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1.
彭咏波  马永平  唐伟  夏永鹏  邱宗荫 《生殖与避孕》2007,27(12):764-767,771
目的:探讨2-DE技术在人类正常与弱精不育者精浆全蛋白中筛选差异蛋白的应用。方法:先用IPG-DALT技术对12例弱精不育与5例正常精浆蛋白进行分离,银染后,经PDQUest2D比较二者间差异蛋白点。结果:在不育精浆中高丰度存在而正常精浆中低表达的有55个蛋白斑点(3个在正常缺失),在正常精浆中存在而在不育精浆中低表达的有43个(3个在不育缺失)。结论:成功建立了正常与弱精不育精浆蛋白的2-DE图谱并分析表达差异蛋白,为弱精症精浆差异表达蛋白的质谱鉴定奠定了基础。  相似文献   

2.
腺嘌呤诱导不育大鼠精子中精蛋白mRNA的研究   总被引:6,自引:0,他引:6  
目的 :研究正常生育大鼠和腺嘌呤诱导的不育大鼠附睾精子中精蛋白及其 m RNA的含量以及它们的相关性。方法 :大鼠喂服腺嘌呤制备不育动物模型 ,收集对照组和不育组大鼠附睾精子 ,提取总碱性核蛋白 ( TNBP) ,电泳分析 ;提取总 RNA,用 RT- PCR分析精蛋白 m RNA相对含量。结果 :不育组大鼠附睾精子中精蛋白含量明显低于正常组 ( P<0 .0 1 ) ,对精蛋白 m RNA的分析也得到了相似结果 ,正常组和不育组相比较 ,有非常显著差异 ( P<0 .0 0 1 )。结论 :精子中精蛋白 m RNA的相对含量能反映出精子中精蛋白的相对含量 ,为男性不育症的基因诊断提供了新思路  相似文献   

3.
目的:应用同位素标记相对和绝对定量(i TRAQ)联合液相色谱-串联质谱(LC-MS/MS)技术筛选和鉴定正常组与子痫前期组胎盘组织差异蛋白,寻找潜在的子痫前期生物标记物。方法:收集2014年12月至2015年6月青岛大学附属医院产科收治的重度子痫前期患者30例(子痫前期组)和正常足月妊娠患者30例(正常组)。剖宫产术后取部分胎盘组织,提取总蛋白,对总蛋白进行变性、还原及酶解,i TRAQ标记后用质谱仪进行鉴定,得到表达差异蛋白。结果:(1)共鉴定到差异蛋白234个,子痫前期组较正常组表达丰度相差1.5倍以上(上调比值1.50或下调比值0.67),差异有统计学意义的蛋白质点共24个,其中14个蛋白点较正常组表达上调,10个点表达下调。结论:i TRAQ联合LC-MS/MS技术能有效筛选出子痫前期胎盘组织差异蛋白。  相似文献   

4.
目的 寻求与重度子痫前期发生相关的蛋白,为重度子痫前期发病机制研究奠定基础.方法 选择2008年1~3月在天津医科大学总医院产科住院分娩的重度子痫前期患者及正常妊娠妇女各8例,采集正常孕妇和重度子痫前期患者的胎盘组织,提取总蛋白后进行双向电泳技术进行分离.蛋白质凝胶图像经PDQuest软件分析后,获得差异蛋白质点.对差异蛋白质点进行时间飞行质谱(MALDI-TOF-MS)鉴定.结果 与正常妊娠相比,重度子痫前期患者胎盘组织丰度差异表达相差2倍以上,并经Student's t-test检验差异有统计学意义的蛋白质点有14个(P<0.05),其中9个点在重度子痫前期组表达上调,另5个点表达下调.这些蛋白广泛参与细胞信号转导、细胞凋亡、应激反应、细胞结构、能量代谢等生理过程.结论 重度子痫前期的发生中,胎盘组织可能存在独特的蛋白表达模式.  相似文献   

