人髓母细胞瘤干细胞样细胞的分离鉴定及放疗对其增殖影响的体外实验研究

【作者】 汤旭群；

【导师】 毛颖； 姚瑜； 赵曜；

【作者基本信息】 复旦大学 ， 神经外科学， 2010， 博士

【摘要】 [目的]体外培养人髓母细胞瘤细胞,并从中分离鉴定人髓母细胞瘤干细胞样细胞;观察干细胞样细胞的生物学特性,包括形态学、CD133阳性比例、单克隆形成率、增殖曲线及细胞周期分布等；分析人髓母细胞瘤复发前后干细胞样细胞的存在、分布特点及细胞增殖状态,比较原发髓母细胞瘤和复发髓母细胞瘤中干细胞样细胞的增殖特性；体外实验观察放疗对人髓母细胞瘤干细胞样细胞和非干细胞样细胞增殖曲线和克隆形成率的影响,并从细胞周期分布变化,周期蛋白表达和DNA损伤修复角度分析髓母干细胞样细胞的放射生物学特性。[方法]用无血清培养液(含EGF、bFGF、LIF和B27)培养5例原代髓母细胞瘤和1例髓母细胞瘤细胞株D283,观察其生长特性；流式细胞仪检测髓母细胞系D283和原代髓母细胞无血清培养下不同时间点的CD133阳性细胞比率,以及不同培养条件下(无血清和含血清培养)细胞的形态学特点和CD133阳性比率；单克隆形成实验观察其增殖能力,免疫荧光染色法观察其干细胞样标志物的表达及血清培液诱导其多向分化情况,以初步鉴定髓母干细胞样细胞；用流式细胞仪分选无血清培养的髓母细胞株D283和原代髓母细胞,将其分成CD133阳性细胞群和CD133阴性细胞群,比较两群细胞在无血清培养条件下的生长形态、干细胞标志物表达和增殖情况；免疫缺陷鼠(NOD/SCID)前臂皮下注射观察两群细胞的致瘤性；继发肿瘤再次体外无血清培养,观察其生长特性,检测其CD133阳性细胞比率,用H & E和免疫组化染色法观察肿瘤细胞的组织学特点及GFAP,Ki67,及P53等表达情况,并与原代髓母细胞瘤比较,以鉴定髓母干细胞样细胞。用免疫组化和免疫荧光法观察5例原发髓母细胞瘤及其复发后干细胞样标志物(CD133、Nestin、DCX、PSA-NCAM及TUC-4)的表达情况；用核增殖抗原Ki67 (MIB1)免疫组化染色法,观察原发髓母细胞瘤及其复发后肿瘤细胞的增殖状态,并比较两者的区别；采用核增殖抗原Ki67和干细胞样标志物的免疫荧光双染法,观察髓母细胞瘤干细胞样细胞在肿瘤复发前后的增殖情况,并比较两者的差别。XTT法分别检测无血清培养的未分选髓母细胞、CD133阳性和CD133阴性三组细胞的增殖情况,并绘制各组的生长曲线；6孔板克隆形成实验观察各组细胞的克隆形成率；PI法检测CD133阳性和CD133阴性细胞的细胞周期,比较两组细胞的周期分布特点；未分选的髓母细胞及分选后的CD133阳性和CD133阴性细胞分别予以体外照光,流式细胞仪检测照光后48小时未分选髓母细胞的CD133阳性细胞比率,并与照光前比较；XTT法分别检测CD133阳性和CD133阴性细胞照光后的增殖情况,绘制生长曲线并与照光前比较；6孔板克隆形成实验观察照光后CD133阳性和CD133阴性细胞的克隆形成率,并与照光前比较；PI法观察照光后24小时CD133阳性和CD133阴性细胞的细胞周期分布情况,并与照光前比较；Western Blot法检测照光前后CD133阳性和阴性细胞的周期蛋白表达情况,并比较它们之间的差异；AnnexinV流式检测法和彗星电泳法观察CD133阳性细胞照光前后的凋亡变化和DNA损伤修复情况,并与CD133阴性细胞群比较。[结果]细胞体外培养发现原代髓母细胞瘤在无血清培液中悬浮生长,呈神经球样细胞团,该群细胞中6%±0.58%的细胞能自我增殖,形成单细胞克隆球；D283无血清培养后也有悬浮生长的神经球样结构形成,其单克隆形成率为21.43%±1.69%。分选后的CD133阳性细胞在无血清培液中能形成悬浮肿瘤球,CD133阴性细胞大部分呈贴壁生长,无肿瘤球形成。CD133阳性细胞在NOD-SCID鼠皮下形成的继发肿瘤体外再次无血清培养后能形成悬浮肿瘤球,形态同原代髓母细胞。流式细胞仪检测发现5例原代髓母细胞的CD133阳性比率均不高,最高为4.74%±0.48%,随着无血清培养时间的延长,其CD133阳性细胞比率开始呈上升趋势,以后保持不变；用含血清培液培养1周后,其CD133阳性比率均有不同程度的下降。血清培液中D283的CD133阳性比率在不同时间点维持不变,均在20%左右；D283无血清培养后,其不同时间点的CD133阳性比率均维持在17%左右。CD133阳性细胞所致继发肿瘤的CD133阳性比率同原代肿瘤细胞,CD133阴性细胞所致瘤的CD133阳性比率接近0；无血清培养的髓母细胞体外照光后24小时其CD133阳性率有所升高。免疫荧光和免疫组化染色发现无血清培养的D283和原代髓母细胞可以表达多种干细胞标志物；髓母细胞瘤干细胞样细胞可以分为静息型和增殖型两种,且大都为静息型；原代髓母细胞血清诱导分化后可表达GFAP、β-tubulin和CNPase等分化标志物；D283用含血清培液培养后仍表达干细胞标志物,但不表达GFAP、β-tubulin和CNPase;分选后的CD133阳性细胞能表达其它神经干细胞标志物,CD133阳性细胞群也可分为静息型和增殖型两种；5例原发髓母细胞瘤及其复发肿瘤标本中均存在干细胞样标志物(包括CD133、Nestin、DCX、PSA-NCAM及TUC-4等)的表达,其中CD133和Nestin有特征性的表达区域,被成为“血管周围龛(perivascular nich)";原发髓母细胞瘤的CD133表达弱,复发后其CD133阳性率有所升高；5例原发髓母细胞瘤中Ki67均呈高表达(proliferative index>10%),复发后Ki67仍为高表达(原位复发)或更高表达(远隔部位复发)；人髓母细胞瘤中存在处于增殖状态的干细胞样细胞,并且复发后髓母细胞瘤干细胞样细胞的增殖性更强。体内致瘤实验发现104及105个CD133阳性细胞能在NOD-SCID鼠皮下致瘤,H & E染色提示继发肿瘤与原发肿瘤的组织学特征一致；107个CD133阴性细胞也能成瘤,但肿瘤体积较小生长较慢。XTT法描绘细胞生长曲线时发现CD133阳性细胞在无血清培液中的生长速度较未分选髓母细胞和CD133阴性细胞快；CD133阳性细胞的增殖能力在3Gy照光后72小时内被抑制,但72小时后有所恢复。6孔板克隆形成实验发现CD133阳性细胞的克隆形成率(32.56%±2.66%)高于未分选髓母细胞和CD133阴性细胞(分别为20.01%±1.49和5.89%±6.76%)；CD133阳性细胞的克隆形成率在3Gy照光后仅有轻微的下降(从照光前的32.56%±2.66%下降到照光后的30.61%±0.93%)。