【作者】 马四海；

【导师】 王任直；

【作者基本信息】 北京协和医学院 ， 外科学， 2011， 博士

【摘要】 垂体腺瘤(pituitary adenoma)是最多发的颅内肿瘤之一,其解剖检出率为25%。该肿瘤的生物学性状及临床表现存在较大个体差异。部分垂体腺瘤生长缓慢,无明显临床症状,只在体检时被发现,无需特殊治疗。而另有部分垂体腺瘤,常规手术、放疗及垂体药物(例如,多巴胺受体激动剂及生长抑素类似物)均难以将其治愈,病变反复发作、持续进展,少部分出现肿瘤组织的远位转移,即垂体腺癌,其发病率为0.2%。在筛选难治性垂体腺瘤替代治疗措施的过程中,替莫唑胺(temozolomide, TMZ)已被部分临床试验报道疗效确切。但另有部分报道未发现TMZ对该类肿瘤发挥稳定疗效,其对垂体腺瘤抑制作用的分子机制仍不明确。本实验研究以垂体腺瘤原代细胞及细胞系为平台,探讨TMZ对垂体腺瘤的效用及相关分子生物学机制,并报道一例不典型垂体腺瘤的替莫唑胺临床治疗情况。第一部分垂体腺瘤细胞的体外培养目的：分析影响垂体腺瘤细胞体外生存的因素,探讨适于垂体腺瘤细胞维持生物学性状的体外培养方案,改良人垂体腺瘤细胞体外生存条件,为临床及基础实验研究提供可靠的体外实验平台。方法：取无功能垂体腺瘤手术标本33例,行体外分离、接种及培养。并就细胞的体外生存时间行生存相关因素分析。肿瘤细胞接种于Laminin、Matrigel、PPL Collagen及普通培养板界面,观察各细胞培养界面对垂体肿瘤细胞活力的影响。结果：Kaplan Meier生存分析发现细胞体外存活时间与患者年龄、性别、肿瘤类型、肿瘤是否复发、肿瘤侵袭与否、术前是否接受过放疗及Ki-67 LI水平等均没有明显相关(P>0.05)。二维及三维培养界面无功能垂体腺瘤原代细胞活力对比分析显示,Matrigel、Laminin、Collagen组细胞活力明显大于细胞培养板裸面培养的细胞(Matrige、Laminin组vs. NC, P<0.01; Collagen组vs. NC, P<0.001)。而Lysine组与对照组相比没有明显差异(P>0.05)。Matrigel及Laminin组细胞活力大于其它各培养界面的细胞(P<0.001, vs. Lysine; P<0.01 vs.Collagen)。collagen组细胞活力则高于Lysine组(P<0.05)。Matrigel与Laminin组细胞活力没有统计学差异(P>0.05)。因此,各细胞培养界面的无功能垂体腺瘤细胞活力大小排序为：Matrigel、Laminin组>Collagen组>Lysine及普通培养板裸面组。结论：体外培养的无功能垂体腺瘤细胞生长缓慢。其体外生存时间与患者年龄、性别、肿瘤类型、肿瘤是否复发、肿瘤侵袭与否、术前是否接受过放疗及Ki-67 LI水平等均没有明显相关。Laminin及Matrigel培养界面可以较PPL及Collagen三维培养界面更好地提升垂体腺瘤细胞的活力,可为垂体腺瘤体外实验研究提供活力更接近在体环境的细胞平台。第二部分替莫唑胺对促性腺激素垂体腺瘤细胞的效用及机制目的：常规手术、化疗及放疗多难以治愈侵袭性垂体腺瘤,新的替代疗法亟待研发。临床已尝试将替莫唑胺扩大适应症用于治疗侵袭性垂体腺瘤,但临床疗效报道存在分歧。本实验首次研究替莫唑胺对促性腺激素腺瘤细胞活力及激素分泌的抑制效果及可能机制。方法：梯度浓度的替莫唑胺(31.25-1000μM)或溶剂对照孵育aT3-1细胞0-72 h后,CCK-8法检测细胞活力,并依据细胞生长抑制曲线获得IC5o值。收集IC5o浓度替莫唑胺处理的细胞样本行流式细胞技术、TUNEL染色及投射电子显微镜检测,对细胞周期改变及凋亡程度进行统计学分析。使用Caspase-Glo(?)试剂盒检测肿瘤细胞caspase-3/7,-8及-9的活性改变。Western blotting法检测凋亡相关蛋白Bcl-2及PARP的表达情况。Centaur(?) XP免疫测定系统检测细胞培养基上清促性腺激素水平变化。结果：替莫唑胺剂量及时间依赖性抑制aT3-1细胞活力(P<0.01 vs. control,250μM,24 h)并导致肿瘤细胞周期S期延长及G2/M期阻滞(P<0.05 vs. control,250μM,24 h)。替莫唑胺孵育24 h可明显诱发早期细胞凋亡(P<0.001 vs. control,250μM),透射电镜及TUNEL染色发现肿瘤细胞核团缩、染色质浓聚等细胞凋亡形态学改变。替莫唑胺(250,μM)孵育24 h将aT3-1细胞caspase-3/7的活性提升了6倍,caspase-9活性提升了7倍,caspase-8活性提升了3倍,且显著负调抗凋亡蛋白Bcl-2的表达(P< 0.001 vs. control)并促进PARP的蛋白裂解。细胞培养基上清的FSH及LH水平在替莫唑胺孵育后明显下降(P<0.01 vs. control,250μM,24h)。结论：替莫唑胺稳定抑制促性腺激素腺瘤细胞活力并下调促性腺激素水平,诱导垂体肿瘤细胞周期阻滞,且通过调控死亡受体及线粒体凋亡信号通路促进肿瘤细胞凋亡。替莫唑胺可能成为侵袭性垂体腺瘤的可靠替代疗法。第三部分替莫唑胺对人原代垂体腺瘤细胞的药效及其与肿瘤MGMT表达水平的相关性分析目的：观察替莫唑胺对人原代垂体腺瘤细胞的药效,并检验MGMT表达水平对肿瘤TMZ敏感性指示作用的准确度。方法：取垂体腺瘤手术标本26例,行体外分离、接种及培养。原代细胞平台上行TMZ药物敏感试验,计算每株细胞的TMZ IC50数值。免疫组化法检测细胞来源肿瘤的MGMT表达水平,并将其与细胞的TMZ IC50数值进行Spearman相关分析。结果：TMZ处理的26例原代垂体腺瘤细胞中,24例(92.31%,2例无功能垂体腺瘤除外)表现TMZ剂量依赖性活力抑制；9例(34.620%,3例GH腺瘤,6例无功能垂体腺瘤)出现小于最大给药浓度的IC50值。细胞来源患者的垂体腺瘤组织MGMT免疫组织化学染色结果显示,9例(34.62%,4例GH腺瘤,1例泌乳素腺瘤,4例无功能垂体腺瘤)为低表达,MGMT表达比率小于10%；14例(53.85%,12例无功能垂体腺瘤,2例生长激素腺瘤)为中度表达,MGMT表达比率大于10%但小于50%；3例(11.54%,均为无功能垂体腺瘤)为高表达,MGMT表达比率大于50%。对26例垂体腺瘤TMZ IC50数据集及MGMT表达比率值的Spearman相关性分析结果显示,二者的相关系数为-0.094,P>0.05,即TMZ IC50及MGMT表达比率之间没有明显相关关系。结论：TMZ能够对约30%-40%的垂体腺瘤发挥稳定抑瘤作用。TMZ对垂体腺瘤细胞的IC50及肿瘤MGMT表达比率之间不存在明显相关关系,提示MGMT不能精确地指示垂体腺瘤对TMZ的敏感性。TMZ的在体疗效仍有待临床研究进一步阐明。第四部分替莫唑胺治疗难治性垂体腺瘤一例目的：探讨难治性垂体腺瘤的有效替代治疗方法。方法：采用手术、伽马刀及再次手术治疗一例难治性垂体腺瘤患者。根据其原代肿瘤细胞的TMZ药敏试验结果,选用TMZ治疗两个疗程,总结分析其疗效。结果：由于肿瘤生长迅速,常规手术、放疗及药物治疗均难以控制难治性垂体腺瘤的生长及病情进展。肿瘤组织病理检测结果提示,Ki-67 LI约40%-50%,MGMT表达属低水平。TMZ对该患者原代肿瘤细胞的IC50小于最大给药浓度,提示肿瘤细胞对TMZ敏感。患者服用替莫唑胺两个疗程,肿瘤停止快速生长,MRI示肿瘤出现液化坏死。结论：对于常规手术、放疗及药物治疗无效的难治性垂体腺瘤,替莫唑胺化疗可能是一种有效替代治疗方法。更多还原

