类固醇受体辅激活因子SRC-3在小鼠电离辐射所致造血损伤中的调控保护作用及机制研究

发布时间：2018-06-16 04:08

本文选题：SRC-3 + 基因敲除小鼠　；参考：《第三军医大学》2013年博士论文

【摘要】：造血组织对电离辐射极为敏感，是辐射损伤的主要靶器官之一。辐射射线可导致造血细胞大量凋亡或死亡，使细胞周期改变，细胞增殖迟缓，导致机体出现严重造血功能障碍。与此同时，电离辐射还可降低骨髓中基质细胞的数目和分泌细胞因子的能力，进一步阻碍辐射后造血机能的恢复。因此，尽量保护残存造血细胞的数量、促进照后造血细胞的增殖和维持照后造血微环境的稳定，是治疗辐射后造血损伤的有效措施。而如何同时在造血细胞和基质细胞中实现多途径、多靶点的辐射后保护，是治疗急性放射病造血损伤时新的思考。类固醇受体辅激活因子-3(steroid receptor coactivator-3, SRC-3)是SRC家族中重要的一员，能够与多种核受体和转录因子相互作用，参与激活多条信号通路，完成相应靶基因的转录。SRC-3不仅能在机体中发挥了广泛的生物学功能；更作为促瘤因子参与促进多种肿瘤细胞的增殖和生长，以及对抗化疗药物引起的凋亡。已有报道证实，SRC-3在恶性血液细胞中高表达，并能发挥促增殖和对抗药物所致凋亡的作用。因此我们推断，SRC-3可能在外因所致的造血细胞损伤中发挥一定作用。目前，对SRC-3参与体内造血调控的机制尚不清楚，关于在辐射所致造血损伤中的作用更是未见报道。为了验证SRC-3是否参与了小鼠辐射后造血损伤效应，并探明其相关机制，我们以SRC-3基因敲除型和野生型小鼠为实验对象开展了以下实验。我们通过海外合作的方式从美国休斯敦贝勒医学院引进SRC-3基因敲除小鼠的亲代小鼠，并在成功繁殖和进行基因表型鉴定后，获得一批理想的SRC-3基因敲除型和野生型实验用小鼠。在对所得实验小鼠按照不同基因型分组后，我们以60Co γ射线分别进行了6.0Gy和4.5Gy的全身照射(total body irradiation,TBI)，制造了致死剂量照后和亚致死剂量照后两种不同的动物辐射损伤模型。为了明确SRC-3在小鼠电离辐射所致造血损伤中的效应及探讨其相关机制，我们分别从体内、体外两方面展开实验。首先，我们评估了SRC-3~(-/-)小鼠和野生型小鼠在辐射后不同的造血损伤效应，包括整体效应，如小鼠一般情况、死亡率、体重等指标；外周血细胞、骨髓造血组织、胸腺和脾脏淋巴组织的损伤情况；并检测了小鼠血清中造血相关细胞因子（IGF-1，IL-3，IL-6, TPO）的水平变化，验证了SRC-3在小鼠体内辐射后造血损伤和造血恢复过程中均能发挥防护作用。其次，我们以小鼠细胞作为实验对象，分两部分探讨了SRC-3参与调控和保护小鼠辐射后造血损伤的机制：1)以两种不同基因型小鼠的骨髓有核细胞(bone marrow nucleated cells, BMNCs)作为研究对象，在辐射前和辐射后各时相点，检测了SRC-3对小鼠BMNCs细胞的凋亡、增殖、细胞周期、相关增殖通路的影响，以及对凋亡相关分子(NF-κB,p53,Bcl-2,Bax)和周期调控因子(cyclin A，E, D1，CDK2，CDK4，p21,p53)的调控作用，阐明了SRC-3在辐射后的小鼠BMNCs中的调控和保护作用。2)以两种不同基因型小鼠的骨髓基质细胞(bone marrow stromal cells, BMSCs)作为研究对象，在辐射前和辐射后各时相点，检测了SRC-3对小鼠BMSCs细胞的增殖活性、成纤维细胞集落形成单位(colony-forming unit of fibroblast, CFU-F)形成能力、增殖相关p-AKT蛋白表达量，和上清中VCAM-1，IGF-1，IL-3，IL-6和TPO水平的影响，明确了SRC-3在小鼠BMSCs细胞中的调控作用。所得结果总结如下：主要结果： 1.以SRC-3+/-杂合子小鼠为亲代交配繁殖得到一定数目的野生型(SRC-3+/+)、杂合子(SRC-3+/-)和基因敲除的纯合子(SRC-3~(-/-))小鼠。通过PCR和Western blot方法分别对子代小鼠进行基因表型和蛋白表型的鉴定后，将所得SRC-3~(-/-)小鼠SRC-3+/+小鼠分组供实验用。蛋白表型鉴定结果发现SRC-3~(-/-)小鼠中SRC-3基因已经彻底敲除，其骨髓、脾脏和胸腺组织中未测得SRC-3蛋白的表达；而野生型小鼠中骨髓细胞的SRC-3蛋白的表达水平显著高于其在脾脏和胸腺中的表达水平(P0.05)。 2. SRC-3~(-/-)小鼠与同龄野生型小鼠比较，具有体重轻、身材短小、发育迟缓，成年雌鼠性成熟障碍，不易受孕等特点。