骨髓间充质干细胞对裸鼠肾癌细胞系A498生物学特性影响的研究

The research on the biological characteristics of renal carcinoma cell line A498 of bone marrow mesenchymal stem cells

目的 研究骨髓间充质干细胞对肾癌细胞系A498其生物学特性的影响。方法 培养肾癌A498细胞及原代分离小鼠骨髓间充质干细胞,采用CM-Dil标记骨髓间充质干细胞活性和增殖能力。取36只雄性裸鼠进行实验,采用随机对照方法将裸鼠分为对照组和实验组,每组18例。利用A498细胞系建立裸鼠皮下移植瘤模型,当肿瘤移植瘤长为0.5-0.8 cm时,对照组注入生理盐水,实验组注入骨髓间充质干细胞。比较两组荷瘤小鼠间生存时间差异及生物学特性;利用Tranwell细胞转移模型,比较处理组A498细胞转移能力的改变。结果 人肾癌A498细胞成功接种在裸鼠背侧皮下,20 d后皮下移植瘤长径为0.5-0.8 cm,成瘤率为100%。骨髓间充质干细胞组皮下注射3-15 d内体积增殖速度较快,与生理盐水组比较差异具有统计学意义（P〈0.05）;荧光显微镜下显示：骨髓间充质干细胞在绿色荧光激发下细胞呈现橙红色,细胞质与细胞核呈现类圆形。细胞移植3 d后,CM-Dil主要位于针道附近,多为圆形;移植6 d后,骨髓间充质干细胞向周围迁移,且圆形细胞数目减少。移植15 d后,骨髓间充质干细胞红色明亮度下降;随着时间的不断延长,两组裸鼠生存周期呈现下降趋势,骨髓间充质干细胞组周期死亡率,显著高于生理盐水组（P〈0.05）;骨髓间充质干细胞组注射3 d后肿瘤细胞异型性明显,胞浆比较丰富,肿瘤中间血管相对较多;注射6 d后,肿瘤细胞周围存在炎细胞浸润;生理盐水组注射3 d后肿瘤细胞呈弥漫性生长,瘤细胞体积变大,间质内微血管结构清楚,炎性细胞相对较多;骨髓间充质干细胞与肾癌细胞系A498共同培养后,能够显著能加细胞转移能力（P〈0.05）。结论 骨髓间充质干细胞属于非成体干细胞,具备自我更新和多向分化潜能,能促进A498肾癌裸鼠瘤的生长,增加肿瘤的恶性程度,且细胞生物学特性良好。研究骨髓间充质干细胞对肾癌细胞系A498的影响能为临床治疗提供依据和参考,具有较高的临床应用价值。

Objective To study the biological characteristics of bone marrow mesenchymal stem cells on renal carcinoma cell line A498 and provide the basis for clinical. Methods Renal cell carcinoma A498 cells and bone marrow mesenchymal stem cells were cultured, and inter -CM - Dil were used to label and prolifer bone marrow mesenchymal Stem cell activity. 36 mice were analyzed and randomly divided into a control group and a bone marrow mesenchymal stem cell group, 18 cases in each group. Subcutaneous tumor model in nude mice were build. When the tumor xenografts length reached 0. 5-8 cm, the control group were injected with saline, mesenchymal stem cells were injected into the group of bone marrow mesenchymal stem cells. The survival time and biological characteristics of the two groups were compared. The tran-swell assay was performed to compare the difference of cocultured A498 cell between the two groups. Results Human renal carcinoma A498 cells were successfully inoculated subcutaneously in nude backside. Long diameter of subcutaneous tumor reached 0. 5-8 cm after 20 d, tumor formation rate was 100% . Proliferation rate in research group in 3 -15 d was faster than that of control group with statistically significant difference （ P 〈0. 05） with the saline group differences; fluorescence microscopy showed： bone marrow mesenchymal stem cells in the green fluorescence excitation cell showed orange - red shape, the cytoplasm and the nucleus render round. 3 d after cell transplantation, CM - Dil was main near the needle and mostly round ; 6 d after transplantation, mesenchymal stem cells migrate to the surrounding, and the number of round cells were reduced. After transplanting 15 d, brightness of red bone marrow mesenchymal stem cells decreased ; With the continuous extension of time, two groups of mice survival period show a downward trend, mesenchymal stem cells cycle group mortality was significantly higher than that of saline group （ P 〈0.05）. After injection of tumor cells 3 d, atypia in Mesenchymal stem cell group was significant, cytoplasm was rich and the blood vessels in the middle of the tumor was rich ; after injection 6 d, inflammatory cells around the tumor cells were observed ; 3 d after injection of tumor cells in the saline group, diffuse growth of large tumor cells, clear interstitial capillary structure, more inflammatory cells were showed. Bone marrow mesenchymal stem cells co - cultured renal carcinoma cell line A498 can improve the capability of cell metastasis. Conclusion Bone marrow mesenchymal stem cells are adult stem cells with self - renewal and differentiation potential, and can promote A498 renal cell carcinoma in nude mice tumor growth, increase the degree of malignancy. The biological characteristics of the cells is good, the currentstudy can provide the basis and reference for clinical treatment with high clinical value.