高表达SDF-1人脐血源基质细胞经PECAM-1介导调控巨核细胞增殖迁移的机制研究
发布时间：2018-09-05 15:33
【摘要】：临床上,患者进行造血干细胞移植及大剂量放化疗后,体内较长时期血小板减少且恢复较慢。其中,巨核细胞损伤导致的血小板减少,除输注血小板外,尚缺乏有效的治疗手段,并且随着血小板输注的增多,也增加了输血相关感染性疾病和潜在性的移植物抗宿主病(graft versus host disease,GVHD)的发生。巨核细胞发育以及血小板的生成是一个复杂的生物学过程,包括造血干细胞发育为巨核祖细胞(megakaryocytic progenitors cell, MKPC),MKPC又进一步分化和成熟为MK,并释出血小板。研究者发现利用造血干/祖细胞进行定向诱导分化、体外扩增巨核细胞,再将扩增的巨核细胞输注给患者,可能有助于解决骨髓移植后血小板恢复较慢的临床问题,减少血小板的输注。 造血基质细胞作为造血微环境(hematopoietic inductive microenvironment,HIM)的主要成分,可以分泌多种细胞因子,促进巨核细胞增殖分化、成熟产板。因此,从修复或重建骨髓造血微环境正常功能入手治疗巨核细胞损伤,是一个值得探索的领域。以往对基质细胞的研究多集中在人骨髓基质细胞(human bone marrow stromal cells,hBMSCs),但是由于hBMSCs的数量及增殖分化潜能随年龄增加而下降、采集骨髓增加供者痛苦和风险,另外由于自体移植中患者自身造血微环境异常,而异体移植又存在组织相容性等诸多问题,限制了hBMSCs在临床上的广泛运用。人脐血中的造血干细胞较外周血和骨髓更原始,具有来源广泛,采集方便,免疫原性弱和长期造血重建的特点,已成为新的造血干细胞来源。 那么,在人脐血中是否存在着造血基质细胞?以及其具体的生物学特点有待探究。课题组长期从事人脐血源基质细胞(human umbilical cord blood-derived stromal cells,hUCBDSCs)及脐血造血微环境的研究,首次发现并证实人脐血中存在基质细胞的前体细胞,能通过特定的细胞因子组合使hUCBDSCs得以有效扩增;以hUCBDSCs为滋养层的体外扩增体系对脐血CD34~+细胞具有明显的扩增作用,可促进巨核细胞集落生成单位(CFU-Meg)的形成;体内试验中,hUCBDSCs促进小鼠辐照后CFU-Meg形成和血小板恢复的作用明显优于hBMSCs。对于上述hUCBDSCs能够促进巨核细胞发育这一生物学现象,本室在国内外杂志上已经做了详尽的报道,但是对于这一生物现象的具体机制尚不清楚。 血小板生成素(thrombopoietin,TPO)是巨核细胞发育以及血小板成熟的重要诱导因子,有关TPO调控巨核细胞发育和血小板生成的研究也有系列报道,但有研究发现TPO治疗后存在产生抗凝血抗体、加重出血等危险。另有文献报道,TPO-/-小鼠体内巨核祖细胞虽然减少,但是残留的巨核细胞和血小板在形态和功能上并没有受到损害,并且基质细胞衍生因子(stromal cell derived factor,SDF-1)仍然可以促进残余血小板的成熟和释放。Dhanjal和Wu两个实验室于2007年先后发现,PECAM-1-/-小鼠体内巨核细胞无法沿SDF-1浓度梯度迁移,最终导致其成熟障碍。SDF-1主要由基质细胞分泌产生,属于趋化因子CXC亚家族,在造血干/祖细胞的增殖、分化、迁移和归巢中发挥重要作用。那么在巨核细胞发育过程中是否存在着SDF-1/PECAM-1调控轴?其具体机制又是什么?基于此,我们提出“高表达SDF-1人脐血源基质细胞在PECAM-1协同下促进巨核细胞增殖、迁移”这一假设。本课题在构建巨核细胞/hUCBDSCs共培养模型的基础上,以SDF-1/PECAM-1为切入点,观察hUCBDSCs体外促进巨核细胞增殖和迁移的作用;围绕PECAM-1的上下游蛋白/信号通路,深入探讨hUCBDSCs促进巨核细胞发育的可能机制,有望为临床上运用hUCBDSCs治疗巨核细胞损伤、促进血小板恢复这一新的细胞治疗手段提供理论依据和实验基础。 方法: 1.人脐血源基质细胞(hUCBCSCs)共培养影响巨核细胞PECAM-1的表达实验培养hUCBDSCs和HEL细胞;建立Transwell HEL细胞/hUCBDSCs共培养模型;ELISA检测hUCBDSCs分泌SDF-1的情况;CCK-8检测人脐血源基质细胞hUCBDSCs对HEL细胞增殖的影响;细胞迁移实验检测人脐血源基质细胞hUCBDSCs对HEL细胞的迁移的影响;RT-PCR检测HEL细胞PECAM-1在mRNA水平的表达;免疫荧光组化和Western blot检测HEL细胞PECAM-1的蛋白表达水平。 2. SDF-1/PECAM-1在巨核细胞发育中的机制探讨 分两部分:第一节,SDF-1/PECAM-1慢病毒RNAi载体的构建 siRNA的设计,vshRNA载体的构建,慢病毒包装,慢病毒感染靶细胞,RNAi的效率检测。 第二节,SDF-1/PECAM-1在巨核细胞增殖迁移中的机制探讨 SDF-1和PECAM-1分别敲低后,RT-PCR、Western blot检测HEL细胞中PECAM-1的表达变化;SDF-1和PECAM-1分别敲低后,RT-PCR、免疫荧光组织化学检测HEL细胞中CXCR4的表达变化;CCK-8检测RNAi后HEL细胞的增殖变化情况;细胞迁移实验检测RNAi后细胞迁移情况;Western blot检测SHP-2蛋白(Src homology 2 domain-containing tyrosine phosphatase)的表达;Western blot检测PI3K/Akt,MAKP/ERK两信号通路中Akt,ERK磷酸化蛋白的表达变化。 结果: 1.镜下观察人脐血源基质细胞和HEL细胞。