正常情况下，与野生型比较，SRC-3~(-/-)小鼠体重和血清中IGF-1水平显著偏低，两者比较存在统计学差异(P0.05)。并且发现，SRC-3~(-/-)小鼠的外周血中WBC、RBC和PLT计数和骨髓有核细胞计数均略低于野生型；胸腺指数、脾脏指数略高于野生型，但两者比较无统计学差异。 3.给予致死剂量TBI后，SRC-3~(-/-)小鼠显现出更严重的整体损伤效应，表现为一般情况更差、体重下降明显，30天生存率更低，与野生型比较存在统计学差异(P0.05)，提示SRC-3~(-/-)小鼠机体抗辐射能力更弱。 4.给予亚致死剂量TBI后，SRC-3~(-/-)小鼠中外周血血象更低，骨髓有核细胞计数和造血集落形成单位计数更少，与野生型比较存在统计学差异(P0.05)。同时骨髓病理切片提示所见辐射后SRC-3~(-/-)小鼠的骨髓更空旷。提示SRC-3~(-/-)小鼠辐射后显现出更加严重的外周血和骨髓的造血损伤效应。值得一提的是，SRC-3~(-/-)小鼠的巨核系造血细胞在辐射后损伤尤重，在照射后11天极低点至随后恢复过程中的各时相点，均可观察到SRC-3~(-/-)小鼠的外周血PLT计数、病理切片中骨髓腔巨核细胞数目和体外培养巨核系集落形成单位显著低于野生型，两者比较存在统计学差异(P0.05)。 5.给予亚致死剂量TBI后，在各个时相点可观察到，SRC-3~(-/-)小鼠的脾脏指数和胸腺指数均略高于野生型，并与野生型比较无统计学差异。说明SRC-3~(-/-)小鼠脾脏和胸腺的辐射后损伤程度明显低于骨髓损伤程度，因此SRC-3~(-/-)小鼠的淋巴组织在辐射后的损伤效应与骨髓组织有所不同。 6.给予亚致死剂量TBI后，SRC-3~(-/-)小鼠血清中IGF-1水平在照后各时相点持续处于较低水平，与野生型比较存在统计学差异(P0.05)。SRC-3~(-/-)小鼠血清中造血因子IL-3和IL-6在照后第7天显著均低于野生型，两者比较存在统计学差异(P0.05)。血清中细胞因子TPO水平则未发现存在组间差异。 7.用流式法测定两组小鼠的BMNCs的凋亡率和所含Sca-1+细胞比例后发现，在正常情况下，两种小鼠BMNCs的凋亡率和所含Sca-1+细胞比例无统计学差异。而辐射后SRC-3~(-/-)小鼠的BMNCs中凋亡率显著升高和Sca-1+细胞比例显著降低，与野生型比较存在统计学差异(P0.05)。同时，用Western blot方法测得辐射后SRC-3~(-/-)小鼠BMNCs中的凋亡蛋白p53和Bax表达显著升高，抗凋亡蛋白NF-κB (p65)和Bcl-2表达显著降低，与野生型比较存在统计学差异(P0.05)。提示SRC-3~(-/-)小鼠的BMNCs在辐射后凋亡增多、残留数目较少；其增高的凋亡敏感性与凋亡相关蛋白的表达量异常有关。 8.用流式法测定两组小鼠的BMNCs的细胞周期和增殖指数后发现，在正常情况下，两种小鼠BMNCs的细胞周期分布和增殖指数无统计学差异。而辐射后SRC-3~(-/-)小鼠的BMNCs表现出G0/G1期显著升高，S期阻滞的特点，同时增殖指数也显著低于野生型(P0.05)。进一步研究发现，辐射后SRC-3~(-/-)小鼠BMNCs中正性周期调控因子，包括CyclinD1、CyclinE、 CyclinA、CDK2、CDK4的表达水平显著降低；负性周期调控因子p53和p21的蛋白表达量显著升高，与野生型比较有统计学差异(P0.05)。并且，SRC-3~(-/-)小鼠BMNCs中p-AKT蛋白表达量在辐射后下降，表明SRC-3~(-/-)小鼠AKT增殖信号通路在辐射后活性降低。以上结果提示SRC-3~(-/-)小鼠的BMNCs在辐射后存在细胞周期分布异常和增殖障碍的特点，其中周期调控因子的表达水平和的AKT信号通路活性辐射后降低，是导致辐射后造血恢复缓慢的重要原因。 9.用CKK-8法测定体外培养所得小鼠BMSCs的增殖活性后发现，正常情况下，SRC-3~(-/-)小鼠的增殖活性显著低于野生型(P0.05)；而辐射后两者组间差异更加显著(P0.01)。并且，SRC-3~(-/-)小鼠的CFU-F形成能力在照前和照后均显著低于野生型，两者比较有统计学差异(P0.05)。另外，我们还检测了小鼠BMSCs中调控增殖的AKT信号通路活性，发现照前两组小鼠BMSCs中p-AKT蛋白表达量水平接近；但在照后SRC-3~(-/-)小鼠BMSCs中p-AKT蛋白表达量显著低于野生型，两组比较有统计学差异(P0.05)。