BioVector? M207 (Mel207) Human Malignant Melanoma Cell Line / M207 (Mel207) 人惡性黑色素瘤細(xì)胞系

一 產(chǎn)品基本信息與遺傳學(xué)背景

• 細(xì)胞名稱：M207（常寫作 Mel207），人惡性黑色素瘤細(xì)胞。

• 物種來源：人源（Homo sapiens）。該細(xì)胞系是國際黑色素瘤轉(zhuǎn)化醫(yī)學(xué)研究中的重要基礎(chǔ)模型，也是美國 MD 安德森癌癥中心細(xì)胞系項(xiàng)目（MD Anderson Cell Lines Project）的代表性腫瘤株系之一（Cellosaurus 登錄號：CVCL_D747）。

• 遺傳學(xué)與分子突變特征（核心科研背景）：

  • NRAS 突變（關(guān)鍵特征）：M207 細(xì)胞攜帶經(jīng)典的 NRAS 基因激活熱點(diǎn)突變（p.Gln61Leu / c.182A>T）。大約有 15%–20% 的皮膚黑色素瘤由 NRAS 突變驅(qū)動。該突變導(dǎo)致下游的 MAPK 信號通路（RAS-RAF-MEK-ERK）和 PI3K/Akt/mTOR 通路發(fā)生強(qiáng)烈的、非配體依賴性的結(jié)構(gòu)性持續(xù)激活。

  • EGFR 突變/變異：攜帶表皮生長因子受體 EGFR 基因激酶區(qū)缺失突變（p.Leu747_Pro753delinsSer），這在黑色素瘤群體中較為獨(dú)特，常被用于探究黑色素瘤中受體酪氨酸激酶（RTK）旁路對常規(guī)療法的耐藥貢獻(xiàn)。

  • PTEN 雜合缺失：具有抑癌基因 PTEN 的雜合性缺失（Heterozygous Deletion）。PTEN 的部分失活進(jìn)一步解除了對 PI3K 通路的負(fù)反饋抑制，協(xié)同增強(qiáng)了該細(xì)胞的增殖與抗凋亡能力。

  • BRAF 狀態(tài)：屬于 BRAF 野生型（BRAF-wildtype）。該特征使其與臨床最常見的 $BRAF^{V600E}$ 突變細(xì)胞系（如 A375、M14）形成鮮明的對照。

• 生長動力學(xué)與特性：貼壁生長（Adherent），細(xì)胞形態(tài)多呈現(xiàn)典型的多角形、梭形或不規(guī)則的上皮樣（Epithelial-like）交織排布。其倍增時間（Doubling time）約為 25.2 小時，屬于分裂擴(kuò)增較為旺盛的侵襲性腫瘤模型。

• 生物安全級別：1-2 級（BSL-1/2）。操作應(yīng)在標(biāo)準(zhǔn)的二級生物安全柜內(nèi)嚴(yán)格按照無菌細(xì)胞房規(guī)范進(jìn)行。

(注：在早期部分眼科文獻(xiàn)或舊數(shù)據(jù)庫分類中，該編號偶爾與眼葡萄膜黑色素瘤株系 Mel270 發(fā)生拼寫混淆，但根據(jù)國際細(xì)胞鑒定委員會（ICLAC）與 Cellosaurus 權(quán)威認(rèn)定，M207 是一株明確攜帶 NRAS 突變的惡性黑色素瘤細(xì)胞系。)

二 核心科研價值與轉(zhuǎn)化醫(yī)學(xué)應(yīng)用

M207 細(xì)胞在攻克黑色素瘤藥物耐藥性及聯(lián)合靶向方案設(shè)計中扮演了關(guān)鍵角色：

1. NRAS 突變型黑色素瘤靶向治療與聯(lián)合用藥篩選：

   臨床上針對 $BRAF^{V600E}$ 突變的黑色素瘤已有高效的維羅非尼（Vemurafenib/PLX4032）等靶向藥，但這些藥物對 NRAS 突變的 M207 細(xì)胞天然耐藥。因此，M207 被廣泛用作“反向?qū)φ罩辍保糜陂_發(fā)專門針對 NRAS 突變?nèi)后w的廣譜 RAF 抑制劑（Pan-RAF Inhibitor）聯(lián)合 MEK 抑制劑（如 TAK733、Trametinib）的協(xié)同致死毒殺矩陣，或評價全新的 ERK 激酶小分子阻斷劑。

