The major histocompatibility complex （MHC） is a dynamic genetic region with an essential role in the adaptive immunity of jawed vertebrates. The MHC polymorphism is affected by many processes such as birth-and- deat...The major histocompatibility complex （MHC） is a dynamic genetic region with an essential role in the adaptive immunity of jawed vertebrates. The MHC polymorphism is affected by many processes such as birth-and- death evolution, gene conversion, and concerted evolution. Studies investigating the evolution of MHC class I genes have been biased toward a few particular taxa and model species. However, the investigation of this region in nonavian reptiles is still in its infancy. We present the first characterization of MHC class I genes in a species from the family Lacertidae. We assessed genetic diversity and a role of selection in shaping the diversity of MHC class I exon 4 among 37 individuals of Eremias multiocellata from a population in Lanzhou, China. We generated 67 distinct DNA sequences using cloning and sequencing methods, and identified 36 putative functional variants as well as two putative pseudogene-variants. We found the number of variants within an individual varying between two and seven, indicating that there are at least four MHC class I loci in this species. Gene duplication plays a role in increasing copy numbers of MHC genes and allelic diversity in this species. The class I exon 4 sequences are characteristic of low nucleotide diversity. No signal of recombination is detected, but purifying selection is detected in β2-microglobulin interaction sites and some other silent sites outside of the function-constraint regions. Certain identical alleles are shared by Eremias multiocellata and E. przewalskii and E. brenchleyi, suggesting trans-species polymorphism. The data are compatible with a birth-and-death model of evolution.展开更多
AIM： TO investigate the role of MHC class Ⅱ in the modulation of gastric epithelial cell apoptosis induced by H pylori infection. METHODS： After stimulating a human gastric epithelial cell line with bacteria or ago...AIM： TO investigate the role of MHC class Ⅱ in the modulation of gastric epithelial cell apoptosis induced by H pylori infection. METHODS： After stimulating a human gastric epithelial cell line with bacteria or agonist antibodies specific for MHC class Ⅱ and CD95, the quantitation of apoptotic and anti-apoptotic events, including caspase activation, BCL-2 activation, and FADD recruitment, was performed with a fluorometric assay, a cytometric bead array, and confocal microscopy, respectively. RESULTS： Pretreatment of N87 cells with the anti-MHC class Ⅱ IgM antibody RFD1 resulted in a reduction in global caspase activation at 24 h of H pylori infection. When caspase 3 activation was specifically measured, crosslinking of MHC class Ⅱ resulted in a marked reduced caspase activation, while simple ligation of HHC class Ⅱ did not. Crosslinking of HHC class Ⅱ also resulted in an increased activation of the anti-apoptosis molecule BCL-2 compared to simple ligation. Confocal microscope analysis demonstrated that the pretreatment of gastric epithelial cells with a crosslinking anti-HHC class Ⅱ IgH blocked the recruitment of FADD to the cell surface. CONCLUSION： The results presented here demonstrate that the ability of MHC class Ⅱ to modulate gastric epithelial apoptosis is at least partially dependent on its crosslinking. Furthermore, while previous research has demonstrated that MHC class Ⅱ signaling can be proapoptotic during extended ligation, we have shown that the crosslinking of this molecule has anti-apoptotic ef-fects during the earlier time points of Hpylori infection. This effect is possibly mediated by the ability of MHC class Ⅱ to modulate the activation of the pro-apoptotic receptor Fas by blocking the recruitment of the accessory molecule FADD, and this delay in apoptosis induction could allow for prolonged cytokine secretion by Hpyloriinfected gastric epithelial cells.展开更多
