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Antigene peptide nucleic acid specifically inhibits MYCN expression in human neuroblastoma cells leading to cell growth inhibition and apoptosis Roberto Tonelli,Stefania Purgato,Consuelo Camerin,Raffaele Fronza,Fabrizio Bologna,Simone Alboresi,Monica Franzoni,Roberto Corradini,Stefano Sforza,Andrea Faccini,Jason M. Shohet,Rosangela Marchelli,and Andrea Pession Department of Pediatrics, University of Bologna,Sant'Orsola-Malpighi Hospital, Bologna, Italy; Department of Organic and Industrial Chemistry, University of Parma, Parma,Italy; and 3Center for Cell and Gene Therapy, Texas Children's Cancer Center, Baylor College of Medicine, Houston, Texas Abstract We developed an antigene peptide nucleic acid for selective inhibition of MYCN transcription in neuroblastoma cells, targeted against a unique sequence in the antisense DNA strand of exon 2 of MYCN and linked at its NH2 terminus to a nuclear localization signal peptide.

抗原肽核酸抑制MYCN具体表达的神经母细胞瘤细胞,导致细胞生长的抑制和凋亡罗伯托利,斯特凡Purgato ,苏埃洛卡默琳,拉菲尔Fronza ,梅奥尼博洛尼亚,西蒙娜Alboresi ,莫妮卡弗兰索尼,罗伯托Corradini ,斯特凡诺斯福尔扎,安德烈Faccini ,杰森先生Shohet , Rosangela Marchelli ,和安德烈Pession儿科,博洛尼亚大学,圣' Orsola -马尔皮基医院,博洛尼亚,意大利;有机部和工业大学化学帕尔马,帕尔马,意大利和3Center的细胞和基因治疗,德州儿童癌症中心, Baylor医学院,得克萨斯州休斯敦摘要我们开发了一种抗原肽核酸的选择性抑制神经母细胞瘤MYCN转录细胞,针对一个独特的序列反义DNA链的第2外显子,并与在MYCN 其氨基端为核定位信号肽。

In this study, we utilized NCCmelan5 and NCCmelb4 melanoblast, two melanoblast cell lines which were previously used to investigate the treatment of vitiligo, to investigate the anti-apoptotic effects of LED (630nm), and ruled out others causes of repigmentation including proliferation, mitochondrial metabolism, melanin production, migration distances of MBs after irradiation, to formulate the theoretical basis LED's treatment of vitiligo.

在此研究中,我们使用研究白斑常用的NCCmelb4 and NCCmelan5两株黑色素母细胞珠来研究LED 光源对黑色素母细胞的抗凋亡的细胞保护功能,并且排除其对於细胞直接造成细胞增生,细胞移动,或黑色素增加的机转,希望为其治疗白斑的应用提供依据。

The primary sporogenous cells divided into the second sporogenous cells which would turn into microspore mother cells in a microsporangium. The cytokinesis in meiosis of the microspore mother cells was of modified simultaneous type, forming the decussate, isobilateral or T-shaped tetrads.

初生造孢细胞分裂形成次生造孢细胞,次生造孢细胞再转化为小孢子母细胞,小孢子母细胞减数分裂的胞质分裂为修饰性同时型,四分体排列方式为交叉型、对称型或"T"型,成熟花粉粒二细胞型,开花时散出。

The primary sporogenous cells divided into the second sporogenous cells which would turn into microspore mother cells in a microsporangium. The cytokinesis in meiosis of the microspore mother cells was of modified simultaneous type, forming the decussate, isobilateral or T-shaped tetrads.

初生造孢细胞分裂形成次生造孢细胞,次生造孢细胞再转化为小孢子母细胞,小孢子母细胞减数分裂的胞质分裂为修饰性同时型,四分体排列方式为交叉型、对称型或&T&型,成熟花粉粒二细胞型,开花时散出。

The size of follicle begin primordial follicle to tertiary follicle(1.5~3mm).The number of mitochondrion increased,and the cristae increased.Gogi body change to type,cortical granule increased,and moved behind cytolemma.The proportion of small,middle and large follicles on ovary were 92.51%(92.51±5.87),4.60%(4.60±4.91) and 2.88%(2.88±2.22) respectively.

卵泡在发育过程中,卵泡细胞形成的突起与卵母细胞始终保持密切的联系:卵母细胞从原始卵泡到直径1.5~3mm的三级卵泡,细胞质中线粒体数量由少到多,线粒体内的嵴不断增加,高尔基体由不典型到变成典型,皮质颗粒由开始形成到形成较多的皮质颗粒,并逐渐地迁移到卵母细胞膜下。

The results showed that: at metaphase I, the homoeologous chromosome pairing among different F1 hybrids ranged from 2.0 to 11.4 bi-valents formed by homoeologous chromosomes per pollen mother cell, and very few multivalents, and even very few bivalents were formed by two chromosomes within one genome rather than homoeologous chromosomes in some PMCs; at anaphase I, all biva-lents were disjoined and most univalents were divided.

