9-顺式维甲酸协同γ-干扰素诱导神经母细胞瘤分化、凋亡及生长抑制的实验研究

目的 观察9-顺式维甲酸（9-cis retinoic acid，9-cis RA）联合γ干扰素（gammm interferon，γ-IFN）在体外诱导神经母细胞瘤（neuroblastoma，NB）SK-N-SH细胞分化、凋亡过程中细胞形态、周期的变化，探讨其协同作用的效果及其机制。方法 将细胞分为A（对照组）、B（9-cisRA用药组）、C（γ-IFN用药组）和D（9-cisRA和γ-IFN联合用药组）4组，在处理后适当的时间分别观察细胞形态学变化，进行分化神经节细胞计数，用Hoechst-PI荧光染色法观察细胞的凋亡和死亡，四甲基偶氮唑盐（MTT）法测定NB细胞抑制率，流式细胞仪（flow cytometry，FCM）检测细胞周期分布及其凋亡和死亡。结果 联合用药组细胞形态学分化明显，神经节细胞分化率显著高于其他各组（P〈0．01）；MTT法测定显示联合用药能显著提高NB细胞抑制率并明显优于单独用药；Hoechst-PI荧光染色及FCM钙依赖性磷脂结合蛋白V和碘化丙啶（annexin V／PI）染色法检测一致显示联合用药对于诱导NB细胞早期凋亡和坏死具有显著的协同作用；FCM细胞周期动力学分析发现联合用药组C0-G1期细胞比率增加，S期细胞比率减少。结论 9-cisRA联合γ-IFN在体外对NB细胞的分化、凋亡和生长抑制能产生明显的协同作用，为其联合应用于临床提供理论依据和指导。     

1,25(OH)2D3对载脂蛋白E缺乏鼠主动脉内皮细胞增生与凋亡及一氧化氮合酶表达的影响

目的 了解1,25(OH)2D3对载脂蛋白E缺乏鼠(apoE-/-)主动脉内皮细胞(EC)增生、凋亡及内皮型一氧化氮合酶(eNOS)表达的影响.方法 apoE-/-鼠主动脉EC分离培养,MTT法观察1,25(OH)2D3影响apoE-/-鼠EC生长,TUNEL检测细胞凋亡,RT-PCR检测Bc1-2、Fas mRNA和eNOS mRNA表达.结果 细胞对照组与无水乙醇对照组EC增生率差异无统计学意义[(0.162±0.031)vs.(0.158±0.006),p＞0.05],1,25(OH)2D3在10-4、10-5、10-6、10-7、10-8 mol/L时EC增生率高于对照组(P＜0.01),但不同浓度1,25( OH)2D3作用组之间差异无统计学意义[分别为(0.189±0.013)、(0.285±0.011)、(0.296±0.026)、(0.284±0.017)、(0.233±0.010),P ＞0.05],选定10-6mol/L 1,25(OH)2D3为研究浓度,干预分离培养apoE-/-鼠主动脉EC.1,25( OH)2D310-6mol/L组、细胞对照组、无水乙醇对照组凋亡指数分别为(15.14±3.19)、(18.94 ±4.22)、(19.27±4.58),Bcl-2 mRNA分别为(0.78±0.16)、(0.46±0.21)、(0.42±0.17),Fas mRNA分别为(0.43±0.12)、(0.79±0.21)、(0.81±0.20),eNOS mRNA分别为(0.56±0.16)、(0.39±0.13)、(0.35±0.11).25(OH)2D3干预组EC凋亡指数、Fas mRNA、eNOS mRNA降低,Bcl-2 mRNA增高(P均＜0.01),细胞对照组与无水乙醇对照组比较差异无统计学意义(P＞0.05).相关分析发现在1,25(OH)2D3干预组,eNOS表达量与凋亡指数、Fas mRNA呈负相关(r=-0.676、-0.758),与Bcl-2表达呈正相关(r =0.762),差异有统计学意义(P均＜0.01).结论 1,25(OH)2D3刺激apoE-/-鼠主动脉EC增生、抑制主动脉EC凋亡,影响凋亡相关基因Bcl-2、Fas mRNA表达,上调eNOS mRNA表达.     

