分选卵巢癌腹水中癌细胞的实验研究

Establishment of methodology of sorting tumor cells from ovarian cancer peritoneal fluids

摘要:

目的 探讨免疫荧光细胞化学法（ICC）联合荧光原位杂交法（FISH）分离卵巢癌腹水中癌细 胞的可行性。方法 复制卵巢癌腹水模型，分阴性对照组和4 个阳性组（A、B、C、D 组），即4 ml 良性腹 腔冲洗液中分别有0、5、10、20 和40 个标记线粒体绿色荧光（Mito-Tracker Green）的卵巢癌SKOV3 细胞， 每组样本制备3 份。复制卵巢癌裸鼠原位移植模型，分阳性组和阴性对照组，每组6 只BALB/c 裸鼠，阳性组 裸鼠在卵巢接种SKOV3 细胞，各组分别在接种后4、6 和8 周取2 只裸鼠收集腹水。采用磁激活细胞分选法 富集癌细胞，用ICC-FISH 鉴别分离样本中的癌细胞，以8 号染色体探针（CEP8）信号>2 个为FISH 阳性标准， 规定DAPI+/EBA-1+/Mito-Tracker Green+/CEP8+ 细胞为检测癌细胞。结果 腹水模型：阴性对照组和阳 性组均可见EBA-1 阳性细胞，阳性组均有标记的SKOV3 细胞，癌细胞的回收率为20% ～ 50%，12 个阳性样 本中9 个样本的检测率为100%。裸鼠模型：在200 倍显微镜视野下，4、6 和8 周每个视野的SKOV3 细胞数 最多分别为3、8 和15 个。结论 将卵巢癌腹水磁富集后，ICC-FISH 可以准确地识别其中的癌细胞，该方法 为研究和治疗卵巢癌提供了新思路。

Abstract:

Objective To investigate the viability of the protocol to sort tumor cells from ovarian cancer peritoneal fluids by combining immunocytochemistry (ICC) and fluorescence in situ hybridization (FISH). Methods The ovarian cancer peritoneal fluid model was established and divided into a negative control group and four positive groups by adding 0, 5, 10, 20 and 40 ovarian cancers SKOV3 cells marked by Mito-Tracker Green into 4 ml benign peritoneal washing fluid respectively, 3 duplications per group. The nude mouse model bearing orthotopically transplanted human ovarian cancer SKOV3 cells was established, including the negative control group and the positive group, with 6 BALB/c nude mice per group and 2 nude mice at each time point. Peritoneal fluids of the nude mice in the positive group were collected at the 4th, 6th and 8th w after transplanting SKOV3 cells under the capsule of the left ovary. SKOV3 cells were enriched from the peritoneal fluids by magnetic activated cell sorting (MACS). Then, the cancer cells were identified exactly by EBA-1-ICC and CEP8-FISH (Chromosome Enumeration Probe). The cells with the characteristic of DAPI+/EBA-1+/Mito-Tracker Green+/CEP8+ were classified as detected cancer cells by the positive standard of CEP8 >2. Results In the peritoneal fluid model, the EBA-1-positive cells were found in the negative control group and all SKOV3 cell-positive groups, the marked SKOV3 cells were found in all SKOV3 cell-positive groups and their recovery rates were 20%-50%, the detection rates in 9 of 12 SKOV3 cell-positive samples were 100%. In the nude mouse model, under every 20×10 microscopic field, the maximum number of SKOV3 cancer cells was 3, 8 and 15 respectively at the 4th, 6th and 8th w. Conclusions The protocol of combining ICC and FISH can exactly identify cancer cells from ovarian cancer peritoneal fluids which have been enriched by MACS. The technique provides new ideas for studying ovarian cancers.