以上结果说明SRC-3~(-/-)小鼠BMSCs的增殖能力和CFU-F形成能力在照射前降低，辐射后程度加重；而照射后SRC-3~(-/-)小鼠BMSCs中AKT增殖信号通路活性降低可能是导致该损害加重的一个重要原因。 10.用ELISA法测定小鼠BMSCs细胞培养上清液中的细胞因子水平后发现，正常情况下，SRC-3~(-/-)小鼠BMSCs上清中VCAM-1和IGF-1水平偏低，与野生型比较存在统计学差异(P0.05)。而辐射损伤后，SRC-3~(-/-)小鼠BMSCs上清中VCAM-1、IGF-1、IL-3和IL-6水平均显著低于野生型，两者比较具有统计学差异(P0.05)，说明SRC-3~(-/-)小鼠BMSCs分泌细胞因子的能力显著低于野生型。结论： 1.缺乏SRC-3可增加致死剂量TBI后小鼠死亡率，表明SRC-3可以影响辐射后小鼠的存活率，，在整体水平上提高小鼠对辐射损伤的防护能力。 2.缺乏SRC-3可导致亚致死剂量TBI后，小鼠骨髓损伤更重、造血恢复更缓慢。因此，SRC-3能减轻小鼠辐射后骨髓造血损伤程度，促进造血恢复，特别是对巨核系造血影响更加明显。 3. SRC-3与维持血清中IGF-1、IL-3和IL-6水平有密切关系。缺乏SRC-3可导致照射前后血清中的细胞因子IGF-1显著降低，以及照射后血清中IL-3和IL-6水平严重不足。 4. SRC-3可保护辐射后小鼠BMNCs中存活的Sca-1+细胞和降低凋亡细胞，并通过激活NF-κB (p65)蛋白和抑制p53蛋白辐射后的活性，降低小鼠BMNCs辐射后凋亡敏感性。 5. SRC-3可促进辐射后小鼠BMNCs的增殖和周期进程，降低G0/G1期比例，减少S期阻滞。其机制与上调正性周期调控因子CyclinD1、CyclinE、 CyclinA、CDK2、CDK4和下调负性周期调控因子p21和p53的表达水平，激活AKT信号通路有关。 6.缺乏SRC-3可导致正常情况下小鼠BMSCs的增殖活性、CFU-F形成能力，以及BMSCs上清中VCAM-1和IGF-1的水平显著下降。不仅如此，还可降低辐射后小鼠BMSCs中的p-AKT蛋白表达和上清中IL-3和IL-6的水平。因此SRC-3促进基质细胞的增殖和分泌细胞因子的能力；通过调控造辐射后基质细胞的数目和细胞因子的水平，维护造血微环境的稳定来实现对辐射后造血的防护。
[Abstract]:The hematopoietic tissue is very sensitive to ionizing radiation, which is one of the main target organs of radiation damage. Radiation rays can cause a large number of apoptosis or death of hematopoietic cells, change the cell cycle and slow the proliferation of cells, and cause serious hematopoietic dysfunction. At the same time, ionizing radiation can also reduce the number and secretion of stromal cells in the bone marrow. The ability of cell factors further hinders the recovery of hematopoietic function after radiation. Therefore, it is an effective measure to protect the number of residual hematopoietic cells, promote the proliferation of hematopoietic cells after irradiation and maintain the stability of the hematopoietic microenvironment after irradiation, which is an effective measure for the treatment of hematopoietic injury after radiation. The radiation protection after target is a new thinking for treating hematopoietic injury of acute radiation sickness.