鼠尾草酸增加1,25(OH)2D3诱导HL-60细胞分化及其机制研究

目的：已证实1, 25(OH)2D3可以诱导HL-60细胞向成熟单核细胞分化,但其不足之处是高钙血症和形成耐药株。该实验应用鼠尾草酸（CA）联合1, 25(OH)2D3研究对HL-60细胞分化的影响及引起细胞内活性氧（ROS）水平和细胞内钙离子浓度的改变。方法：应用鼠尾草酸,1,25（OH）2D3单独和联合应用处理HL-60细胞,共分为5组：空白对照组;低浓度1,25（OH）2D3（1 nmol/L）组、鼠尾草酸组;联合处理组（10 μmol/L鼠尾草酸和1 nmol/L 1,25（OH）2D3）;高浓度1,25（OH）2D3（100 nmol/L）组。每24 h监测1次,通过四唑氮蓝(MTT)法观察细胞生长,共72 h;收集处理后72 h的HL-60细胞,通过光学显微镜观察细胞形态,用流式细胞仪（FCM）检测不同处理组细胞周期及单核细胞分化标记CD14的表达,ROS和细胞内钙离子浓度。结果：72 h后,联合处理组HL-60细胞与空白对照组相比,细胞增殖明显受抑制（Ab= 0.560±0.020; P<0.01）,细胞形态具有成熟单核细胞特征,CD14的表达率升高（57.62%;P<0.01）,G0/G1 期细胞显著增多, ROS水平下降［（52.67±10.76）%,P＜0.01］;联合处理组细胞内钙离子水平同空白对照组比较差异无显著性（115.64±17.74 nmol/L,P>0.05）,但与高浓度1,25（OH）2D3组相比细胞内钙离子水平明显下降(P<0.01)。结论：鼠尾草酸可增强1,25（OH）2D3对HL-60细胞的诱导分化、增强抑制HL-60细胞增殖作用,细胞阻滞于G0/G1期,细胞内活性氧水平降低,这可能与细胞的诱导分化机制有关,且这一联合作用不增高细胞内钙离子水平。     

1,25-(OH)2D3抑制脂多糖诱导的脐血CD4+T细胞IL-13和IL-17的表达

目的 本研究旨在探讨生命早期脂多糖(LPS)对脐血CD4+T细胞白介素-13(IL-13)和白介素-17(IL-17)表达的影响及1,25-(OH)2D3对其表达的干预作用,为维生素D的临床合理应用及其对哮喘等变态反应性疾病的防治提供理论依据。方法 选取顺产的足月新生儿12例,断脐后立即取胎盘端脐静脉血50 mL,采用密度梯度离心法分离脐血单个核细胞(CBMCs),磁珠分选CD4+T细胞后依据不同的处理方法分为空白刺激组、LPS(10 μg/mL)单独刺激组和LPS(10 μg/mL)+1,25-(OH)2D3(10-8 mmol/L)共刺激组。培养72 h后采用双抗夹心酶联免疫吸附试验(ELISA)和Real-Time PCR分别检测培养上清液中IL-13和IL-17水平及CD4+T细胞中IL-13和IL-17 mRNA表达。结果 与空白刺激组相比,LPS单独刺激组培养上清液中IL-13和IL-17水平及CD4+T细胞中IL-13和IL-17 mRNA表达水平明显升高(均P<0.01),而1,25-(OH)2D3处理可降低其表达水平(均P<0.05),但仍高于空白刺激组(均P<0.01)。结论 LPS可促进脐血CD4+T细胞IL-13和IL-17的表达;1,25-(OH)2D3对于LPS诱导的脐血CD4+T细胞IL-13和IL-17表达具有抑制作用,提示1,25-(OH)2D3可能在变应原致敏的早期发挥一定保护作用。     

