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骨髓间充质干细胞对肾癌裸鼠皮下移植瘤的影响

发布时间:2018-11-24 14:59
【摘要】:目的:研究骨髓间充质干细胞(mesenchymal stem cells, MSC)对A498肾癌裸鼠移植瘤的影响。方法:培养肾癌A498细胞和人MSC,用CM-Dil标记MSC并对标记后MSC细胞的活性和增殖能力进行比较,构建A498肾癌裸鼠皮下移植瘤模型,实验裸鼠共36只,当裸鼠移植瘤长径达0.5-0.8cm,将其随机分为2组:一组注射CM-Dil标记的MSC (MSC组),另一组注射生理盐水(NS对照组),其中,每组各有6只用于观察生存期。从接种肿瘤的第1天起,隔天观察裸鼠饮食、排便、活动状况以及肿瘤的生长情况:在注射MSC之后的不同时间段(3天,6天,15天,30天)用游标卡尺测量裸鼠皮下移植瘤的长径、短径,并计算皮下移植瘤体积,记录注射当天开始至裸鼠死亡的时间,计算各组裸鼠平均生存时间,比较两组裸鼠生存时间差异。分别在MSC注射接种后的3天,6天,15天,30天,将MSC组和NS对照组裸鼠各处死3只,留取MSC组皮下移植瘤组织冰冻切片,在荧光显微镜下,观察CM-Dil标记的MSC在不同的时间点裸鼠肿瘤组织中的分布情况;苏木素-伊红(hematoxylin-esosin,HE)染色后行HE染色病理学观察。取各组小鼠上述时间点的肿瘤组织切片,用RT-PCR和Western Blot分别检测Twist基因和蛋白的表达情况。结果:CM-Dil标记的MSC活性和增殖与未标记的MSC无明显改变;MSC组裸鼠皮下移植瘤在注射后15天内增长缓慢,与NS对照组比较有显著差异(P0.05)。在注射MSC的15天后,MSC组皮下移植瘤增长速度减慢,15天与30天时,两组裸鼠肿瘤体积均较前增大,但差异无统计学意义(P0.05);MSC组裸鼠的生存期延长;荧光冰冻切片追踪裸鼠肿瘤组织中CM-Dil标记的MSC,荧光显微镜下观测到CM-Dil标记的MSC在绿色荧光激发下,细胞膜呈明亮的橙红色,细胞质与细胞核呈类圆形暗区,随着时间的推移,荧光亮度逐渐变弱,荧光范围也逐渐缩小;HE染色结果显示MSC组炎症反应较NS对照轻;Twist基因以及Twist蛋白在MSC组的表达明显比NS组少,差异有统计学意义(P0.05)。 结论:骨髓间充质十二细胞移植显著抑制A498肾癌裸鼠移植瘤的生长,并延迟受体裸鼠的生存时间,预示着骨髓间充质干细胞在肾癌的治疗中巨大的潜力和前景;同时,Twist的表达在这一过程中农现出显著的相关性,有望成为肿瘤发生和发展等生物学行为的重要指标,将有可能成为骨髓间充质干细胞治疗肿瘤的新靶点。
[Abstract]:Objective: to study the effect of bone marrow mesenchymal stem cell (mesenchymal stem cells, MSC) on transplanted tumor of A 498 renal cell carcinoma in nude mice. Methods: cultured A498 cells and human MSC, were labeled with MSC with CM-Dil, and the activity and proliferative ability of MSC cells were compared. A subcutaneous transplanted tumor model of A498 renal cell carcinoma in nude mice was established. There were 36 nude mice. When the diameter of transplanted tumor in nude mice was 0.5-0.8 cm, they were randomly divided into two groups: one group was injected with MSC (MSC labeled with CM-Dil) and the other group was injected with normal saline (NS control group). Six rats in each group were used to observe the survival time. From the first day of tumor inoculation, the nude mice were observed diet, defecation, activity and tumor growth on the following day: 3 days, 6 days, 15 days after MSC injection. The length and short diameter of subcutaneous transplanted tumor in nude mice were measured with Vernier caliper, the volume of subcutaneous transplanted tumor was calculated, the time from the day of injection to the death of nude mice was recorded, and the average survival time of nude mice in each group was calculated. The survival time of nude mice was compared between the two groups. At 3 days, 6 days, 15 days and 30 days after MSC inoculation, 3 nude mice in MSC group and NS control group were killed, and frozen sections of subcutaneous transplanted tumor tissue in MSC group were taken. The distribution of CM-Dil labeled MSC in tumor tissues of nude mice at different time points was observed. Hematoxylin-eosin (hematoxylin-esosin,HE) staining was followed by HE staining. RT-PCR and Western Blot were used to detect the expression of Twist gene and protein respectively. Results: the activity and proliferation of MSC labeled by CM-Dil had no significant change compared with unlabeled MSC, and the growth of subcutaneous transplanted tumor in MSC group was slow within 15 days after injection, which was significantly different from that of NS control group (P0.05). 15 days after MSC injection, the growth rate of subcutaneous transplanted tumor in MSC group slowed down. At 15 and 30 days, the tumor volume of nude mice in both groups increased, but the difference was not statistically significant (P0.05) the survival time of nude mice in); MSC group was prolonged. Fluorescence frozen sections were used to trace the CM-Dil labeled MSC, fluorescence microscope in nude mice tumor tissues. Under green fluorescence excitation, the membrane of CM-Dil labeled MSC was bright orange-red, and the cytoplasm and nucleus were round dark areas. With the passage of time, the fluorescence brightness becomes weaker, and the fluorescence range is gradually reduced. The results of HE staining showed that the inflammatory reaction in MSC group was lighter than that in NS control group. The expression of Twist gene and Twist protein in MSC group was significantly lower than that in NS group (P0.05). Conclusion: bone marrow mesenchymal 12 cell transplantation can significantly inhibit the growth of xenografts of A498 renal cell carcinoma in nude mice and delay the survival time of recipient nude mice, which indicates the great potential and prospect of bone marrow mesenchymal stem cells in the treatment of renal cell carcinoma. At the same time, the expression of Twist in this process showed a significant correlation, which is expected to become an important indicator of biological behavior, such as tumorigenesis and development, and may become a new target of bone marrow mesenchymal stem cells in the treatment of tumor.
【学位授予单位】:厦门大学
【学位级别】:硕士
【学位授予年份】:2014
【分类号】:R737.11

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