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目的:建立可稳定传代的肾透明细胞癌小鼠肺转移模型,并在此基础上初步筛选出肾透明细胞癌特异性鼠肺转移细胞亚株,为后续研究奠定基础。方法:选用本实验室前期建立的肾透明细胞癌细胞株RCC05-TXJ,体外扩增培养制成细胞悬液后注射于NOD-SCID小鼠颈背部皮下,成瘤后取出瘤组织接种于另外NOD-SCID小鼠的肾包膜下,待有肉眼可见瘤块后,处死小鼠并取出肿瘤组织接种于新NOD-SCID小鼠的肾包膜下,反复数轮,直至出现稳定的鼠肺转移模型。此时取模型鼠肺转移组织,一部分继续接种于新鼠肾包膜下,反复数轮,尝试以转移组织诱导肺转移;另一部分进行原代培养,细胞经体外扩增纯化后制成悬液接种于新鼠肾包膜,数轮循环至产生肺转移,筛选能产生稳定鼠肺转移的细胞亚株。整个筛选过程均观察记录原位成瘤、淋巴及肺转移形成情况,并对肿瘤组织进行病理组织学观察及CA9(肾癌标志)、CD133(干细胞标志)表达检测。结果:以RCC05-TXJ细胞株接种所致NOD-SCID小鼠颈背部皮下肿瘤组织作为移植瘤材接种鼠肾包膜在第2轮即出现鼠肺转移,此后3～6轮以鼠肺转移组织移植肾包膜下,均能成功诱导转移。以鼠肺转移组织原代培养RCC细胞均获成功,第6轮原代培养细胞扩增纯化后制成的细胞悬液(RCC05-TXJ-L)接种于鼠肾包膜下,出现特异性肺转移,重复2轮实验结果稳定。RCC05-TXJ-L细胞具有生长速度快(传代时间约为2d)、原位成瘤时间短(1周左右)、接种后转移性较强、CA9及CD133表达升高等特点。结论:RCC05-TXJ在NOD-SCID小鼠体内多轮反复筛选可建立稳定的肾细胞癌鼠肺转移动物模型;在此基础上筛选出的肺转移性细胞亚株RCC05-TXJ-L可稳定诱导NOD-SCID鼠产生肺部转移。
OBJECTIVE: To establish a lung metastasis model of mouse renal clear cell carcinoma which can be stably passaged, and based on which a preliminary screening of renal clear cell carcinoma-specific mouse lung metastatic subtypes was established, which laid the foundation for further study. Methods: Renal cell line RCC05-TXJ established in our laboratory was expanded and cultured in vitro. The cell suspension was injected into the back of the neck of NOD-SCID mice subcutaneously. After the tumor was removed, the tumor tissues were inoculated into other NOD -SCID mice under the renal capsule, until visible tumor blocks, the mice were sacrificed and tumor tissue was removed and inoculated under the renal capsule of new NOD-SCID mice, repeated several rounds until a stable rat lung metastasis model. At this point take the model mouse lung metastasis, part of the continued inoculation in the rat renal capsule, repeated rounds, try to transfer tissue to induce lung metastasis; the other part of the primary culture, the cells were amplified by in vitro purification made suspension Inoculated in the nephritis kidney capsule, a few cycles to produce lung metastasis, screening can produce stable mouse lung metastasis subline. Throughout the screening process were observed in situ tumor formation, lymph and lung metastasis, and histopathological observation of tumor tissue and CA9 (renal cell carcinoma marker), CD133 (stem cell marker) expression detection. Results: Inoculation of RCC05-TXJ cell line NOD-SCID mice back of the neck subcutaneous tumor tissue as a tumor material inoculated rat kidney capsule in the second round of mouse lung metastasis occurred, after 3 to 6 rounds of lung metastasis Transplanted renal capsule, can successfully induce metastasis. Primary culture of RCC cells with murine lung metastasis were successful. The sixth round of primary cultured cells were expanded and purified and the cell suspension (RCC05-TXJ-L) was inoculated into the rat renal capsule with specific lung Transfer, repeat two rounds of experimental results stable. RCC05-TXJ-L cells with fast growth (passage time of about 2d), in situ tumor formation time is short (about 1 week), after inoculation strong metastasis, CA9 and CD133 expression increased characteristics. CONCLUSIONS: RCC05-TXJ can be used to establish a stable animal model of lung metastasis of rat renal cell carcinoma by multiple rounds of repeated screening in NOD-SCID mice. Based on this, RCC05-TXJ can stably induce lung metastatic subgroup RCC05-TXJ-L NOD-SCID mice produce lung metastases.