BioVector? RBL20 人視網(wǎng)膜母細(xì)胞瘤細(xì)胞株

BioVector? RBL20 Human Retinoblastoma Cell Line

第一部分 中文說明

一 產(chǎn)品基本信息與遺傳學(xué)背景

• 細(xì)胞名稱：BioVector? RBL20

• 保藏機(jī)構(gòu)貨號：DSMZ ACC 944

• 物種來源：人類 (Homo sapiens)

• 組織與疾病背景：該細(xì)胞株建立于1988年，分離自一名患有雙側(cè)視網(wǎng)膜母細(xì)胞瘤（Bilateral retinoblastoma）的16個月大幼兒患者的惡性原發(fā)腫瘤組織。視網(wǎng)膜母細(xì)胞瘤是一種高度惡性的兒童神經(jīng)外胚層眼內(nèi)惡性腫瘤。

• 核心致癌與抑癌基因突變特征（RB1 滅活特征）：

  • 作為經(jīng)典的視網(wǎng)膜母細(xì)胞瘤模型，該細(xì)胞系伴有雙股抑癌基因 RB1（Retinoblastoma 1） 的病理性完全滅活。

  • 具體的突變分子特征為：第一條等位基因攜帶經(jīng)典的剪切位點(diǎn)變異 IVS6+1G>T，而另一條等位基因則發(fā)生了雜合性丟失（Loss of Heterozygosity, LOH）。這一雙擊（Two-hit）遺傳學(xué)事件導(dǎo)致了細(xì)胞內(nèi) RB1 腫瘤抑制蛋白的徹底缺失，從而使細(xì)胞周期進(jìn)入無限制性循環(huán)，驅(qū)動視網(wǎng)膜神經(jīng)上皮層的惡性克隆演進(jìn)。

• 生物安全級別：1級（BSL-1）。

二 細(xì)胞形態(tài)學(xué)與培養(yǎng)環(huán)境

• 形態(tài)學(xué)特征：展現(xiàn)典型的視網(wǎng)膜母細(xì)胞瘤神經(jīng)外胚層分化特征。在倒置顯微鏡下，細(xì)胞通常呈小圓形或多角形，且高度傾向于聚集形成大小不一、松散或緊密的球狀細(xì)胞懸浮成團(tuán)簇、類器官樣小球（Spherical cluster aggregate clumps in suspension），極少以單細(xì)胞分散存在。

• 生生模式：懸浮生長（通常成團(tuán)塊、球狀懸?。?/p>

• 群體倍增時間（Doubling Time）：大約 48 小時左右。

• 標(biāo)準(zhǔn)完全培養(yǎng)基配方：

  • 基礎(chǔ)培養(yǎng)基：80% 至 90% BioVector? RPMI-1640 培養(yǎng)基。

  • 維持添加：10% 至 20% 優(yōu)質(zhì)熱滅活胎牛血清（h.i. FBS）。

  • 1% Penicillin-Streptomycin 雙抗溶液。

• 物理培養(yǎng)參數(shù)：37攝氏度恒溫、5% 二氧化碳、空氣飽和濕度。

三 細(xì)胞傳代與復(fù)蘇標(biāo)準(zhǔn)操作步驟

1. 初始接種與常規(guī)傳代操作（Subculturing Protocol）：

   • 日常傳代：由于細(xì)胞高度傾向于形成球狀密集團(tuán)簇懸浮生長，在傳代時需要物理性地輔助吹打，以促進(jìn)細(xì)胞內(nèi)部的水分和營養(yǎng)自平衡。

   • 物理處理：當(dāng)細(xì)胞球體變得過大、內(nèi)部開始發(fā)暗（提示可能出現(xiàn)中心區(qū)壞死），或每毫升活細(xì)胞密度達(dá)到飽和階段時必須傳代。直接將包含細(xì)胞球的懸液移至離心管中，以 150 g 離心 5 分鐘收集。吸除舊基后，加入適量新鮮預(yù)熱的完全培養(yǎng)基。

   • 重懸與分散：使用無菌吸管或微量移液器輕輕反復(fù)吹打細(xì)胞團(tuán)塊，使密集的球狀結(jié)構(gòu)物理分散為較小的多細(xì)胞微團(tuán)或單細(xì)胞懸液。常規(guī)傳代比例為 1比2 至 1比4，每 3 到 4 天常規(guī)處理一次。

2. 凍存細(xì)胞復(fù)蘇：

   • 快速將凍存管自液氮罐中取出，移入 37 攝氏度 BioVector? 水浴鍋中持續(xù)輕柔搖晃解凍，在 1 到 2 分鐘內(nèi)令其極速融化。

   • 將解凍的細(xì)胞漿液緩慢移入含有 5 毫升 預(yù)熱完全培養(yǎng)基的無菌離心管中，以 150 g 離心 5 分鐘。

   • 徹底吸除含有 DMSO 凍存液的上清，使用新鮮的完全培養(yǎng)基（初次復(fù)蘇強(qiáng)烈建議使用 20% 的 FBS 濃度進(jìn)行早期高營養(yǎng)維持）重懸細(xì)胞沉淀。接種于培養(yǎng)器皿中，放置于箱內(nèi)靜置。

四 核心科研應(yīng)用方向

1. 視網(wǎng)膜母細(xì)胞瘤發(fā)生機(jī)理與 RB1 信號調(diào)控研究：BioVector? RBL20 是國際上用于探索兒童雙側(cè)視網(wǎng)膜母細(xì)胞瘤（Hereditary/Bilateral RB）發(fā)病機(jī)制以及 RB1 基因非編碼區(qū)/剪切突變（IVS6+1G>T）病理毒性效應(yīng)極其標(biāo)準(zhǔn)且特異的內(nèi)源性缺陷工具模型。

2. 小分子靶向藥物、細(xì)胞周期調(diào)控劑與表觀遺傳學(xué)篩查：由于該細(xì)胞天然缺失 RB1 蛋白，它是篩選能繞過 RB1 軸而直接抑制下游 E2F 活性的小分子拮抗劑、CDK4/6 抑制劑抵抗性分析、以及檢測能夠糾正或抑制特定剪切突變靶向藥物效能的理想體外藥理學(xué)反應(yīng)底物。

3. 轉(zhuǎn)錄組學(xué)、非編碼 RNA 功能分析與眼科罕見病腫瘤數(shù)據(jù)庫對接：該細(xì)胞已被納入國際先進(jìn)的眼科/神經(jīng)外胚層腫瘤 RNA-Seq 高通量數(shù)據(jù)庫系統(tǒng)。主要用于分析特定癌基因在神經(jīng)視網(wǎng)膜分化停滯中的調(diào)控藍(lán)圖，并作為建立視網(wǎng)膜母細(xì)胞瘤疾病生物標(biāo)記物譜（Biomarker profiling）的重要基礎(chǔ)質(zhì)控參考。

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.

BioVector NTCC質(zhì)粒載體菌株細(xì)胞蛋白抗體基因保藏中心

電話:400-800-2947

工作QQ/微信同號:1843439339

網(wǎng)址

http://www.nedfriskphoto.com