1,25-(OH)2D3对糖尿病大鼠肾脏Nephrin蛋白表达的影响

目的探讨1,25-(OH)2D3对糖尿病(DM)大鼠肾脏Nephrin蛋白表达的影响。方法Wistar大鼠分为对照组、DM组、1,25-(OH)2D3组。建立糖尿病大鼠模型,实验12周末观察各组血尿、蛋白尿、肾组织光镜电镜、免疫组织化学,采用反转录聚合酶链反应(RT-PCR)检测其肾皮质NephrinmRNA及蛋白的表达。结果与对照组及1,25-(OH)2D3组相比,DM组大鼠尿红细胞及尿蛋白排泄明显增多,肾小球细胞数亦增加(P<0.01,0.05),NephrinmRNA及蛋白质表达明显下调(Pa<0.01)。1,25-(OH)2D3组肾小球细胞数、NephrinmRNA及蛋白质表达与对照组相比无统计学差异(Pa>0.05)。结论1,25-(OH)2D3可减轻血尿、蛋白尿,上调NephrinmRNA及蛋白质的表达而有肾保护作用。     

PRDX6与阿糖胞苷联合作用于HL-60细胞的体外实验研究

目的研究过氧化氧化还原蛋白6(PRDX6)与阿糖胞苷(Ara-C)联合作用对HL-60细胞抑制率的影响,并进一步分析产生该影响的可能机制。方法以不同浓度Ara-C作用于HL-60细胞后,显微镜下观察细胞形态改变;与不同药物作用后,以MTT法检测各组细胞的生长抑制率,RT-PCR技术检测各组细胞PRDX 1～6 mRNA的表达。结果不同浓度Ara-C作用后各组细胞均发生明显的形态学变化,且随着Ara-C浓度的增加,HL-60细胞数目明显减少,细胞形态学变化愈加明显;与对照组比较,单用PRDX 6对HL-60细胞的抑制率为(2.6±0.005)%,差异无显著性(P>0.05),单用Ara-C及联合用药组对HL-60细胞的抑制率分别为(29.8±0.289)%、(33.7±0.222)%,差异有显著性(P<0.05);与单用Ara-C组比较,联合用药组对HL-60细胞的抑制率差异有显著性(P<0.05);与对照组比较,单用PRDX 6组PRDX 1～6 mRNA的表达量差异无显著性(P>0.05);与单用Ara-C组比较,联合用药组PRDX 1～2 mRNA、PRDX 4～5 mRNA表达量明显减少,差异有显著性(P<0.05);PRDX 3 mRNA与PRDX 6 mRNA在各组间的表达均无明显变化,差异无显著性(P>0.05)。结论 Ara-C对HL-60细胞的生长抑制作用具有浓度依赖性,联合应用PRDX 6可提高Ara-C对HL-60细胞的抑制率,其原因可能与PRDX 1～2 mRNA、PRDX4～5 mRNA表达量减少有关。     

三氧化二砷对维甲酸耐药的神经母细胞瘤细胞SK-N-AS中HoxC9表达的影响

目的 本研究通过检测三氧化二砷(ATO)处理前后维甲酸(RA)耐药的SK-N-AS细胞中HoxC9以及EZH2的表达,初步探寻ATO促进RA耐药的NB细胞分化的具体机制.方法 用CCK-8试剂盒测定ATO对NB细胞SK-N-AS增殖抑制率,倒置相差显微镜下观察细胞生长情况,并测量神经突触总长度值.利用质谱技术和非标定量...     