PI法细胞周期检测发现CD133阳性细胞的G0/G1期比例(61.50%±2.31%)高于CD133阴性细胞(54.72%±0.97%)；照光后CD133阳性细胞的G0/G1期比例上升到了67.67%±3.34%。Western Blot法检测细胞周期蛋白的表达情况时发现照光前Cyclin B在CD133阳性细胞中的表达量较低,照光后其表达量有显著增加。流式细胞仪AnnexinV凋亡检测法发现3Gy照光后24小时CD133阳性细胞的凋亡细胞比例较CD133阴性细胞低(前者为2.33%±0.06%,后者达到了9.33%±1.16%)。彗星电泳法(Comet assay)发现5Gy照光后48小时CD133阳性细胞的“彗星尾”大部分已经消失,CD133阴性细胞的拖尾现象仍存在。[结论]无血清培养的髓母细胞中存在干细胞样细胞,它们能形成悬浮肿瘤球、自我增殖、多向分化以及在NOD-SCID鼠皮下成瘤；CD133阳性的髓母细胞富集了髓母干细胞样细胞；髓母干细胞样细胞可分为静止群和活跃群,且静止群较多,这是干细胞样细胞难根治,易复发的原因之一；原代髓母细胞CD133阳性比率在无血清培养条件下开始呈上升趋势,含血清培养后又逐渐下降,说明干细胞因子及微环境对髓母干细胞样细胞的维持至关重要。原发和复发髓母细胞瘤中均有脑肿瘤干细胞样细胞的存在；髓母细胞瘤干细胞样细胞在肿瘤复发后表现出更强的增殖能力。CD133阳性髓母细胞具有较强的增殖能力；体外照光后CD133阳性细胞的强增殖性和克隆形成率相比CD133阴性细胞没有受到明显抑制,CD133阳性细胞具有放疗抵抗特性。其具体的机制可能与CD133阳性细胞照光后的细胞周期再分布,周期蛋白表达,抗凋亡和DNA损伤修复能力有关。更多还原

【Abstract】 Aims:To isolate TSCs from MB first, and then characterize TSCs including morphology, ratio of CD133+cells, clonogenecity, growth curve and cell cycle distribution. To examine the existence of tumor stem-like cells(TSCs) in primary and recurrent medulloblastoma(MB) and analyze their distribution characteristics and proliferative status. To compare the proliferative index of MB-TSCs before and after recurrence. To analyze the proliferative features of TSCs in human MB after radiotherapy in vitro and explore their radiobiological characteristics in terms of cell cycle distribution, Cyclin expression and DNA damage response.Materials and methods:Five primary MBs obtained from surgical patients and one cell line(D283) were cultured in serum free medium(SFM, containing EGF、bFGF、LIF and B27). The ratio of CD133+cells in D283 and primary MB were analyzed with flowcytometry at different times and different culture condition. Proliferation, expression of stem cell markers and multilineage differentiation of these cells were investigated by clone formation assays and immunofluorescence. CD133+and CD133-MB cells were sorted by fluorescence activated cell sorting. Cell morphology, growth curve, clonogenecity and expressions of stem cell markers in SFM were compared between this two populations. Cells were injected into the armpit of NOD-SCID mouse subcutaneously to determine their tumorgenecity. To characterize the secondary tumor, cells were disassociated again and the ratio of CD133+cells were analyzed with flowcytometry. HE and immunohistochemistry with Ki67, GFAP and p53 were performed.The data used were obtained from five consecutive patients with MBs who subsequently experienced tumor recurrence from 2004 to 2007 in our department and completed the same treatment schedule, including surgery, radiation, and adjuvant chemotherapy. Then we performed the immunohistochemical and immunofluorent assays to analyze the expression of TSC proteins(including CD133、Nestin、DCX、PSA-NCAM and TUC-4) and proliferative features of TSCs in primary and recurrent MBs. Differences were calculated using paired t test for data from