5.
目的:研究与子宫内膜癌淋巴转移相关的蛋白质。方法:提取正常子宫内膜、淋巴结阴性和淋巴结阳性的子宫内膜癌组织总蛋白,用双向凝胶电泳分离蛋白质,用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)分析鉴定差异蛋白点。结果:在淋巴结阴性和淋巴结阳性的子宫内膜癌中,共筛选出11个差异蛋白,与淋巴结阴性组比较,淋巴结阳性内膜癌中,热休克蛋白27、3种肌动蛋白、S100钙结合蛋白A9、丙酮酸激酶、α烯醇化酶、白蛋白和组氨酸三聚体核苷结合蛋白1表达上调,天冬酰胺酶样1蛋白和酯酶D/甲酰-谷胱甘肽水解酶下调。结论:淋巴结阳性和阴性子宫内膜癌的双向凝胶电泳图谱之间存在较明显差异。经鉴定的9种蛋白质可能在子宫内膜癌发生淋巴转移过程中发挥作用。  相似文献   

6.
良性转归葡萄胎与恶性转变葡萄胎的比较蛋白质组学研究   总被引:3,自引:0,他引:3  
目的探讨良性转归葡萄胎与恶性转变葡萄胎的差异表达蛋白质鉴定方法。方法对北京协和医院妇产科2005年9月至2006年12月的7例良性转归葡萄胎及11例恶性转变葡萄胎进行比较蛋白质组学研究,将提取的总蛋白利用双向凝胶电泳(2-DE)分离,对凝胶上的蛋白点进行比较分析,找出明显差异表达的蛋白点,采用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS),获得肽质量指纹图(PMF),在数据库中鉴定差异蛋白。结果(1)良性转归葡萄胎与恶性转变葡萄胎组织的双向凝胶电泳图谱上大多数蛋白点有很好的相关性,比较分析后共筛选出32个差异点。(2)对差异点进行肽质量指纹图分析,获得理想匹配的蛋白质17个,鉴定出一些细胞骨架蛋白,应激相关蛋白,凋亡相关蛋白,与信号转导、细胞增殖及分化相关的蛋白,其中11个可能与葡萄胎恶性转变相关。结论应用蛋白质组学技术成功鉴定了17个差异蛋白,其中11个可能与葡萄胎恶性转变相关,为进一步筛选用于评价葡萄胎预后的标志物奠定了基础。  相似文献   

7.
目的:通过检测钙黏蛋白1(CIB1)在少弱精子症患者精子中的表达,探讨其与少弱精子症发病机制的关系。方法:收集25例少弱精子症患者精液标本,其中少弱精子组13例和弱精子组12例,另同期收集正常对照组19例。分别采用半定量PCR(RT-PCR)及荧光定量PCR(qRT-PCR)方法和Western印迹检测,并比较各组精子中CIB1和周期蛋白依赖性激酶1(CDK1)的mRNA及蛋白表达量。结果:1RT-PCR和qRT-PCR检测表明:在少弱精子组、弱精子组和正常对照组的CIB1mRNA的相对表达量均呈上升趋势,而CDK1 mRNA的相对表达量均呈下降趋势,CIB1 mRNA与CDK1 mRNA在3组间两两比较,除qRT-PCR检测中少弱精子组与弱精子组差异无统计学意义(P0.05),其余组间比较差异均有统计学意义(P0.01)。2CIB1蛋白的相对表达量在少弱精子组、弱精子组和正常对照组中呈上升趋势,3组间两两比较,差异均有统计学意义(P0.05;P0.01);而CDK1蛋白的相对表达量呈下降趋势,其中少弱精子组与正常对照组比较,差异有统计学意义(P0.05)。结论:CIB1可能通过CDK1通路参与少弱精子症的发病机制。  相似文献   

8.
卵巢上皮性癌细胞转移相关蛋白的比较蛋白组学分析   总被引:7,自引:0,他引:7  
Su D  Xu SH  Gu LH 《中华妇产科杂志》2005,40(9):619-622
目的筛选卵巢上皮性癌(卵巢癌)转移相关蛋白,进一步探讨卵巢癌的转移机制。方法以卵巢癌细胞系HO-8910和高转移卯巢癌细胞系HO-8910PM为研究对象,采用比较蛋白组学的研究技术(双向电泳、质谱分析),筛选两个细胞系中差异表达的蛋白质。结果高转移细胞系HO-8910PM与卵巢癌细胞系HO-8910比较,共出现了21个差异表达蛋白质点,其中16个蛋白质点表达上调,5个蛋白质点表达下调。21个差异表达蛋白质点有17个被鉴定,并分为7大类,即锌指蛋白、钙结合蛋白、DNA修复及合成蛋白、细胞调节蛋白、细胞代谢相关蛋白、细胞信号和传导蛋白及细胞表面抗原。结论卵巢癌细胞系HO-8910和高转移卵巢癌细胞系HO-8910PM的蛋白质表达存在明显差异。  相似文献   

