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1.
目的探讨人骨髓基质细胞(HBMSC)对脐血(CB)单个核细胞(MNC)体外扩增的支持作用及寻找最佳的收获时机。方法将从CB标本中分离出的MNC接种于已建立的无血清培养体系(含或不含HBMSC及细胞因子支持),在0、6、10及14d检测MNC数、CD34 细胞数及集落形成单位(CFU)数。结果在体外培养过程中,各时间点相同条件下,有HBMSC层支持组的各项测量指标均较其他组高(P<0.05),其中HBMSC联合外源性细胞因子组扩增效果最佳。体外培养6~10d时,以上各项指标基本上达到最高值。结论HBMSC联合外源性细胞因子能实现CB在体外的有效扩增,此体系可能是扩增造血干、祖细胞的较理想方案。体外培养6~10d是收获的最佳时间。  相似文献   

2.
人脐血内皮祖细胞体内促进血管重建的实验研究   总被引:3,自引:0,他引:3  
目的:通过观察人脐血内皮祖细胞(EPC)在裸鼠体内缺氧状态下分化成内皮细胞的现象,初步研究其促进血管重建的作用。方法:根据EPC表面标记CD133,采用免疫磁珠法分离培养EPC。设计EPC裸鼠成瘤实验,观察瘤体内血管增生情况;将荧光标记的EPC注射于裸鼠腹部皮瓣中,观察EPC参与皮瓣血管重建情况;将EPC接种于聚羟基乙酸(PGA)中,移植于裸鼠皮下,观察EPC参与血管重建。结果:原代EPC贴壁后呈梭形,从第7天开始数量明显增加,呈克隆状生长。透射电镜观察到成熟内皮细胞最具特征性的细胞器——weibel-Palade小体。裸鼠成瘤实验:抗人vWF免疫荧光显示大量EPC参与肿瘤血管生成;裸鼠皮瓣实验:荧光标记EPC参与裸鼠皮瓣血管生成;PGA移植实验:抗人vWF免疫酶组织化学染色显示EPC参与血管生成。结论:EPC参与血管重建,具有促进血管新生,加速缺血组织血管化的作用。  相似文献   

3.
Obtaining of mesenchymal progenitor cells from the human umbilical cord   总被引:1,自引:0,他引:1  
BACKGROUND: Mesenchymal progenitor cells (MPCs or mesenchymal stem cells, MSC) have the capability for differentiation into various lineages of mesenchymal tissue. MPCs are widely distributed in a variety of tissues in the adult human body and also present in the fetal environment. However, MPCs are a rare population in these tissues. In this study we evaluated the possibility that MPCs or cells with MPC-like potency are present in the umbilical cord (UC). METHODS: Term UCs were collected and stored in sterile saline solution. The UCs (10 cm) were cut into 1 cm length, the vessels were striped manually and the tissue immersed in an enzyme cocktail for 3 h at 37 degrees C. The isolated umbilical cord mesenchymal progenitor cells (UCMPCs) were pelleted by low speed centrifugation, suspended and cultured. RESULTS: (1) Umbilical cord mesenchymal progenitor cells (UMPCs) could be isolated in sufficient quantities and (2) could be cultured easily. (3) These cells demonstrated a fibroblast-like phenotype. (4) They could be expanded in culture and induced to form several different types of cells. (5) In immunochemistry these cells express mesenchymal markers (CD 13, CD 105) but not haematopoetic lineage markers (CD 14 and CD 34). CONCLUSION: Our observation suggested that MPCs are present in human umbilical cord. Instead, it should be considered a valuable resource for the isolation of potent cells for cell-based therapies, especially in general and pediatric surgery.  相似文献   

