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目的筛选卵巢上皮性癌(卵巢癌)淋巴道定向高转移恶性肿瘤细胞亚群,并对其生物学特性进行鉴定。方法将卵巢癌细胞株 SKOV3细胞接种于裸鼠爪垫皮下,待植入细胞在裸鼠体内成瘤并发生转移时,取其淋巴结转移灶中癌细胞再次接种于裸鼠爪垫皮下传代,每代均取出淋巴结转移灶中癌细胞进行培养,通过单细胞分离(克隆)培养或局部淋巴结转移灶筛选的方法建立连续传代细胞系,重复传3代后,通过裸鼠接种实验观察各代裸鼠的淋巴结转移率。利用细胞生长曲线、HE 染色、染色体分析、流式细胞分析、裸鼠接种、免疫组化等方法鉴定各代细胞的生物学特性。结果所建立的连续传代1～3代的细胞系,分别命名为 SKOV3-PM1、SKOV3-PM2和 SKOV3-PM3细胞,癌细胞在裸鼠体内传3代后,裸鼠的淋巴结转移率为100%(10/10),原代 SKOV3细胞的淋巴结转移率为10%(1/10),两者比较,差异有统计学意义(P<0.05);SKOV3-PM3细胞形成的淋巴结转移灶的数目(26/10)较 SKOV3细胞多(1/10,P<0.01);且转移灶多位于淋巴结。细胞染色体分析显示,SKOV3细胞染色体数目大多数分布在83～89条,SKOV3-PM3细胞染色体数目大多数分布在91～105条。SKOV3与 SKOV3-PM3细胞染色体数目比较,差异有统计学意义(P<0.05);转移灶中上皮膜抗原(EMA)表达呈阳性,证实转移细胞株保持着卵巢癌细胞的生物学特性;SKOV3-PM3细胞倍增时间为22.7 h,SKOV3细胞为49.6 h,两者比较,差异有统计学意义(P<0.05)。流式细胞分析显示,SKOV3-PM1、SKOV3-PM2、SKOV3-PM3细胞的淋巴结转移灶中癌细胞的 DNA 合成期和分裂期细胞的比例分别为24.2%、29.4%和36.7%,明显高于 SKOV3细胞的21.5%(P<0.05)。结论本研究成功建立了卵巢癌淋巴道定向高转移细胞系,为研究卵巢癌的淋巴结浸润转移的发病机制提供了良好的实验材料。
Objective To screen ovarian epithelial carcinoma (ovarian cancer) lymphatic metastatic malignant tumor cell subgroup and to identify its biological characteristics. Methods The ovarian cancer cell line SKOV3 was inoculated subcutaneously into the nude mouse paw pads. When the implanted cells were tumorigenic and metastasized in nude mice, the cancerous cells in the metastatic lymph node were re-inoculated subcutaneously in the nude mouse paw. Each time, On behalf of the cancer cells in lymph node metastasis were removed for culture, single cell isolation (cloning) or local lymph node metastasis screening method to establish a continuous passage cell line, after repeated passage 3 generations, nude mice inoculation experiment observed the generation of nude mice Lymph node metastasis rate. The biological characteristics of each cell line were identified by cell growth curve, HE staining, chromosome analysis, flow cytometry analysis, nude mice inoculation and immunohistochemistry. Results The established cell lines of passage 1 to passage 3 were named as SKOV3-PM1, SKOV3-PM2 and SKOV3-PM3 respectively. After 3 passages of cancer cells in nude mice, the rate of lymph node metastasis in nude mice was 100% (10/10). The rate of lymph node metastasis in primary SKOV3 cells was 10% (1/10), the difference was statistically significant (P <0.05). The number of metastatic lymph nodes in SKOV3-PM3 cells 26/10) than SKOV3 cells (1/10, P <0.01), and the metastasis mostly located in lymph nodes. Chromosome analysis revealed that the majority of chromosomes in SKOV3 cells ranged from 83 to 89, while the majority of chromosomes in SKOV3-PM3 cells ranged from 91 to 105. The number of chromosomes in SKOV3 and SKOV3-PM3 cells was significantly different (P <0.05), and the positive expression of epithelial membrane antigen (EMA) in metastatic cells confirmed that the metastatic cell lines maintained the biological characteristics of ovarian cancer cells. The number of SKOV3- PM3 cell doubling time was 22.7 h, SKOV3 cells was 49.6 h, the difference between the two was statistically significant (P <0.05). Flow cytometry analysis showed that the ratio of DNA synthesis and mitosis of cancer cells in lymph node metastasis of SKOV3-PM1, SKOV3-PM2 and SKOV3-PM3 cells were 24.2%, 29.4% and 36.7% respectively, which was significantly higher than that of SKOV3 21.5% of cells (P <0.05). Conclusion This study successfully established lymphatic metastatic ovarian cancer cell lines, in order to study the pathogenesis of lymph node metastasis of ovarian cancer provides a good experimental material.