AIM： To investigate the role of MHC class Ⅱ in the modulation of gastric epithelial cell apoptosis induced by H pylori infection. METHODS： After stimulating a human gastric epithelial cell line with bacteria or ago...AIM： To investigate the role of MHC class Ⅱ in the modulation of gastric epithelial cell apoptosis induced by H pylori infection. METHODS： After stimulating a human gastric epithelial cell line with bacteria or agonist antibodies specific for MHC class Ⅱ and CD95, the quantitation of apoptotic and anti-apoptotic events, including caspase activation, BCL-2 activation, and FADD recruitment, was performed with a fluorometric assay, a cytometric bead array, and confocal microscopy, respectively. RESULTS： Pretreatment of N87 cells with the anti-MHC class ⅡIgM antibody RFD1 resulted in a reduction in global caspase activation at 24 h of H pylori infection. When caspase 3 activation was specifically measured, crosslinking of MHC class Ⅱ resulted in markedly reduced caspase activation, while simple ligation of MHC class Ⅱ did not. Crosslinking of MHC class Ⅱ also resulted in an increased activation of the anti-apoptosis molecule BCL-2 compared to simple ligation. Confocal microscope analysis demonstrated that the pretreatment of gastric epithelial cells with a crosslinking anti-MHC class Ⅱ IgM blocked the recruitment of FADD to the cell surface. CONCLUSION： The ability of MHC class Ⅱ to modulate gastric epithelial apoptosis is at least partially dependent on its crosslinking. The crosslinking of this molecule has anti-apoptotic effects during the earlier time points of Hpylori infection. This effect is possibly mediated by the ability of MHC class Ⅱ to modulate the activation of the pro-apoptotic receptor Fas by blocking the recruitment of the accessory molecule FADD, and this delay in apoptosis induction could allow for prolonged cytokine secretion by Hpylori-infected gastric epithelial cells.展开更多
Using human umbilical vein endothelial cells (HUVEC) and porcine aortic endothelial cells (PAEC) as target cells, human peripheral blood NK cells (PBNK) and NK92 cells as effector cells, the differential cytotoxicitie...Using human umbilical vein endothelial cells (HUVEC) and porcine aortic endothelial cells (PAEC) as target cells, human peripheral blood NK cells (PBNK) and NK92 cells as effector cells, the differential cytotoxicities of NK cells to allo- and xeno-endothelial cells were studied. The influence of MHC class I molecules on the cytotoxicity of human NK cells was assayed using acid treatment, and blockades of MHC class I antigens, CD94 and KIR (NKB1). The results indicated that the killing of PAEC by the two kinds of NK cells is higher than that of HUVEC. After acid-treatment, the cytotoxicity of the two kinds of NK cells to PAEC and HUVEC is significantly enhanced, but the magnitude of the enhancement is different. The enhancement of NK killing to acid treated HUVEC is much greater than that to PAEC. Blockade of CD94 mAb did not alter the NK cytotoxicity, while blockade of NKB1 mAb enhanced the cytotoxicity of PBNK to HUVEC and PAEC by 95% and 29% respectively. The results above suggested that the differential recognition of MHC I molecules of xeno-endothelial cells by human NK cells could be the major reason for higher NK cytotoxicity to PAEC. KIR might be the primary molecule that transduced inhibitory signals when endothelial cells were injured by NK cells.展开更多
The worldwide declines in amphibian populations have largely been caused by infectious fungi and bacteria. Given that vertebrate immunity against these extracellular pathogens is primarily functioned by the major hist...The worldwide declines in amphibian populations have largely been caused by infectious fungi and bacteria. Given that vertebrate immunity against these extracellular pathogens is primarily functioned by the major histocompatibility complex（MHC） class Ⅱ molecules, the characterization and the evolution of amphibian MHC class Ⅱ genes have attracted increasing attention. The polymorphism of MHC class Ⅱ genes was found to be correlated with susceptibility to fungal pathogens in many amphibian species, suggesting the importance of studies on MHC class Ⅱ genes for amphibians. However, such studies on MHC class Ⅱ gene evolution have rarely been conducted on amphibians in China. In this study, we chose Omei treefrog（Rhacophorus omeimontis）, which lived moist environments easy for breeding bacteria, to study the polymorphism of its MHC class