急性HBV感染模型中BNK细胞的免疫调节机制研究

发布时间：2018-04-23 00:00

本文选题：HBV + NK细胞　；参考：《中国科学技术大学》2012年博士论文

【摘要】：HBV属于嗜肝DNA病毒科,是有包膜病毒并含有大约3.2kb的环状双链DNA基因组。HBV感染可导致有或无临床表征的肝炎。虽然已经研发出预防性的高效疫苗,但是全球仍有大约3.5亿人是慢性HBV感染者并且有发展为晚期肝脏疾病以及肝癌的风险。HBV的有效清除依赖于患者的年龄和免疫状态,免疫应答低下导致持续性或者慢性HBV感染。因此,宿主的免疫应答在HBV感染过程中具有重要的作用。一直以来,免疫学家们认为适应性免疫系统在HBV的清除和致病方面具有重要的作用,相比之下,作为天然免疫系统重要成分的NK细胞在HBV感染中的作用却不是很清楚。虽有文献表明清除小鼠的NK细胞导致HBV耐受的现象,以及急性HBV患者清除HBV的感染伴随着体内NK细胞活化的现象,但是对于HBV感染后NK细胞如何发挥抗HBV感染功能则知之甚少。近来,有很多关于NK细胞对适应性免疫系统调节的报道。但是HBV感染过程中NK细胞是否具有对适应性免疫系统的调节功能也不是很清楚。因而,NK细胞在HBV感染过程中是否具有重要的作用,以及NK细胞是否参与对后续适应性免疫应答的调节,以及两大免疫系统如何相互协调介导HBV的清除?这正是我们所关心的问题。因此,本论文的研究目的在于研究HBV清除的免疫学机制,探讨HBV感染过程中是否需要NK细胞的参与,以及重点探索NK细胞是否介导对适应性免疫细胞的活化,以期为临床慢性HBV患者的治疗以及疫苗的研制提供新的策略。在本论文中对小鼠尾静脉高压注射pAAV/HBV1.2质粒,建立了小鼠急性HBV感染模型。利用该模型我们深入探讨了HBV清除的免疫学机制以及NK细胞如何诱导适应性免疫系统启动而清HBV感染。根据CD8-/-小鼠血清HBV相关抗原的检测以及体外阻断CD8+T细胞后上清中细胞因子的分泌情况评估CD8+T细胞在HBV清除中的作用；采用流式细胞术检测NK细胞的活化,以及利用NK细胞清除和转输实验证明NK细胞如何参与抗HBV感染；通过对缺陷小鼠的特定细胞清除及过继转输实验检测NK细胞对CD8+T细胞的影响；清除CD4-/-小鼠的NK细胞研究其是否参与NK细胞所介导的抗病毒及促进CD8+T细胞的活化；利用转输实验及流式细胞术检测NK细胞对CD4+T细胞的调节；通过抗体阻断实验进一步证明DCs亦受到NK细胞的调节。另外,通过肝内注射及尾静脉高压注射Ad-EGFP实验,以及手术摘除实验、体内外分析肝脏淋巴结细胞等实验验证肝脏淋巴结与肝脏的关系。利用免疫放射法(IRMA)定量检测小鼠血清中HBsAg, HBeAg, anti-HBs, HBV DNA以及ALT水平,以及H-E染色和免疫组化分析来WT小鼠以及缺陷小鼠中病毒的清除特点；CBA检测培养上清中细胞因子的变化；流式细胞术检测NK细胞、CD8+T细胞、CD4+T细胞以及DCs的表型变化以及胞内细胞因子的表达情况。通过上述一系列的研究方法,我们取得如下的实验结果： 1.CD8+T细胞参与无细胞病变性在HBV清除过程中具有重要的作用通过对C57BL/6小鼠高压尾静脉注射20μgpAAV/HBV质粒,检测血清HBsAg, HBeAg, anti-HBs, HBV DNA以及ALT水平。结果表明HBV在感染小鼠体内能够实现HBV的复制,且表达HBV相关抗原,WT小鼠可在HBV感染后3-4周内清除病毒感染,但是不引起肝细胞损伤和淋巴细胞浸润,即为无细胞病变性感染模型,类似于人类急性HBV感染。为了进一步检测HBV的清除是否有免疫系统的参与,我们检测CD8-/-小鼠在该模型中的特点,提示CD8-/-小鼠血清HBsAg和HBeAg均明显高于WT小鼠。分离WT小鼠脾脏的淋巴细胞,体外用a-CD8抗体阻断CD8+T细胞的作用,导致脾脏淋巴细胞IFN-γ及TNF-α分泌能力均明显降低,上述结果表明提示CD8+T细胞对于急性HBV感染模型中HBV的清除具有重要的作用。 2.NK细胞来源的IFN-y促进HBV的清除我们在该模型中发现HBV质粒高压注射后第3天,脾脏NK细胞明显增加,且CD69表达亦明显增加。NK细胞在HBV质粒高压注射后第5天和第7天IFN-γ表达明显增加。为进一步研究NK细胞在HBV清除中的作用,我们清除WT小鼠体内的NK细胞,肝组织病理染色结果表明NK细胞清除小鼠的肝组织HBcAg表达以及血清HBsAg及HBeAg表达明显高于对照小鼠。同样NK细胞缺陷小鼠Nfi13-/-小鼠在HBV质粒高压注射后亦不能清除HBV感染,而且分别转输WT小鼠的NK细胞和GKO小鼠的NK细胞至Nfi13-/-小鼠,则发现转输WT小鼠的NK细胞其血清HBsAg低于转输GKO小鼠NK细胞组。另外对NK细胞清除小鼠尾静脉注射mIFN-γ可以明显降低NK细胞清除所导致血清HBsAg的升高。因此,上述结果表明NK细胞来源的IFN-γ是参与HBV清除的主要功能。 3.NK细胞来源的IFN-y促进抗HBV CD8+T细胞免疫应答上述结果提示HBV清除过程中既需要CD8+T细胞的参与,又依赖于NK细胞的作用。那么二者之间是否存在调节作用呢?