9.
重度子痫前期患者血清差异蛋白质组学研究   总被引:1,自引:0,他引:1  
目的:利用蛋白质组学技术寻找敏感性高、特异性好的血清标志物作为重度子痫前期的诊断依据。方法:采用双向凝胶电泳与质谱联用的方法筛选子痫前期重度患者和健康孕妇血清中的差异蛋白质。2-DE分离两组血清蛋白质,图像分析软件识别差异蛋白质点,电喷雾串联质谱(ESI-Q-TOF MS/MS)鉴定差异蛋白质,Western blot检测差异蛋白质—P物质在两组血清中的表达情况。结果:采用双向凝胶电泳与质谱识别了8个差异蛋白质点,鉴定出3种差异蛋白:凝血酶、P物质及白蛋白。Western blot结果显示,子痫前期重度患者血清中P物质的表达水平明显低于健康孕妇。结论:凝血酶、P物质及白蛋白可能参与了重度子痫前期的发生、发展过程,检测它们的水平对子痫前期重度的诊断可能具有一定的参考价值。  相似文献   

10.
目的 建立子宫腺肌病病灶组织的蛋白质表达图谱,筛选并鉴定差异表达的蛋白质.方法 收集2007年1月至10月北京协和医院妇产科收治的因子宫腺肌病行子宫全切除术患者的子宫肌层病灶组织(腺肌病组)及因宫颈上皮内瘤变和宫颈癌行子宫全切除术患者的正常子宫肌层组织(对照组)各5份,采用双向凝胶电泳技术分别建立两组的蛋白质表达图谱;利用图像分析软件进行比较分析,寻找差异表达的蛋白质点;应用基质辅助激光解吸电离飞行时间质谱和生物信息学方法鉴定差异表达的蛋白质,并进行功能分析.结果 子宫腺肌病组织考马斯亮蓝染色图谱中平均含(512±36)个蛋白质点,以其中1块凝胶为参考胶进行匹配,不同凝胶之间蛋白质点的匹配率为83.7%;银染图谱中平均含(762±54)个蛋白质点,不同凝胶之间蛋白质点的匹配率为81.1%.与对照组比较,腺肌病组中有15个恒定差异表达的蛋白质点,其中10个被成功鉴定.这些蛋白质的功能涉及细胞骨架、氧化反应、凋亡和免疫反应等.结论 子宫腺肌病组织在细胞骨架、氧化反应、凋亡、免疫反应等过程中存在异常,这些过程可能参与了子宫腺肌病的发病.  相似文献   

11.
目的探讨电压调控的钾离子(K~+)通道Slo1、Slo3与其γ调节亚基LRRC26、LRRC52在不同活力男性精子中的mRNA水平。方法收集精液参数正常者与特发性弱精子症患者的精子提取物,采用实时荧光定量PCR技术测定电压调控的K~+通道Slo1、Slo3和其γ调节亚基LRRC26、LRRC52 mRNA相对表达量,并分析其与精子活力的相关性。结果 Slo1、LRRC26、LRRC52 mRNA在特发性弱精子症患者精子中的表达水平(0.47±0.14,0.56±0.08,0.55±0.14)明显低于参数正常男性(0.99±0.21,1.16±0.24,1.00±0.21),差异均具有统计学意义(P0.05);Slo3的表达在参数正常男性精子中(1.03±0.18)亦高于特发性弱精子症者(0.71±0.19),但差异无统计学意义(P0.05)。结论电压调控的K~+通道中Slo1与γ调节亚基LRRC26、LRRC52 mRNA的低表达与精子活力低下相关。  相似文献   

