BioVector? Hep-12 人肝細胞癌復發(fā)株（富含腫瘤起始細胞）

BioVector? Hep-12 Human Recurrent Hepatocellular Carcinoma Cell Line Technical Datasheet

第一部分：中文說明

一、 產(chǎn)品基本信息與遺傳學背景

• 細胞名稱：Hep-12 人肝細胞癌復發(fā)株

• 系統(tǒng)學 Accession：Cellosaurus CVCL_A1RS

• 保藏登記號：中國微生物菌種保藏管理委員會普通微生物中心（CGMCC No. 3204）

• 物種來源：人類 (Homo sapiens)

• 組織背景：該細胞株衍生自一名 47 歲中國男性原發(fā)性肝細胞癌（HCC）患者手術切除后的復發(fā)腫瘤組織。與之互為配對的同源株為 Hep-11（源自該患者的原發(fā)腫瘤組織）。

• 遺傳與表觀特征：

  • 病毒轉化/整合：細胞基因組內伴有明確的乙型肝炎病毒（HBV）基因整合，證實其病毒介導的轉化背景。

  • 關鍵突變：攜帶經(jīng)典的 TP53 基因突變：p.Arg249Ser (c.747G>T)，該突變是 HBV 相關性或黃曲霉毒素暴露肝癌的典型標志性熱點突變。

  • 腫瘤干性（Stemness）：Hep-12 在生物學研究中被證實高度富含腫瘤起始細胞（Tumor-Initiating Cells, TICs / 肝癌干細胞 CSCs）。其具有明顯的干細胞樣特性，在體外展現(xiàn)出極強的無血清懸浮成球（Tumor Spheres）與自更新能力。

• 生物安全級別：2級（BSL-2）。因其含有 HBV 整合片段且源自人類惡性腫瘤，所有操作均須在二級生物安全柜中進行。

二、 細胞形態(tài)學與培養(yǎng)環(huán)境

• 形態(tài)學特征：主要表現(xiàn)為上皮樣圓形或類圓形細胞。作為復發(fā)株，其在常規(guī)培養(yǎng)體系中相較于原發(fā)株具有更強的自發(fā)聚集成多細胞腫瘤球體（Spheres）的傾向，這與其富含的 TICs 屬性緊密相關。

• 倍增時間（Doubling Time）：約 53 小時，其體外生長受干性維持機制調控，速度較為溫和。

• 標準完全培養(yǎng)基配方：

  • 基礎培養(yǎng)基：高糖 DMEM 或 RPMI-1640 基礎肉湯。

  • 常規(guī)維持添加：10%–15% 優(yōu)質胎牛血清（FBS）+ 1× 谷氨酰胺 + 1% 雙抗（青霉素-鏈霉素）。

  • 干性富集培養(yǎng)（非貼壁成球模式，可選）：若需維持并增強其 TICs 的未分化狀態(tài)，可更換為無血清干細胞培養(yǎng)基：DMEM/F12 + 20 ng/mL EGF + 20 ng/mL bFGF + 1× B27 Supplement。

• 物理培養(yǎng)參數(shù)：37°C 恒溫、5% 二氧化碳（$CO_2$）、飽和空氣濕度培養(yǎng)箱。

三、 細胞傳代與復蘇標準操作步驟

1. 常規(guī)傳代操作（周期 4–6 天，視細胞密度而定）：

   • 鑒于 Hep-12 具有懸浮成球與貼壁混雜生長的特性，傳代時先將上清培養(yǎng)基連同懸浮的細胞球小心吸入離心管中。

   • 向留有貼壁細胞的瓶內加入少量無菌 PBS 輕輕洗滌，吸出 PBS。加入 0.25% Trypsin-EDTA 消化液，37°C 孵育 2–3 分鐘。

   • 顯微鏡下觀察到細胞開始解離，加入含血清的完全培養(yǎng)基終止消化。用移液槍溫和吹打，將細胞團塊徹底打散成單細胞懸液，以防團塊中心發(fā)生缺氧壞死。

   • 將消化的細胞與最初吸出的懸浮細胞球合并，于 1000 RPM (約 200g) 離心 5 分鐘。棄上清，用新鮮完全培養(yǎng)基重懸，按 1:2 至 1:3 比例接種至新的培養(yǎng)瓶中。

2. 凍存細胞復蘇：

   • 將冷凍管從液氮中取出，立即投入 37°C 水浴中快速搖動融化（1–2 分鐘內）。

   • 將融化的細胞懸液移至含 5 mL 預熱培養(yǎng)基的離心管中，1000 RPM 離心 5 分鐘以去除殘留的 DMSO。

   • 棄上清，用完全培養(yǎng)基重懸，接種于培養(yǎng)瓶中。復蘇后的前 24 小時內盡量避免劇烈搖動培養(yǎng)瓶，以利其粘附和沉降。

四、 核心科研應用方向

1. 肝癌干細胞（CSCs/TICs）靶向研究：作為極為罕見的“原發(fā)-復發(fā)”同源配對模型（Hep-11 / Hep-12），用于研究肝癌細胞向干性狀態(tài)演變、惡性克隆演化及癌癥復發(fā)的免疫逃逸機制。