我们清除WT小鼠体内NK细胞,发现CD8+T细胞数目明显减低；肝脏和脾脏CD8+T细胞CD44+CD62L-CD8+T表达以及分泌IFN-γ明显减低；且NK细胞清除小鼠的肝脏HBV-特异性CD8+T细胞的活化明显减低。为了明确NK细胞分泌的IFN-γ促进CD8+T细胞的免疫应答,我们清除CD8-/-小鼠体内NK细胞,转输CFSE标记的CD8+T细胞,并同时向NK细胞清除CD8-/-小鼠体内分别转输WT小鼠的NK细胞以及GKO(Ifn-/-)小鼠的NK细胞,我们发现CFSE-CD8+T细胞在清除NK细胞的受体中的增殖以及分泌IFN-γ的能力与对照小鼠(不清除NK细胞组)相比均明显下降。转输WT小鼠的NK细胞则能促进CFSE-CD8+T细胞的增殖以及分泌IFN-γ。相比之下,转输GKO小鼠NK细胞其促进CFSE-CD8+T细胞的增殖及分泌IFN-γ的能力相对较弱。另外,转输CFSE-CD8+T胞至GKO小鼠体内,并对一些受体小鼠转输WT小鼠的NK细胞,结果表明转输WT小鼠的NK细胞的受体小鼠,其肝脏和脾脏中CFSE-CD8+T细胞的增殖及分泌IFN-γ明显增强。综上所述,NK细胞分泌的IFN-γ促进CD8+T细胞的免疫应答。 4.NK细胞促进抗HBV-CD8+T细胞的再次免疫应答接下来,分选HBV质粒高压注射后第5天的脾脏CD8+T细胞并标记CFSE,分别转输至NK细胞清除小鼠及对照小鼠体内,24小时后对受体小鼠高压注射HBV质粒。发现NK细胞参于脾脏抗HBV CD8+T细胞的再次免疫应答。为了进一步佐证此观点,我们又分选了HBV质粒高压注射后第14天的肝脏CD8+T细胞并标记CFSE,分别转输至NK细胞清除小鼠及对照小鼠体内。同样发现NK细胞清除小鼠的肝脏和脾脏中CFSE-CD8+T细胞的增殖及分泌IFN-γ的能力明显下降。因此,NK细胞参于来自于肝脏或脾脏的抗HBV CD8+T细胞的再次免疫应答。 5.CD4+T细胞在NK细胞抗HBV感染及抗HBV CD8+T细胞活化中的作用为了检测CD4+T细胞是否在NK细胞控制HBV的感染及调节CD8+T细胞的活化中发挥作用,我们检测Ragl-/-和CD4-/-小鼠清除HBV的情况,结果表明Ragl-/-和CD4-/-小鼠体内虽然存在NK细胞,但是其不能发挥抗HBV作用。进一步清除Rag1-/-和CD4-/-小鼠中NK细胞,发现血清HBsAg的亦无明显变化。而WT小鼠中NK细胞可控制HBV的感染。进一步实验发现CD4-/-小鼠体内NK细胞不能促进CD8+T细胞分泌IFN-γ,因为WT小鼠的NK细胞明显促进HBV特异性CD8+T细胞分泌IFN-γ。清除CD4-/-小鼠体内的NK细胞也不能明显改变CD4-/-小鼠体内CD8+T细胞分泌IFN-γ。因此我们认为CD4-/-小鼠的NK细胞间接抗HBV作用,以及间接调节CD8+T细胞。且NK细胞的抗病毒作用依赖于CD4+T细胞的存在。上述结果表明,CD4+T细胞不仅介导NK细胞的抗HBV感染,也参与其对CD8+T细胞的调节。 6.CD4+T细胞辅助产生抗HBV CD8+T细胞免疫应答上述结果表明CD4+T细胞参与HBV的清除,我们进一步检测CD4-/-小鼠清除HBV的能力,结果表明CD4-/-小鼠血清HBsAg和HBeAg均明显高于WT小鼠,而且HBV质粒高压注射后第8周也不能清除HBV感染。提示CD4+T细胞也参与HBV的清除过程。转输小鼠的CD8+T细胞至Rag1-/-小鼠并不能帮助该小鼠清除HBV感染,提示CD8+T细胞抗HBV感染需要CD4+T细胞的辅助。另外,体外阻断小鼠脾脏CD4+T细胞的作用,亦导致脾脏淋巴细胞分泌IFN-y下调。因此,CD8+T细胞发挥抗HBV感染功能需要CD4+T细胞辅助。 7.NK细胞促进CD4+T细胞免疫应答既然CD4+T细胞辅助CD8+T细胞抗病毒免疫应答,而且NK细胞也促进CD8+T细胞的免疫应答。CD4+T细胞与NK细胞之间可能存在调节关系。比较CD4-/-小鼠与WT小鼠中NK细胞的功能,结果表明CD4-/-小鼠中NK细胞的功能正常。进一步检测NK细胞是否调节CD4+T细胞的功能,转输CFSE标记的CD4+T细胞至CD4-/-小鼠体内,发现NK细胞清除导致CFSE-CD4+T细胞在受体小鼠的肝脏和脾脏的增殖及IFN-γ分泌明显减低。且分离来自于NK细胞清除小鼠的肝脏CD4+T细胞,体外经Core129肽段刺激,产生分泌IFN-y的HBV-特异性-CD4+T细胞的能力亦明显减低。以上结果表明NK细胞不受到CD4+T细胞的调节,且NK细胞促进CD4+T细胞的抗HBV免疫应答。 8.NK细胞分泌的IFN-γ促进CDllb+DCs免疫应答 NK细胞清除小鼠脾脏CDllb+DCs明显减低,且CDllb+DCs表面CD86和CD80的表达也明显下降,且脾细胞IL-18,IL-15和IFN-α的mRNA表达水平明显下调。尾静脉注射IFN-y以清除小鼠体内的IFN-y,发现该小鼠脾脏CD11b+DCs的表达亦明显下降下降,且脾细胞IL-15和IFN-α的mRNA表达水平亦下调。因此,NK细胞分泌的IFN-y亦促进脾脏CD11b+DCs的活化,其可能介导后续适应性免疫应答的活化。 9.肝脏淋巴结对于HBV免疫应答发生的影响另外,通过对小鼠肝内注射以及高压尾静脉注射Ad-EGFP,发现肝脏门部右侧的淋巴结可以引流肝脏EFGP+细胞,提示肝脏淋巴结引流肝脏的细胞。并且发现肝脏淋巴结能产生抗HBV特异性免疫应答。