BioVector? YK-M2 人急性髓系白血病細(xì)胞株

BioVector? YK-M2 Human Acute Myeloid Leukemia 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? YK-M2 人急性髓系白血病細(xì)胞

• 系統(tǒng)學(xué) Accession：Cellosaurus CVCL_A726

• 保藏機(jī)構(gòu)貨號：DSMZ ACC 949

• 物種來源：人類 (Homo sapiens)

• 性別與年齡：男性，65歲（Japanese 種群背景）

• 組織與疾病背景：該細(xì)胞株于2011年建立，源自一名患有急性髓系白血病部分成熟型（Acute Myeloid Leukemia, FAB分型為 M2 型）的65歲日本男性患者的骨髓（Bone marrow）外周血單核細(xì)胞組織。

• 核心致癌分子與突變特征：

  • RUNX1-RUNX1T1 融合基因（即 AML1-ETO 融合）：該細(xì)胞株由于伴有染色體隱匿性易位 $t(8;21)(q22;q22)$，導(dǎo)致轉(zhuǎn)錄因子 RUNX1（舊稱 AML1）與編碼核輔阻遏物的 RUNX1T1（舊稱 ETO）基因發(fā)生融合。該融合蛋白通過阻斷造血分化核心靶基因的轉(zhuǎn)錄，是 AML-M2 型白血病中最核心的致病分子標(biāo)志物之一。

  • 復(fù)雜的繼發(fā)性突變譜：除了經(jīng)典的 $t(8;21)$ 外，通常還伴有其他協(xié)同突變（如 KIT、FLT3 或表觀遺傳調(diào)節(jié)因子的變異），共同驅(qū)使細(xì)胞實(shí)現(xiàn)惡性無限增殖和分化阻斷。

• 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)典型的原始白血病髓系細(xì)胞形態(tài)。顯微鏡下胞體呈中等大小、圓形或卵圓形，核質(zhì)比高，在培養(yǎng)液中以分散懸浮的單細(xì)胞形式生長。

• 生長模式：完全懸浮生長。

• 密度依耐性與敏感特征：

  • 該細(xì)胞對低接種濃度和環(huán)境波動(dòng)極其敏感。當(dāng)細(xì)胞密度過低時(shí)（如低于每毫升 $2.0 \times 10^5$ 個(gè)活細(xì)胞），細(xì)胞由于缺乏自分泌生長因子的相互支持，會(huì)迅速進(jìn)入停滯期并傾向于發(fā)生大規(guī)模自發(fā)性凋亡。

  • 在復(fù)蘇早期，細(xì)胞生長通常較為遲緩，極易形成團(tuán)塊狀或輕微結(jié)塊現(xiàn)象，屬于該株的正常生理表型。

• 標(biāo)準(zhǔn)完全培養(yǎng)基配方：

  • 基礎(chǔ)培養(yǎng)基：BioVector? RPMI-1640 培養(yǎng)基。

  • 維持添加：

    • 15% 到 20% BioVector? 優(yōu)質(zhì)熱滅活胎牛血清（h.i. FBS）。高濃度優(yōu)質(zhì)血清是維持該株存活和抑制復(fù)蘇早期衰退的關(guān)鍵。

    • 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. 常規(guī)傳代操作：

   • 核心維持密度：在日常擴(kuò)增過程中，務(wù)必將活細(xì)胞密度嚴(yán)格控制在 每毫升 $3.0 \times 10^5$ 至 $1.5 \times 10^6$ 個(gè)活細(xì)胞 之間。

   • 當(dāng)細(xì)胞密度接近每毫升 $1.5 \times 10^6$ 個(gè)上限，或者培養(yǎng)基由于乳酸大量蓄積而明顯變黃時(shí)，必須進(jìn)行傳代。

   • 將細(xì)胞懸液轉(zhuǎn)移至無菌離心管中，以 150 g 至 200 g 離心 5 分鐘，徹底棄去舊培養(yǎng)基。

   • 接種策略：加入新鮮預(yù)熱的完全培養(yǎng)基重懸，推薦起始接種密度控制在每毫升 $3.0 \times 10^5$ 到 $4.0 \times 10^5$ 個(gè)活細(xì)胞。常規(guī)傳代比例通常為 1比2 至 1比3，每 2 到 3 天需要補(bǔ)液或傳代一次。