ELISA检测到hUCBDSCs,较之hBMSCs,能表达较高量的SDF-1,特别是在第7天细胞融合时分泌量达到峰值,约3.5ng/ml;hUCBDSCs对HEL细胞的趋化作用强于hBMSCs(p0.05);在和hUCBDSCs共培养后HEL细胞增殖加快,在第4天和第7天时,其增殖的OD值均高于对照组(p0.05);从RT-PCR、Western blot及免疫荧光染色实验结果得到,hUCBDSCs共培养促进HEL细胞的PECAM-1的表达。 2.利用RNAi的理论和方法制备出靶向SDF-1和PECAM-1基因的两个慢病毒载体,经RT-PCR、Western blot检测两个载体工作正常,能有效敲低目的基因。 3. SDF-1/PECAM-1在巨核细胞增殖迁移中的机制探讨 3.1 HEL细胞和hUCBDSCs共培养后,其PECAM-1在mRNA水平和蛋白水平均表达上调,当hUCBDSCs敲低SDF-1后,其作为滋养层细胞再和HEL细胞共培养,导致HEL细胞PECAM-1表达下调;另一面,当HEL细胞的PECAM-1敲低后,再和正常的hUCBDSCs共培养,其表面的PECAM-1表达仍然下降; 3.2不论是mRNA水平还是蛋白水平,HEL细胞的PECAM-1的敲低并不会影响其CXCR4的表达(p0.05); 3.3 HEL细胞和hUCBDSCs共培养后,hUCBDSCs对HEL细胞的增殖和迁移作用均增强(p0.01)。而当hUCBDSCs的SDF-1敲低后,共培养后hUCBDSCs对HEL细胞的增殖和迁移均受到抑制(p0.01);另一方面,当HEL细胞的PECAM-1敲低后,再进行共培养后,HEL细胞的增殖和迁移也同样受到了抑制(p0.01); 3.4 HUCBDSCs共培养后增强HEL细胞SHP-2的表达;而SDF-1和PECAM-1的敲低抑制HEL细胞SHP-2蛋白的表达; 3.5 HUCBDSCs共培养后增强HEL细胞Akt和ERK的磷酸化;而SDF-1和PECAM-1的敲低抑制HEL细胞Akt和ERK的磷酸化。 结论: 1. HUCBDSCs,较之hBMSCs,能分泌较高水平的SDF-1,促进巨核细胞PECAM-1的表达; 2.在巨核细胞/人脐血源基质细胞共培养体系中,存在着SDF-1/PECAM-1联合信号调控,从而促进巨核细胞的增殖和迁移; 3. SDF-1/PECAM-1联合通过激活pI3K/Akt,MAKP/ERK信号通路,促进巨核细胞的增殖和迁移。
[Abstract]:In clinic, thrombocytopenia and recovery of platelets in patients after hematopoietic stem cell transplantation and high-dose radiotherapy and chemotherapy are slow. Among them, thrombocytopenia caused by megakaryocyte injury is lack of effective treatment besides platelet transfusion, and with the increase of platelet transfusion, it also increases the incidence of transfusion-related infectious diseases and The development of megakaryocytes and platelet formation is a complex biological process, including the development of hematopoietic stem cells into megakaryocytic progenitors cells (MKPC), the further differentiation and maturation of MKPC into MK and the release of hemorrhagic platelets. It was found that directional induction of differentiation by hematopoietic stem/progenitor cells, expansion of megakaryocytes in vitro, and infusion of expanded megakaryocytes into patients may help to solve the clinical problem of slow platelet recovery after bone marrow transplantation and reduce platelet transfusion.
Hematopoietic stromal cells (HSCs), as the main component of hematopoietic microenvironment (HIM), can secrete a variety of cytokines, promote the proliferation and differentiation of megakaryocytes and mature platelets. Previous studies on stromal cells have focused on human bone marrow stromal cells (hBMSCs). However, the number and proliferation and differentiation potential of hBMSCs decrease with age. Bone marrow collection increases donor pain and risk. In addition, the patient's