进一步实验表明手术摘除肝脏淋巴结导致大约30%的HBV耐受小鼠出现,而摘除脾脏却不影响小鼠HBV感染情况。进一步确证了HBV感染模型中肝脏淋巴结发生抗HBV的免疫应答。检测HBV感染后肝脏淋巴结细胞表型,发现HBV感染后CD8+T细胞及DCs发生增殖。因此,肝脏淋巴结可以发生针对HBV特异性免疫应答。综上所述,我们对成年雄性小鼠高压尾静脉注射pAAV/HBV1.2质粒,建立模拟人类急性HBV感染的小鼠HBV感染模型。通过研究我们详细阐述了HBV清除的免疫学机制,明确了NK细胞在HBV的清除中的重要作用,证明了NK细胞分泌的IFN-γ介导了后续适应性免疫的活化进而促进HBV的清除。另外,我们亦发现肝脏淋巴结在HBV感染中具有重要的作用,肝脏淋巴结能产生抗HBV特异性CD8+T细胞,为更好的解释HBV感染中的免疫学机制提供理论依据。
[Abstract]:HBV belongs to the liver of the liver DNA virus, which has a enveloped virus and contains approximately 3.2kb of the cyclic double chain DNA genome.HBV infection that can lead to or without clinical characterization of hepatitis. Although a prophylactic and efficient vaccine has been developed, about 350 million people worldwide are chronic HBV infected and have the development of advanced liver disease and the wind of liver cancer. The effective clearance of risk.HBV depends on the age and immune state of the patient, and the low immune response leads to persistent or chronic HBV infection. Therefore, the host immune response plays an important role in the HBV infection process.
Immunologists have long believed that the adaptive immune system plays an important role in the scavenging and pathogenesis of HBV. In contrast, the role of NK cells as an important component of the natural immune system is not very clear in HBV infection. Although the literature shows that the elimination of NK cells in mice leads to HBV tolerance and the acute HBV patients. The infection of HBV is associated with the activation of NK cells in the body, but little is known about how NK cells play an anti HBV infection after HBV infection. Recently, there are many reports about the regulation of the adaptive immune system of NK cells. But whether NK cells have the function of regulating the adaptive immune system in the course of HBV infection is not the same as that of the adaptive immune system. It is clear, therefore, whether NK cells have an important role in HBV infection, and whether NK cells are involved in the regulation of subsequent adaptive immune responses, and how the two major immune systems coordinate with each other to mediate the clearance of HBV, which is what we are concerned about.