4.
Hematopoietic stem cell transplantation (HSCT) is the treatment of choice for children and certain adults with malignant and nonmalignant hematologic disease. Since viral infections are the major problem, this study examined those that might potentially be transmitted to HSCT recipients via bone marrow (BM) versus umbilical cord blood (UCB). BM progenitor cells, peripheral blood leukocytes, and plasma samples were collected from 30 allogenic BM donors. Umbilical cord blood hematopoietic stem cells and plasma samples were also collected from 34 UCB donors. Viral DNA extracted and purified from collected specimens was processed using nested polymerase chain reactions (PCR) to detect human parvovirus B19 (HPV B19), human herpesvirus-6 (HHV-6), varicella-zoster virus (VZV), human cytomegalovirus (HCMV), and Epstein-Barr virus (EBV). The prevalences of HCMV DNA in collected BM progenitor cells versus UCB hematopoietic stem cells were 73% versus 23%, respectively. Conversely, HHV-6 DNA was not detected in any collected specimen by simple PCR. Distribution of the other investigated virus DNAs except EBV DNA was similar in specimens collected from both groups. EBV DNA was not determined in UCB hematopoietic stem cells. The results indicate that the risk of viral transmission to BM transplant recipients via UCB hematopoietic stem cells is less than that with BM progenitor cells.  相似文献   

5.
The purpose of this study was investigation of the potential to isolate mesenchymal stem cells (MSCs) from human umbilical cord blood (UCB) and differentiate them into epithelial cells in mouse skin tissues. Mononuclear cells (MNCs) from UCB (UCB-MNCs) were isolated and induced to MSCs in culture. UCB-MSCs were transfected with pEGFP and labeled with PKH26 dye. eGFP-transfected and PKH26-labeled UCB-derived MSCs were purified by flow cytometry to gate purified eGFP(+) PKH26(+) cells and transplanted at a single clone level into injured nude Balb/C mice by tail vein injection. The phenotype of cultured UCB-MSCs, the expression of human cytokeratins and the expression of eGFP(+) PKH26(+) cells in mouse skin tissues were examined by flow cytometry. Human HLA-1 antigen and cytokeratin 10 (CK10) were detected by direct immunofluorescence on mouse skin tissue sections and flow cytometry. Sry gene (sex-determining region of Y chromosome) was detected by PCR reaction. The results showed that MSCs were isolated from UCB and had heterogeneous morphology and growth potential. Moreover, UCB-derived MSCs localized into mouse skin tissues and differentiated into skin epithelial cells confirmed by in vivo cell tracking and human antigen detection. At two weeks after transplant, a number of eGFP(+) PKH26(+) cells were detected in recipient mouse skin tissues. The detection of sry gene and HLA-1 antigen further confirmed that the human UCB-derived cells were present in recipient mouse skin tissues. Human cells localizing to mouse skin and differentiating into skin epithelial cells were demonstrated by cytokeratins (CK) 8 and 10 expression during flow cytometry, and CK10 expression on injured skin tissue section by direct immunofluorescence. CONCLUSION: UCB-derived MSCs localized to injured skin in vivo and differentiated into epithelial phenotypes. The results demonstrate that UCB-derived MSCs contribute to skin tissue regeneration in vivo and may be an ideal cell source for therapy of skin epithelial tissue injury, including burns.  相似文献   

6.
目的 利用生物反应器大规模扩增人脐血造血干/祖细胞,并通过动物移植实验检验该方法的有效性.方法 采集抗凝脐血10份,分离出单个核细胞(MNC),分别进行生物反应器扩增培养和静态扩增培养.检测扩增前后细胞表面CD34、CD38、CD133、CD184和CD62L分子的表达,并进行造血干/祖细胞集落的培养.取非肥胖糖尿病重症联合免疫缺陷小鼠,以X射线照射后,分为4组,其中MNC组小鼠注射未经扩增培养的MNC;静态扩增组小鼠注射经过静态扩增培养的细胞;反应器扩增组小鼠注射经过生物反应器扩增培养的细胞;空白对照组小鼠注射生理盐水.移植后6周处死存活小鼠,收集骨髓细胞,检测其中CD45+、CD3+、CD19+和CD33+细胞的含量以及人特异的Cart-Ⅰ和Alu基因的表达.结果 生物反应器扩增前MNC为(1.2~2.8)×108个,扩增后为(3.7~12.6)×108个,扩增后的细胞数明显高于静态扩增培养者(P<0.01).经生物反应器扩增后所形成的红系集落形成单位、粒-巨噬细胞集落形成单位数明显高于经静态扩增者(P<0.05).移植6周后,空白对照组小鼠均死亡,MNC组存活率为35%,静态扩增组存活率为30%,反应器扩增组存活率为62.9%,后者明显高于前二者(P<0.05).各组存活小鼠骨髓细胞中均检测到Alu基因和Cart-Ⅰ基因的表达以及人源CD33+、CD45+、CD3+及CD19+细胞.结论 利用生物反应器可大规模扩增人脐血造血干/祖细胞,所得细胞能植入小鼠体内,并能获得造血功能重建.  相似文献   