own hematopoietic microenvironment is abnormal in autologous transplantation, and allogeneic transplantation exists. Human umbilical cord blood hematopoietic stem cells (HBMSCs) are more primitive than peripheral blood and bone marrow, and have the characteristics of extensive sources, convenient collection, weak immunogenicity and long-term hematopoietic reconstruction. HBMSCs have become a new source of hematopoietic stem cells.
So, whether there are hematopoietic stromal cells in human umbilical cord blood and its specific biological characteristics need to be explored. Somatic cells can effectively amplify hUCBDSCs by specific cytokine combinations; the in vitro amplification system with hUCBDSCs as trophoblast has obvious effect on the proliferation of cord blood CD34~+ cells and promotes the formation of megakaryocyte colony-forming unit (CFU-Meg); in vivo experiments, hUCBDSCs can promote the formation of CFU-Meg and platelets in irradiated mice. Recovery is obviously superior to hBMSCs. For the biological phenomenon that hUCBDSCs can promote megakaryocyte development, our laboratory has done a detailed report in domestic and foreign journals, but the specific mechanism of this biological phenomenon is still unclear.
Thrombopoietin (TPO) is an important inducer of megakaryocyte development and platelet maturation. There are a series of reports on the regulation of TPO on megakaryocyte development and platelet formation, but some studies have found that there is a risk of anti-coagulation antibody and aggravation of bleeding after TPO treatment. Although megakaryocyte progenitor cells were reduced, the morphology and function of residual megakaryocytes and platelets were not impaired, and stromal cell derived factor (SDF-1) could still promote the maturation and release of residual platelets. SDF-1 is produced mainly by stromal cells and belongs to the CXC subfamily. It plays an important role in the proliferation, differentiation, migration and homing of hematopoietic stem/progenitor cells. Based on this, we proposed the hypothesis that human umbilical cord blood stromal cells with high expression of SDF-1 can promote the proliferation and migration of megakaryocytes in synergy with PECAM-1. The possible mechanism of hUCBDSCs promoting megakaryocyte development around the upstream and downstream protein/signaling pathway of PECAM-1 will provide theoretical and experimental basis for the clinical application of hUCBDSCs in the treatment of megakaryocyte injury and platelet recovery.