1,25-(OH)2D3对局灶节段性肾小球硬化大鼠尿足细胞排泄及肾小球WT-1分布的影响

目的 探讨1,25-(OH)2D3对局灶节段性肾小球硬化(FSGS)大鼠尿足细胞(UPC)排泄及肾小球WT-1蛋白分布的影响.方法 24只SD大鼠分为3组:对照组、FSGS组、1,25-(OH)2D3组.采用左肾摘除,阿霉素重复注射诱导FSGS大鼠模型.1,25-(OH)2D3组在第1次给予阿霉素后1周埋植渗透性微量泵按0.03 ng/(g·d)皮下给予1,25-(OH)2D3.实验10周末检测尿白蛋白排泄率(UAER)、尿转化生长因子-β1(TGF-β1)及结缔组织生长因子(CTGF).免疫荧光法检测尿沉渣足细胞特异性标志蛋白Wflm's tumor-1(WT-1),观察肾小球WT-1的分布.尿WT-1荧光细胞即为尿液足细胞.结果 FSGS组UPC、UAER、TGF-β1、CTGF、肾小球细胞数及细胞外基质(ECM)/肾小球毛细血管襻面积均较对照组明显升高(P<0.01).与FSGS组相比,1,25-(OH)2D3组UAER、ECM/肾小球毛细血管襻面积显著降低(P<0.01),UPC、TGF-β1、肾小球细胞数亦降低(P<0.05),CTGF无统计学意义(P>0.05).肾小球荧光染色示WT-1在对照组正常,FSGS组呈节段性缺失,1,25-(OH)2D3组缺失较轻.UPC与UAER呈正相关(r.=0.42,P<0.05),TGF-β1与CTGF亦呈正相关(r.=0.47,P<0.05).结论 尿液中脱落足细胞检测可作为判断.FSGS病情活动性的标志之一.1,25-(OH)2D3可减轻FSGS大鼠UPC、UAER、TGF-β1的排泄,抑制肾小球细胞数及ECM增殖,恢复肾组织WT1-表达而有肾保护作用.     

1,25-(OH)2D3对哮喘小鼠肺内TIM-4表达的影响

目的：建立小鼠哮喘气道重塑模型,观察1,25-(OH)2D3对哮喘小鼠气道结构及T细胞免疫球蛋白域粘蛋白域蛋白-4（T cell immunoglobulin mucin protein-4, TIM-4）表达的影响。方法：30只BALB/c雌性小鼠随机分为对照组、哮喘组和1,25-(OH)2D3干预组,采用卵清蛋白致敏、激发建立哮喘模型。取小鼠肺组织,采用苏木精-伊红染色观察气道重塑情况,并应用RT-PCR法和免疫组化法检测肺内TIM-4 mRNA和蛋白的表达。结果：哮喘组发生典型的气道重塑改变,与对照组比较,TIM-4表达水平升高（105±9 vs 42±5,P<0.05）;1,25-(OH)2D3干预组较哮喘组气道重塑有所改善,TIM-4表达降低（78±6）,差异有统计学意义（P<0.05）。结论：TIM-4可能参与小鼠气道重塑过程;新型免疫调节剂1,25-(OH)2D3可下调哮喘小鼠肺内TIM 4的表达,改善气道重塑。     

活性维生素D及其类似物对足细胞的保护作用

1,25(OH)2D3产生不足是慢性肾脏病(CKD)的主要特征之一,不仅导致钙磷代谢异常及骨矿化障碍,而且加速肾疾病的进展。1,25(OH)2D3及其类似物可减轻CKD患者的蛋白尿,减轻动物模型足细胞的损伤、凋亡和脱落,促进裂孔隔膜蛋白的表达,维持肾小球滤过屏障的完整性。1,25(OH)2D3保护足细胞机制与抑制足细胞肾素-血管紧张素系统、阻断Wnt/β-Catenin和转化生长因子(TGF)-β1信号通路有关。     

Effects of 1,25-Dihydroxyvitamin D3 and Retlnolc Acid on the Proliferation and Cell Cycle Phase Distribution of Neuroblastoma SK-N-SH Cells

The hormone 1,25-(OH)₂D₃ has been shown to modulate cell proliferation and induce differentiation in several normal and malignant cell lines.In this work, we examined the effect of the hormone on the neuroblastoma SK-N-SH cell line.The steroid did not influence cell growth and cell cycle distribution, while retinoic acid inhibited proliferation and induced an accumulation of the cells in the G₀/G₁ phase of the cell cycle.1,25-(OH)₂D₃ did not alter cell morphology.The activities of the 1-alpha- and 24-hydro aylases were low and not regulated by the hormone.The level of the total 1,25-(OH)₂D₃ receptor was low.We conclude that the lack of effect of 1,25-(OH)₂D₃ on the SK-N-SH cell line is related to the low level of the 1,25-(OH)₂D₃ receptor.     