Ⅱ genes and the underlying evolutionary mechanisms. We amplified the entire MHC class ⅡB exon 2 sequence in the R. omeimontis using newly designed primers. We detected 102 putative alleles in 146 individuals. The number of alleles per individual ranged from one to seven, indicating that there are at least four loci containing MHC class ⅡB genes in R. omeimontis. The allelic polymorphism estimated from the 102 alleles in R. omeimontis was not high compared to that estimated in other anuran species. No significant gene recombination was detected in the 102 MHC class ⅡB exon 2 sequences. In contrast, both gene duplication and balancing selection greatly contributed to the variability in MHC class ⅡB exon 2 sequences of R. omeimontis. This study lays the groundwork for the future researches to comprehensively analyze the evolution of amphibian MHC genes and to assess the role of MHC gene polymorphisms in resistance against extracellular pathogens for amphibians in China.展开更多
Background Previous studies showed that anti MHC-II monoclone antibody （MAb） only had partial inhibiting effect of alloreactive mixed lymphocyte reaction （MLR） in vitro and it was unsteady and non-persistent. The ...Background Previous studies showed that anti MHC-II monoclone antibody （MAb） only had partial inhibiting effect of alloreactive mixed lymphocyte reaction （MLR） in vitro and it was unsteady and non-persistent. The aim of this research was to determine whether radioactive isotope 188Re marked MHC-II antibody could benefit the allograft acceptance in transplantation as compared to normal MHC-II antibody. Methods 188Re was incorporated to 2E9/13F（ab＇）2 which is against swine MHC class II antigen （MAb-~88Re）. Porcine peripheral blood mononuclear （PBMC） cells were examined for proliferation and cytokine mRNA expression after stimulation with MHC-II MAb or MAb-188Re. Results The proliferative response of recipient PBMCs in mixed lymphocyte reaction （MLR） to donor alloantigen showed that the stimulation index of MAb-188Re group was significantly lower than the MHC-II MAb group and control （P 〈0.05）. mRNA expression of interleukin 2, interferon y and tumor necrosis factor a （type 1 cytokines） was lower in MAb-188Re group than the MHC-II MAb group, while interleukin 10 （type 2 cytokines） was higher in MAb-~aSRe group in the first 24 hours. Conclusion MAb-188Re could help the graft acceptance by inhibiting T cell proliferation, lowering the expression of type 1 cytokines and elevating the type 2 cytokines produced by PBMC.展开更多
[ Objective] To prepare the monoclonal antibody against chicken major histocompatibility complex class II molecules （MHC II）. [ Method ] The prokaryotic expression of the gene fragments of exons 2 -6 encoding alpha ...[ Objective] To prepare the monoclonal antibody against chicken major histocompatibility complex class II molecules （MHC II）. [ Method ] The prokaryotic expression of the gene fragments of exons 2 -6 encoding alpha chain of MHC II and exons 3 -6 encoding beta chain of MHC II were performed based on its protein sequences. After BALB/c mice were immunized with the purified fusion proteins, the mouse spleen cells were fused with mouse myeloma cells SP2/0. Then the positive hybridoma cells were screened and detected by indirect enzyme-linked immunosorbent assay （ELISA）. [ Result] One hybridoma cell strain secreting monoclonal antibody against alpha chain and two strains secreting monoclonal antibody against beta chain were obtained. These three hybridoma cell strains were named as MHC II alpha-4, MHC II betas-2 and MCH II betas-31, respectively. Their titers of ascites in indirect ELISA were 1 ： 256 000, 1 ： 256 000 and 1 ： 1 280 000, respectively. These antibodies could specifically recog- nize MHC II alpha chain or beta chain in western blotting. [ Conclusion] Three obtained hybridoma stains can stably produce the monoclonal antibody against chicken MHC class II molecules.展开更多