Method:
1. Human umbilical cord blood stromal cells (hUCBCSCs) were co-cultured to influence the expression of megakaryocyte PECAM-1. hUCBDSCs and HEL cells were cultured in vitro. Transwell HEL cells / hUCBDSCs co-cultured model was established; SDF-1 secreted by hUCBDSCs was detected by ELISA; proliferation of HEL cells was detected by human umbilical cord blood stromal cells hUCBDSCs was detected by CCK-8; The migration of HEL cells was influenced by hUCBDSCs, the expression of PECAM-1 in HEL cells was detected by RT-PCR, and the expression of PECAM-1 in HEL cells was detected by immunofluorescence histochemistry and Western blot.
The mechanism of 2. SDF-1/PECAM-1 in megakaryocyte development
It is divided into two parts: Section 1, construction of SDF-1/PECAM-1 lentiviral RNAi vector.
SiRNA design, vshRNA vector construction, lentivirus packaging, lentivirus infection target cells, RNAi efficiency detection.
The second section, the mechanism of SDF-1/PECAM-1 in the proliferation and migration of megakaryocytes.
After SDF-1 and PECAM-1 were knocked down respectively, the expression of PECAM-1 in HEL cells was detected by RT-PCR and Western blot; after SDF-1 and PECAM-1 were knocked down, the expression of CXCR4 in HEL cells was detected by RT-PCR and immunofluorescence histochemistry; the proliferation of HEL cells was detected by CCK-8 after RNAi; and the migration of HEL cells after RNAi was detected by cell migration test. The expression of SHP-2 (Src homology 2 domain-containing tyrosine phosphatase) was detected by blot, and the expression of Akt and ERK phosphorylated proteins in PI3K/Akt and MAKP/ERK signaling pathways were detected by Western blot.
Result:
1. Microscopic observation of human umbilical cord blood stromal cells and HEL cells. ELISA detection of hUCBDSCs, compared with hBMSCs, can express a higher amount of SDF-1, especially on the 7th day of cell fusion secretion reached a peak, about 3.5 ng/ml; hUCBDSCs chemotaxis on HEL cells stronger than hBMSCs (p0.05); after co-culture with hUCBDSCs, HEL cells proliferation accelerated, and on the 4th day and On the 7th day, the proliferative OD value was higher than that of the control group (p0.05). The results of RT-PCR, Western blot and immunofluorescence staining showed that the co-culture of hUCBDSCs promoted the expression of PECAM-1 in HEL cells.
2. Two lentiviral vectors targeting SDF-1 and PECAM-1 genes were prepared by using the theory and method of RNAi. RT-PCR and Western blot showed that the two vectors worked well and knocked down the target gene effectively.
The mechanism of 3. SDF-1/PECAM-1 in the proliferation and migration of megakaryocytes
3.1 After co-culture of HEL cells and hUCBDSCs, the expression of PECAM-1 was up-regulated at both mRNA and protein levels. When hUCBDSCs knocked down SDF-1, it was co-cultured with HEL cells as trophoblast cells, resulting in the down-regulation of PECAM-1 expression in HEL cells. On the other hand, when HEL cells were knocked down by PECAM-1, PECAM-1 was co-cultured with normal hUCBDSCs and the PECAM-1 surface of HEL cells was observed. Da still fell.
3.2 The expression of CXCR4 was not affected by the knockdown of PECAM-1 in HEL cells at both mRNA and protein levels (p0.05).
3.3 After co-culture of HEL cells and hUCBDSCs, the proliferation and migration of HEL cells were enhanced by hUCBDSCs (p0.01). When the SDF-1 of hUCBDSCs was knocked down, the proliferation and migration of HEL cells were inhibited by hUCBDSCs after co-culture (p0.01); on the other hand, when the PECAM-1 of HEL cells was knocked down, the proliferation and migration of HEL cells were inhibited by co-culture. It was also inhibited (P0.01).
3.4 HUCBDSCs co-cultured HEL cells enhanced the expression of SHP-2, while SDF-1 and PECAM-1 knockdown inhibited the expression of SHP-2.
3.5 HUCBDSCs co-cultured HEL cells enhanced the phosphorylation of Akt and ERK, while SDF-1 and PECAM-1 knockdown inhibited the phosphorylation of Akt and ERK.
Conclusion:
1. HUCBDSCs, compared with hBMSCs, can secrete a higher level of SDF-1 and promote the expression of PECAM-1 in megakaryocytes.
2. In the megakaryocyte/human umbilical cord blood-derived stromal cells co-culture system, SDF-1/PECAM-1 combined with signal regulation can promote the proliferation and migration of megakaryocytes.