The steroid receptor coactivator -3 (steroid receptor coactivator-3, SRC-3) is an important member of the SRC family. It can interact with a variety of nuclear receptors and transcription factors and participate in activating multiple signal pathways. The transcriptional.SRC-3 of the corresponding target gene can not only play a wide biological function in the body, but also act as a tumor stimulating factor. It has been reported that SRC-3 is highly expressed in malignant blood cells and can play a role in promoting proliferation and antagonism to drug induced apoptosis. Therefore, we infer that SRC-3 may play a role in the injury of hematopoietic cells caused by external causes. The mechanism of SRC-3's involvement in the regulation of hematopoiesis in vivo is not clear. There is no report about the role of the hematopoiesis in radiation induced hematopoiesis. In order to verify whether SRC-3 participates in the effect of postradiation hematopoiesis in mice, and the related mechanisms are explored, the following experiments have been carried out on SRC-3 gene knockout and wild type mice.
We introduced SRC-3 gene knockout mice from the Baylor College of Medicine, Houston, in the way of overseas cooperation. After successful reproduction and genetic phenotype identification, a group of ideal SRC-3 knockout and wild type experimental mice were obtained. After grouping the experimental mice in accordance with different genotypes, we used 60Co The total body radiation of 6.0Gy and 4.5Gy (total body irradiation, TBI) was carried out by gamma ray, and two different animal radiation damage models were made after lethal dose illumination and sublethal dose irradiation.
In order to identify the effect of SRC-3 on the hematopoietic damage induced by ionizing radiation in mice and to explore its related mechanisms, we carried out the experiment from two aspects in vivo and in vitro. First, we evaluated the effects of different hematopoiesis in SRC-3~ (- / -) mice and wild type mice after radiation, including the overall effect, such as the general condition, mortality and weight of mice. The damage of peripheral blood cells, bone marrow hematopoietic tissue, thymus and spleen lymphatic tissue, and the level changes of hematopoiesis related cytokines (IGF-1, IL-3, IL-6, TPO) in the serum of mice were detected, and the protective effect of SRC-3 on hematopoiesis and recovery of blood after radiation in mice was verified. Secondly, we were small. As the experimental object, the mouse cells were divided into two parts to explore the mechanism of SRC-3 participation in regulating and protecting the hematopoiesis after radiation in mice: 1) the bone marrow nucleated cells (bone marrow nucleated cells, BMNCs) of two different genotypes mice were used as the research object. The SRC-3 effect on the death of BMNCs cells in mice before and after radiation was detected. The effects of apoptosis related molecules (NF- kappa B, p53, Bcl-2, Bax) and cyclical regulators (cyclin A, E, D1, CDK2, CDK4, p21), as well as the regulation and protection of the two different genotypic mice Marrow stromal cells, BMSCs), as the research object, detected the proliferation activity of SRC-3 on mouse BMSCs cells before and after radiation, and the formation of colony forming unit of fibroblasts (colony-forming unit of fibroblast, CFU-F). The effect of Ping on the regulation of SRC-3 in mouse BMSCs cells was clarified.