目的探讨骨肉瘤组织中MHCⅠ类链相关蛋白A(MHC class Ⅰ chain-related A,MICA)、MMP-9和NF-κB的表达及相互间的关系,为研究骨肉瘤组织中MICA蛋白表达和脱落机制提供组织学依据。方法应用免疫组织化学方法检测66例骨肉瘤、11例骨母细胞...目的探讨骨肉瘤组织中MHCⅠ类链相关蛋白A(MHC class Ⅰ chain-related A,MICA)、MMP-9和NF-κB的表达及相互间的关系,为研究骨肉瘤组织中MICA蛋白表达和脱落机制提供组织学依据。方法应用免疫组织化学方法检测66例骨肉瘤、11例骨母细胞瘤,8例骨化性纤维瘤和6例正常骨组织中MICA蛋白的表达情况,并检测66例骨肉瘤组织中MMP-9和NF-κB蛋白的表达情况。结果(1)MICA蛋白在骨肉瘤组织、骨母细胞瘤、骨化性纤维瘤和正常骨组织的表达率分别为51.6%(34/66)、9%(1/11)、0(0/8)、0(0/6),骨肉瘤组织中MICA表达与骨母细胞瘤(P=0.01)、骨化性纤维瘤(P<0.01)和正常骨组织(P<0.05的差异有显著性。(2)骨肉瘤组织中MMP-9和NF-κB表达率分别为55%(36/66)和73%(48/66);NF-κB与MICA(r=0.373,P<0.01)和MMP-9(r=0.536,P<0.01)的表达呈正相关关系。结论MICA蛋白可作为骨肉瘤诊断的分子标志物;NF-κB可能参与MICA蛋白表达和脱落的分子机制,NF-κB可作为骨肉瘤免疫治疗的候选靶点。展开更多
基金supported by the Science and Technology Project for Outstanding Youths in Life Science (KSCX2-EW-Q-6) from the Chinese Academy of SciencesNational Natural Science Foundation of China (31272281)
文摘The major histocompatibility complex （MHC） is a dynamic genetic region with an essential role in the adaptive immunity of jawed vertebrates. The MHC polymorphism is affected by many processes such as birth-and- death evolution, gene conversion, and concerted evolution. Studies investigating the evolution of MHC class I genes have been biased toward a few particular taxa and model species. However, the investigation of this region in nonavian reptiles is still in its infancy. We present the first characterization of MHC class I genes in a species from the family Lacertidae. We assessed genetic diversity and a role of selection in shaping the diversity of MHC class I exon 4 among 37 individuals of Eremias multiocellata from a population in Lanzhou, China. We generated 67 distinct DNA sequences using cloning and sequencing methods, and identified 36 putative functional variants as well as two putative pseudogene-variants. We found the number of variants within an individual varying between two and seven, indicating that there are at least four MHC class I loci in this species. Gene duplication plays a role in increasing copy numbers of MHC genes and allelic diversity in this species. The class I exon 4 sequences are characteristic of low nucleotide diversity. No signal of recombination is detected, but purifying selection is detected in β2-microglobulin interaction sites and some other silent sites outside of the function-constraint regions. Certain identical alleles are shared by Eremias multiocellata and E. przewalskii and E. brenchleyi, suggesting trans-species polymorphism. The data are compatible with a birth-and-death model of evolution.
基金Supported by the National Institutes of Health Grants DK50669, DK56338 and National Institutes of Health T32 AI007536-06 Training Grant
文摘AIM： TO investigate the role of MHC class Ⅱ in the modulation of gastric epithelial cell apoptosis induced by H pylori infection. METHODS： After stimulating a human gastric epithelial cell line with bacteria or agonist antibodies specific for MHC class Ⅱ and CD95, the quantitation of apoptotic and anti-apoptotic events, including caspase activation, BCL-2 activation, and FADD recruitment, was performed with a fluorometric assay, a cytometric bead array, and confocal microscopy, respectively. RESULTS： Pretreatment of N87 cells with the anti-MHC class Ⅱ IgM antibody RFD1 resulted in a reduction in global caspase activation at 24 h of H pylori infection. When caspase 3 activation was specifically measured, crosslinking of MHC class Ⅱ resulted in a marked reduced caspase activation, while simple ligation of HHC class Ⅱ did not. Crosslinking of HHC class Ⅱ also resulted in an increased activation of the anti-apoptosis molecule BCL-2 compared to simple ligation. Confocal microscope analysis demonstrated that the pretreatment of gastric epithelial cells with a crosslinking anti-HHC class Ⅱ IgH blocked the recruitment of FADD to the cell surface. CONCLUSION： The results presented here demonstrate that the ability of MHC class Ⅱ to modulate gastric epithelial apoptosis is at least partially dependent on its crosslinking. Furthermore, while previous research has demonstrated that MHC class Ⅱ signaling can be proapoptotic during extended ligation, we have shown that the crosslinking of this molecule has anti-apoptotic ef-fects during the earlier time points of Hpylori infection. This effect is possibly mediated by the ability of MHC class Ⅱ to modulate the activation of the pro-apoptotic receptor Fas by blocking the recruitment of the accessory molecule FADD, and this delay in apoptosis induction could allow for prolonged cytokine secretion by Hpyloriinfected gastric epithelial cells.