2. 復發(fā)耐藥與腫瘤免疫治療：利用其高富含 TICs 的表型，高通量篩選針對肝癌干細胞特異性表面抗原的靶向藥物、小分子小分子抑制劑，或作為開發(fā)針對復發(fā)性肝癌新型免疫療法的細胞模型。

3. HBV 相關肝癌發(fā)病機制：利用其明確的 HBV 整合背景與經(jīng)典的 TP53 突變表型（p.Arg249Ser），深度解析病毒因子與宿主抑癌基因失活在推動肝癌復發(fā)病程中的協(xié)同分子病理機制。

PART 2: ENGLISH SECTION

I. General Information and Genetic Background

• Cell Line Name: Hep-12

• Cellosaurus Accession: CVCL_A1RS

• Repository Registration: International Depositary Authority, China General Microbiological Culture Collection Center; CGMCC No. 3204

• Species Origin: Human (Homo sapiens)

• Tissue Origin: Derived from the recurrent tumor tissue of a 47-year-old Chinese male patient diagnosed with adult hepatocellular carcinoma (HCC). Its autologous parental counterpart is Hep-11, isolated from the primary tumor tissue of the exact same individual.

• Oncogenic & Stemness Profiles:

  • Viral Integration/Transformation: The genomic structure harbors host-integrated Hepatitis B Virus (HBV) DNA sequences, which serve as the transformant driver.

  • Key Mutation: Possesses the definitive hotspot TP53 mutation: p.Arg249Ser (c.747G>T), which is strongly correlated with HBV-driven hepatocarcinogenesis and specific environmental mutagen exposures.

  • Tumor-Initiating Dynamics: Extensively characterized as being highly enriched with Tumor-Initiating Cells (TICs / Cancer Stem Cells, CSCs). Hep-12 cells exhibit distinct stem cell-like properties, rendering them a premier model for exploring self-renewal and tumor-sphere generation in vitro.

• Biosafety Level: BSL-2. Given the stable integration of HBV fragments and its malignant human provenance, all experimental procedures must be conducted within certified Class II Biosafety Cabinets.

II. Morphological Attributes and Cultivation Media

• Morphology: Displays an epithelial-like shape.Reflecting its recurrent derivation and high-stemness phenotype, it exhibits a strong tendency to form dense, floating multicellular tumor-spheres under standardized culture parameters.

• Population Doubling Time: Approximately 53 hours, reflecting a measured, stemness-regulated kinetic profile.

• Standard Complete Growth Medium Formulation:

  • Basal Medium: High-glucose DMEM or RPMI-1640 broth.

  • Routine Maintenance Supplements: 10%–15% high-quality Fetal Bovine Serum (FBS) + 1× L-Glutamine + 1% Penicillin-Streptomycin.

  • TIC Enrichment Culture (Optional Sphere Assay): To strictly maintain or expand its undifferentiated progenitor state, transition cells into serum-free stem-cell media consisting of: DMEM/F12 + 20 ng/mL recombinant human EGF + 20 ng/mL bFGF + 1× B27 Supplement.

• Physical Incubation Thresholds: Regulated at 37°C under an atmospheric layer of 5% Carbon Dioxide ($CO_2$) and saturated relative humidity.

III. Subculturing and Thawing Protocols

1. Routine Passaging Schedule (4–6 Day Cycles):

   • Because Hep-12 propagates via a mixed pattern of adhered single cells and floating multicellular clusters, harvest the supernatant containing suspended spheres into a sterile tube first.

   • Rinse the remaining attached layer with sterile PBS, aspirate, and introduce 0.25% Trypsin-EDTA solution before incubating at 37°C for 2–3 minutes.

   • Once microscopic inspection indicates initial cell rounding, neutralize the enzyme using serum-supplemented complete medium. Gently but thoroughly pipette the suspension to break apart dense cellular clusters into a single-cell slurry to maintain viability.

   • Combine the trypsinized suspension with the harvested floating spheres and centrifuge at 1000 RPM (~200g) for 5 minutes. Decant the supernatant, resuspend the pellet in fresh complete growth media, and split at a 1:2 to 1:3 ratio into new flasks.

2. Cryovial Thawing and Recovery:

   • Retrieve the cryovial from liquid nitrogen storage and immediately plunge it into a 37°C water bath, agitating continuously until the matrix liquefies (within 1–2 minutes).

   • Transfer the thawed cells into a tube filled with 5 mL of pre-warmed medium and centrifuge at 1000 RPM for 5 minutes to clear out the toxic DMSO cryoprotectant.

   • Discard the supernatant, gently resuspend the sediment in complete growth medium, and seed into the flask. To facilitate optimal cell anchoring, avoid moving or agitating the culture vessels during the initial 24 hours post-thaw.

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