基于Wnt/β-catenin信号通路研究前列消癥汤干预前列腺癌干细胞的生物学机制

Stem cells play an important role in the occurrence and development of prostate cancer. Wnt/β-catenin signal pathway plays a critical role in maintaining the quantity and speciality of cancer stem cells.Qian lie xiao zheng decoction is an effective formula used by professor Liu youfang from Guanganmen hospital that treats prostate cancer. It can improve prostate cancer clinical symptoms and inhibit the proliferation of prostate cancer cells to PC-3M.Pilot studies indicate that Chinese medicine can inhibit Wnt/β-catenin signal pathway and affect the proliferation and differentiation of cancer stem cells. Based on this, The project puts forward a scientific hypothesis that the decoction by negative controlling activity and expression of Wnt/β-catenin downstream signal pathway could regulate biological behavior of prostate cancer stem cells.To verify the hypothesis, selecting prostate cancer kind of stem cells as research object, taking induction of Wnt/β-catenin-critical signal pathway which sustains the character of cancer stem cells as key point, combining both in vivo and in vitro experiments ,taking cell culture and molecular biology technology, this project researches Qianliexiaozheng decoction's influence to cancer stem cells form, molecule maker, infiltrating ability, transitive ability of and activity of Wnt/β-catenin, thereby reveals mechanism that Chinese medicine can regulate cancer stem cells through Wnt/β-catenin signal pathway. Which can evaluate objectively curative effect mechanism of curing by Chinese medicine and also provide a base of using medicine clinically and evaluating curative effect objectively.

干细胞在前列腺癌的发生、发展中扮演重要角色，Wnt/β-catenin信号通路（Wnt通路）在维持肿瘤干细胞的数量、特性方面发挥重要作用。前列消癥汤是广安门医院刘猷枋教授治疗前列腺癌的经验方，可改善前列腺癌患者临床症状，抑制前列腺癌细胞PC-3M增殖。前期研究显示：中医药可抑制Wnt通路，影响肿瘤干细胞分化、增殖。基于此，课题提出前列消癥汤可通过负性调控Wnt下游信号通路活性与表达，而调控前列腺癌干细胞生物学行为的科学假说。为验证假说，本课题选取前列腺癌类干细胞为研究对象，以维持肿瘤干细胞特性的关键通路Wnt通路为切入点，体内、外实验相结合，采用细胞培养和分子生物学技术，研究前列消癥汤对肿瘤干细胞形态、分子标记、浸润和迁移能力及对Wnt/β-catenin活性的影响，揭示中医药通过Wnt信号通路对肿瘤干细胞的调控机制，客观评价中医药治疗前列腺癌的疗效机制，为临床用药和疗效客观评价提供基础。

课题完成工作分为三部分，（1）观察前列消癥汤对激素非依赖性前列腺癌 PC-3 细胞增殖及侵袭力的影响；（2）采用侧群细胞分选法（SP法）和免疫表型法对前列腺癌PC-3细胞分选干细胞；（3）采用免疫磁珠法从PC-3细胞中分选出干细胞。 . 试验1用不同浓度前列消癥汤分别作用前列腺癌 PC-3 细胞24，48和72小时，用细胞增殖试验（CCK8法）测量细胞增殖，细胞流式技术测量细胞周期分布情况，Transwell法检测细胞侵袭能力。结果：前列消癥汤含药血清能抑制 PC-3 细胞增殖，减弱PC-3细胞的侵袭力。试验2采用侧群细胞分选法和免疫表型法分选干细胞，多次试验结果显示：前列腺癌PC-3细胞中CD133+和CD44+的细胞含量极低，甚至难以检测。分析原因：（1）SP法分选干细胞失败原因：可能因为PC-3细胞株中不表达ABCG2。（2）免疫表型法分选干细胞失败原因：前列腺癌PC-3细胞株多由患者体内不同转移病灶中分离建系，可能由于PC-3细胞建系前所生存的微环境不同，导致其干细胞表型发生了变化。试验3 采用免疫磁珠分选法分选干细胞，初步研究结果发现，该法分选CD133+和CD44+细胞在PC-3细胞中的含量分别为97.12%和96.89%。这表明，采用免疫磁珠分选法能够成功从PC-3细胞系分选出干细胞。. 取得的成果：已在核心期刊发表论文3篇（其中1篇已录用，待发表）。培养了1名博士研究生，1名博士后（传承）。