Therefore, the purpose of this study is to study the immunological mechanism of HBV scavenging, to explore the need for the participation of NK cells in the process of HBV infection, and to explore whether NK cells can mediate the activation of adaptive immune cells, in order to provide a new strategy for the treatment of chronic HBV patients and the development of the vaccine.
In this paper, pAAV/HBV1.2 plasmids were injected into the tail vein of mice, and a mouse model of acute HBV infection was established. Using this model, we explored the immunological mechanism of HBV clearance and how the NK cells induce the initiation of the adaptive immune system to clear the HBV infection. The detection of HBV related antigens in the serum of CD8-/- mice and the blocking of C in vitro The secretion of cytokines in the supernatant of D8+T cells was used to evaluate the role of CD8+T cells in the clearance of HBV. Flow cytometry was used to detect the activation of NK cells and the use of NK cell clearance and transfer experiments to demonstrate how NK cells were involved in anti HBV infection; NK cells were detected by specific cell scavenging and adoptive transfer experiments on the defective mice. The effect on CD8+T cells; scavenging the NK cells of CD4-/- mice to investigate whether it participates in the anti-virus mediated by NK cells and promotes the activation of CD8+T cells; the transfer test and flow cytometry are used to detect the regulation of NK cells to CD4+T cells; and the antibody blocking experiment further proves that DCs is also regulated by NK cells. In addition, through the liver, the liver is regulated by the liver. The relationship between liver lymph nodes and liver was verified by Ad-EGFP experiment with internal injection and high pressure of caudal vein, operation extirpation and liver lymph node cells. The level of HBsAg, HBeAg, anti-HBs, HBV DNA and ALT in the serum of mice was detected by immuno radiation (IRMA), and H-E staining and immunohistochemical analysis were used for WT. The scavenging characteristics of virus in mice and deficient mice; the change of cytokine in the culture supernatant by CBA; flow cytometry to detect NK cells, CD8+T cells, CD4+T cells and the phenotype changes of DCs and the expression of intracellular cytokines.