Main results:
1. a certain number of wild type (SRC-3+/+), heterozygote (SRC-3+/-) and gene knockout homozygote (SRC-3~ (/ -)) mice were obtained by parental mating of SRC-3+/- heterozygote mice. After identification of gene phenotypes and protein forms of offspring mice by PCR and Western blot, the SRC-3+/+ mice were divided into SRC-3~ (- / -) mice. The results of protein phenotypic identification found that the SRC-3 gene in SRC-3~ (- / -) mice had been completely knocked out, and the expression of SRC-3 protein was not detected in the bone marrow, spleen and thymus tissues, while the expression level of SRC-3 protein in the bone marrow cells in the wild type mice was significantly higher than that in the spleen and thymus (P0.05).
2. SRC-3~ (- / -) mice, compared with the same age wild type mice, have the characteristics of light weight, short stature, retardation, adult female sexual maturity and not easily conceived. Under normal conditions, the weight of SRC-3~ (- / -) mice and the IGF-1 level in serum were significantly lower than those in the wild type, and there were statistical differences (P0.05). And SRC-3~ (- (-)) The number of WBC, RBC, PLT and bone marrow nucleated cells in the peripheral blood of the mice were slightly lower than that in the wild type, and the thymus index and spleen index were slightly higher than those of the wild type, but there was no statistical difference between them.
3. after the lethal dose of TBI, the SRC-3~ (- / -) mice showed a more severe overall damage effect, showing a worse general condition, a significant decrease in weight, a lower 30 natural survival rate, and a statistical difference from the wild type (P0.05), suggesting that the anti radiation ability of the SRC-3~ (- / -) mice was weaker.
4. after the sublethal dose of TBI, the peripheral blood hemogram of the SRC-3~ (- / -) mice was lower, the count of nucleated cells in the bone marrow and the number of the hematopoietic colony forming units were less, and there was a statistical difference between the wild type and the wild type (P0.05). Meanwhile, the bone marrow pathological sections suggested that the bone marrow of the SRC-3~ (- / -) mice after the radiation was more open. It suggested that the SRC-3~ (- / -) mice were irradiated after the radiation. It is worth mentioning that the SRC-3~ (- / -) megakaryocyte hematopoietic cells are particularly damaged after radiation, and the PLT count of the peripheral blood of SRC-3~ (- / -) rats can be observed at 11 days after irradiation, and the marrow cavity megakaryocyte in pathological sections can be observed. Cell number and colony forming unit of megakaryocyte in vitro were significantly lower than those in wild type, and there was a significant difference between them (P0.05).
5. after the sublethal dose of TBI was given, the spleen index and thymus index of SRC-3~ (- / -) mice were slightly higher than that in the wild type, and there was no statistical difference compared with the wild type. It showed that the degree of post radiation injury in spleen and thymus of SRC-3~ (- / -) mice was lower than that of bone marrow injury, so the lymphatic group in SRC-3~ (- / -) mice The damage effect after irradiation is different from that of bone marrow.
6. after the sublethal dose of TBI was given, the level of IGF-1 in the serum of SRC-3~ (- / -) mice remained at a low level at each time point after illumination, and there was a statistical difference between the wild type and the wild type (P0.05). The serum hematopoietic factor IL-3 and IL-6 in the serum of.SRC-3~ (- / -) mice were significantly lower than the wild type at the seventh day after illumination, and there was a statistical difference between the two (P0.05) serum (P0.05). There was no difference in the level of cytokine TPO between the two groups.
7. the rate of apoptosis and the proportion of Sca-1+ cells contained in the BMNCs of the two groups of mice were measured by flow method. There was no significant difference between the apoptosis rate and the proportion of the Sca-1+ cells in the two mice of BMNCs. The apoptosis rate of BMNCs in SRC-3~ (- / -) mice after radiation was significantly increased and the proportion of Sca-1+ cells decreased significantly. The statistical difference (P0.05). At the same time, the expression of apoptotic protein p53 and Bax in BMNCs of SRC-3~ (- / -) mice was significantly increased after radiation, and the expression of NF- kappa B (p65) and Bcl-2 was significantly reduced in SRC-3~ (/ -) mice BMNCs, and there was a statistical difference between the apoptotic protein and the wild type (P0.05). The higher sensitivity of apoptosis is related to the abnormal expression of apoptosis related proteins.
