BioVector® Hep-12 人肝细胞癌复发株（富含肿瘤起始细胞）

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

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：Hep-12 人肝细胞癌复发株

• 系统学 Accession：Cellosaurus CVCL_A1RS

• 保藏登记号：中国微生物菌种保藏管理委员会普通微生物中心（CGMCC No. 3204）

• 物种来源：人类 (Homo sapiens)

• 组织背景：该细胞株衍生自一名 47 岁中国男性原发性肝细胞癌（HCC）患者手术切除后的复发肿瘤组织。与之互为配对的同源株为 Hep-11（源自该患者的原发肿瘤组织）。

• 遗传与表观特征：

  • 病毒转化/整合：细胞基因组内伴有明确的乙型肝炎病毒（HBV）基因整合，证实其病毒介导的转化背景。

  • 关键突变：携带经典的 TP53 基因突变：p.Arg249Ser (c.747G>T)，该突变是 HBV 相关性或黄曲霉毒素暴露肝癌的典型标志性热点突变。

  • 肿瘤干性（Stemness）：Hep-12 在生物学研究中被证实高度富含肿瘤起始细胞（Tumor-Initiating Cells, TICs / 肝癌干细胞 CSCs）。其具有明显的干细胞样特性，在体外展现出极强的无血清悬浮成球（Tumor Spheres）与自更新能力。

• 生物安全级别：2级（BSL-2）。因其含有 HBV 整合片段且源自人类恶性肿瘤，所有操作均须在二级生物安全柜中进行。

二、 细胞形态学与培养环境

• 形态学特征：主要表现为上皮样圆形或类圆形细胞。作为复发株，其在常规培养体系中相较于原发株具有更强的自发聚集成多细胞肿瘤球体（Spheres）的倾向，这与其富含的 TICs 属性紧密相关。

• 倍增时间（Doubling Time）：约 53 小时，其体外生长受干性维持机制调控，速度较为温和。

• 标准完全培养基配方：

  • 基础培养基：高糖 DMEM 或 RPMI-1640 基础肉汤。

  • 常规维持添加：10%–15% 优质胎牛血清（FBS）+ 1× 谷氨酰胺 + 1% 双抗（青霉素-链霉素）。

  • 干性富集培养（非贴壁成球模式，可选）：若需维持并增强其 TICs 的未分化状态，可更换为无血清干细胞培养基：DMEM/F12 + 20 ng/mL EGF + 20 ng/mL bFGF + 1× B27 Supplement。

• 物理培养参数：37°C 恒温、5% 二氧化碳（$CO_2$）、饱和空气湿度培养箱。

三、 细胞传代与复苏标准操作步骤

1. 常规传代操作（周期 4–6 天，视细胞密度而定）：

   • 鉴于 Hep-12 具有悬浮成球与贴壁混杂生长的特性，传代时先将上清培养基连同悬浮的细胞球小心吸入离心管中。

   • 向留有贴壁细胞的瓶内加入少量无菌 PBS 轻轻洗涤，吸出 PBS。加入 0.25% Trypsin-EDTA 消化液，37°C 孵育 2–3 分钟。

   • 显微镜下观察到细胞开始解离，加入含血清的完全培养基终止消化。用移液枪温和吹打，将细胞团块彻底打散成单细胞悬液，以防团块中心发生缺氧坏死。

   • 将消化的细胞与最初吸出的悬浮细胞球合并，于 1000 RPM (约 200g) 离心 5 分钟。弃上清，用新鲜完全培养基重悬，按 1:2 至 1:3 比例接种至新的培养瓶中。

2. 冻存细胞复苏：

   • 将冷冻管从液氮中取出，立即投入 37°C 水浴中快速摇动融化（1–2 分钟内）。

   • 将融化的细胞悬液移至含 5 mL 预热培养基的离心管中，1000 RPM 离心 5 分钟以去除残留的 DMSO。

   • 弃上清，用完全培养基重悬，接种于培养瓶中。复苏后的前 24 小时内尽量避免剧烈摇动培养瓶，以利其粘附和沉降。

四、 核心科研应用方向

1. 肝癌干细胞（CSCs/TICs）靶向研究：作为极为罕见的“原发-复发”同源配对模型（Hep-11 / Hep-12），用于研究肝癌细胞向干性状态演变、恶性克隆演化及癌症复发的免疫逃逸机制。

2. 复发耐药与肿瘤免疫治疗：利用其高富含 TICs 的表型，高通量筛选针对肝癌干细胞特异性表面抗原的靶向药物、小分子小分子抑制剂，或作为开发针对复发性肝癌新型免疫疗法的细胞模型。

3. HBV 相关肝癌发病机制：利用其明确的 HBV 整合背景与经典的 TP53 突变表型（p.Arg249Ser），深度解析病毒因子与宿主抑癌基因失活在推动肝癌复发病程中的协同分子病理机制。

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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