1. 实验动物选择与预处理1. Animal selection & pre-treatment
动物筛选标准Selection criteria
- 性成熟雌性爪蟾，体重 ≥90 g，泄殖孔松弛且无感染迹象。Sexually mature females ≥90 g body weight; cloacal region relaxed and free of infection.
- 近 6 个月内未经历取卵手术；检疫隔离 ≥7 天。No surgery within the past 6 months; minimum 7-day quarantine.
- 剔除体表溃疡、异常浮游或摄食抑制个体。Exclude animals with skin ulcers, abnormal floating or reduced feeding.
麻醉方案Anaesthesia
- 将爪蟾置于含碎冰的 0.1× MMR 缓冲液（4 ℃）中 30–45 min；麻醉终点：翻正反射消失，四肢肌肉松弛。Place in 0.1× MMR on crushed ice (4 ℃) for 30–45 min; endpoint: loss of righting reflex and limb muscle relaxation.
- 低温麻醉降低代谢率，减少手术应激，无需化学麻醉剂残留担忧。Cold anaesthesia lowers metabolic rate and reduces surgical stress without concerns about chemical residues.
2. 手术操作规范2. Surgical procedure
器械灭菌Instrument sterilisation
- 剪刀、镊子、缝合针线高压灭菌（121 ℃，20 min）或 75% 乙醇浸泡 30 min。Autoclave scissors, forceps and sutures (121 ℃, 20 min) or soak in 75% ethanol for 30 min.
切口与取卵Incision & excision
- 下腹部正中线（避开腹主动脉分支）纵向切开皮肤及肌层 0.5–1 cm。Make a 0.5–1 cm longitudinal incision through skin and muscle on the ventral midline (avoiding the aortic branches).
- 用钝头镊轻拉卵巢瓣，剪取所需卵母细胞团块（单次取卵量 ≤卵巢总量的 1/3）；置于 OR2 溶液中。Gently pull the ovary lobe with blunt forceps; excise required clumps (≤1/3 of total ovary per surgery) and transfer to OR2 solution.
术后缝合与护理Suture & recovery
- 肌层连续缝合（5-0 可吸收缝线），皮肤间断缝合（6-0 尼龙线）。Continuous muscle suture (5-0 absorbable), interrupted skin suture (6-0 nylon).
- 术后置于浅水容器（水深 ≤2 cm，20–22 ℃），头部暴露水面自然复苏；术后 24 h 内禁食，监测伤口愈合。Recover in shallow water (≤2 cm deep, 20–22 ℃) with head above surface; withhold food for 24 h and monitor wound healing.
3. 卵母细胞采集与 OR2 保存液3. Collection & OR2 storage solution
OR2 溶液配方（pH 7.8 ± 0.1）OR2 solution (pH 7.8 ± 0.1)
| 成分Component | 浓度（mmol/L）Concentration (mmol/L) | 功能Function |
|---|---|---|
| NaCl | 41.0 | 维持渗透压Osmolarity |
| KCl | 1.0 | 稳定膜电位Membrane potential |
| MgCl₂ | 0.5 | 防止卵母细胞粘连Prevent clumping |
| HEPES | 2.5 | 缓冲 pHpH buffer |
4 ℃ 保存，有效期 ≤2 h（取卵后立即使用）。OR2 不含 Ca²⁺，可防止卵泡膜收缩、减少组织损伤。Store at 4 ℃; use within 2 h of collection. Ca²⁺-free OR2 prevents follicle membrane contraction and minimises tissue damage.
- 将卵母细胞团用显微剪剪成含 5–10 个细胞的小块；OR2 溶液洗涤 5 次（100×g，1 min）至上清澄清；剔除破裂或褪色细胞。Trim clumps to 5–10 cells with microscissors; wash 5× in OR2 (100×g, 1 min) until supernatant is clear; discard ruptured or discoloured oocytes.
4. 胶原酶酶解分离4. Collagenase digestion
| 参数Parameter | 设定值Value |
|---|---|
| 胶原酶浓度（Sigma C0130）Collagenase (Sigma C0130) | 0.5–2.0 mg/mL in OR2 |
| 温度Temperature | 22–25 ℃ |
| 振荡频率Shaking | 80 rpm（水平摇床） |
| 消化时间Digestion time | 30–45 min（镜下监测卵泡膜脱落） |
| 终止方法Stop method | 10% FBS in OR2，洗涤 3× |
每 10 min 轻柔吹打一次；当 ≥80% 卵母细胞游离时立即终止，过度消化损伤卵膜。Pipette gently every 10 min; stop immediately when ≥80% oocytes are free — over-digestion damages the vitelline membrane.
5. 卵母细胞培养5. Oocyte culture
- 消化后的卵母细胞转入 OR2（含矿物油覆盖防止蒸发）的 60 mm 培养皿，17 ℃ 恒温培养箱（湿度 70%，无 CO₂），培养时间 ≤24 h。Transfer digested oocytes to a 60 mm dish with OR2 (mineral oil overlay); incubate at 17 ℃ (70% humidity, no CO₂) for ≤24 h.
- 实验所需选取 V–VI 期卵母细胞（动物极色素均匀，直径 ≥1.2 mm）。Select stage V–VI oocytes for experiments (uniform animal-pole pigment, diameter ≥1.2 mm).
6. 质量控制6. Quality control
| 项目Parameter | 标准范围Acceptable range | 检测频率Frequency |
|---|---|---|
| 存活率（台盼蓝）Viability (trypan blue) | ≥90% | 每批次Per batch |
| 胶原酶活性Collagenase activity | 200–400 U/mg | 每新批次Per new lot |
| OR2 渗透压OR2 osmolarity | 180–200 mOsm/kg | 每周Weekly |
异常处理：卵母细胞大量破裂 → 降低胶原酶浓度或缩短消化时间；术后感染 → 隔离个体，皮下注射恩诺沙星（5 mg/kg）。Troubleshooting: mass lysis → reduce collagenase concentration or shorten digestion; post-op infection → isolate animal, subcutaneous enrofloxacin (5 mg/kg).
7. 重复取卵管理与生物安全7. Repeated surgery & biosafety
- 术后恢复期 ≥6 个月，期间提供高蛋白饲料（粗蛋白 ≥45%）；单只雌蟾终生取卵 ≤6 次。Recovery interval ≥6 months with high-protein feed (crude protein ≥45%); ≤6 total surgeries per female.
- 废弃卵巢组织经 10% 福尔马林固定后高压灭菌处置（121 ℃，30 min）；操作后彻底洗手。Dispose of waste ovarian tissue by fixation in 10% formalin followed by autoclaving (121 ℃, 30 min); wash hands thoroughly.
所有手术须取得所在机构 IACUC 批准，全程遵循无菌原则与动物福利规定。All surgeries require IACUC approval; maintain aseptic technique and animal welfare standards throughout.
参考文献References
- Goldin AL (1992) Maintenance of Xenopus laevis and oocyte injection. Methods Enzymol 207:266–279.
- Sigel E (1990) Use of Xenopus oocytes for the functional expression of plasma membrane proteins. J Membr Biol 117(3):201–221.
- Sive HL, Grainger RM, Harland RM (2000) Early Development of Xenopus laevis: A Laboratory Manual. Cold Spring Harbor Laboratory Press.