the same patient and Student’s t test for data from separate groups.The growth curves of unsorted MB cells, CD133+and CD133-cells were drafted using XTT assays. The 6-well palte clone formation assay was used to measure their clonogenecity. The cell cycle of each group was analyzed by flowcytometry using propidium iodide staining. Ionizing radiation(IR) treatment of each group was performed using linear accelerator Varian LINAC 600C at 0.4 Gy/min. The ratios of CD133+cells before and 48 hours after IR were analyzed with flowcytometry. The growth curves and clonogenecity of CD133+and CD133-cells after IR were compared with those before IR respectively. The cell cycle of each group after IR was also analyzed by flowcytometry using propidium iodide staining. The quantity of Cyclin B and Cyclin E before and after IR was measured using Western Blot assay. The IR-induced apoptosis was analyzed by flowcytometry using AnnexinV staining. The DNA damage response of each group after IR was analyzed using comet assay.Results:MB cells cultured in the SFM could propagate and form tumorspheres. Colony efficiency of D283 and primary MB(BT2) was 21.43%±1.69%and 6%±0.58% respectively. The highest CD133 ratio of primary MBs was 4.74%±0.48%from BT2. The CD133 ratio of MB cells cultured in SFM increased with time in the first one week and then remained stationary levels. It began to decrease after MB cells were cultured in serum contained medium(SCM). The CD133 ratio of D283 remained about 20%in SFM and 17%in SCM at different times. Many stem cell markers such as CD133, nestin, SOX2, PSA-NCAM and TUC-4 could be detected in MB cells cultured in SFM. Most MB-TSCs were quiescent while the other were proliferative. MB cells could differentiate to multiple lineages by detecting expression of GFAP、β-tubulin and CNPase after they were cultured in SCM. D283 in SCM could still express stem cell markers and never expressed GFAP、β-tubulin and CNPase. The separated CD133+cells could form tumorspheres in SFM and express kinds of stem cell markers while most CD133-cells were adherent and could not form tumorspheres in SFM. CD133+cells could also be categorized into quiescent and proliferative groups.104 and 105 CD133+MB cells could undergo tumorgenesis subcutaneously in NOD-SCID mouse. Secondary tumors exhibited similar biological features to primary tumors.107 CD133-MB cells also displayed tumorgenesis in NOD-SCID mouse but the secondary tumors were smaller and grew slowly.Of the 5 patients,2 had recurrence at the primary site and 3 had a distant recurrence. TSC markers such as CD133, nestin. DCX, PSA-NCAM, and TUC-4 were expressed no matter in the five primary or recurrent MBs. CD133 and nestin were expressed in a specific region called perivascular niche (PVN). All the 10 tumor specimens had a high proliferative index(PI), regardless