7.
脐血间充质干细胞移植对大鼠局灶性脑缺血的影响   总被引:5,自引:1,他引:4  
目的 从人脐血中分离纯化间充质干细胞(MSC) ,观察其移植对大鼠大脑中动脉栓塞后神经功能恢复的影响及细胞的存活、迁移向神经细胞分化的情况。方法 雄性SD大鼠45只,用线栓法建立大鼠大脑中动脉栓塞(MCAO)模型,大鼠随机分为3组:MSC移植组、单核细胞组和生理盐水组。移植后1、7、14、2 1、2 8d采用改良神经功能损害评分(mNSS)观察大鼠神经功能恢复情况,应用免疫组织化学和免疫荧光双标记技术检测5溴 2脱氧尿核苷(BrdU )标记的MSC细胞的存活、迁移及其胶质纤维酸性蛋白(GFAP)和神经元特异性核蛋白(NeuN)的表达。结果 人脐血MSC细胞移植可显著提高大鼠局灶性脑缺血后神经功能的恢复(P <0 .0 5 )。移植的MSC细胞可在大鼠脑组织中存活,并向缺血区域迁移,11.67%MSC细胞表达GFAP ,3 .72 %MSC细胞表达NeuN。结论 人脐血中含有MSC细胞并可促进局灶性脑缺血大鼠的神经功能恢复,移植细胞可在大鼠脑缺血区域中存活、迁移并向星形胶质细胞或神经元分化  相似文献   

8.
脐血间充质干细胞的生物学特性及其成骨细胞定向诱导   总被引:5,自引:1,他引:5  
目的:研究人间充质干细胞(Mesenchymal Stem Cells,MSCs)的定向分化为成骨细胞的条件及其成骨活性.方法:采用标准Ficoll-Hypaque技术分离脐血和成人骨髓MSCs,以地塞米松、β-磷酸甘油和维生素C为辅剂定向诱导成骨细胞,碱性磷酸酶、骨矿化结节和Ⅰ型胶原作为成骨细胞鉴定与活性评价的指标.结果:两种MSCs的细胞形态和生物学特性无明显差异.定向成骨细胞诱导后,成骨细胞标志性产物碱性磷酸酶、骨矿化结节和Ⅰ型胶原均得到了稳定的表达,并显著高于未诱导MSCs.结论:脐血和成人骨髓MSCs在适当的条件下均可向成骨细胞定向转化.  相似文献   

9.
脐带间质干细胞的研究进展   总被引:1,自引:1,他引:0  
沈华  沈尊理 《中国美容医学》2007,16(8):1158-1160
间充质干细胞(mesenchymal stemcells,MSCs)最初是特指骨髓基质细胞,后来研究发现在胎儿肝脏、肺脏、心脏等实质脏器及脐带血中均提取到了与骨髓基质细胞生物学特性相似、免疫表型相同的干细胞,因此将间质组织来源的与骨髓MSCs生物学性状相似,具有多向分化潜能的细胞统称为间充质干细胞。  相似文献   

10.
人脐血间充质干/祖细胞诱导为心肌样细胞的实验研究   总被引:10,自引:0,他引:10  
目的 了解脐血间充质干 /祖细胞用于心肌细胞再生的可行性 ,并探讨其最佳的诱导培养条件。方法 将脐血单个核细胞置于低血清 (2 % )DMEM培养基中生成贴壁细胞层 ,依传代方法 ,用相同培养条件进行扩增 ,扩增后的贴壁细胞置入心肌诱导培养液中 ,并添加 5 氮杂胞苷进行诱导分化、采用心肌特异性收缩蛋白 肌钙蛋白T染色鉴定被诱生的心肌样细胞。结果 脐血间充质干 /祖细胞克隆在脐血单个核细胞中出现频率为 0 .5× 10 -6,在传 2 0代时 ,可有效扩增 1.3× 10 7倍 ,诱导后 ,70 %的脐血间充质干 /祖细胞分化为心肌样细胞。结论 采用上述扩增与诱导条件 ,脐血间充质干 /祖细胞可得到有效扩增 ,并可高效向心肌样细胞分化。  相似文献   

