77、siR3转染组为18.83±1.79.结论 CXCR4-siRNA表达载体通过降低CXCR4基因的蛋白表达能显著抑制神经母细胞瘤细胞的体外侵袭能力,有望为神经母细胞瘤的基因治疗开辟新途径.

Abstract：

Objective To explore the effect of silencing chemokine receptor type 4 (CXCR4) by siRNA on the invasion capability of neuroblastoma cell line SH-SY5Y in vitro. Methods Three siRNAs targeting CXCR4 were chemically synthesized and transfected into SH-SY5Y cells. The transfection efficiency was observed under fluorescence microscope. CXCR4 expression at mRNA and protein levels were detected by semi-quantitative RT-PCR and Western blotting. The invasion capability of the cells was evaluated by Boyden Chamber in vitro. Results Compared with control groups, after the SH-SY5Y cells being transfeeted with the three CXCR4 targeting siRNAs, CXCR4 mRNA in transfected cells significantly decreased (0. 32 ± 0. 09, 0. 35 ± 0. 13 and 0. 33 ± 0. 11 vs 0. 58 ± 0. 13, P＜0. 05 ), CXCR4 protein detected by immunohistochemistry was decreased (75. 98 ± 4. 81, 75. 52 ± 3. 95and 76. 35 ± 6. 51 vs 92. 196 ± 3. 89, P＜0. 01 ), CXCR4 protein detected by Western blotting was also decreased (0. 1103 ± 0. 0023, 0. 1203 ± 0. 0015 and 0. 1308 ± 0. 0018 vs 0. 4832 ± 0. 0012, P＜0. 01 ).The invasion capability of the SH-SY5Y cells was decreased 48 hours after the cells were transfected (25.48±2.81, 30.89±2.77 and 18.83± 1.79 vs 53. 11 ±3.72, P＜0.05). Conclusions Silencing CXCR4 by siRNA decreases the invasion capability of SH-SY5Y cells.     

急性脑损伤患儿下丘脑促肾上腺皮质激素释放因子水平的变化

Objective To explore the changes of corticotropin releasing factor (CRF) levels secreted by hypothalamus neuron in children with acute brain injury. Methods Fifty-one intracranial-infection children with brain injury and 11 intracranial-noninfection children with brain injury were chosen from pediatric intensive care unit of our hospital. Severities of their brain damage were evaluated by Glasgow score,and CRF level in cerebrospinal fluid (CSF) and serum TNF-α and IL-6 levels were measured by radioimmunoassay. Results There was no significant difference of Glasgow scores between the intracranial infection group and intracranial-noninfection group ( P = 0. 302 6 ), CSF CRF level of intracranial infection group was significantly lower than that of intracranial-noninfection group ( P ＜ 0. 01 ), serum TNF-α and IL-6 levels of intracranial infection group were significantly higher than those of intracranial-noninfection group ( P ＜ 0. 01,P ＜0. 001 ). As comparing to the children with Glasgow score of 6 ～ 7, the levels of CSF CRF and serum TNF-α and IL-6 in children with Glasgow score of 4 ～ 5 were significantly increased ( P ＜ 0. 05, P ＜ 0. 001 ).Conclusion CSF CRF level of the children with acute brain injury is changing, which may be concerned with the secretion of hypothalamus CRF neuron stimulated by TNF-α, IL-6 and hypoxia stress in children with brain injury.     

足月新生儿肺通气功能肺力学改变与心功能的关系

目的 探讨足月新生儿肺通气功能、肺力学及心功能的变化规律及其相关性.方法 200例足月儿,据日龄分为A、B、C、D四组,其日龄分别为0～24 h、～72 h、～1周、～28 d.采用德国耶格公司Master screen Paed型肺功能仪测定肺通气功能及单次阻断法测定气道阻力、肺顺应性.肺功能主要参数:潮气量(TV)、分钟通气量(MV)、呼吸系统顺应性(Crs)、呼吸系统阻力(Rrs).采用美国SonoSite 180 PLUS彩色多普勒超声诊断仪检测心功能.心功能主要参数:每搏输出量、心输出量(CO).结果 A、B、C、D四组的TV分别为20.2±3.8、21.1±3.7、22.3±4.5、23.9±4.9(ml),C、D组TV与A组比较,D组TV与B组比较,差异有显著性(P＜0.05).Rrs、Crs组间差异无显著性(P＞0.05).A、B、C、D四组CO分别为0.93±0.23、0.93±0.23、1.02±0.21、1.08±0.27(L/min),D组CO与A、B组比较差异有显著性(P＜0.05).CO与Rrs呈负相关(r=-0.16,P＜0.05),与MV、TV、Crs呈正相关(r分别为0.50、0.54、0.13,P均＜0.05).结论 足月儿生后72 h后肺通气功能趋于稳定.左心输出量于生后1周明显增加.肺通气功能和肺力学指标是影响心功能的因素.