Cytotoxicity of lymphokine-activated killer cells against human neuroblastoma cells: modulation by neuroblast differentiation

The cytolytic activity of lymphokine-activated killer (LAK) cells against human neuroblastoma (NB) cells was investigated using the continuous NB cell lines, IMR-32, Kelly, and two subclones of SK-N-SH, SH-SY5Y (neuroblastic phenotype), and SH-EP (non-neuronal phenotype). NB cells were found to be sensitive targets of LAK. Of the SK-N-SH subclones, the neuroblasts, SH-SY5Y, were more susceptible to LAK killing than were the non-neuronal cells, SH-EP. Pretreatment of the targets SH-SY5Y and SH-EP with the differentiating agents, retinoic acid (RA, 10 microM), herbimycin A (236 nM), or nerve growth factor (10 ng/ml), did not substantially alter LAK killing. Furthermore, these differentiating agents did not measurably affect LAK activity during the cytolysis assay or with 1-h preincubation of the LAK effectors. However, co-incubation of the LAK cultures over the 3-day activation period with RA (1 microM) or PGE2 (1 microM) inhibited cytolysis by 80%, suggesting that these agents interfere with an early activation step of LAK. These results support the potential use of LAK treatment for neuroblastoma, in combination with differentiation agents that do not affect neuroblastoma sensitivities toward LAK cells. However, some differentiation agents, (e.g., RA) and endogenous prostaglandins (e.g., PGE2) may interfere with LAK activation.     

The vitamin A analogues: 13-cis retinoic acid, 9-cis retinoic acid, and Ro 13-6307 inhibit neuroblastoma tumour growth in vivo

BACKGROUND: Neuroblastoma, a childhood tumour of the sympathetic nervous system, may undergo spontaneous differentiation or regression due to apoptosis after no or minimal therapy. However, the majority of neuroblastomas are diagnosed as metastatic tumours with a poor prognosis in spite of intensive multimodal therapy. Vitamin A and its analogues (retinoic acid, RA) play an important role in normal cel lular differentiation and programmed cell death. RA regulates neuroblastoma growth and differentiation in vitro, and has shown activity against human neuroblastoma in vivo. PROCEDURE: Recently, 9-cis RA was shown to induce apoptosis in vitro in neuroblastoma using a 5 days short-term treatment and subsequent washout. In the present study, nude rats with human neuroblastoma SH-SY5Y xenografts were treated with 13-cis RA (4 mg po daily), 9-cis RA (5 mg po daily) or the novel analogue Ro 13-6307 (0.3 mg po daily) using either a continuous or short-term schedule. RESULTS: ALL three different retinoids decreased neuroblastoma growth significantly in terms of tumour weight after 8-12 days when compared to untreated controls (P < 0.05). Minor signs of toxicity in 13-cis RA treated rats were observed. However, severe toxicity with significant weight loss was seen in all rats treated with 9-cis RA and Ro 13-6307. Toxicity was more pronounced with the continuous regimen. CONCLUSIONS: We conclude that different retinoids reduce neuroblastoma tumour growth in vivo. Drug scheduling and dosage may affect both therapeutic efficacy and toxic side effects. Further in vivo studies are warranted, including pharmacokinetic and molecular analyses, before clinical trials with promising retinoids like 9-cis RA and Ro 13-6307 can be started in children with neuroblastoma.     