【Abstract】 Pituitary adenomas are one of the most common types of intracranial tumors, with autopsy studies demonstrating an incidence of 25%. The biology of pituitary adenomas can vary substantially. Some pituitary adenomas are slow growing, incidentally found, and require no treatment whatsoever. On the other extreme, some pituitary adenomas prove refractory to surgery, radiation, and pituitary-suppressive medications (for example, dopamine receptor agonists and somatostatin analogs) and continue to progress. Pituitary carcinomas occur in 0.2% of patients and may metastasize within the CNS. Temozolomide has proven effective in the treatment of patients with high-grade gliomas. Anecdotal case reports have suggested that temozolomide may have efficacy for some patients with aggressive pituitary adenomas, includ-ing pituitary carcinomas. Low MGMT expression has been linked to a favorable clinical response in patients with aggressive pituitary adenomas undergoing treatment with temozolomide. This study evaluates the potential factors that associate with the invitro survival of pituitary adenoma cells and the response of primary pituitary adenoma cell cultures, gonadotroph adenoma cell lines and a patient suffered from refractory pituitary adenoma to temozolomide.Section One Primary Pituitary Adenoma Cell Culture InvitroObjective:To analyze the impact factors for the in vitro survival of pituitary adenoma cells and select the cell culture regimen that maintain the biological properties of pituitary adenoma cells in vitro so as to improve the survival of human pituitary adenoma cells in vitro and guarantee the quality of the cell line that used in the clinical and experimental research.Methods:33 cases of surgical specimens of non-functional pituitary adenoma were isolated inoculated and cultured in vitro. The survial analysis based on the lifetime of these primary cell lines and different separate criteria was performed to evaluate the relevance between them. Cells seeded on Laminin, Matrigel, PPL, Collagen and general culture plate interface were used to observe the disparity of the effect on cell viability.Results:Kaplan Meier survival analysis showed that there was no significant correlation (P> 0.05) between the survival time of pituitary adenoma cells and the age, sex, tumor type, tumor recurrence, tumor invasion, preoperative radiation therapy or the level of Ki-67 LI. Cell viability analysis, based on non-functional pituitary adenoma cell culture on two-dimensional and three-dimensional interface, showed that the effect of Matrigel, Laminin, Collagen