RNA干扰抑制神经母细胞瘤细胞MYCN基因的初步研究

目的对RNA干扰抑制神经母细胞瘤细胞（LAN-5）MYCN基因表达进行初步研究。方法体外化学合成针对MYCN基因的小干扰RNA（siRNA），经脂质体Lipofectamine 2000包裹后转染神经母细胞瘤细胞，以无关siRNA和未转染的细胞为对照，采用SYBR绿色荧光染料Ⅰ的实时荧光定量RT-PCR检测siRNA的抑制效果。结果脂质体转染siRNA效率可达84.83％，化学合成的siRNA可抑制神经母细胞瘤细胞MYCN基因mRNA表达，转染72h后抑制率达58．3％。结论化学合成的siRNA可以有效抑制神经母细胞瘤MYCN基因的表达，为神经母细胞瘤基因治疗开辟了新的途径。     

RNA干扰对人神经母细胞瘤VEGF-A表达的抑制作用

目的 构建VEGF-A的短发夹RNA真核表达载体,并通过其介导的RNA干扰抑制人神经母细胞瘤细胞株SH-SY5Y VEGF-A的表达.方法 根据VEGF-A的cDNA序列合成两对寡核苷酸片段,退火后分别插入真核表达载体组成重组质粒psc001、psc002,对重组质粒进行PCR、测序鉴定验证并经Western Blot筛选,然后将筛选所得的质粒转染人神经母细胞瘤细胞株SH-SY5Y.转染48h后行Annexin V和PI双重标记流式细胞仪检测转染细胞凋亡情况,ELISA检测VEGF-A蛋白的表达水平.结果 PCR和测序证实寡核苷酸片段已成功插入质粒psc001、psc002中,流式细胞仪检测结果 显示转染的外源基因未引起显著的神经母细胞瘤细胞凋亡,ELISA结果 显示转染后细胞株SH-SYSY的VEGF-A蛋白表达受到显著抑制(P<0.05).结论 成功构建人VEGF-A的短发卡RNA真核表达载体,转染人神经母细胞瘤SH-SY5Y细胞后能显著抑制VEGF-A的表达,但没有引起显著的细胞凋亡(P0.05).     

RNA干扰对人神经母细胞瘤VEGF-A表达的抑制作用

目的 构建VEGF-A的短发夹RNA真核表达载体,并通过其介导的RNA干扰抑制人神经母细胞瘤细胞株SH-SY5Y VEGF-A的表达.方法 根据VEGF-A的cDNA序列合成两对寡核苷酸片段,退火后分别插入真核表达载体组成重组质粒psc001、psc002,对重组质粒进行PCR、测序鉴定验证并经Western Blot筛选,然后将筛选所得的质粒转染人神经母细胞瘤细胞株SH-SY5Y.转染48h后行Annexin V和PI双重标记流式细胞仪检测转染细胞凋亡情况,ELISA检测VEGF-A蛋白的表达水平.结果 PCR和测序证实寡核苷酸片段已成功插入质粒psc001、psc002中,流式细胞仪检测结果 显示转染的外源基因未引起显著的神经母细胞瘤细胞凋亡,ELISA结果 显示转染后细胞株SH-SYSY的VEGF-A蛋白表达受到显著抑制(P<0.05).结论 成功构建人VEGF-A的短发卡RNA真核表达载体,转染人神经母细胞瘤SH-SY5Y细胞后能显著抑制VEGF-A的表达,但没有引起显著的细胞凋亡(P0.05).     

RNA干扰对人神经母细胞瘤VEGF-A表达的抑制作用

目的 构建VEGF-A的短发夹RNA真核表达载体,并通过其介导的RNA干扰抑制人神经母细胞瘤细胞株SH-SY5Y VEGF-A的表达.方法 根据VEGF-A的cDNA序列合成两对寡核苷酸片段,退火后分别插入真核表达载体组成重组质粒psc001、psc002,对重组质粒进行PCR、测序鉴定验证并经Western Blot筛选,然后将筛选所得的质粒转染人神经母细胞瘤细胞株SH-SY5Y.转染48h后行Annexin V和PI双重标记流式细胞仪检测转染细胞凋亡情况,ELISA检测VEGF-A蛋白的表达水平.结果 PCR和测序证实寡核苷酸片段已成功插入质粒psc001、psc002中,流式细胞仪检测结果 显示转染的外源基因未引起显著的神经母细胞瘤细胞凋亡,ELISA结果 显示转染后细胞株SH-SYSY的VEGF-A蛋白表达受到显著抑制(P<0.05).结论 成功构建人VEGF-A的短发卡RNA真核表达载体,转染人神经母细胞瘤SH-SY5Y细胞后能显著抑制VEGF-A的表达,但没有引起显著的细胞凋亡(P0.05).     