基金the National Institutes of Health Grants DK50669 and DK56338. EB was a recipient of a fellowship under National Institutes of Health T32 AI007536-06 Training Grant
文摘AIM： To investigate the role of MHC class Ⅱ in the modulation of gastric epithelial cell apoptosis induced by H pylori infection. METHODS： After stimulating a human gastric epithelial cell line with bacteria or agonist antibodies specific for MHC class Ⅱ and CD95, the quantitation of apoptotic and anti-apoptotic events, including caspase activation, BCL-2 activation, and FADD recruitment, was performed with a fluorometric assay, a cytometric bead array, and confocal microscopy, respectively. RESULTS： Pretreatment of N87 cells with the anti-MHC class ⅡIgM antibody RFD1 resulted in a reduction in global caspase activation at 24 h of H pylori infection. When caspase 3 activation was specifically measured, crosslinking of MHC class Ⅱ resulted in markedly reduced caspase activation, while simple ligation of MHC class Ⅱ did not. Crosslinking of MHC class Ⅱ also resulted in an increased activation of the anti-apoptosis molecule BCL-2 compared to simple ligation. Confocal microscope analysis demonstrated that the pretreatment of gastric epithelial cells with a crosslinking anti-MHC class Ⅱ IgM blocked the recruitment of FADD to the cell surface. CONCLUSION： The ability of MHC class Ⅱ to modulate gastric epithelial apoptosis is at least partially dependent on its crosslinking. The crosslinking of this molecule has anti-apoptotic effects during the earlier time points of Hpylori infection. This effect is possibly mediated by the ability of MHC class Ⅱ to modulate the activation of the pro-apoptotic receptor Fas by blocking the recruitment of the accessory molecule FADD, and this delay in apoptosis induction could allow for prolonged cytokine secretion by Hpylori-infected gastric epithelial cells.
文摘Using human umbilical vein endothelial cells (HUVEC) and porcine aortic endothelial cells (PAEC) as target cells, human peripheral blood NK cells (PBNK) and NK92 cells as effector cells, the differential cytotoxicities of NK cells to allo- and xeno-endothelial cells were studied. The influence of MHC class I molecules on the cytotoxicity of human NK cells was assayed using acid treatment, and blockades of MHC class I antigens, CD94 and KIR (NKB1). The results indicated that the killing of PAEC by the two kinds of NK cells is higher than that of HUVEC. After acid-treatment, the cytotoxicity of the two kinds of NK cells to PAEC and HUVEC is significantly enhanced, but the magnitude of the enhancement is different. The enhancement of NK killing to acid treated HUVEC is much greater than that to PAEC. Blockade of CD94 mAb did not alter the NK cytotoxicity, while blockade of NKB1 mAb enhanced the cytotoxicity of PBNK to HUVEC and PAEC by 95% and 29% respectively. The results above suggested that the differential recognition of MHC I molecules of xeno-endothelial cells by human NK cells could be the major reason for higher NK cytotoxicity to PAEC. KIR might be the primary molecule that transduced inhibitory signals when endothelial cells were injured by NK cells.
基金supported by the National Natural Science Foundation of China(No.31201713,No.31270425 and No.31470442)
文摘The worldwide declines in amphibian populations have largely been caused by infectious fungi and bacteria. Given that vertebrate immunity against these extracellular pathogens is primarily functioned by the major histocompatibility complex（MHC） class Ⅱ molecules, the characterization and the evolution of amphibian MHC class Ⅱ genes have attracted increasing attention. The polymorphism of MHC class Ⅱ genes was found to be correlated with susceptibility to fungal pathogens in many amphibian species, suggesting the importance of studies on MHC class Ⅱ genes for amphibians. However, such studies on MHC class Ⅱ gene evolution have rarely been conducted on amphibians in China. In this study, we chose Omei treefrog（Rhacophorus omeimontis）, which lived moist environments easy for breeding bacteria, to study the polymorphism of its MHC class Ⅱ genes and the underlying evolutionary mechanisms. We amplified the entire MHC class ⅡB exon 2 sequence in the R. omeimontis using newly designed primers. We detected 102 putative alleles in 146 individuals. The number of alleles per individual ranged from one to seven, indicating that there are at least four loci containing MHC class ⅡB genes in R. omeimontis. The allelic polymorphism estimated from the 102 alleles in R. omeimontis was not high compared to that estimated in other anuran species. No significant gene recombination was detected in the 102 MHC class ⅡB exon 2 sequences. In contrast, both gene duplication and balancing selection greatly contributed to the variability in MHC class ⅡB exon 2 sequences of R. omeimontis. This study lays the groundwork for the future researches to comprehensively analyze the evolution of amphibian MHC genes and to assess the role of MHC gene polymorphisms in resistance against extracellular pathogens for amphibians in China.
