骨髓间充质干细胞在前列腺癌骨转移中的作用

发布时间：2018-05-22 08:13

本文选题：前列腺癌 + 间充质干细胞　；参考：《天津医科大学》2017年硕士论文

【摘要】：目的前列腺癌发展到晚期阶段常伴有骨骼的转移,严重影响着患者的生活质量。本研究通过体外细胞实验研究前列腺癌好发骨转移的原因和机制。通过研究骨髓间充质干细胞对于前列腺癌发生骨转移的作用机制,进一步寻找临床预防、治疗前列腺癌骨转移的新对策,从而采取相应的干预措施抑制前列腺癌的复发和转移,最终提高患者的生活质量。方法1.(1)提取小鼠骨髓,根据细胞贴壁生长方式不同,通过全骨髓培养的方法分离并纯化小鼠骨髓间充质干细胞,培养过程中观察细胞的形态学变化。(2)在体外通过诱导剂对间充质干细胞向脂肪细胞、成骨细胞的定向诱导,再行油红O染色、茜素红染色,对间充质干细胞进行鉴定。(3)收集培养间充质干细胞的上清液,标记记为间充质干细胞条件培养基(MSC-CM)。用MSC-CM培养对数生长期的前列腺癌细胞C42细胞1-5天,通过MTT的方法检测各细胞培养孔的吸光值,从而绘制出生长曲线。(4)细胞划痕实验,观察充质干细胞条件培养基处理C42细胞48个小时后细胞的爬片情况,使用Image J软件计算培养细胞的划痕面积。Transwell实验,在上室加入一定数量细胞,下室加入适量的充质干细胞条件培养基,培养24小时后,通过Giemsa染色观察转移至下室细胞的数量。2.(1)利用免疫磁珠细胞分选的方法(MACS)分选出CD133+前列腺癌干细胞,通过RT-PCR和Western Blot实验进一步验证细胞的干性。(2)用DMEM培养基培养CD133+C42细胞1-5天。通过MTT的方法检测各细胞培养孔的吸光值,绘制生长曲线。(3)用间充质干细胞条件培养基(MSC-CM)培养前列腺癌干细胞CD133+C42,分别在第2天及第4天后提取细胞的RNA及蛋白。通过RT-PCR和Western Blot实验观察前列腺癌干细胞表面标志物CD133、CD44的表达情况。结果1.(1)间充质干细胞在培养24小时后开始贴壁,可见短梭形、圆形以及多角形的细胞贴壁生长。3天后细胞突起长短不一。细胞生长第8天细胞密集生长。(2)间充质干细胞诱导形成的脂肪细胞脂质聚集,经过油红O染色,脂滴被染色橘红色,细胞核呈浅蓝色。经诱导形成的成骨细胞形态为多角形,体积增大。经过茜素红染色,产生深红色化合物。(3)生长曲线结果可见MSC-CM组的细胞增殖能力明显增加。(4)细胞划痕实验显示MSC-CM组细胞的划痕面积明显大于对照组。Transwell实验显示MSC-CM组细胞的数量明显大于对照组。2.(1)免疫磁珠细胞分选法(MACS)分选出CD133+C42,通过基因和蛋白水平对其检测,结果符合肿瘤干细胞的生物学特性。(2)MTT实验结果显示,MACS分选的前列腺癌干细胞CD133+C42在体外的增殖能力与未分选的前列腺癌细胞C42比较有明显增强。(3)对照组CD133、CD44表达逐渐减弱,而MSC-CM实验组能够稳定表达。结论1.骨髓间充质干细胞(MSCs)能够显著促进前列腺癌细胞的增殖、迁移和侵袭能力。2.骨髓间充质干细胞(MSCs)能够维持前列腺癌干细胞干性的稳定表达。
[Abstract]:Objective Advanced stage of prostate cancer is often accompanied by bone metastasis, which seriously affects the quality of life of patients. In this study, we studied the cause and mechanism of bone metastasis of prostate cancer by in vitro cell experiment. By studying the mechanism of bone marrow mesenchymal stem cells (BMSCs) on bone metastasis of prostate cancer, we can find a new strategy of clinical prevention and treatment of bone metastasis of prostate cancer, and take corresponding intervention measures to suppress the recurrence and metastasis of prostate cancer. Ultimately improve the patient's quality of life. Methods 1. Mouse bone marrow was isolated and purified by whole bone marrow culture. In vitro, mesenchymal stem cells were induced to adipocytes, osteoblasts were induced by oil red O staining and alizarin red staining. The supernatant of cultured mesenchymal stem cells (MSCs) was collected and labeled as MSCs conditioned medium (MSC-CMM). C42 cells in logarithmic growth phase were cultured with MSC-CM for 1-5 days. The absorptivity of each cell culture pore was measured by MTT method, and the growth curve was drawn. To observe the climbing of C42 cells after 48 hours treatment with the conditioned medium of MSCs, the scratch area of cultured cells was calculated by Image J software, and a certain number of cells were added to the upper chamber. After 24 hours of culture, the number of CD133 prostate cancer cells transferred to the lower chamber was observed by Giemsa staining. The CD133 prostate cancer stem cells were isolated by immunomagnetic bead cell sorting. The dryness of CD133 C42 cells was further verified by RT-PCR and Western Blot experiments. CD133 C42 cells were cultured on DMEM medium for 1-5 days. MTT method was used to detect the absorptivity of each cell culture pore, and the growth curve was drawn. 3) Prostate cancer stem cells (CD133 C42) were cultured in MSCs conditioned medium (MSC-CM). The RNA and protein were extracted on the 2nd and 4th day, respectively. The expression of CD133 and CD44 on prostate cancer stem cells was observed by RT-PCR and Western Blot. Results 1. The mesenchymal stem cells began to adhere to the wall after 24 hours of culture. The short fusiform, round and polygonal cells had different cell processes after 3 days of adherent growth. 2. On the 8th day of cell growth, lipid aggregation of adipocytes induced by mesenchymal stem cells (MSCs) was induced. Lipid droplets were stained orange with light blue nuclei after oil red O staining. The induced osteoblasts were polygonal in shape and enlarged in volume. Dyed with alizarin red, The result of the growth curve showed that the cell proliferation ability of MSC-CM group was obviously increased. (4) Cell scratch test showed that the cell scratch area of MSC-CM group was obviously larger than that of control group. Transwell experiment showed that the number of cells in MSC-CM group was obviously larger than that in MSC-CM group. CD133 C42was isolated by immunomagnetic bead cell sorting method and detected by gene and protein levels. Results according to the biological characteristics of tumor stem cells, the results of MTT assay showed that the proliferation ability of prostate cancer stem cell CD133 C42 isolated by Macs in vitro was significantly enhanced compared with that of non-differentiated prostate cancer cell line C42) the expression of CD133pCD44 in the control group was gradually decreased. The expression of MSC-CM was stable in the experimental group. Conclusion 1. Bone marrow mesenchymal stem cells (MSCs) can significantly promote the proliferation, migration and invasion of prostate cancer cells. Bone marrow mesenchymal stem cells (MSCs) can maintain the dry expression of prostate cancer stem cells.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.25

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