3. SDF-1/PECAM-1 promotes the proliferation and migration of megakaryocytes by activating pI3K/Akt, MAKP/ERK signaling pathway.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363
本文编号：2224700
[Abstract]:In clinic, thrombocytopenia and recovery of platelets in patients after hematopoietic stem cell transplantation and high-dose radiotherapy and chemotherapy are slow. Among them, thrombocytopenia caused by megakaryocyte injury is lack of effective treatment besides platelet transfusion, and with the increase of platelet transfusion, it also increases the incidence of transfusion-related infectious diseases and The development of megakaryocytes and platelet formation is a complex biological process, including the development of hematopoietic stem cells into megakaryocytic progenitors cells (MKPC), the further differentiation and maturation of MKPC into MK and the release of hemorrhagic platelets. It was found that directional induction of differentiation by hematopoietic stem/progenitor cells, expansion of megakaryocytes in vitro, and infusion of expanded megakaryocytes into patients may help to solve the clinical problem of slow platelet recovery after bone marrow transplantation and reduce platelet transfusion.
Hematopoietic stromal cells (HSCs), as the main component of hematopoietic microenvironment (HIM), can secrete a variety of cytokines, promote the proliferation and differentiation of megakaryocytes and mature platelets. Previous studies on stromal cells have focused on human bone marrow stromal cells (hBMSCs). However, the number and proliferation and differentiation potential of hBMSCs decrease with age. Bone marrow collection increases donor pain and risk. In addition, the patient's own hematopoietic microenvironment is abnormal in autologous transplantation, and allogeneic transplantation exists. Human umbilical cord blood hematopoietic stem cells (HBMSCs) are more primitive than peripheral blood and bone marrow, and have the characteristics of extensive sources, convenient collection, weak immunogenicity and long-term hematopoietic reconstruction. HBMSCs have become a new source of hematopoietic stem cells.
So, whether there are hematopoietic stromal cells in human umbilical cord blood and its specific biological characteristics need to be explored. Somatic cells can effectively amplify hUCBDSCs by specific cytokine combinations; the in vitro amplification system with hUCBDSCs as trophoblast has obvious effect on the proliferation of cord blood CD34~+ cells and promotes the formation of megakaryocyte colony-forming unit (CFU-Meg); in vivo experiments, hUCBDSCs can promote the formation of CFU-Meg and platelets in irradiated mice. Recovery is obviously superior to hBMSCs. For the biological phenomenon that hUCBDSCs can promote megakaryocyte development, our laboratory has done a detailed report in domestic and foreign journals, but the specific mechanism of this biological phenomenon is still unclear.
Thrombopoietin (TPO) is an important inducer of megakaryocyte development and platelet maturation. There are a series of reports on the regulation of TPO on megakaryocyte development and platelet formation, but some studies have found that there is a risk of anti-coagulation antibody and aggravation of bleeding after TPO treatment. Although megakaryocyte progenitor cells were reduced, the morphology and function of residual megakaryocytes and platelets were not impaired, and stromal cell derived factor (SDF-1) could still promote the maturation and release of residual platelets. SDF-1 is produced mainly by stromal cells and belongs to the CXC subfamily. It plays an important role in the proliferation, differentiation, migration and homing of hematopoietic stem/progenitor cells. Based on this, we proposed the hypothesis that human umbilical cord blood stromal cells with high expression of SDF-1 can promote the proliferation and migration of megakaryocytes in synergy with PECAM-1. The possible mechanism of hUCBDSCs promoting megakaryocyte development around the upstream and downstream protein/signaling pathway of PECAM-1 will provide theoretical and experimental basis for the clinical application of hUCBDSCs in the treatment of megakaryocyte injury and platelet recovery.
Method:
1. Human umbilical cord blood stromal cells (hUCBCSCs) were co-cultured to influence the expression of megakaryocyte PECAM-1. hUCBDSCs and HEL cells were cultured in vitro. Transwell HEL cells / hUCBDSCs co-cultured model was established; SDF-1 secreted by hUCBDSCs was detected by ELISA; proliferation of HEL cells was detected by human umbilical cord blood stromal cells hUCBDSCs was detected by CCK-8; The migration of HEL cells was influenced by hUCBDSCs, the expression of PECAM-1 in HEL cells was detected by RT-PCR, and the expression of PECAM-1 in HEL cells was detected by immunofluorescence histochemistry and Western blot.