2. 癌癥反相蛋白質(zhì)陣列（RPPA）與功能蛋白質(zhì)組學(xué)：

   作為 MD 安德森癌癥中心黑色素瘤項(xiàng)目的核心分析株，M207 擁有極度完善的反相蛋白質(zhì)微陣列（RPPA）表達(dá)譜數(shù)據(jù)?？蒲腥藛T可直接調(diào)取其在基礎(chǔ)狀態(tài)下或藥物刺激后，數(shù)百種核心激酶（如 p-ERK、p-Akt、p-S6）的磷酸化動力學(xué)圖譜，極其適合結(jié)合生物信息學(xué)研究腫瘤網(wǎng)絡(luò)重構(gòu)。

3. 腫瘤侵襲與微環(huán)境黏附研究：

   由于其攜帶 PTEN 缺陷與 NRAS 強(qiáng)激活，該細(xì)胞表現(xiàn)出極高的體外遷移、侵襲與膠原基質(zhì)穿透能力，常用于構(gòu)建劃痕愈合模型、Transwell 侵襲實(shí)驗(yàn)，以及探究黑色素瘤細(xì)胞與血管內(nèi)皮細(xì)胞、成纖維細(xì)胞之間的旁分泌交互。

三 實(shí)驗(yàn)室細(xì)胞復(fù)蘇、擴(kuò)增傳代與冷凍保存標(biāo)準(zhǔn)步驟

1. 完全培養(yǎng)基配置

M207 細(xì)胞對營養(yǎng)基質(zhì)適應(yīng)性好，推薦配置如下：

• 基礎(chǔ)培養(yǎng)基：RPMI 1640 培養(yǎng)基（推薦）（含 2.0 mM L-Glutamine 谷氨酰胺）。

• 完全添加劑成分：

  • 10% 優(yōu)質(zhì)滅活胎牛血清（FBS）。

  • 1% 滅菌雙抗（Penicillin-Streptomycin, 100 U/mL）。

• 培養(yǎng)環(huán)境：37 攝氏度、5% $CO_2$、恒濕有氧培養(yǎng)箱。

2. 細(xì)胞復(fù)蘇步驟

1. 將配置好的完全培養(yǎng)基在 37 攝氏度水浴中提前預(yù)熱，準(zhǔn)備 15 mL 離心管。

2. 從液氮罐或 -80 攝氏度冰箱中迅速取出 M207 凍存管，立刻投入 37 攝氏度恒溫水浴箱中，輕柔規(guī)律地水平晃動。

3. 在 1 分鐘之內(nèi)令管內(nèi)凍存塊急速融化（待內(nèi)部剩下最后一粒細(xì)小冰芯時即可撈出）。用 75% 酒精擦拭管外壁消毒后移入安全柜。

4. 用移液槍將融化的細(xì)胞懸液吸出，緩慢滴入含有 5 mL 預(yù)熱完全培養(yǎng)基的離心管中，輕柔吹打混勻，防止瞬時滲透壓過高損傷細(xì)胞膜。

5. 以 200 - 300 x g（約 1000 rpm）離心 3 - 4 分鐘，小心吸除含 DMSO 的上清液。

6. 加入 5 mL 新鮮完全培養(yǎng)基重懸沉淀，接種至 T25 培養(yǎng)瓶中，置于 37 攝氏度孵箱中，次日觀察貼壁展平率。

3. 細(xì)胞傳代與消化方法

• 傳代時機(jī)：由于 M207 增殖較為迅速（倍增約 25 小時），當(dāng)細(xì)胞融合度達(dá)到 70% - 80% 時必須及時傳代。若貼壁過密，NRAS 持續(xù)激活導(dǎo)致的局部代謝產(chǎn)物積聚會引發(fā)非特異性接觸抑制或自發(fā)性漂浮。

• 傳代比例：通常按照 1:3 至 1:5 的比例進(jìn)行分瓶傳代，每 2 - 3 天傳代一次。

• 操作步驟：

  1. 吸干瓶內(nèi)舊培養(yǎng)基，用無菌無鈣鎂 PBS 輕輕洗滌細(xì)胞表面 1 - 2 次，徹底洗去血清。

  2. 加入適量 0.25% Trypsin-EDTA 消化液（T25 瓶常規(guī)加入 1 mL），使其均勻浸潤細(xì)胞層。

  3. 放入 37 攝氏度孵箱中消化 1 至 3 分鐘。在倒置顯微鏡下連續(xù)觀察，當(dāng)大部分多角形細(xì)胞回縮變圓、邊界透亮且輕擊瓶壁細(xì)胞開始大量向下滑落時，立即加入 2 倍體積的含血清完全培養(yǎng)基終止消化。