结果表明在中期I阶段,这些杂种一代的近缘染色体联会变化很大,每个花粉母细胞中二价体形成的数目从平均2个到11.4个不等,甚至在某些花粉母细胞中,还发现极少的多价体和非部分同源染色体所形成的单基因组内二价体;在后期I时,所有的二价体分离,同时多数单价体也分离,分离的二价体和分离的单价体都移向两极,从而形成两组染色体;因为这时完整花粉母细胞中分离的二价体在两组染色体中总是对应出现,从而根据半二价体上染色体重组的位置可以分析在二价体的四分体时期发生在非姊妹染色体之间的多种染色体交换类型,如单交换、三线双交换、四线双交换、四线三交换和四线多交换。

Histologically, the seminiferous tubules contained numerous Sertoli cells and more Sertoli-spermatozoa complexes, accompanied by the depletion of Leydig cells with deeply stained nuclei. Mature spermatozoa were stored up in the epididymis, but only a few in the efferent ducts. In the second place was testicular atrophy(32/120; 26.7%). The seminiferous tubules showed moderate to severe inactivity of spermiogenesis with evidence of only spermatogonia, spermatocytes and Sertoli cells. The Leydig cells were obviously decreased in numbers associated with decrease of lipid droplets in their cytoplasms. Testicular hypoplasia was the third disorders(22/120; 18.3%). Only a few spermatogonia and Sertoli cells appeared without any spermiogenesis. The associated changes was decreased in Leydig cells and fibrous hyperplasia in the interstitium.Epididymal stones were sometimes found(12/120; 10%). Grossly, yellowish-white nodules with various sizes and firm in consistency were observed in the epididymis and the front efferent ducts. Microscopically, the epididymal ducts were dilated with voluminous spermatozoa storage, even showed calcification in severe cases. The deposited calcium salts were stained positively by Von Kossa and Alizarin red methods.Amyloidosis was also detected in 10 roosters(8.3%). Eosinophilic, homogeneous, amyloid-like substances were deposited mainly in the testicular interstitium and the periphery of blood vessels. These substances showed positive reaction by Congo red staining. Five roosters(4.2%)had Marek's lesions in the testis, epididymis and peripheral nerves with infiltration of pleomorphic lymphocytes. Only one case showed epithelial necrosis of seminiferous tubules accompanied by fibrous proliferation in the interstitium.

结果发现,在总共搜集的120个病例中,其中因年老所导致的产精力不佳为最多,占38例(31.7%),於镜下可见大量精虫黏附於Sertoli cell的表面,并可见Sertoli cell数量明显增多而Leydig cell明显减少,且其细胞核呈现浓染的现象,而在其副睪中仍可见到成熟精虫蓄留於管腔中,但在其输精管内却只有少量精虫存在;其次为睪丸萎缩,占32例(26.7%),镜下可见中度至重度无造精作用,其生精小管中只见到精母细胞、精原细胞及Sertoli cell存在,但Leydig cell数量明显减少且其细胞质内的脂质也明显减少;睪丸发育不全,占22例(18.3%),於生精小管内只见到精母细胞及少量Sertoli cell存在,不见造精细胞分化,於生精小管间质可见Leydig cell减少并伴随结缔组织增生;副睪结石,占12例(10%),肉眼下可在副睪及输精管前段见到黄白色大小不一的结节,触感坚硬,於镜下可见副睪管扩张并有大量成熟精虫蓄积,严重时可见钙化现象,以Von Kossa及茜素红染色均呈阳性反应;类淀粉沉著症,占10例(8.3%),镜下在睪丸间质及血管周围可见粉红均质样的物质沉积,以刚果红染色成阳性反应;马立克病,占5例(4.2%),镜下可在睪丸、副睪实质及周边神经内均可见到嗜碱性大小不一的淋巴样细胞浸润;睪丸坏死,占1例(0.8%),镜下可见生精小管上皮细胞坏死脱落及间质结缔组织增生。

Membrane potential, intracellular Na+, K+, Cl, H+ activities and Na+, K+ transference number in toad oocytes were measured in vitro by using conventional and ion-sensitive microelectrodes during the insulin-induced oocyte maturation.

近年来,胰岛素及胰岛素样肤类物质在卵母细胞成熟中的作用越来越引起人们的关注。EI一Etr等(1979)用非洲爪蟾卵母细胞作实验,发现胰岛素及其类似物具有促进卵母细胞成熟分裂的作用。

Methods: Oocytes in the GV stage were separated from ovary by squeezing method. In mouse germinal vesicle GV stage, the expression of ATP8 gene in the mitochondria in the single oocyte was detected by RT-PCR, in which, cDNA was synthesized with two methods: one was the single GV-stage oocyte directly to be placed RT, the other was to perform RT after eliminating mtDNA and nucleus DNA with the EeoR Ⅰ enzyme and Dnase. And the product of RT-PCR was cloned and sequenced.

应用挤压法从卵巢中分离获得生发泡期(germinal vesicle, GV)卵母细胞;用RT-PCR检测GV期单个卵母细胞中ATP8基因的表达:其中cDNA的合成分两种方法进行:一是将GV期单个卵母细胞直接进行RT合成cDNA,二是先用DNA酶加EcoR Ⅰ酶祛除mtDNA和核DNA后再进行RT;回收产物构建克隆质粒并测序。

The cumulus cells of OCC were cut off and dispersed by 1 mL syringe. The cumulus cells were co-cultured with the immature oocytes retrieved from the COH cycles after they adherent to the bottom of the dish. The immature oocytes were experienced IVM procedures in different culture media. They were divided into 3 groups(the oocytes at germinal vesicle stage from one woman were allotted to the same group randomly). Group 1: basic culture medium+ human follicular fluid; Group 2: solution A+ cumulus cells; Group 3: solution A+ OCC+ follicle stimulating hormone + epidermal growth factor. Then, the maturation rate, fertilization rate and formation rate of available embryo were observed.

在控制性促排卵周期有未成熟卵母细胞时,将同周期成熟卵丘复合体切出部分卵丘细胞,用1 mL注射器抽打分散细胞,贴壁培养。113个治疗周期中,298枚生发泡期卵母细胞经3种不同培养液体外成熟培养(同一病人的生发泡期卵被随机分到某同一组中):第1组28个周期中73枚:基础培养液+卵泡液;第2组40个周期中115枚:A液+分散贴壁的卵丘细胞;第3组45个周期中110枚:A液+分散贴壁的卵丘细胞+促卵泡生成激素+表皮生长因子。

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