基金This research was supported by a grant from the National Natural Science Foundation of China （No. 30872501）.
文摘Background Previous studies showed that anti MHC-II monoclone antibody （MAb） only had partial inhibiting effect of alloreactive mixed lymphocyte reaction （MLR） in vitro and it was unsteady and non-persistent. The aim of this research was to determine whether radioactive isotope 188Re marked MHC-II antibody could benefit the allograft acceptance in transplantation as compared to normal MHC-II antibody. Methods 188Re was incorporated to 2E9/13F（ab＇）2 which is against swine MHC class II antigen （MAb-~88Re）. Porcine peripheral blood mononuclear （PBMC） cells were examined for proliferation and cytokine mRNA expression after stimulation with MHC-II MAb or MAb-188Re. Results The proliferative response of recipient PBMCs in mixed lymphocyte reaction （MLR） to donor alloantigen showed that the stimulation index of MAb-188Re group was significantly lower than the MHC-II MAb group and control （P 〈0.05）. mRNA expression of interleukin 2, interferon y and tumor necrosis factor a （type 1 cytokines） was lower in MAb-188Re group than the MHC-II MAb group, while interleukin 10 （type 2 cytokines） was higher in MAb-~aSRe group in the first 24 hours. Conclusion MAb-188Re could help the graft acceptance by inhibiting T cell proliferation, lowering the expression of type 1 cytokines and elevating the type 2 cytokines produced by PBMC.
基金supported by the National Natural Science Foundation (30671537)
文摘[ Objective] To prepare the monoclonal antibody against chicken major histocompatibility complex class II molecules （MHC II）. [ Method ] The prokaryotic expression of the gene fragments of exons 2 -6 encoding alpha chain of MHC II and exons 3 -6 encoding beta chain of MHC II were performed based on its protein sequences. After BALB/c mice were immunized with the purified fusion proteins, the mouse spleen cells were fused with mouse myeloma cells SP2/0. Then the positive hybridoma cells were screened and detected by indirect enzyme-linked immunosorbent assay （ELISA）. [ Result] One hybridoma cell strain secreting monoclonal antibody against alpha chain and two strains secreting monoclonal antibody against beta chain were obtained. These three hybridoma cell strains were named as MHC II alpha-4, MHC II betas-2 and MCH II betas-31, respectively. Their titers of ascites in indirect ELISA were 1 ： 256 000, 1 ： 256 000 and 1 ： 1 280 000, respectively. These antibodies could specifically recog- nize MHC II alpha chain or beta chain in western blotting. [ Conclusion] Three obtained hybridoma stains can stably produce the monoclonal antibody against chicken MHC class II molecules.
文摘目的探讨骨肉瘤组织中MHCⅠ类链相关蛋白A(MHC class Ⅰ chain-related A,MICA)、MMP-9和NF-κB的表达及相互间的关系,为研究骨肉瘤组织中MICA蛋白表达和脱落机制提供组织学依据。方法应用免疫组织化学方法检测66例骨肉瘤、11例骨母细胞瘤,8例骨化性纤维瘤和6例正常骨组织中MICA蛋白的表达情况,并检测66例骨肉瘤组织中MMP-9和NF-κB蛋白的表达情况。结果(1)MICA蛋白在骨肉瘤组织、骨母细胞瘤、骨化性纤维瘤和正常骨组织的表达率分别为51.6%(34/66)、9%(1/11)、0(0/8)、0(0/6),骨肉瘤组织中MICA表达与骨母细胞瘤(P=0.01)、骨化性纤维瘤(P<0.01)和正常骨组织(P<0.05的差异有显著性。(2)骨肉瘤组织中MMP-9和NF-κB表达率分别为55%(36/66)和73%(48/66);NF-κB与MICA(r=0.373,P<0.01)和MMP-9(r=0.536,P<0.01)的表达呈正相关关系。结论MICA蛋白可作为骨肉瘤诊断的分子标志物;NF-κB可能参与MICA蛋白表达和脱落的分子机制,NF-κB可作为骨肉瘤免疫治疗的候选靶点。