2. 凍存細(xì)胞復(fù)蘇：

   • 從液氮罐中快速取出冷凍管，立即投入 37攝氏度 BioVector? 水浴鍋中持續(xù)高頻搖動(dòng)使其快速融化，嚴(yán)格控制在 1 到 2 分鐘內(nèi)。

   • 迅速將解凍的細(xì)胞液移入含有 5 到 7 mL 預(yù)熱高血清完全培養(yǎng)基的無菌試管中，150 g 離心 5 分鐘以徹底去除殘留的 DMSO 凍存液。

   • 棄去上清，加入 4 mL 新鮮完全培養(yǎng)基重懸，接種至小培養(yǎng)瓶中，保持相對較高的初始復(fù)蘇細(xì)胞密度（建議 $\ge 5.0 \times 10^5$ cells/mL），并在前 24 小時(shí)內(nèi)盡量避免劇烈晃動(dòng)或頻繁移出培養(yǎng)箱。

四 核心科研應(yīng)用方向

1. $t(8;21)$ / RUNX1-RUNX1T1 陽性白血病的靶向藥物篩選：BioVector? YK-M2 是國際上研究 $t(8;21)$ 核心靶點(diǎn)極為稀缺且珍貴的內(nèi)源性標(biāo)準(zhǔn)細(xì)胞模型。常被用于評估和篩查針對 RUNX1-RUNX1T1 融合蛋白的新型阻斷劑、促其降解的小分子化合物（如組蛋白去乙?；敢种苿?HDACi 的聯(lián)合應(yīng)用），以及針對伴發(fā) KIT 突變靶點(diǎn)的激酶抑制劑藥效學(xué)測試。

2. 髓系分化阻斷與轉(zhuǎn)錄調(diào)控網(wǎng)絡(luò)機(jī)制研究：利用全反式維甲酸（ATRA）、維生素D3（Vitamin D3）或佛波酯（TPA）等誘導(dǎo)劑干預(yù) YK-M2 細(xì)胞，通過定量監(jiān)測其細(xì)胞表面分化抗原（如 CD11b、CD13、CD14、CD33）的切換譜，用于解密 RUNX1-RUNX1T1 融合蛋白在表觀遺傳學(xué)層面上壓制髓系造血分化、誘發(fā)白血病發(fā)生的分子閉環(huán)。

3. AML 難治耐藥性與免疫微環(huán)境逃逸研究：由于該細(xì)胞源自臨床惡性進(jìn)展期髓系病例，常用于建立免疫缺陷小鼠（如 NCG 或 NSG 小鼠）的異種移植白血病模型（PDX-like cell model），在體內(nèi)或體外微環(huán)境壓力下分析急性髓系白血病對常規(guī)化療藥物（如阿糖胞苷 Ara-C、柔紅霉素）產(chǎn)生耐藥的動(dòng)力學(xué)特征及免疫檢查點(diǎn)配體的轉(zhuǎn)錄調(diào)控。

PART 2 ENGLISH SECTION

I General Information and Genetic Background

• Cell Line Name: BioVector? YK-M2

• Synonyms: YKM2, YK-M2

• Cellosaurus Accession: CVCL_A726

• Repository Catalog Number: DSMZ ACC 949

• Species Origin: Human (Homo sapiens)

• Sex and Age of Donor: Male, 65 years old (Japanese population profile)

• Tissue and Disease Background: Established in 2011 from the peripheral blood mononuclear cells / bone marrow aspirate of a 65-year-old Japanese male patient diagnosed with Acute Myeloid Leukemia with maturation (FAB classification: AML-M2 subtype).

• Core Oncogenic Markers & Genomic Traits:

  • RUNX1-RUNX1T1 Gene Fusion ($t(8;21)(q22;q22)$ variants): Characteristically harbors the hallmark reciprocal chromosomal translocation $t(8;21)$, fusing the core binding factor subunit RUNX1 (formerly AML1) to the nuclear corepressor RUNX1T1 (formerly ETO). The resultant chimeric protein acts as a dominant-negative transcriptional repressor that shuts down downstream hematopoiesis master genes, serving as a primary driver mechanism for M2-subtype leukemogenesis.

  • Secondary Mutational Profile: Accompanied by overlapping clonal mutations (e.g., in KIT, FLT3, or epigenetic modifier networks), which cooperatively accelerate proliferation and lock differentiation sequences.