12.
Sperm mitochondrial DNA depletion in men with asthenospermia   总被引:2,自引:0,他引:2  
OBJECTIVE: To determine the content of sperm mitochondrial DNA (mtDNA) in patients with asthenospermia or with poor sperm motility. DESIGN: Analysis of the content of mtDNA as the ratio between the amount of mtDNA and nuclear DNA by using a new concurrent polymerase chain reaction. SETTING: University hospital infertility center. PATIENT(S): Eighty-six men who sought infertility therapy. INTERVENTION(S): Moving characteristics of sperm were examined with a computer-assisted semen analyzer. MAIN OUTCOME MEASURE(S): Sperm samples were classified into two groups, one with asthenospermia and the other with normal moving characteristics. The content of mtDNA in sperm was determined by polymerase chain reaction. We analyzed the mitochondrial mass by MitoTracker Green staining and analyzed DNA content with propidium iodide staining by flow cytometry. RESULT(S): A decrease in sperm mtDNA content was detected in patients with asthenospermia or with poor sperm motility (<20% motility). The relative mtDNA contents in the asthenospermia and normal groups were 7.2 +/- 1.3 (mean +/- SD, n = 23) and 74.1 +/- 2.0 (n = 29), respectively. Lower intensities of propidium iodide staining were detected in patients with asthenospermia or poor motility, but there was no significant difference in MitoTracker Green staining between the sperm with different motility. CONCLUSION(S): We suggest that mtDNA content may serve as a useful indicator of sperm quality and that mtDNA depletion may play an important role in the pathophysiology of some male infertility.  相似文献   

13.
In this study, high resolution two-dimensional (2-D) gel electrophoresis was used to identify human sperm antigens recognized by the sera from infertile women having sperm immobilizing (SI) antibodies. Two-D gel electrophoresis was employed to separate Percoll purified human sperm proteins using isoelectric focusing (IEF), followed by polyacrylamide gel electrophoresis (PAGE). Sperm proteins were transferred to the nitrocellulose membranes and immunoblotted with seven sera from infertile women with high titers of SI antibodies and 6 sera from those without SI antibodies. The blots were compared to the 2-D composite image of human sperm proteins [Sperm Protein Encyclopedia] and sperm surface index and the sperm surface proteins recognized by infertile sera were identified. Fifty-two human sperm surface proteins reacted with sera containing SI antibodies, while 35 of these were reactive with the SI-negative control sera. The average numbers of protein spots reacted with test and control sera were 24.6 and 15.0 respectively. A subset of sperm surface proteins which were unique to the SI antibodies were identified by the following criteria; the sperm protein spots which were highly reactive with the infertile sera containing SI antibodies but not reactive with any of the SI-negative infertile sera. The coordinates of 4 prominent immunoreactive sperm proteins were considered as possibly relevant to antibody mediated female infertility.  相似文献   

14.
人精子蛋白激酶C活性及含量对其活力的影响   总被引:6,自引:2,他引:4  
蛋白激酶 C(PKC)在跨膜信息传递中起着重要的调控作用。已有文献报道 PKC存在于人精子尾部 ,与鞭毛活动力密切相关 ,且参与鞭毛活动力的调节。本实验目的是通过对正常的与活力弱的人精子的 PKC活性及含量进行比较 ,观察 PKC活性及含量对人精子活力的影响。结果表明活力弱的人精子尾部的 PKC活性及含量明显低于正常人精子 ,提示人精子活力低下的原因与 PKC的活性及含量降低有关。  相似文献   

15.
Difference gel electrophoresis (DIGE) of fluorescently labelled human sperm proteins was used to identify diabetes- and obesity-associated changes of the sperm proteome. Semen samples from type 1 diabetics, non-diabetic obese individuals and a reference group of clinically healthy fertile donors were evaluated in a comparative study. The adaptation of a general protein extraction procedure to the solubilization of proteins from isolated progressively motile human spermatozoa resulted in the detection of approximately 2700 fluorescent protein spots in the DIGE images. Comparison of the patients’ sperm proteomes with those of the reference group allowed the identification of 20 spots containing proteins that were present in the sperm lysates at significantly increased or decreased concentrations. In detail, eight of these spots were apparently related to type 1 diabetes while 12 spots were apparently related to obesity. Tryptic digestion of the spot proteins and mass spectrometric analysis of the corresponding peptides identified seven sperm proteins apparently associated with type 1 diabetes and nine sperm proteins apparently associated with obesity, three of which existing in multiple molecular forms. The established proteomic approach is expected to function as a non-invasive experimental tool in the diagnosis of male infertility and in monitoring any fertility-restoring therapy.  相似文献   