RNA干扰对人神经母细胞瘤VEGF-A表达的抑制作用

目的 构建VEGF-A的短发夹RNA真核表达载体,并通过其介导的RNA干扰抑制人神经母细胞瘤细胞株SH-SY5Y VEGF-A的表达.方法 根据VEGF-A的cDNA序列合成两对寡核苷酸片段,退火后分别插入真核表达载体组成重组质粒psc001、psc002,对重组质粒进行PCR、测序鉴定验证并经Western Blot筛选,然后将筛选所得的质粒转染人神经母细胞瘤细胞株SH-SY5Y.转染48h后行Annexin V和PI双重标记流式细胞仪检测转染细胞凋亡情况,ELISA检测VEGF-A蛋白的表达水平.结果 PCR和测序证实寡核苷酸片段已成功插入质粒psc001、psc002中,流式细胞仪检测结果 显示转染的外源基因未引起显著的神经母细胞瘤细胞凋亡,ELISA结果 显示转染后细胞株SH-SYSY的VEGF-A蛋白表达受到显著抑制(P<0.05).结论 成功构建人VEGF-A的短发卡RNA真核表达载体,转染人神经母细胞瘤SH-SY5Y细胞后能显著抑制VEGF-A的表达,但没有引起显著的细胞凋亡(P0.05).     

RNA干扰对人神经母细胞瘤VEGF-A表达的抑制作用

目的 构建VEGF-A的短发夹RNA真核表达载体,并通过其介导的RNA干扰抑制人神经母细胞瘤细胞株SH-SY5Y VEGF-A的表达.方法 根据VEGF-A的cDNA序列合成两对寡核苷酸片段,退火后分别插入真核表达载体组成重组质粒psc001、psc002,对重组质粒进行PCR、测序鉴定验证并经Western Blot筛选,然后将筛选所得的质粒转染人神经母细胞瘤细胞株SH-SY5Y.转染48h后行Annexin V和PI双重标记流式细胞仪检测转染细胞凋亡情况,ELISA检测VEGF-A蛋白的表达水平.结果 PCR和测序证实寡核苷酸片段已成功插入质粒psc001、psc002中,流式细胞仪检测结果 显示转染的外源基因未引起显著的神经母细胞瘤细胞凋亡,ELISA结果 显示转染后细胞株SH-SYSY的VEGF-A蛋白表达受到显著抑制(P<0.05).结论 成功构建人VEGF-A的短发卡RNA真核表达载体,转染人神经母细胞瘤SH-SY5Y细胞后能显著抑制VEGF-A的表达,但没有引起显著的细胞凋亡(P0.05).     

RNA干扰对人神经母细胞瘤VEGF-A表达的抑制作用

目的 构建VEGF-A的短发夹RNA真核表达载体,并通过其介导的RNA干扰抑制人神经母细胞瘤细胞株SH-SY5Y VEGF-A的表达.方法 根据VEGF-A的cDNA序列合成两对寡核苷酸片段,退火后分别插入真核表达载体组成重组质粒psc001、psc002,对重组质粒进行PCR、测序鉴定验证并经Western Blot筛选,然后将筛选所得的质粒转染人神经母细胞瘤细胞株SH-SY5Y.转染48h后行Annexin V和PI双重标记流式细胞仪检测转染细胞凋亡情况,ELISA检测VEGF-A蛋白的表达水平.结果 PCR和测序证实寡核苷酸片段已成功插入质粒psc001、psc002中,流式细胞仪检测结果 显示转染的外源基因未引起显著的神经母细胞瘤细胞凋亡,ELISA结果 显示转染后细胞株SH-SYSY的VEGF-A蛋白表达受到显著抑制(P<0.05).结论 成功构建人VEGF-A的短发卡RNA真核表达载体,转染人神经母细胞瘤SH-SY5Y细胞后能显著抑制VEGF-A的表达,但没有引起显著的细胞凋亡(P0.05).     