Abstract：

Objective To investigate the dynamic change and correlation of the pulmonary ventilative function, mechanic and cardiac function in the term infants. Methods Twenty hundred term infants were divided into A 、B 、C and D groups by age which was 0 ～ 24 h, ～ 72 h, ～ 1 w and 28 d respectively. The lung ventilative and mechanical function were measured respectively by using techniques of tidal breathing flow-volume loop(TBFVL)and the single occlusion. The Master screen Paed-lung function devices of Germanic JAEGER Co. was be used in this study. The parameter of pulmonary function including minute volume(MV) ,tidal volume (TV), respiratory system compliance(Crs) and respiratory system resistance (Rrs). The cardiac function were measured by using SonoSite 180 PLUS color Doppler ultrasonic diagnostic apparatus. The main parameter of cardiac function including cardiac output(CO) and stroke volume(SV). Results The TV of A, B ,C and D group were 20. 2 ± 3.78,21. 1 ± 3.71,22. 3 ± 4. 48 and 23. 9 ±4.90 (ml)respectively, the TV of C and D group were higher than that of A group, and the TV of D group was higher than that of B group (P ＜ 0. 05).There were no significantly difference of Crs, Rrs among A, B, C and D group(P ＞ 0. 05). The CO of A,B,C and D group were 0.93 ±0. 23,0.93 ±0.23,1.02 ±0.21 and 1.08 ±0.27 (L/min) ,the CO of D group was higher than that of A and B groups (P ＜ 0. 05). The CO was negative correlation with Rrs (r = - 0. 16,P ＜ 0. 05) and positive correlation with MV、 TV、 Crs (r was 0. 50、 0. 54、0. 13 respectively, P ＜ 0. 05).Conclusion The lung ventilative function is mature gradually with increasing age. The cardiac output has been obviously improved for postnatal 1 week in the term infants. The pulmonary ventilative function and mechanic parameter are important effective factors of cardiac function.     

小儿肾母细胞瘤中SOCS-1和SOCS-3的表达及其临床意义

目的 探讨小儿肾母细胞瘤中细胞因子信号转导抑制因子-1(SOCS-1)和细胞因子信号转导抑制因子-3(SOCS-3)的表达,以及与小儿肾母细胞瘤不同病理类型之间的内在关系.方法 应用RT-PCR法及Western-Blot法测定10例小儿正常肾脏组织、30例小儿肾母细胞瘤及瘤旁组织中sOCS-1和SOCS-3的表达.结果 与正常小儿肾脏组织(mRNA表达:0.721±0.183、0.694±0.148,蛋白表达:0.746±0.168、0.652±0.183)相比,肾母细胞瘤组织中SOCS-1和SOCS-3 mRNA表达(0.156±0.027、0.103±0.021)及蛋白的表达(0.205±0.079、0.185±0.041)均明显降低(P＜0.01).在肿瘤不同病理类型之间SOCS-1和SOCS-3mRNA的表达(预后良好型:0.175±0.086、0.149±0.100,预后不良型:0.128±0.074、0.091±0.067)有差异(P＜0.05).瘤旁组织SOCS-1 和SOCS-3mRNA的表达(0.572±0.154、0.315±0.137)及蛋白的表达(0.628±0.183、0.439±0.137)与正常肾脏组织有差异(P＜0.05).结论 SOCS-1和SOCS-3的表达下降可能与小儿肾母细胞瘤的发生、发展密切相关,与病理分期也有一定联系.

Abstract：

Objective To investigate the expression levels of suppressor of cytokine signaling-1 and suppressor of cytokine signaling-3 in the Wilms' tumor and to study the inherent relationship between the different pathological types. Methods The expression levels of SOCS-1 and SOCS-3 were assessed by RT-PCR and Western-Blot in ten normal kidney tissues, thirty nephroblastoma specimens and adjacent tissues. Results Compared to the normal kidney tissues(mRNA expressions: 0.721±0.183、0.694±0.148,protein expressions:0.746±0.168、0.652±0.183), the expressions of mRNA (0.156±0.027、0.103±0.021)and protein(0.205±0.079、0.185±0.041)of the SOCS-1 and SOCS-3 in nephroblastoma were significantly lower (P＜0.01). There were statistically significant between mRNA expressions and the different pathological types(FH:0.175±0.086,0、149±0.100,UH:0. 128±0.074、0.091±0.067) (P＜0.05).The difference betweenneuroblastoma and the normal tissues in expressions of mRNA (0. 572 ± 0. 154、0. 315 ± 0. 137)and protein(0. 628± 0. 183、0. 439 ± 0. 137)of the SOCS-1 and SOCS-3 were statistically significant (P＜0. 05). Conclusions The low SOCS-1 and SOCS-3 expressions may have a role in the development and progress of nephroblastoma, and the different pathological types.     