The mechanism of 2. SDF-1/PECAM-1 in megakaryocyte development
It is divided into two parts: Section 1, construction of SDF-1/PECAM-1 lentiviral RNAi vector.
SiRNA design, vshRNA vector construction, lentivirus packaging, lentivirus infection target cells, RNAi efficiency detection.
The second section, the mechanism of SDF-1/PECAM-1 in the proliferation and migration of megakaryocytes.
After SDF-1 and PECAM-1 were knocked down respectively, the expression of PECAM-1 in HEL cells was detected by RT-PCR and Western blot; after SDF-1 and PECAM-1 were knocked down, the expression of CXCR4 in HEL cells was detected by RT-PCR and immunofluorescence histochemistry; the proliferation of HEL cells was detected by CCK-8 after RNAi; and the migration of HEL cells after RNAi was detected by cell migration test. The expression of SHP-2 (Src homology 2 domain-containing tyrosine phosphatase) was detected by blot, and the expression of Akt and ERK phosphorylated proteins in PI3K/Akt and MAKP/ERK signaling pathways were detected by Western blot.
Result:
1. Microscopic observation of human umbilical cord blood stromal cells and HEL cells. ELISA detection of hUCBDSCs, compared with hBMSCs, can express a higher amount of SDF-1, especially on the 7th day of cell fusion secretion reached a peak, about 3.5 ng/ml; hUCBDSCs chemotaxis on HEL cells stronger than hBMSCs (p0.05); after co-culture with hUCBDSCs, HEL cells proliferation accelerated, and on the 4th day and On the 7th day, the proliferative OD value was higher than that of the control group (p0.05). The results of RT-PCR, Western blot and immunofluorescence staining showed that the co-culture of hUCBDSCs promoted the expression of PECAM-1 in HEL cells.
2. Two lentiviral vectors targeting SDF-1 and PECAM-1 genes were prepared by using the theory and method of RNAi. RT-PCR and Western blot showed that the two vectors worked well and knocked down the target gene effectively.
The mechanism of 3. SDF-1/PECAM-1 in the proliferation and migration of megakaryocytes
3.1 After co-culture of HEL cells and hUCBDSCs, the expression of PECAM-1 was up-regulated at both mRNA and protein levels. When hUCBDSCs knocked down SDF-1, it was co-cultured with HEL cells as trophoblast cells, resulting in the down-regulation of PECAM-1 expression in HEL cells. On the other hand, when HEL cells were knocked down by PECAM-1, PECAM-1 was co-cultured with normal hUCBDSCs and the PECAM-1 surface of HEL cells was observed. Da still fell.
3.2 The expression of CXCR4 was not affected by the knockdown of PECAM-1 in HEL cells at both mRNA and protein levels (p0.05).
3.3 After co-culture of HEL cells and hUCBDSCs, the proliferation and migration of HEL cells were enhanced by hUCBDSCs (p0.01). When the SDF-1 of hUCBDSCs was knocked down, the proliferation and migration of HEL cells were inhibited by hUCBDSCs after co-culture (p0.01); on the other hand, when the PECAM-1 of HEL cells was knocked down, the proliferation and migration of HEL cells were inhibited by co-culture. It was also inhibited (P0.01).
3.4 HUCBDSCs co-cultured HEL cells enhanced the expression of SHP-2, while SDF-1 and PECAM-1 knockdown inhibited the expression of SHP-2.
3.5 HUCBDSCs co-cultured HEL cells enhanced the phosphorylation of Akt and ERK, while SDF-1 and PECAM-1 knockdown inhibited the phosphorylation of Akt and ERK.
Conclusion:
1. HUCBDSCs, compared with hBMSCs, can secrete a higher level of SDF-1 and promote the expression of PECAM-1 in megakaryocytes.
2. In the megakaryocyte/human umbilical cord blood-derived stromal cells co-culture system, SDF-1/PECAM-1 combined with signal regulation can promote the proliferation and migration of megakaryocytes.
3. SDF-1/PECAM-1 promotes the proliferation and migration of megakaryocytes by activating pI3K/Akt, MAKP/ERK signaling pathway.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363
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