16.
目的:建立人成熟卵泡液的蛋白质谱图,筛选鉴定卵泡发育/卵母细胞成熟的相关蛋白质。方法:选取48例因男性因素行ICSI-ET治疗的女性为研究对象,收集取卵时获得的人成熟卵泡液,采用双向凝胶电泳-质谱技术分离鉴定人成熟卵泡液蛋白质谱图。结果:获得了较高分辨率的人成熟卵泡液蛋白质谱图,共检测到180多个蛋白质点,筛选鉴定了5个蛋白质点,分别是载脂蛋白E、PRO2044、Hypothetical protein、免疫球蛋白G重链以及转铁蛋白,PRO2044和Hypothetical protein根据蛋白数据库的鉴定分析结果可能是血清白蛋白的修饰产物。转铁蛋白和载脂蛋白E在以往卵泡发育机制的研究罕有报道。结论:蛋白质组学是全面分析人卵泡发育过程中卵泡液蛋白质表达变化的有效手段,筛选鉴定得到的卵母细胞成熟相关蛋白为进一步研究卵母细胞成熟机制提供了理论依据。  相似文献   

17.
目的 探讨蛋白质组学方法 产前筛查孕育唐氏综合征胎儿的孕妇(唐氏综合征孕妇)胎盘组织中差异表达的蛋白标志物.方法 2009年3月至12月首都医科大学附属北京妇产医院确诊的胎儿患唐氏综合征的孕妇10例(唐氏综合征组),同期因其他疾病原因(胎儿染色体检查正常)于孕中期引产的8例孕妇为对照组.用二维荧光差异凝胶电泳技术对两组孕妇产后胎盘组织蛋白进行分离,用Decyder 6.5软件进行图像分析,获得差异表达的蛋白质点;应用基质辅助激光解吸电离-飞行时间质谱(MALDI-TOF-MS)技术对两组中三维丰度比值在1.5倍以上的蛋白质点进行蛋白质谱鉴定,经检索蛋白数据库确定匹配的蛋白质;用蛋白印迹法对所选的差异表达蛋白进行验证,检测差异表达蛋白的相对表达水平.结果 (1)两组胎盘组织中差异表达蛋白:唐氏综合征组和对照组共发现352个差异表达蛋白质点,其中两组比较有统计学意义(P<0.05)的蛋白质点为56个,对其三维丰度比值在1.5倍以上的17个差异蛋白质点行质谱鉴定,其中12个蛋白质点为上调,5个蛋白质点为下调.(2)蛋白质匹配:最终确定可匹配的蛋白质有10种,选取其中差异表达最明显的4种蛋白质,即铜锌型超氧化物歧化酶(SOD1)、热休克蛋白B1(HSP27)、29型内质网蛋白(ERP29)、脂质抗氧化酶6(PRDX6)行相对表达水平检测,唐氏综合征组分别为0.74±0.12、0.29±0.10、0.53±0.16、0.20±0.09,对照组分别为0.51±0.08、0.34±0.16、0.18±0.07、0.35±0.09,两组比较,差异均有统计学意义(P<0.05).结论 唐氏综合征孕妇胎盘组织中存在差异表达蛋白,为产前筛查唐氏综合征提供了待选标志物;但SOD1、HSP27、ERP29、PRDX6是否可作为唐氏综合征筛查的新标志物还需进一步研究.
Abstract:
Objective To discuss protein marks expressed differentially in placenta of Down's syndrome by means of proteomics. Methods We collected placenta of 18 patients(from March 2009 to December 2009 at Beijing Obstetrics and Gynecology Hospital), and divided them into two groups, one was 10 patients with fetal Down's syndrome, the other was normal pregnancies (normal chromosome) with other diseases. We separated proteins expressed in placentas of two groups by two-dimensional difference gel electrophoresis (2D-DIGE), and then analyzed the differential protein spots by software Decyder 6. 5, then,spots differentially expressed by 1.5 fold or more were analyzed by matrix assisted laser desorption ionizationtime of flight-mass spectrometry (MALDI-TOF-MS). In the end, the differential expressional levels of partially identified proteins were validated by western blot analysis. Results (1) Differential proteins of two groups protein spots of placentas separated by 2D-DIGE were analyzed by software Decyder 6. 5 (these colored lights scattered in the image were protein spots), a total of 56 spots out of 352 were differentially expressed (P<0. 05) in two groups. We analyzed 17 protein spots(12 protein spots were over-expressed and 5 protein spots were down-expressed) differentially expressed by 1.5 fold or more by MALDI-TOF-MS.(2) Protein matching after searching protein database, 17 protein spots turn out to be 10 proteins. Four kinds [superoxide dismutase 1 (SOD1), peroxiredoxin 6 (PRDX6), heat shock protein 27 (HSP27),endoplasmic reticulum protein 29 (ERP29)] of them were validated by western blot analysis, the group of fetal Down's syndrome were 0.74 ±0. 12,0.29 ±0. 10,0.53 ±0. 16,0.20 ±0. 09,the group of normal pregnancies were 0. 51 ±0. 08,0. 34 ± 0. 16,0. 18 ± 0. 07,0. 35 ± 0. 09, the results confirmed the observed changes in proteomics. Conclusions Compared with normal pregnancies, there were differential proteins expressed in placenta of Down's syndrome. This approach might provide new screening markers in use for prediction of Down's syndrome, however, further study should be done to make these 4 proteins (SOD1,HSP27, ERP29, PRDX6) be new screening markers.  相似文献   

