【作者】 张鹏；

【导师】 龚建平；

【作者基本信息】 华中科技大学 ， 普通外科学， 2006， 博士

【摘要】 目的:由于技术限制,此前还较少有人直观地展现非同步化条件下化疗药物诱导细胞凋亡的细胞周期特异性。但是,细胞周期同步化被证明是对细胞状态的一种干扰,可能导致“假象”研究结果。在这部分研究中,我们将首先建立化疗药物作用于对数生长期细胞系的细胞周期特异性检测模型。方法:以处于对数生长期的人类急性淋巴细胞性白血病细胞系(MOLT-4细胞)为研究对象,分别用喜树碱(CPT)、阿糖胞苷(Ara-C)、替尼泊甙(VM-26)、甲氨蝶呤(MTX)、泰素(Taxol)、长春新碱(VCR)等常用细胞周期特异性化疗药物(Cell-cycle specific agents, CCSA)以不同剂量、不同作用时间加以处理,以API法检测细胞凋亡的细胞周期特异性。以分选后激光共聚焦荧光显微镜技术对API法的结果加以进一步验证。从而比较观察出各种药物诱导细胞周期特异性凋亡的最佳条件,为进一步研究建立模型。结果:CCSA作用于对数生长期的细胞系,在较低浓度及较短时间内仅引起细胞周期阻滞效应或(和)少量细胞周期特异性凋亡,在一定浓度及作用时间范围内出现明显的细胞周期特异性凋亡,超出这个范围后,往往引起广泛的无周期特异性的细胞死亡。喜树碱0.2μg/ml,阿糖胞苷100μg/ml,替尼泊甙50μg/ml,甲氨蝶呤40μg/ml作用4-6小时出现S期特异性凋亡;泰素1μg/ml、长春新碱1μg/ml作用9小时出现G2/M期凋亡。结论:CCSA诱导的典型的早期细胞周期特异性凋亡在一定条件范围内可被API技术检测。在以对数生长的Molt-4细胞为模型时,这种特异性与目前一般认为的一致。目的:利用第一部分研究建立的研究模型,检测在靶细胞生长状态发生改变后,化疗药物诱导细胞凋亡的细胞周期特异性,探讨化疗药物作用于体内外肿瘤的细胞周期特异性有无改变。方法:建立“高密度状态”培养的Molt-4细胞模型,观察药物引起的细胞周期特异性凋亡,比较其与以对数生长期模型的差异。在此基础上,以临床急性淋巴细胞性白血病为研究对象,观察药物在体内、外的不同效应。结果:当作用于“高密度状态”的Molt-4细胞时,CPT、Ara-C、MTX、VM-26、VCR与Taxol诱导的细胞凋亡主要都集中于G0/G1期,少部分在S期。当作用于临床急性淋巴细胞性白血病标本时, CPT、Ara-C、MTX、VM-26与VCR诱导的细胞凋亡主要都集中于G0/G1期,少部分在S期,Taxol则在S期。在对一例进行初次化疗的急性淋巴细胞性白血病患者的观察中,发现单用阿糖胞苷诱导G0/G1期及少量S期特异性凋亡,阿糖胞苷联合泰素在体内诱导的细胞周期特异性凋亡与此相同。而这些结果与对数生长期模型都不相同。结论:在体内外不同生长状态下,化疗药物诱导细胞凋亡的细胞周期特异性会发生改变。化疗药物作用于“高密度状态”的Molt-4细胞以及作用于临床急性淋巴细胞性白血病标本所诱导的凋亡的细胞周期特异性类似,而且与患者化疗时体内的结果相符,进一步证实“高密度状态”的Molt-4细胞是体外培养细胞系模拟体内条件的较佳模型。我们的发现同时还提示,目前常用的化疗药物细胞周期特异性分类表可能与药物在体内的实际效应存在较大出入,可能需要重新评估。更多还原

【Abstract】 Object:Due to technical limitations, there was very few paper published directly exhibiting the drug-induced cell-cycle specific apoptosis by non-synchronous methods. But the cell cycle synchronization has been proved to disturb the cell growing status and might lead to pseudo results. In this part of present research, we will establish a model of exponentially growing molt-4 cells, to test the cell-cycle specificity of chemotherapeutic agent in vitro.Methods: The human acute lymphocyte leukemia cell line molt-4 was cultured to an exponentially growing status. Six commonly used cell-cycle specific agents (CCSA), including camptothecin (CPT), cytarabine (Ara-C), teniposide (VM-26), methotrexate (MTX), vincristine (VCR) and paclitaxel (Taxol), were added into the cell culture at different concentrations and incubated for 4 to 9 hours respectively. The drug-induced cell-cycle specific apoptosis was detected by API assay. For further verification of the API assay, post-sorting laser scan confocal microscopy (PSC) was used.Results: At very low concentration with very short period of incubating time, CCSAs only induced cell cycle blocking effect and very few cells went to apoptosis. At higher concentration with appropriate incubating period, typical cell-cycle specific apoptosis appeared. But at concentration even higher than that or with longer incubating time, cells commited to die in all phases of the cell cycle. 0.2μg/ml CPT, 100μg/ml Ara-C, 50μg/ml VM-26 and 40μg/ml MTX induced S phase specific apoptosis at 4-6 hours after the administration, 1μg/ml Taxol and 1μg/ml VCR induced G2/M phase specific apoptosis at 9 hours. Conclusions: The cell-cycle specific apoptosis induced by CCSAs could be typically detected by the API assay under certain conditions. The cell-cycle specificities of the 6 tested drugs in exponential model were consistent with those of common belief. Objects: Based on the result of the first part of this research, the cell-cycle specificities of chemotherapeutic agents were detected in models of different cell growing status. We wanted to know whether the specificity would be different or not under such condition.Methods: The model of“high-density cultured”molt-4 cells was established and was compared with the exponential model. In order to observe the in vivo effect of CCSAs, the clinical specimens of acute lymphocyte leukemia cells (the ex vivo model) were used and a leukemia patient (the in vivo model) was investigated during her first time chemotherapy.Results: When incubated with high-density cultured molt-4 cells, CPT, Ara-C, MTX, VM-26, VCR and Taxol all induced apoptosis mainly in G0/G1 phase with a small proportion in S phase. In the clinical specimens (ex vivo model), CPT, Ara-C, MTX, VM-26 and VCR again induced apoptosis mainly in G0/G1 phase with a small proportion in S phase, while Taxol only in S phase. During the first day of chemotherapy of the patient, Ara-C induced G0/G1 and a little S phase specific apoptosis. The result of Ara-C plus Taxol together in the second day was almost the same with the first day.Conclusions: The cell-cycle specificity of CCSA might change in different growing status of the target cells. The cell-cycle specific apoptosis induced in high-density cultured molt-4 was similar to that in clinical specimens, and was also consistent with that in vivo. Our observation further confirmed that high-density cultured cell line might be a better model to mimic the in vivo effect than the exponential model. It also suggested that the current classification of cell-cycle specific agent might not suitable for in vivo application and might need to be reassessed.更多还原