group on cell viability was significantly greater than the bare surface of cell culture plates (Matrigel, Laminin group vs. NC, P<0.01; Collagen group vs. NC, P<0.001). There was no significant difference (P>0.05) when Lysine compared with the control group. Cell viability of the cells cultured on Laminin and Matrigel was significantly higher than other cells cultured on other interfaces (P<0.001, vs. Lysine; P <0.01 vs.Collagen). Cell viability collagen group were higher than those Lysine group (P <0.05) while there was no significant difference between Matrigel and Laminin group (P>0.05). Therefore, the sequence of the effect of different cell culture interfaces on pituitary adenoma cell viability was Matrigel, Laminin group> Collagen group> Lysine and general culture plate bare surface group.Conclusion:The in vitro cultured non-functional pituitary adenoma cells growed slowly. There was no significant correlation (P> 0.05) between the survival time of pituitary adenoma cells and the age, sex, tumor type, tumor recurrence, tumor invasion, preoperative radiation therapy or the level of Ki-67 LI. Laminin and Matrigel culture interface can be more powerful than Collagen and PPL to enhance the vitality of the pituitary adenoma cells, and may guarantee a qualified platform for in vitro research.Second Two Effect of Temozolomide on Cell Viability in Gonadotroph Adenoma Cell LinesBackground:Invasive pituitary adenomas are usually refractory to routine neurosurgery, radiosurgery or medications, and alternative therapies are expected. The effects of temozolomide (TMZ) on the inhibiton of gonadotroph adenoma cell viability and hormone secretion were evaluated.Methods:Cell viability and IC50 values were evaluated afterαT3-1 cells were treated with TMZ (31.25-1000μM) or vehicle for 0-72 h. Cell cycle changes and extent of apoptosis were detected using flow cytometry, TUNEL and TEM. Molecular mechanism of TMZ was investigated by Caspase-Glo(?) assay and immunoblotting. Gonadotropin secretion were assessed by immunoassay system.Results:TMZ dose-and time-dependently suppressed cell proliferation (P<0.01 vs. control,250μM,24 h) and induced S-phase accumulation and G2/M-phase arrest (P< 0.05 vs. control,250μM,24 h). Early apoptotic cells increased following a 24-h TMZ incubation (P<0.001 vs. control,250μM), consistent with TEM and TUNEL detection that exhibited morphological features of apoptosis. TMZ (250μM) increased the level of caspase-3/-7 6-fold, caspase-9 7-fold, and caspase-8 3-fold after a 24-h incubation, while attenuated Bcl-2 expression (P<0.001 vs. control) and raised the proteolysis of PARP. Both FSH and LH level were significantly decreased by TMZ (P<0.01 vs. control,250μM,24 h).Conclusions:TMZ inhibited cell proliferation and hormone secretion, and induced cell cycle arrest and apoptotic cell death in gonadotroph adenoma cells via both death receptor