18.

Purpose

Sperm DNA fragmentation has been suggested as a marker for infertility diagnosis and prognosis. Hence, understanding its impact on male physiology and post-genomic pathways would be clinically important. We performed the proteomics and functional enrichment analyses of viable spermatozoa from ejaculates with low and high sperm DNA fragmentation to identify protein expression and pathways altered in association with sperm DNA fragmentation.

Methods

Sperm DNA fragmentation using the Comet assay and the Komet 6.0.1 software was assessed in raw samples from 89 subjects from a human reproduction service. The Low and High sperm DNA fragmentation groups were formed according to the Olive Tail Moment variable. Spermatozoa proteins from these groups were pooled and analyzed by a shotgun proteomic approach (2D nanoUPLC-ESI-MSE). Differentially expressed proteins were used for a functional enrichment study.

Results

Two hundred and fifty-seven proteins were identified or quantified in sperm from the Low and High sperm DNA fragmentation groups. Of these, seventy-one proteins were exclusively or overexpressed in the Low group, whereas twenty-three proteins were exclusively or overexpressed in the High group. One hundred and sixty-three proteins were conserved between these groups. We also functionally related the differentially expressed proteins in viable spermatozoa from the groups. Processes such as triacylglycerol metabolism, energy production, protein folding, response to unfolded proteins, and cellular detoxification were found to be altered in these cells.

Conclusions

Sperm DNA fragmentation is associated with differential protein expression in viable spermatozoa. These proteins may potentially be used as biomarkers for sperm DNA integrity.  相似文献   

19.

Purpose

To compare proteomic profiles of spermatozoa from patients with varicocele and poor sperm quality before and after varicocelectomy.

Methods

This work was designed as a prospective and observational study. The study was based on 20 men with varicocele grade 3 and poor sperm quality undergoing varicocelectomy at the Fertility Unit of Royan institute in 2009. Two semen samples were collected, one before varicocelectomy and the other after surgery. Protein separation was done by two-dimensional protein electrophoresis, and analyzed by gel densitometry and mass spectrometry. Differential sperm protein expression levels were measured by gel densitometry.

Results

Comparison of the sperm parameters showed that sperm motility and concentration were increased after varicocelectomy. At the level of protein, a total of 3 protein spots were identified whose expression was significantly lower in sperm samples before varicocelectomy compared with after surgery including heat shock protein A5 (HSPA5), superoxide dismutase 1 (SOD1) and δ-subunit of the catalytic core of mitochondrial adenosine triphosphate synthase (ATP5D).

Conclusions

High grade varicocoele affects sperm protein expression presumably because of increasing testicular temperature. These proteins play essential roles in sperm production, DNA integrity protection, and sperm motility. This novel study demonstrates that varicocelectomy can improve both sperm quality and proteins expression.  相似文献   

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