  4. 用移液槍輕柔平穩(wěn)地吹打瓶壁，將細(xì)胞吹散成單細(xì)胞懸液。收集至離心管中，300 x g 離心 3 分鐘，棄上清。

  5. 加入新鮮完全培養(yǎng)基，吹勻后按比例接種到新的培養(yǎng)容器中。

4. 細(xì)胞冷凍保存

• 凍存液配方：90% 完全培養(yǎng)基（或純高品質(zhì) FBS） + 10% 細(xì)胞級 DMSO。

• 凍存操作：冷凍前確保細(xì)胞處于對數(shù)生長活躍期（融合度約 75% 且活力達(dá) 90% 以上）。離心收集細(xì)胞，用配置好的凍存液重懸，調(diào)整最終細(xì)胞密度至 $1.5 \times 10^6$ 至 $3 \times 10^6\text{ cells/vial}$。裝入凍存管后立即放入程序降溫盒（如 Mr. Frosty 梯度降溫盒，維持 -1 攝氏度/分鐘的慢凍速度），置于 -80 攝氏度過夜。次日必須迅速轉(zhuǎn)移至 -196 攝氏度液氮罐中長期冷凍保存，以防止細(xì)胞活力衰退。

Part 2 English Section

I General Information and Genetic Architecture

• Cell Line Designation: M207 (frequently indexed as Mel207), Human Malignant Melanoma Cell Line.

• Species Origin: Homo sapiens (Human).This lineage serves as a vital model for translational melanoma oncology and represents a core matrix within the MD Anderson Cancer Center Cell Lines Project (Cellosaurus Accession: CVCL_D747).

• Genomic and Mutational Profiles (Critical Research Framework):

  • NRAS Hyper-activation (Canonical Driver): M207 harbors a classical, canonical activating hotspot mutation in NRAS (p.Gln61Leu / c.182A>T). Approximately 15%–20% of cutaneous melanomas are driven by oncogenic NRAS variants. This driver leads to robust, ligand-independent constitutive hyper-activation of downstream MAPK cascades (RAS-RAF-MEK-ERK) and PI3K/Akt/mTOR pathways.

  • EGFR Variant Co-occurrence: Features an epidermal growth factor receptor EGFR kinase domain deletion mutation (p.Leu747_Pro753delinsSer). This configuration is rare in cutaneous melanoma, making M207 an excellent model to explore how receptor tyrosine kinase (RTK) feedback bypass loops contribute to therapeutic resistance.

  • PTEN Heterozygous Loss: Characterized by a heterozygous deletion of the tumor suppressor PTEN. Partial PTEN inactivation removes key negative feedback controls over the PI3K network, synergizing with the NRAS mutation to accelerate cell proliferation and evade apoptosis.

  • BRAF Wildtype Matrix: Characterized as BRAF-wildtype, making it an essential negative control line when compared against clinical $BRAF^{V600E}$ models (such as A375 or M14).

• Growth Kinetics & Morphology: Adherent monolayer. Displays distinct polygonal, spindle-shaped, or irregular epithelial-like interlaced cellular morphologies. It exhibits highly active proliferation kinetics with a documented doubling time of approximately 25.2 hours.

• Biosafety Matrix: Classified under Biosafety Level 1 or 2 (BSL-1/2). Standard handling protocols should proceed within certified Class II Biosafety Cabinets under sterile clinical culture conditions.

(Note: Historical literature occasionally notes minor typographical confusion between M207 and the uveal melanoma cell line Mel270. However, current authenticated data from ICLAC and Cellosaurus clarify that M207 is a distinct cutaneous malignant melanoma cell line harboring an NRAS mutation.)

II Strategic Research Value and Translational Fields

The M207 cell line provides an invaluable in vitro platform for mapping kinase pathway re-routing and developing combination targeted oncology regimens:

1. Targeted Therapeutics and Combination Screening for NRAS-Mutant Melanoma:

   While $BRAF^{V600E}$ melanomas respond highly to vemurafenib (PLX4032), these standard single-agent inhibitors are intrinsically ineffective against NRAS-driven M207 cells. Consequently, M207 serves as a benchmark model to evaluate novel Pan-RAF inhibitors combined with MEK inhibitors (e.g., TAK733, Trametinib), or to screen newly synthesized small-molecule ERK inhibitors to induce synthetic lethality.