Differential gene regulation by 9-cis and all-trans retinoic acid in neuroblastoma cells

BACKGROUND: 9-cis retinoic acid (RA) is more effective than all-trans RA at inducing neuroblastoma differentiation in vitro, and has distinct biological properties with respect to its ability to promote apoptosis in N-type neuroblastoma cells. The cellular effects of 9-cis RA may, in part, result from activation of retinoid X receptor (RXR) homodimers. If this hypothesis is correct, 9-cis RA may control the expression of a different subset of retinoid-regulated genes compared to all-trans RA. PROCEDURE: We have therefore used differential mRNA display to identify genes differentially expressed in neuroblastoma cells in response to all-trans and 9-cis RA. RESULTS: The majority of cDNAs differentially expressed in response to all-trans or 9-cis RA matched to nonredundant Genbank sequences or EST database sequences. Differential-display profiles were similar in SH SY 5Y and SH S EP cells, clonal derivatives of the mixed neuroblastoma cell line SK N SH, although there were apparent differences between these cell lines with respect to the retinoid-regulation of specific RT-PCR cDNA fragments. CONCLUSIONS: These data support the view that 9-cis and all-trans RA act via different receptor mechanisms.     

Prenatal administration of retinoic acid increases the trophoblastic insulin-like growth factor 2 protein expression in the nitrofen model of congenital diaphragmatic hernia

Background

The high mortality rate in congenital diaphragmatic hernia (CDH) is attributed to pulmonary hypoplasia (PH). Insulin-like growth factor 2 (IGF2) is an important regulator of fetal growth. The highest levels of IGF2 expression are found in the placenta, which are negatively regulated by decidual retinoid acid receptor alpha (RARα). It has been demonstrated that prenatal administration of retinoic acid (RA) suppresses decidual RARα expression. Previous studies have further shown that prenatal administration of RA can reverse PH in nitrofen-induced CDH model. In IGF2 knockout animals, low levels of IGF2 are associated with decreased placental growth and PH. We therefore hypothesized that nitrofen decreases trophoblastic IGF2 expression and prenatal administration of RA increases it through decidual RARα in the nitrofen-induced CDH model.

Methods

Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). RA was given intraperitoneally on D18, D19 and D20. Fetuses were harvested on D21 and divided into three groups: control, CDH and nitrofen+RA. Immunohistochemistry was performed to evaluate decidual RARα and trophoblastic IGF2 expression. Protein levels of IGF2 in serum, intra-amniotic fluid and left lungs were measured by enzyme-linked immunosorbent assay.

Results

Significant growth retardation of placenta and left lungs was observed in the CDH group compared to control and nitrofen+RA group. Markedly increased decidual RARα and decreased IGF2 immunoreactivity were found in the CDH group compared to control and nitrofen+RA group. Significantly decreased IGF2 protein levels were detected in serum, intra-amniotic fluid and left lungs in the CDH group compared to control and nitrofen+RA group.

Conclusion

Our findings suggest that nitrofen may disturb trophoblastic IGF2 expression through decidual RARα resulting in retarded placental growth and PH in the nitrofen-induced CDH. Prenatal administration of RA may promote lung and placental growth by increasing trophoblastic IGF2 expression.     

SPONTANEOUS APOPTOSIS AND RETINOIC ACID RECEPTOR INCIDENCE IN NEUROBLASTOMAS AND PERIPHERAL NEUROECTODERMAL TUMORS

Twelve cases of neuroblastoma (NB) (7 boys and 5 girls) and 4 cases of primitive peripheral neuroectodermal tumor (PNET) (3 boys and 1 girl) were investigated for the presence of apoptosis and retinoic acid receptor (RAR) by immunhistochemical method. The apoptotic index in NB was zero or 1% in 8 children and relatively low (2-4.8%) in the other 4 cases, while it was higher (4.1-10.5%) in PNET. The RAR index determined by immunoperoxidase reaction in NB was zero or 3% in 5 cases and 9-34% in 7 children. RAR index in PNET was 16-68% in all the 4 cases. Good correlation (r=.47 according to Pearson-Bravis) was found between the number of RAR and spontaneus apoptosis. These results suggest that the RAR index in untreated NB and PNET shows great individual variation since its determination is necessary for the evaluation of the efficacy of retinoic acid treatment.