1.CD8+T cells play an important role in the scavenging process of HBV.
The serum HBsAg, HBeAg, anti-HBs, HBV DNA and ALT levels were detected by injecting 20 mu gpAAV/HBV plasmids into the high pressure tail vein of C57BL/6 mice. The results showed that HBV could realize the replication of HBV and expressed HBV antigen in the infected mice. The WT mice could remove the virus infection within 3-4 weeks after the infection, but did not cause liver cell damage and drench. In order to further detect the involvement of the immune system in the clearance of HBV, we detected the characteristics of the CD8-/- mice in this model, suggesting that the serum HBsAg and HBeAg of the CD8-/- mice were significantly higher than those of the WT mice in order to further detect the involvement of the immune system in the scavenging of the CD8-/- mice. The lymphocyte of the spleen of WT mice was separated from the lymphocytes of the WT mice. The effect of a-CD8 antibody on CD8+T cells was blocked by external use, and the secretion of IFN- gamma and TNF- alpha in the spleen lymphocytes were significantly reduced. The results suggest that CD8+T cells play an important role in the clearance of HBV in the acute HBV infection model.
2.NK cell derived IFN-y promotes the clearance of HBV
In this model, we found that the spleen NK cells increased obviously at third days after high pressure injection of HBV plasmids, and the expression of CD69 increased obviously in.NK cells in fifth days and seventh days after HBV plasmids injection. To further study the role of NK cells in HBV clearance, we scavenging NK cells in the WT mice and pathological staining of liver tissue. The color results showed that the expression of HBcAg in the liver tissue of NK cells and the expression of HBsAg and HBeAg in the serum were significantly higher than those of the control mice. Similarly, the Nfi13-/- mice of the NK cell defect mice could not clear the HBV infection after the HBV plasmid high pressure injection, and then transferred to the NK cells of the WT mice and the NK cells of the GKO mice to the mice. The serum HBsAg of NK cells in WT mice was lower than that of the GKO mouse NK cell group. In addition, the increase of serum HBsAg caused by the clearance of NK cells by mIFN- gamma in the tail vein of NK cells was significantly reduced. Therefore, the above results showed that IFN- gamma of NK cell sources was the main function of participation in HBV clearance.
3.NK cell derived IFN-y promotes immune response to HBV CD8+T cells
These results suggest that HBV scavenging process requires the involvement of CD8+T cells and the role of NK cells. Then, is there any regulatory effect between the two? We scavenging NK cells in WT mice, finding the number of CD8+T cells significantly reduced, and the CD62L-CD8+T expression of CD44+ in the liver and spleen CD8+T cells and the decrease of IFN- gamma; and In order to clear the IFN- gamma secreted by NK cells to promote the immune response of CD8+T cells, we scavenged NK cells in the CD8-/- mice and transferred CFSE labeled CD8+T cells to clear the IFN- gamma secreted by NK cells. Meanwhile, we removed the CFSE labeled CD8+T cells from the CD8-/- mice and removed the CD8+T cells from the CD8-/- mice to the NK cells. Fn-/-) NK cells in mice, we found that the proliferation of CFSE-CD8+T cells in the scavenging of NK cells and the ability to secrete IFN- gamma were significantly lower than those of the control mice (not scavenging NK cells). The NK cells transferred from WT mice could promote the proliferation of CFSE-CD8+T cells and secrete IFN- gamma. The proliferation of CFSE-CD8+T cells and the ability to secrete IFN- gamma were relatively weak. In addition, the transfer of CFSE-CD8+T cells to GKO mice and the transfer of some receptor mice to NK cells of WT mice showed that the proliferation and secretion of IFN- gamma in the liver and spleen of the recipient mice of the NK cells in the liver and spleen were significantly enhanced. The IFN- gamma secreted by NK cells promotes the immune response of CD8+T cells.
4.NK cells promote reimmunization against HBV-CD8+T cells
Then, the splenic CD8+T cells were selected for fifth days after high pressure injection of HBV plasmids and labeled with CFSE, then transferred to NK cells to clear the mice and the control mice. After 24 hours, the HBV plasmid was injected into the recipient mice by high pressure. It was found that NK cells were used to re immunization of the spleen against HBV CD8+T cells. In order to further support this view, we were also selected. The liver CD8+T cells of fourteenth days after high pressure injection of HBV plasmid were labeled with CFSE and transferred to NK cells to scavenging mice and control mice respectively. It was also found that NK cells cleared the proliferation of CFSE-CD8+T cells and the ability to secrete IFN- gamma in the liver and spleen of mice. Therefore, NK cells were involved in the anti HBV CD8 from the liver and spleen. The immune response of +T cells.
