1. 目的Purpose
规范非洲爪蟾（Xenopus laevis）卵母细胞的手术取出、胶原酶消化与体外培养标准流程，适用于电生理研究、外源基因异源表达与蛋白质功能分析。Standardise the surgical excision, collagenase digestion and in vitro culture of Xenopus laevis oocytes for electrophysiology, heterologous expression and protein functional analysis.
2. 试剂与溶液Reagents & solutions
| 试剂Reagent | 规格Spec | 储存Storage |
|---|
| 胶原酶 I 型Collagenase type I | Sigma C0130 | −20 ℃ 分装避光 |
| MS-222 麻醉剂MS-222 anaesthetic | Sigma E10521 | 4 ℃ 避光 |
| 青霉素–链霉素Pen-Strep | Gibco 15140-122 | −20 ℃ |
| 丙酮酸钠Sodium pyruvate | Sigma P2256 | 室温干燥RT, dry |
1.8 Ca²⁺ ND96 培养液（pH 7.4）1.8 Ca²⁺ ND96 culture medium (pH 7.4)
| 成分Component | 浓度（mM）Concentration (mM) |
|---|
| NaCl | 96 |
| KCl | 2 |
| MgCl₂ | 1 |
| CaCl₂ | 1.8 |
| 丙酮酸钠Sodium pyruvate | 2.5 |
| HEPES | 5 |
| 青霉素Penicillin | 100 IU/mL |
| 链霉素Streptomycin | 100 μg/mL |
用 1 M NaOH 调 pH 至 7.4；0.22 μm 滤膜过滤；4 ℃ 保存，有效期 7 天。CaCl₂ 最后加入以防沉淀。0 Ca²⁺ ND96：去除 CaCl₂，其余成分相同，用于消化全程。Adjust to pH 7.4 with 1 M NaOH; filter through 0.22 μm; store at 4 ℃ for up to 7 days. Add CaCl₂ last to prevent precipitation. 0 Ca²⁺ ND96: omit CaCl₂, otherwise identical; use throughout digestion.
3. 卵巢取出3. Ovarian excision
麻醉Anaesthesia
- 冰麻法：将爪蟾置于碎冰（4 ℃）中埋没 30 min，检测后肢反射消失后操作。Ice anaesthesia: bury frog in crushed ice (4 ℃) for 30 min; confirm loss of hind-limb reflex before proceeding.
- MS-222 法：0.1% MS-222（pH 7.0）溶液中浸泡 10 min。MS-222: immerse in 0.1% MS-222 (pH 7.0) for 10 min.
手术操作Surgical procedure
- 将爪蟾腹面朝上固定于冰预冷托盘；在左下腹切开约 1 cm 切口（皮肤→肌层），用镊子轻取卵巢叶，剪取所需量后置于 0 Ca²⁺ ND96。Fix frog ventral-side up on ice-cooled tray; make a ~1 cm incision (skin → muscle layer) in lower left abdomen; gently grasp ovary lobe with forceps, excise required amount and transfer to 0 Ca²⁺ ND96.
- 分层缝合（肌层用 5-0 可吸收缝线，皮肤用 6-0 尼龙线）；术后置于浅水（22 ℃，含 50 IU/mL 青霉素）中复苏，观察 48 h。Suture in layers (5-0 absorbable for muscle, 6-0 nylon for skin); recover in shallow water (22 ℃, 50 IU/mL penicillin); observe for 48 h.
4. 卵母细胞分离与胶原酶消化4. Oocyte isolation & collagenase digestion
- 用钟表镊将卵巢组织撕成 10–20 个细胞的小团，在 0 Ca²⁺ ND96 中洗涤 5 次（100×g，1 min）至上清澄清。Tease ovary tissue into clumps of 10–20 cells with watchmaker forceps; wash 5× in 0 Ca²⁺ ND96 (100×g, 1 min) until supernatant is clear.
- 加入消化液（1 mg/mL 胶原酶 I 型，0 Ca²⁺ ND96，20 mL），22 ℃ 摇床（70 rpm）消化；每 10 min 轻柔吹打 1 次（前 30 min），之后每 5 min 监测一次消化进度。Add digestion solution (1 mg/mL collagenase I in 0 Ca²⁺ ND96, 20 mL); rock at 70 rpm, 22 ℃; pipette gently every 10 min for the first 30 min, then monitor every 5 min.
- 终止标准：≥80% 卵母细胞游离（滤泡膜脱落），立即加入 10× 体积 0 Ca²⁺ ND96 终止；洗涤 3 次（100×g）后转入 1.8 Ca²⁺ ND96 培养液。Stop when ≥80% of oocytes are free (follicle membranes detached): add 10× volume 0 Ca²⁺ ND96 immediately; wash 3× (100×g) then transfer to 1.8 Ca²⁺ ND96.
5. 卵母细胞筛选（Dumont 分期）5. Oocyte selection (Dumont staging)
| 分期Stage | 形态特征Morphology | 直径（μm）Diameter (μm) | 适用实验Application |
|---|
| I–II | 透明或半透明，无明显色素Transparent/pale, no pigment | 50–450 | 早期发育研究Early development |
| III–IV | 局部色素，动物极开始分化Partial pigment, animal pole forming | 450–1000 | 基因显微注射Gene microinjection |
| V–VI | 动物极深棕，植物极乳白，赤道亮环清晰Dark animal pole, pale vegetal pole, clear equatorial band | 1000–1300 | 电生理 / 蛋白表达Electrophysiology / expression |
仅选用 V–VI 期、表面光滑无损伤、两半球界限清晰的健康卵母细胞；剔除破裂、褪色或残留滤泡膜的个体。Select only healthy stage V–VI oocytes with smooth surface and clear hemisphere border; discard ruptured, discoloured or follicle-contaminated oocytes.
6. 培养与质量控制6. Culture & quality control
- 培养条件：18–20 ℃ 恒温培养箱；1.8 Ca²⁺ ND96（含双抗）；每日更换培养液。Incubation: 18–20 ℃; 1.8 Ca²⁺ ND96 with antibiotics; change medium daily.
- 存活率：台盼蓝染色（0.4%，5 min）死亡率 ≤5%；静息膜电位 ≥−40 mV（微电极验证）。Viability: trypan blue (0.4%, 5 min) death rate ≤5%; resting membrane potential ≥−40 mV (microelectrode verification).
- 消化全程用 0 Ca²⁺ ND96，防止 Ca²⁺ 激活蛋白酶损伤细胞；单只雌蛙取卵间隔 ≥6 个月，累计取卵 ≤5 次。Use 0 Ca²⁺ ND96 throughout digestion to prevent Ca²⁺-activated protease damage; one female per surgery every ≥6 months, ≤5 total surgeries.
手术操作须遵循 IACUC 批准的实验方案；废弃生物组织经 10% 福尔马林固定后高压灭菌（121 ℃，30 min）处置；操作后彻底洗手。Surgery must follow an IACUC-approved protocol. Dispose of biological waste by fixation in 10% formalin followed by autoclaving (121 ℃, 30 min). Wash hands thoroughly afterwards.
参考文献References
- Goldin AL (1992) Maintenance of Xenopus laevis and oocyte injection. Methods Enzymol 207:266–279.
- Dumont JN (1972) Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals. J Morphol 136:153–180.
- Sive HL, Grainger RM, Harland RM (2000) Early Development of Xenopus laevis: A Laboratory Manual. Cold Spring Harbor Laboratory Press.