8. the cell cycle and proliferation index of BMNCs in two groups of mice were measured by flow method. The cell cycle distribution and proliferation index of the two BMNCs mice were not statistically different in normal conditions. The BMNCs of SRC-3~ (- / -) mice after radiation showed a significant increase in the G0/G1 stage and the characteristics of the retardation of the S phase, and the proliferation index was also significantly lower than that in the wild. Type (P0.05). Further study found that the positive periodic regulators in the SRC-3~ (/ -) mouse BMNCs after radiation, including CyclinD1, CyclinE, CyclinA, CDK2, CDK4, were significantly reduced, and the protein expression of the negative cyclical regulator p53 and p21 increased significantly, and was statistically different from the wild type (P0.05). Moreover, SRC-3~ (- / -) mice The expression of p-AKT protein in s decreased after radiation, indicating that the SRC-3~ (- / -) mouse AKT proliferation signal pathway decreased after radiation. The above results suggest that BMNCs in SRC-3~ (/ -) mice has the characteristics of abnormal cell cycle distribution and proliferation disorder after radiation, in which the expression level of the cell and the active radiation of the AKT signaling pathway in the SRC-3~ (- / -) mice Reduction is an important reason for the slow recovery of hematopoiesis after radiation.
9. the proliferation activity of BMSCs in vitro cultured in vitro was measured by CKK-8. The proliferation activity of SRC-3~ (- / -) mice was significantly lower than that of the wild type (P0.05) in normal conditions, while the difference between the two groups was more significant (P0.01). And the CFU-F formation ability of SRC-3~ (- / -) mice was significantly lower than that in the wild type before and after illumination. There was a statistically significant difference (P0.05). In addition, we also detected the activity of AKT signaling pathway in the mouse BMSCs, and found that the p-AKT protein expression level in the two groups of mice was close to the level of the p-AKT protein expression in the BMSCs mice, but the p-AKT protein expression in the SRC-3~ (- / -) mice was significantly lower than that of the wild type in the SRC-3~ (- / -) mice after illumination, and there was a statistically significant difference (P0.05). The results showed that the proliferation ability and CFU-F formation ability of BMSCs in SRC-3~ (- / -) mice decreased before irradiation and increased after radiation, and the decrease of AKT proliferation pathway activity in SRC-3~ (/ -) mice after irradiation may be an important cause of the aggravation of the damage.
10. the level of cytokine in the culture supernatant of BMSCs cells in mice was measured by ELISA. Under normal conditions, the level of VCAM-1 and IGF-1 in the BMSCs supernatant of SRC-3~ (- / -) mice was lower than that in the wild type (P0.05). The average of VCAM-1, IGF-1, IL-3 and water in the BMSCs supernatant of SRC-3~ (- / -) mice was significantly lower after radiation injury. In wild type, there was a statistically significant difference between them (P0.05), indicating that SRC-3~ (- / -) mice had significantly lower BMSCs secreting cytokines than the wild type.
Conclusion:
1. the lack of SRC-3 can increase the mortality of mice after the lethal dose of TBI, indicating that SRC-3 can affect the survival rate of irradiated mice and improve the protective ability of mice to radiation injury on the whole level.
2. after the lack of SRC-3 can lead to sublethal dose of TBI, the damage of bone marrow in mice is heavier and the recovery of hematopoiesis is more slow. Therefore, SRC-3 can reduce the degree of hematopoietic injury after radiation in mice and promote the recovery of hematopoiesis, especially in megakaryocyte hematopoiesis.
3. SRC-3 is closely related to the maintenance of serum levels of IGF-1, IL-3 and IL-6. The lack of SRC-3 can lead to a significant decrease in the serum cytokine IGF-1 before and after irradiation, and the severe deficiency of the serum IL-3 and IL-6 levels in the serum after irradiation.
4. SRC-3 can protect the surviving Sca-1+ cells and the apoptotic cells in BMNCs after radiation, and reduce the apoptosis sensitivity of mice after BMNCs radiation by activating NF- kappa B (p65) protein and inhibiting the activity of p53 protein after radiation.
5. SRC-3 can promote the proliferation and cycle progression of BMNCs in mice after irradiation, reduce the proportion of G0/G1 phase, and reduce S phase arrest.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R818

【参考文献】