RNAi抑制Wnt1信号通路对人神经母细胞瘤SH-SY5Y细胞增殖抑制的实验研究

Objective To detect the expression of Wnt1 in human neuroblastoma cell line SH-SYSY, and to observe the effect of Wntl signaling pathway on the proliferation of the cells. Methods PCR, Western blotting and immunofluorescence technology were used to test the expression of Wnt1 in human neuroblastoma SH-SY5Y cells. RNAi technology was used to observe its inhibitory effect on the growth of the cells. SiRNA targeting Wnt1 was transfected into SH-SY5Y cells. The protein expression of Wntl and β-catenin were detected by Western blotting at 48 hours after transfec-tion. The quantity and the morphologic changes of the cells were observed by light microscope. The proliferative rate of SH-SY5Y cells after RNAi transfection was studied by MTT assay. Results Wnt1 expressed well in human neuroblastoma SH-SYSY cells. And the cells were successfully transfected with siRNA targeting Wntl mediated by Lipofectamine in vitro. The levels of the Wnt1 proteins ex-pression by western blotting in negative control, vector, nonsense-siRNA and Wnt-1 RNAi groups were 0. 341±0. 064,0. 401±0. 078,0. 328±0. 034,0. 041±0. 015, respectively. As the downstream molecular in the Wntl signaling pathway, β-catenin protein expression were 0. 275±0. 054, 0. 299± 0. 060, 0. 271±0. 034, 0. 037±0. 018, respectively. Quantification analysis revealed that Wnt-1 spe-cifie RNAi reduced Wnt-1 and β-catenin protein significantly(P<0. 01 ), while there was no difference in the negative control, vector, nonsense-siRNA groups (P>0. 05). The morphological changes of the SH-SYSY cells after 24h and 48h of transfection were observed under the light microscope. The density of the cells were obviously decreased in the specific Wnt1-siRNA group at 24 hours post-trans-fection, the formation of lamellipodium were blocked, and the time of focal adhesive clone were de-layed, apoptotic bodies were observed, especially at 48 hours post-transfection, accompanying with lots of floating, round and dead cells. However, in the other three groups the SH-SY5Y cells grew well and stretched out many lamellipodium and no obvious floating and dead cells were seen. Metabolic activity at 0, 12, 24, 36 and 48 hour after transfection was determined by MTT assay. The cell viability was reduced significantly after treatment with specific Wnt-1 siRNA as compared with the negative control or vector or non-specific siRNA group (P<0. 01). In contrast, transfection of the negative control, vector or non-specific siRNA had no obvious effect on cell proliferation(P>0. 05). Conclusions Wnt1 can express in human neuroblastoma SH-SYSY cells. Wnt1 signaling pathway may play a very important role in the proliferation of Human neuroblastoma SH-SYSY cells.     

脓毒症患儿营养状况的评估

Objective To investigate the severity of infection which is related with serum albumin,cytokines(IL-6、IL-8 and TNF-α)and the concentration of amino acids,it can be helpful to early find malnutrition in sepsis children and provide theories for nutrition support.Methods This prospective study was performed on hospitalized children who were classified as sepsis group(n = 52)and severe sepsis group(n =41).Control group included 300 healthy children.The serum albumin,cytokines(IL-6,IL-8 and TNF-α)and the concentration of amino acids in different groups were measured before the nutrition support.Results Severe sepsis group were all with hypoproteinemia.The concentration of IL-6,IL-8 and TNF-α in severe sepsis group was(193.95±74.11)ng/L、(481.33±186.58)ng/L、(21.00±9.43)ng/L respectively,which were significantly higher than those of sepsis group and control group(P ＜0.01).The concentration of aspartic acid,argine and glycine in severe sepsis group was(23.6±8.5)μmol/L、(6.1±4.7)μmol/L、(101.4± 60.6)μmol/L respectively,which were significantly lower than those of sepsis group and control group(P ＜ 0.01).Conclusion The concentration of aspartic acid,argine and glycine which influence immunologic responses was significantly lower in severe sepsis group with hypoproteinemia,so early nutritional support can improve clirtical outcome.     

胎羊单侧输尿管不完全性梗阻后肾脏足细胞表型转化及PAX2、VEGF的表达

Objective To investigate the phenotypic change of the podocytes and paired box gene 2 (PAX2) and vascular endothelial growth factor (VEGF) expressions in fetal lambs' kidneys with unilateral partial ureteral obstruction. Methods Sixteen fetal lambs were randomly assigned to the obstruction and control group. The obstruction group included 12 lambs, whose superior segment ureters were tied with splited silastic tube to induce unilateral partial ureteral obstruction between 75and 85 gestational days. The control group had 4 fetal lambs subjected to sham operation. After birth,the kidneys of the lambs were harvested to study the phenotypic change of the podocytes as well as expressions of PAX2 and VEGF in the kidneys. Results Nine out of the 12 lambs of the obstruction group were delivered alive. All 4 sham operated lambs were delivered alive. Renal cortex cysts of varying sizes, interstitial fibrosis, a decrease in glomeruli number, and severe podocyte foot process fusion (4. 20± 1.08% vs 86. 79 ± 1.66%) were found in the obstructed kidneys compared with the control kidneys. The PAX2 expression in obstructed kidneys was significantly higher than that of the control kidneys (1.43 ± 0. 09 vs 2. 44 ± 0. 09, P＜0. 05). The VEGF expression was significantly decreased compared with the control kidneys (0. 80 ± 0. 15 vs 0. 33 ± 0. 14, P＜0. 05). Conclusions The changes of the phenotype and PAX2 and VEGF expressions may play roles in the pathogenesis of obstructive nephropathy.