Role of 5.CD4+T cells in NK cells against HBV infection and HBV CD8+T cell activation
In order to detect whether CD4+T cells play a role in controlling HBV infection in NK cells and regulating the activation of CD8+T cells, we detected the scavenging of HBV in Ragl-/- and CD4-/- mice. The results showed that although NK cells existed in Ragl-/- and CD4-/- mice, they could not play an anti HBV act. It was found that there was no significant change in serum HBsAg, while NK cells in WT mice could control the infection of HBV. Further experiments found that NK cells in CD4-/- mice did not promote the secretion of IFN- gamma in CD8+T cells, because NK cells in WT mice obviously promoted the secretion of HBV CD8+T cells. - / - / - CD8+T cells secreted IFN- gamma. Therefore, we think that the NK cells in CD4-/- mice are indirect anti HBV and indirectly regulate CD8+T cells. And the antiviral effect of NK cells depends on the existence of CD4+T cells. The results show that CD4+T cells not only mediate the HBV dyeing of NK cells, but also participate in the regulation of CD8+T cells.
6.CD4+T cells assist in producing anti HBV CD8+T cell immune responses
The above results showed that CD4+T cells were involved in the clearance of HBV. We further detected the ability of CD4-/- mice to scavenge HBV. The results showed that the serum HBsAg and HBeAg in the CD4-/- mice were significantly higher than those of WT mice, and the HBV plasmid could not clear the HBV infection for eighth weeks after the high pressure injection of the HBV plasmid. Cell to Rag1-/- mice did not help the mice to scavenging HBV infection, suggesting that the anti HBV infection of CD8+T cells needed CD4+T cell assistance. In addition, blocking the role of CD4+T cells in the spleen of mice in vitro also resulted in the secretion of IFN-y in the spleen lymphocytes. Therefore, CD8+T cells needed CD4+T cell assistance to resist HBV infection.
7.NK cells promote CD4+T cell immune response
Since CD4+T cells assist the immune response of CD8+T cells, and NK cells also promote the immune response of CD8+T cells, there may be a regulatory relationship between.CD4+T and NK cells. Compare the function of NK cells in CD4-/- mice and WT mice. The results show that the work of NK cells in CD4-/- mice is normal. The function of T cells, transfer of CFSE labeled CD4+T cells to CD4-/- mice, it was found that NK cell clearance resulted in the proliferation of CFSE-CD4+T cells in the liver and spleen and the secretion of IFN- gamma in the receptor mice. The separation from NK cells removed the liver CD4+T cells from mice and stimulated by Core129 peptide segment in vitro, producing IFN-y HBV- specificity. The ability of sex -CD4+T cells was also significantly reduced. The above results showed that NK cells were not regulated by CD4+T cells, and NK cells promoted the anti HBV immune response of CD4+T cells.
IFN- cells secreted by 8.NK cells promote CDllb+DCs immune response
The expression of CDllb+DCs in the spleen of mice decreased significantly by NK cells, and the expression of CD86 and CD80 on the CDllb+DCs surface also decreased obviously, and the mRNA expression level of IL-18, IL-15 and IFN- a in the spleen cells decreased obviously. The tail vein was injected with IFN-y to clear the IFN-y in the mice. The expression level of N- mRNA is also down regulated. Therefore, the IFN-y secreted by NK cells also promotes the activation of CD11b+DCs in the spleen, which may mediate the activation of adaptive immune response.
9. effects of hepatic lymph nodes on HBV immune response
In addition, the lymph nodes on the right side of the hepatic portal were found to drain the liver EFGP+ cells on the right side of the hepatic portal, suggesting that the liver lymph nodes drain the liver cells by using the intrahepatic injection of the liver and the high pressure tail vein for Ad-EGFP. The liver lymph nodes can produce the anti HBV specific immune response. About 30% of the HBV tolerant mice appeared, but the spleen did not affect the HBV infection in mice.

【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392

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