RNA干扰对人神经母细胞瘤VEGF-A表达的抑制作用

目的 构建VEGF-A的短发夹RNA真核表达载体,并通过其介导的RNA干扰抑制人神经母细胞瘤细胞株SH-SY5Y VEGF-A的表达.方法 根据VEGF-A的cDNA序列合成两对寡核苷酸片段,退火后分别插入真核表达载体组成重组质粒psc001、psc002,对重组质粒进行PCR、测序鉴定验证并经Western Blot筛选,然后将筛选所得的质粒转染人神经母细胞瘤细胞株SH-SY5Y.转染48h后行Annexin V和PI双重标记流式细胞仪检测转染细胞凋亡情况,ELISA检测VEGF-A蛋白的表达水平.结果 PCR和测序证实寡核苷酸片段已成功插入质粒psc001、psc002中,流式细胞仪检测结果 显示转染的外源基因未引起显著的神经母细胞瘤细胞凋亡,ELISA结果 显示转染后细胞株SH-SYSY的VEGF-A蛋白表达受到显著抑制(P<0.05).结论 成功构建人VEGF-A的短发卡RNA真核表达载体,转染人神经母细胞瘤SH-SY5Y细胞后能显著抑制VEGF-A的表达,但没有引起显著的细胞凋亡(P0.05).     

RNA干扰对人神经母细胞瘤VEGF-A表达的抑制作用

目的 构建VEGF-A的短发夹RNA真核表达载体,并通过其介导的RNA干扰抑制人神经母细胞瘤细胞株SH-SY5Y VEGF-A的表达.方法 根据VEGF-A的cDNA序列合成两对寡核苷酸片段,退火后分别插入真核表达载体组成重组质粒psc001、psc002,对重组质粒进行PCR、测序鉴定验证并经Western Blot筛选,然后将筛选所得的质粒转染人神经母细胞瘤细胞株SH-SY5Y.转染48h后行Annexin V和PI双重标记流式细胞仪检测转染细胞凋亡情况,ELISA检测VEGF-A蛋白的表达水平.结果 PCR和测序证实寡核苷酸片段已成功插入质粒psc001、psc002中,流式细胞仪检测结果 显示转染的外源基因未引起显著的神经母细胞瘤细胞凋亡,ELISA结果 显示转染后细胞株SH-SYSY的VEGF-A蛋白表达受到显著抑制(P<0.05).结论 成功构建人VEGF-A的短发卡RNA真核表达载体,转染人神经母细胞瘤SH-SY5Y细胞后能显著抑制VEGF-A的表达,但没有引起显著的细胞凋亡(P0.05).     

RNA干扰对人神经母细胞瘤VEGF-A表达的抑制作用

目的 构建VEGF-A的短发夹RNA真核表达载体,并通过其介导的RNA干扰抑制人神经母细胞瘤细胞株SH-SY5Y VEGF-A的表达.方法 根据VEGF-A的cDNA序列合成两对寡核苷酸片段,退火后分别插入真核表达载体组成重组质粒psc001、psc002,对重组质粒进行PCR、测序鉴定验证并经Western Blot筛选,然后将筛选所得的质粒转染人神经母细胞瘤细胞株SH-SY5Y.转染48h后行Annexin V和PI双重标记流式细胞仪检测转染细胞凋亡情况,ELISA检测VEGF-A蛋白的表达水平.结果 PCR和测序证实寡核苷酸片段已成功插入质粒psc001、psc002中,流式细胞仪检测结果 显示转染的外源基因未引起显著的神经母细胞瘤细胞凋亡,ELISA结果 显示转染后细胞株SH-SYSY的VEGF-A蛋白表达受到显著抑制(P<0.05).结论 成功构建人VEGF-A的短发卡RNA真核表达载体,转染人神经母细胞瘤SH-SY5Y细胞后能显著抑制VEGF-A的表达,但没有引起显著的细胞凋亡(P0.05).     

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.     

A sequential treatment algorithm for infants with stage 4s neuroblastoma and massive hepatomegaly

Infants with 4s neuroblastoma (NB) and massive hepatomegaly have a guarded prognosis and mortality approaches 30%. We report on eight patients with 4s NB and massive hepatomegaly treated with multiple modalities. One patient had spontaneous tumor regression. Three patients had progressive disease and responded to chemotherapy. Four patients progressed despite intravenous chemotherapy, of whom two died, and two were salvaged with hepatic intra-arterial chemoembolization. Treatment of infants with stage 4s NB with massive hepatomegaly should be individualized based on disease course. A sequential approach with observation, intravenous chemotherapy, and intra-arterial chemoembolization, may improve the outcome of these infants.     

Successful treatment of progressive stage 4s hepatic neuroblastoma in a neonate with intra-arterial chemoembolization

Stage 4s neuroblastoma (NB) is a unique entity seen in infants less than 1 year of age, with metastatic disease confined to liver, skin, or bone marrow. Despite metastatic spread, stage 4s NB has a favorable outcome. An exception to this is seen in neonates who present with progressive enlargement of the liver with secondary respiratory compromise and liver failure. We describe a 4-week-old neonate who presented with 4s NB, with a rapidly enlarging liver, resulting in respiratory and hepatic failure, who had a rapid, sustained, and ongoing response to chemoembolization of the hepatic artery. This approach is feasible at this age, and may be effective in improving the outcome in this group of patients.     

