BioVector® RBL20 人视网膜母细胞瘤细胞株

BioVector® RBL20 Human Retinoblastoma Cell Line

第一部分 中文说明

一 产品基本信息与遗传学背景

• 细胞名称：BioVector® RBL20

• 保藏机构货号：DSMZ ACC 944

• 物种来源：人类 (Homo sapiens)

• 组织与疾病背景：该细胞株建立于1988年，分离自一名患有双侧视网膜母细胞瘤（Bilateral retinoblastoma）的16个月大幼儿患者的恶性原发肿瘤组织。视网膜母细胞瘤是一种高度恶性的儿童神经外胚层眼内恶性肿瘤。

• 核心致癌与抑癌基因突变特征（RB1 灭活特征）：

  • 作为经典的视网膜母细胞瘤模型，该细胞系伴有双股抑癌基因 RB1（Retinoblastoma 1） 的病理性完全灭活。

  • 具体的突变分子特征为：第一条等位基因携带经典的剪切位点变异 IVS6+1G>T，而另一条等位基因则发生了杂合性丢失（Loss of Heterozygosity, LOH）。这一双击（Two-hit）遗传学事件导致了细胞内 RB1 肿瘤抑制蛋白的彻底缺失，从而使细胞周期进入无限制性循环，驱动视网膜神经上皮层的恶性克隆演进。

• 生物安全级别：1级（BSL-1）。

二 细胞形态学与培养环境

• 形态学特征：展现典型的视网膜母细胞瘤神经外胚层分化特征。在倒置显微镜下，细胞通常呈小圆形或多角形，且高度倾向于聚集形成大小不一、松散或紧密的球状细胞悬浮成团簇、类器官样小球（Spherical cluster aggregate clumps in suspension），极少以单细胞分散存在。

• 生生模式：悬浮生长（通常成团块、球状悬浮）。

• 群体倍增时间（Doubling Time）：大约 48 小时左右。

• 标准完全培养基配方：

  • 基础培养基：80% 至 90% BioVector® RPMI-1640 培养基。

  • 维持添加：10% 至 20% 优质热灭活胎牛血清（h.i. FBS）。

  • 1% Penicillin-Streptomycin 双抗溶液。

• 物理培养参数：37摄氏度恒温、5% 二氧化碳、空气饱和湿度。

三 细胞传代与复苏标准操作步骤

1. 初始接种与常规传代操作（Subculturing Protocol）：

   • 日常传代：由于细胞高度倾向于形成球状密集团簇悬浮生长，在传代时需要物理性地辅助吹打，以促进细胞内部的水分和营养自平衡。

   • 物理处理：当细胞球体变得过大、内部开始发暗（提示可能出现中心区坏死），或每毫升活细胞密度达到饱和阶段时必须传代。直接将包含细胞球的悬液移至离心管中，以 150 g 离心 5 分钟收集。吸除旧基后，加入适量新鲜预热的完全培养基。

   • 重悬与分散：使用无菌吸管或微量移液器轻轻反复吹打细胞团块，使密集的球状结构物理分散为较小的多细胞微团或单细胞悬液。常规传代比例为 1比2 至 1比4，每 3 到 4 天常规处理一次。

2. 冻存细胞复苏：

   • 快速将冻存管自液氮罐中取出，移入 37 摄氏度 BioVector® 水浴锅中持续轻柔摇晃解冻，在 1 到 2 分钟内令其极速融化。

   • 将解冻的细胞浆液缓慢移入含有 5 毫升 预热完全培养基的无菌离心管中，以 150 g 离心 5 分钟。

   • 彻底吸除含有 DMSO 冻存液的上清，使用新鲜的完全培养基（初次复苏强烈建议使用 20% 的 FBS 浓度进行早期高营养维持）重悬细胞沉淀。接种于培养器皿中，放置于箱内静置。

四 核心科研应用方向

1. 视网膜母细胞瘤发生机理与 RB1 信号调控研究：BioVector® RBL20 是国际上用于探索儿童双侧视网膜母细胞瘤（Hereditary/Bilateral RB）发病机制以及 RB1 基因非编码区/剪切突变（IVS6+1G>T）病理毒性效应极其标准且特异的内源性缺陷工具模型。

2. 小分子靶向药物、细胞周期调控剂与表观遗传学筛查：由于该细胞天然缺失 RB1 蛋白，它是筛选能绕过 RB1 轴而直接抑制下游 E2F 活性的小分子拮抗剂、CDK4/6 抑制剂抵抗性分析、以及检测能够纠正或抑制特定剪切突变靶向药物效能的理想体外药理学反应底物。

3. 转录组学、非编码 RNA 功能分析与眼科罕见病肿瘤数据库对接：该细胞已被纳入国际先进的眼科/神经外胚层肿瘤 RNA-Seq 高通量数据库系统。主要用于分析特定癌基因在神经视网膜分化停滞中的调控蓝图，并作为建立视网膜母细胞瘤疾病生物标记物谱（Biomarker profiling）的重要基础质控参考。

PART 2 ENGLISH SECTION

I General Information and Genetic Background

• Cell Line Name: BioVector® RBL20

• Repository Catalog Number: DSMZ ACC 944

• Species Origin: Human (Homo sapiens)

• Tissue and Disease Background: Established in 1988 from the primary malignant tumor mass of a 16-month-old infant patient diagnosed with bilateral retinoblastoma. Retinoblastoma represents a highly aggressive intraocular neuroectodermal malignancy typically occurring in early childhood.