• Biosafety Level: BSL-1.

II Morphological Attributes and Cultivation Media

• Morphology: Displays classic primitive myeloid blastic architecture. Under standard light microscopy, cells present medium-sized, round or oval profiles with prominent nucleoli and high nuclear-to-cytoplasmic volume ratios, expanding strictly as an unattached single-cell suspension.

• Growth Mode: Suspension growth.

• Density Dependency & Sensitivity Indicators:

  • Highly vulnerable to low biomass initial settings. If the cellular seed density plummets below critical thresholds ($< 2.0 \times 10^5$ viable cells/mL), proliferation ceases due to the exhaustion of essential autocrine growth factors, frequently leading to culture crash or mass apoptosis.

  • During immediate post-thaw phases, cell expansion typically lags, and temporary cell clumping or micro-aggregation may manifest, which represents an intrinsic physiological feature of this specific line.

• Standard Complete Growth Medium Formulation:

  • Basal Medium: BioVector? RPMI-1640 medium.

  • Routine Supplements:

    • 15% to 20% premium BioVector? Heat-Inactivated Fetal Bovine Serum (h.i. FBS). Sustained high serum titers are mandatory to preserve baseline fitness and buffer against post-thaw viability decline.

    • 1% Penicillin-Streptomycin solution.

• Physical Incubation Thresholds: Regulated strictly at 37 degrees Celsius under an atmospheric layer of 5% Carbon Dioxide and saturated air humidity.

III Subculturing and Thawing Protocols

1. Routine Passaging Schedule:

   • Core Density Window: Maintain the active suspension profile tightly bounded between $3.0 \times 10^5$ and $1.5 \times 10^6$ viable cells/mL.

   • Subculture the vessel when the biomass density reaches or approaches the $1.5 \times 10^6$ cells/mL baseline, or when the phenol red parameters shift heavily towards yellow due to lactic acid accumulation.

   • Pellet the suspension via centrifugation at 150 g to 200 g for 5 minutes, and draw off the spent broth.

   • Seeding Target: Resuspend the cells in fresh pre-warmed complete medium. Crucially, never re-seed fresh cultures at densities lower than $3.0 \times 10^5$ viable cells/mL. The recommended split sequence averages 1:2 to 1:3, demanding medium replenishment or subculturing every 2 to 3 days.

2. Cryovial Thawing and Recovery:

   • Retrieve the cryovial from storage and submerge it into a 37 degrees Celsius BioVector? water bath with rapid agitation until completely liquefied within 1 to 2 minutes.

   • Promptly transfer the cell suspension into a sterile tube carrying 5 to 7 mL of pre-warmed complete growth medium and spin at 150 g for 5 minutes to fully purge residual DMSO.

   • Decant the supernatant, gently resuspend the cell pellet in fresh, serum-enriched complete BioVector? medium, and plate. Maintain a high cell density baseline ($\ge 5.0 \times 10^5$ cells/mL) during the immediate post-thaw phase and minimize physical disturbance for the first 24 hours.

IV Strategic Research Applications

1. t(8;21) / RUNX1-RUNX1T1 Target Therapeutic Screening: BioVector? YK-M2 stands as an excellent, scarce endogenous reference model for exploring therapeutic interventions against $t(8;21)$-driven AML. It is widely utilized to screen small-molecule inhibitors targeting the RUNX1-RUNX1T1 multimerization domain, evaluate molecular glue degraders, or test combination regimens incorporating HDAC inhibitors and secondary KIT kinase blockers.

2. Elucidating Myeloid Differentiation Blockades and Transcription Networks: Deployed alongside chemical differentiation vectors (such as All-Trans Retinoic Acid ATRA, Vitamin D3, or TPA) to track alterations in immunophenotypic clusters (measuring variations in myeloid antigens CD11b, CD13, CD14, and CD33), helping to decode the epigenetic mechanisms by which the fusion complex arrests hematopoietic maturation.

3. AML Refractory Resistance and Xenograft Modeling: Since it originates from a clinically advanced myeloid malignancy case, this line is heavily used to construct cell-line-derived xenograft (CDX) leukemia models in immunodeficient mice (e.g., NSG or NCG strains). It serves to track in vivo resistance kinetics against conventional chemotherapeutic agents like Cytarabine (Ara-C) or Daunorubicin, while profiling immune checkpoint ligand evasion mechanisms.

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