泌乳素腺瘤干细胞样细胞的鉴定及其耐药机制研究

Dopamine agonists (DA) are the primary therapy for prolactinomas. However, prolactinomas recurrence after DA withdrawal was still a key clinical problem. Recently, the studies revealed that tumor recurrence originated from radio- and chemo-resistant tumor stem cell. Our previous study shown that expression of neural stem/progenitor cell proteins in prolactinomas by immunocytochemistry. Tumor cells dissected from prolactinomas and cultured in vitro formed neurosphere-like colonies in the presence of bFGF and EGF. CD133 positive cells sorted from neurosphere-like colonies owned colony-forming ability. Tumor cell expressed dopamine D2 receptor (D2R) and was sensitive to DA therapy, on the contrary, CD133 positive cells did not express D2R and was resistant to DA therapy. This research will identify tumor stem like cells by tumor transplanted nude mice model. After that we assess the response of tumor stem like cells to DA therapy by neural colony-forming cell assay and tumor formation test. And then, we confirm the effect of D2R on the response of tumor stem like cells to DA therapy by modulation of D2R expression using lentivirus vector. Furthermore, We analyze the molecular feature and the mechanism of drug resistance of tumor stem like cells by gene chip, bioinformatics, modulation of gene expression and immunocoprecipitation. This research may contribute to resolving the clinical question of prolactinomas recurrence after DA withdrawal.

垂体泌乳素腺瘤首选多巴胺受体激动剂治疗，但其停药后复发是目前临床治疗的难题。最近研究表明肿瘤复发主要与耐受放疗和化疗的肿瘤干细胞相关。我们前期研究发现，泌乳素腺瘤标本上存在神经干/前体细胞标记物阳性的细胞，进而在肿瘤新鲜标本中分离培养出类似神经球的肿瘤细胞球，从中分选的CD133阳性细胞不仅拥有良好的克隆形成能力，而且具有不同于一般肿瘤细胞的特性：不表达D2R，且耐受溴隐亭干预。为进一步明确肿瘤干细胞样细胞及其耐药机制，本项目将首先采用裸鼠种植模型进行肿瘤干细胞样细胞鉴定，然后运用克隆形成分析及种植成瘤实验评估肿瘤干细胞样细胞对药物治疗的反应，进而通过慢病毒载体调节D2R表达，验证其对肿瘤干细胞样细胞药物反应的影响，并采用基因芯片、生物信息学、基因表达调节及免疫共沉淀等技术确认肿瘤干细胞样细胞的分子特性及耐药机制，为今后临床上解决停药后肿瘤复发的治疗难题奠定基础。

垂体泌乳素腺瘤是最常见的功能性垂体腺瘤，约占所有垂体腺瘤的40%-66%。目前，垂体泌乳素腺瘤首选多巴胺受体激动剂（DA）治疗，但需长期用药及停药后复发仍是目前临床治疗的难题。最近研究表明肿瘤复发主要与耐受放疗和化疗的肿瘤干细胞相关。本课题对泌乳素腺瘤标本进行免疫荧光检测发现存在干细胞标记物CD133、nestin表达阳性细胞，并成功从人垂体泌乳素腺瘤及大鼠垂体泌乳素腺瘤MMQ细胞株中分离培养出肿瘤干细胞样细胞。继而，本课题（1）采用CCK-8法及流式细胞凋亡分析技术检测发现MMQ细胞对DA治疗敏感，而肿瘤干细胞样细胞球细胞耐受DA治疗，但肿瘤干细胞样细胞球分化的肿瘤细胞又重新恢复对DA治疗的敏感性；(2)进一步采用免疫荧光检测发现MMQ细胞上CD133（-），多巴胺2型受体（D2R）高表达，而肿瘤干细胞样细胞球CD133（+）， D2R低或不表达，但肿瘤干细胞样细胞球分化的细胞又恢复D2R高表达，三种细胞D2R的表达与其对DA治疗敏感性相对应；（3）对泌乳素腺瘤标本进行免疫荧光检测未发现有CD133和D2R共染的细胞；（4）MMQ细胞经药物浓度梯度干预后出现CD133阳性细胞比例明显增高，而D2R阳性细胞比例显著降低，出现肿瘤干细胞样细胞富集；上述结果表明泌乳素腺瘤干细胞样细胞耐受多巴胺受体激动剂治疗，而D2R低或不表达是耐药的重要机制。课题组进一步对3组MMQ细胞和3组分选的CD133阳性细胞进行芯片检测分析，发现差异表达基因1098个，其中表达水平差异大于2倍的有562个，表达上调的有268个，表达下调的有294个，主要分布在肿瘤相关信号、MAPK信号、细胞骨架蛋白调控、细胞周期，葡萄糖代谢和P53调控等相关通路。本课题研究明确了泌乳素腺瘤干细胞样细胞耐药相关机制及分子特性，为进一步逆转肿瘤干细胞耐药研究奠定基础，为今后解决停药后复发的临床治疗难题提供新的策略。