• Core Oncogenic Signatures & RB1 Inactivation Profile:

  • Characterized as an authentic reference standard for retinoblastoma modeling, this cell line exhibits complete biallelic inactivation of the RB1 (Retinoblastoma 1) tumor suppressor gene.

  • The specific genetic molecular blueprint involves a pathognomonic splice-site mutation IVS6+1G>T on the first allele, coupled with a concurrent Loss of Heterozygosity (LOH) event wiping out the counter allele.This definitive "two-hit" mechanism abolishes structural RB1 protein synthesis, triggering unrestricted cell cycle progression and drive clonal expansion.

• Biosafety Level: BSL-1.

II Morphological Attributes and Cultivation Media

• Morphology: Displays classical neuroectodermal features characteristic of high-grade retinoblastoma variants. Under phase-contrast microscopy, cells present as small round or polygonal units that highly tend to cluster together, organizing into multicellular spherical aggregates and tight suspension clumps, with a minimal presence of isolated single cells.

• Growth Mode: Strict suspension growth (growing predominantly as multicellular clustered spheroids).

• Population Doubling Time: Approximately 48 hours.

• Standard Complete Growth Medium Formulation:

  • Basal Medium: 80% to 90% BioVector® RPMI-1640 medium.

  • Routine Supplements: 10% to 20% premium BioVector® Heat-Inactivated Fetal Bovine Serum (h.i. FBS).

  • 1% Penicillin-Streptomycin solution.

• Physical Incubation Parameters: Maintained continuously at 37 degrees Celsius under a humidified atmospheric setting of 5% Carbon Dioxide.

III Subculturing and Thawing Protocols

1. Routine Passaging Schedule:

   • Subculturing Routine: Because this line expands primarily as heavy, dense suspension clusters, routine handling demands careful mechanical dissociation to ensure optimal nutrient and gas diffusion across the core matrices.

   • Biomass Processing: Subculture when the floating spheroids grow excessively large, exhibit dark/necrotic centers, or when nutrient consumption prompts a sharp color shift in the medium. Pellet the clustered mass by centrifuging at 150 g for 5 minutes. Decant the spent broth and replenish with fresh, pre-warmed complete medium.

   • Mechanical Dissociation: Gently pipette the clustered cell sediment up and down using a sterile pipette to break down dense spherical frameworks into smaller micro-aggregates or a semi-single cell suspension. Re-distribute the cells into fresh vessels at recommended split sequence configurations between 1:2 and 1:4 every 3 to 4 days.

2. Cryovial Thawing and Recovery:

   • Retrieve the cryovial from liquid nitrogen storage parameters and immediately submerge it into a 37 degrees Celsius BioVector® water bath with gentle continuous agitation, securing complete liquefaction within 1 to 2 minutes.

   • Transfer the cell slurry slowly into a sterile centrifuge tube carrying 5 mL of pre-warmed complete growth medium and spin down at 150 g for 5 minutes.

   • Decant the supernatant completely to clean out toxic residual DMSO traces, and gently resuspend the cell pellet in fresh complete growth medium (utilizing an elevated 20% FBS concentration during immediate post-thaw phases is highly recommended to stimulate cluster propagation). Plate into cultivation vessels and leave undisturbed in the incubator.

IV Strategic Research Applications

1. Dissecting Retinoblastoma Pathogenesis and RB1 Splice-Site Deregulation: BioVector® RBL20 serves as a standard endogenous reference platform to investigate pediatric bilateral retinoblastoma pathogenesis and analyze the downstream functional consequence of the specific IVS6+1G>T splice-site mutation.

2. Targeted Small-Molecule Screening and Cell Cycle Bypass Evaluation: Lacking a functional RB1 axis, this cell line functions as a specialized assay tool to screen novel small-molecule inhibitors targeting downstream E2F complexes, map resistance profiles to conventional CDK4/6 inhibitors, and evaluate therapeutics designed to bypass or correct aberrant pre-mRNA splicing configurations.

3. High-Throughput Transcriptomics and Biomarker Mapping: Fully characterized within international high-throughput retinoblastoma RNA-Seq data pipelines, RBL20 is actively leveraged to decipher the gene expression landscapes governing neuroretinal differentiation arrest.It serves as a vital biological control for validating candidate diagnostic biomarkers in pediatric ocular oncology.

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