Congenital dumbbell neuroblastoma

A rare case of congenital dumbbell neuroblastoma with paraplegia and sphincter involvement is described. Magnetic resonance imaging (MRI) clearly demonstrated both intraspinal and right paravertebral tumors in a patient who showed signs of paraplegia of the lower extremities at birth. The patient was treated by laminectomy and intraspinal tumor excision followed by retroperitoneal tumor excision. Mild chemotherapy consisting of low-dose cyclophosphamide and vincristine was carried out postoperatively for 6 months. Radiation therapy was not employed. At 13 months of age the patient showed no evidence of residual or recurrent tumor, and the paralysis of the right lower extremity was greatly improved. Offprint requests to: S. Uemoto     

Congenital cervical neuroblastoma

Primary congenital cervical neuroblastomas are very rare. A history of upper aerodigestive compromise with Horner syndrome can be of value for the early diagnosis of this lesion. Congenital neuroblastomas usually have a favorable outcome. Like all the other clinically relevant groups of neuroblastomas, management should take into consideration the biological findings of each tumor, which predicts outcome than other clinical findings.     

4S neuroblastoma: the long-term outcome

BACKGROUND: Stage 4S neuroblastoma is associated with a high rate of spontaneous maturation and involution, with survival rates of 70-90%. There is little long-term follow-up data describing the disease status or late effects. The aim of this study was to assess the clinical outcome and imaging findings in long-term survivors of 4S neuroblastoma. METHODS: The patient population was identified from a single centre over 26 years. Twenty-five of 31 consecutive patients were long-term survivors. Five died from disease progression and one from cerebral palsy related complications. All survivors underwent clinical examination. Abdominal ultrasound scanning, liver function tests, hepatitis viral screen, and urinary catecholamines were performed. RESULTS: The mean age at diagnosis was 8 +/- 9 weeks with a mean age when studied of 11 years and 10 months +/- 8 years. Twenty of 25 had no significant clinical findings, three had disease associated clinical abnormalities (neurological, multiple subcutaneous nodules). Three patients had treatment related effects (small testes, urethral stricture, radiation induced soft tissue hypoplasia, post-surgical Horners syndrome). Persistant adrenal enlargement and calcification was noted in three patients. Twelve patients had abnormal liver ultrasound findings ranging from mildly coarse echotexture to structural changes without evidence of hepatic dysfunction or infection. Treatment did not correlate with abnormal hepatic ultrasound findings. CONCLUSIONS: The majority of long-term survivors of stage 4S neuroblastoma have no clinically or radiologically significant sequelae but do have residual abnormalities. These findings have implications for subsequent management of unrelated medical conditions in this patient group.     

神经母细胞瘤的凋亡研究

研究临床神经母细胞瘤凋亡情况,了解其与预后的关系。方法取神经母细胞瘤石腊切片,运用原位DNA末端标记法,检测凋亡细胞,计算凋亡细胞占肿瘤细胞百分比,求得凋亡指数。结果预后好的神经母细胞瘤大于预后差的神经母细胞瘤的凋亡指数。若凋亡指数大于9％,提示自发性消退。结论凋亡指数对神经母细胞瘤判断预后、指导治疗有重要价值。     

Cytomegalovirus-associated stage 4S neuroblastoma relapsed stage 4

Neuroblastoma is one of the most frequent solid tumors in childhood, rarely recurrent after five years from diagnosis. Cytomegalovirus (CMV), a major pathogen causing congenital birth defects and severe opportunistic diseases, has been shown to have teratogenic, immunodepressive and oncogenic properties. The case of a girl with stage 45 neuroblastoma diagnosed at three months and relapsed as stage 4 five years later is reported. In both circumstances, active CMV infection was revealed by positive CMV-specific IgM and IgA antibodies, CMV-DNAemia and CMV culture. At three months, the patient presented with subcutaneous nodules, hepatosplenomegaly and increased aminotransferase levels, and the opsoclonus-myoclonus syndrome. Mental retardation developed later on. At 5 years, relapsed neuroblastoma was preceded by a mononucleosis-like syndrome concomitant with active CMV infection and decreased levels of immune cells and natural killer activity. Clinical, virologic, and immunologic findings suggest an immune-mediated pathogenic role for CMV in this tumor. © 1995 Wi1ey-Liss Inc.