2. Reverse-Phase Protein Array (RPPA) & Functional Proteomics:

   As a benchmark entity in the MD Anderson Melanoma Project database, M207 has deeply mapped Reverse-Phase Protein Array (RPPA) expression profiles. Investigators can retrieve calibrated quantitative data tracking hundreds of signaling hubs (such as p-ERK, p-Akt, p-S6) under baseline or drug-stressed states, making it ideal for systems biology modeling.

3. Tumor Invasion and Microenvironmental Mechanics:

   Driven by combined PTEN deficiency and oncogenic NRAS, this cell line exhibits high migratory, invasive, and extracellular matrix (ECM) penetrative capacities. It is routinely integrated into scratch wound-healing models, Transwell invasion assays, and studies decoding paracrine cross-talk between melanoma cells, endothelial sheets, and cancer-associated fibroblasts (CAFs).

III Thawing, Proliferation, Passaging, and Cryopreservation Routines

1. Formulating the Complete Growth Medium

M207 adapts seamlessly to standard nutrient baselines. The optimized recipe includes:

• Basal Medium: RPMI 1640 Growth Medium (Highly Recommended) (pre-fortified with 2.0 mM L-Glutamine).

• Complete Supplements:

  • 10% Premium Heat-Inactivated Fetal Bovine Serum (FBS).

  • 1% Sterilized Penicillin-Streptomycin cocktail (100 U/mL final working threshold).

• Environment: 37°C under a humidified atmosphere containing 5% $CO_2$.

2. Cryovial Thawing Routine

1. Pre-warm the formulated complete growth medium inside a 37°C water bath.

2. Extract the M207 cryovial from liquid nitrogen and instantly submerge it into the 37°C water bath with continuous horizontal agitation.

3. Achieve complete liquefaction rapidly within 1 minute (remove when a minimal frozen core remains). Swab the exterior thoroughly with 75% ethanol before transferring it into the biosafety hood.

4. Pipette the cell suspension gently and drop it slowly into a 15 mL tube containing 5 mL of pre-warmed complete growth medium to mitigate sudden osmotic shock.

5. Centrifuge at 200–300 × g (~1000 rpm) for 3–4 minutes, then cleanly aspirate the DMSO-laden supernatant fluid.

6. Replenish with 5 mL of fresh complete medium, smoothly resuspend the pellet via gentle pipetting, and transfer into a T25 culture flask. Incubate at 37°C with 5% $CO_2$ and check attachment profiles the following day.

3. Subculturing and Cell Passaging Guide

• Confluency Cutoff: Due to its rapid doubling time (~25 hours), passaging must occur precisely when the monolayer reaches 70%–80% confluency. Avoid overgrowth, as extreme cell-packing and NRAS-driven metabolic shifts can trigger non-specific detachment or cell death.

• Split Ratios & Seeding Density: Split vessels utilizing standard split ratios of 1:3 to 1:5 every 2 to 3 days.

• Step-by-Step Harvesting Routine:

  1. Aspirate spent medium and rinse the cell layer 1–2 times with sterile, calcium/magnesium-free PBS to remove residual serum proteins that neutralize trypsin.

  2. Add an appropriate volume of 0.25% Trypsin-EDTA Solution (approx. 1 mL for standard T25 flasks) to submerge the monolayer.

  3. Incubate at 37°C for 1 to 3 minutes. Monitor continuously under an inverted microscope; as soon as the polygonal cell boundaries round up and detach upon gentle mechanical tapping, instantly add a double volume of serum-containing complete growth medium to quench enzymatic activity.

  4. Gently pipette the vessel walls to yield a homogeneous single-cell suspension. Centrifuge at 300 × g for 3 minutes, then discard the supernatant fluid.

  5. Disperse the pellet into fresh complete medium, split according to target ratios, and allocate into new culture vessels.

4. Cryopreservation Protocol

• Freezing Medium Matrix: 90% Complete Growth Medium (or pure premium FBS) supplemented with 10% analytical-grade cell-culture DMSO.

• Freezing Routine: Harvest active, log-phase cultures showing >90% cell viability metrics. Centrifuge and resuspend the cell mass to hit a final concentration baseline of $1.5 \times 10^6$ to $3 \times 10^6\text{ cells/vial}$. Aliquot into sterile cryovials and transfer immediately into a standardized controlled-rate freezing box (e.g., Mr. Frosty container, ensuring a constant thermal drop profile of -1°C/minute). House at -80°C overnight, and shift the vials into the liquid nitrogen vapor phase (-196°C) the following day for long-term preservation.

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