of primary or recurrent MBs. In addition, the PI was even higher in the group of patients with recurrent MB at a distant site (p<0.05), while the PI remained almost the same in the group of patients with primary recurrence. Moreover, the Ki67/nestin-, Ki67/DCX-and Ki67/TUC-4-positive cells were significantly increased in recurrent MB at both primary and distant site, whereas TSCs in primary medulloblastoma showed much lower proliferative features(p<0.05).CD133+MB cells grew faster than the unsorted and CD133-cells in terms of their growth curves. Colony efficiency of CD133+cells(32.56%±2.66%) were higher than unsorted and CD133-cells(20.01%±1.49 and 5.89%±6.76% respectively). The G0/G1 ratio in CD133+cell group(61.50%±2.31%) were higher than that in CD133-cell group(54.72%±0.97%). The CD133 ratio increased obviously 24 hours after MB cells were treated with IR in vitro. The proliferative capacity of CD133+cells were inhibited temporarily within 72 hours after IR, but they recovered soon afterwards. Colony efficiency of CD133+cells treated with IR decreased slightly(from 32.56%±2.66%to 30.61%±0.93%). The ratio of G0/G1 phase in CD133+cell group increased after IR(from 61.50%±2.31%to 67.67%±3.34%). The quantity of Cyclin E expressed in CD133+cell group were more than that in CD133-cell group and it decreased after IR. The quantity of Cyclin B expressed in CD133+cells were less than that in CD133-cells and it increased significantly after IR. The ratio of apoptosis induced by IR in CD133+cell group (2.33%±0.06%) was less than in CD133-cell group(9.33%±1.16%). The "comet tails" in CD 133+cell group vanished remarkably 48 hours after IR, while they could still be seen in most irradiated CD133-cells.Conclusions:MB cells in SFM can propagate, differentiate to multiple lineage and undergo tumorgenesis. CD133+cells are enriched with MB-TSCs. Most MB-TSCs are in quiescent status which are not sensitive to IR. The stem cell niche, including cytokines and focal microenvironment is important to the maintenance of TSCs. Present serum free culture condition need to be further explored to accommodate to TSCs’thriving in vitro.The tumorigenesis of MBs and their recurrence might be related to TSCs. TSCs may have strong proliferative capacity and be resistant to radiotherapy. In addition, proliferating TSCs in MB may be associated with its metastases or dissemination. More proliferating TSCs in medulloblastomas denote worse prognosis.CD133+cells are resistant to IR comparing to CD133-cells. This may be attributed to their redistribution of cell cycle, differential expression of Cyclins, anti-apoptosis capacity and preferential DNA damage response. The results may be helpful for the further study of radiobiological characteristics of CD133+cells in the future and improvement of curative effect of radiotherapy.更多还原