and mitochondrial pathway, suggesting that it might represent a useful medical management of invasive gonadotroph adenomas.Section Three Effect of Temozolomide on Human Pituitary Adenoma Cells and Its Relationship with the Expression of MGMTObjective:To observe the effect of temozolomide on human pituitary adenoma cells and test the indicative efficacy of MGMT expression on the sensitivity of TMZ for pituitary tumors.Methods:Surgical specimens of 26 patients with pituitary adenoma underwent isolation, inoculation and cell culture. Drug sensitivity test for TMZ on primary pituitary adenoma cells was performed, and the IC50 values was calculated for each cell line. Immunohistochemical detection for MGMT expression of these tumors was performed to correlate with those TMZ IC50 values by Spearman correlation analysis.Results:Among 26 TMZ-treated primary pituitary adenoma cells,24 cases (92.31%, except for 2 cases of nonfunctioning pituitary adenomas cells) demonstrated dose-dependent inhibitory activity,9 cases (34.62%,3 cases of GH adenomas,6 cases of nonfunctioning pituitary adenomas) demonstrated minor IC50 values that less than the maximum dose of TMZ. MGMT immunohistochemistry results showed that 9 cases (34.62%,4 cases of GH adenomas,1 case of prolactinomas,4 cases of nonfunctioning pituitary adenomas) showed low MGMT expression, less than 10%; 14 cases (53.85%, 12 cases of nonfunctioning pituitary adenomas,2 cases of GH adenomas) showed moderate MGMT expression, greater than 10% but less than 50%; 3 cases (11.54%, non-functional pituitary adenomas) showed high level MGMT expression, MGMT expression ratio greater than 50%. Spearman correlation analysis for TMZ IC50 data and the value of MGMT expression ratio showed that the correlation coefficient was-0.094, P>0.05, namely the TMZ IC50 value and the rate of MGMT expression were not significantly correlated.Conclusion:TMZ was able to steadily inhibit about 30%-40% of the primary pituitary adenoma cell lines. There was no significant correlation between TMZ IC50 value of pituitary adenoma cells and tumor MGMT expression ratio, suggesting that MGMT expression can not accurately indicate the sensitivity of pituitary adenoma to TMZ. Further research was needed to analyze the mechanism of TMZ tolerance.Section Four Effect of Temozolomide on a Refractory Pituitary AdenomaObjective:To explore the effective treatment for refractory pituitary adenomas.Method:Based on MGMT expression and sensitive test of the primary pituitary adenoma cells to Temozolomide, we treated a refractory pituitary adenoma patient.Result:MGMT expression in this case was at low level, and primary pituitary adenoma cells were sensitive to temozolomide in vitro. The tumor volume decreased after the first cycle Temozolomide therapy.Conclusion:Temozolomide may be a salvage therapy for those refractory pituitary adenomas with low MGMT expression.更多还原