11.
This study aimed to determine whether mesenchymal stem cells (MSCs) derived from umbilical cord blood (UCB) would promote cutaneous wound healing. MSCs from human UCB were isolated and identified. The characteristics of the isolated MSCs' growth and proliferation were assayed in vitro. The MSCs labeled with 5‐bromodeoxyuridine (BrdU) were applied on fresh cutaneous mice wounds. The healing rates were surveyed. The distribution and the differentiation into keratinocytes of the labeled MSCs in the wound tissue were checked by immunohistochemistry staining. The isolated MSCs could grow and proliferate well in vitro. The isolated MSCs from UCB could be labeled by 5‐bromodeoxyuridine successfully. The MSCs derived from UCB could enhance the healing of mice skin defect wounds, and it was found that the implanted MSCs could differentiate into keratinocyte in the wound tissue. It was demonstrated that MSCs from UCB can be isolated and proliferated successfully. The local administration of MSCs derived from UCB improves skin defect wound healing in mice.  相似文献   

12.
体外诱导人脐血间充质干细胞向软骨细胞分化的初步研究   总被引:4,自引:1,他引:3  
目的:探讨人脐带血间充质干细胞存在及体外向成软骨细胞分化的可能性。方法:将无菌条件下采集健康产妇脐带血,用相对密度为1.077g/ml的人淋巴细胞分离液分离脐血单个核细胞,利用间充质干细胞贴壁生长的特性获得MSCs,流式细胞仪检测其表面抗原标志。联合应用TGF-β1和IGF-Ⅰ及含有1×ITS+1的高糖DMEM进行诱导培养,并于诱导前及诱导后第21天留取细胞,甲苯胺蓝染色,免疫细胞化学法检测II型胶原的表达,分析是否诱导出成软骨细胞。结果:脐血MSC强表达CD44、CD105,不表达CD34、CD45。诱导21天甲苯胺蓝染色阳性,特异性表达II型胶原。结论:人足月脐带血中存在MSCs,在TGF-β1等生长因子诱导下可以向具有软骨细胞表型和功能的细胞分化。  相似文献   

13.
目的 探讨人脐血间充质干细胞移植对大鼠脊髓损伤后骨密度的影响,为脊髓损伤后骨质疏松的临床治疗奠定基础。 方法 建立大鼠脊髓损伤模型,48只SD大鼠随机分为3组:假手术组(Sham)、脊髓损伤组(SCI)、hUCMSCs移植组(SCI + hUCMSCs),每组16只。分别于术后0周、6周和12周应用双能X线法测定大鼠腰椎及股骨骨密度。结果 0周时,3组之间股骨及腰椎骨密度均无明显差异(P>0. 05) ;6周时,与假手术组比较SCI组及移植组骨密度均明显下降(P<0. 05),而移植组较SCI组有所改善(P<0. 05) ;12周时,与假手术组比较SCI组骨密度明显下降(P <0.01),移植组骨密度未见明显降低(P >0.05),移植组较SCI组明显改善(P<0.01)。结论 hUCMSCs移植有助于改善大鼠脊髓损伤后股骨及腰椎骨密度,hUCMSCs移植可能用于治疗脊髓损伤骨质疏松,为脊髓损伤后骨质疏松的治疗提供新思路。  相似文献   

14.
Umbilical cord blood (UCB) is recognized as an enriched source of endothelial progenitor cells (EPCs) with potential therapeutic value. Because cryopreservation is the only reliable method for long-term storage of UCB cells, the clinical application of EPCs depends on our ability to acquire them from cryopreserved samples; however, the feasibility of doing so remains unclear. In this study we demonstrate that EPCs can be isolated from cryopreserved UCB-derived mononuclear cells (MNCs). The number of outgrowth EPC colonies that emerged in culture from cryopreserved samples was similar to that obtained from fresh UCB. Furthermore, EPCs obtained from cryopreserved MNCs were phenotypically and functionally indistinguishable from freshly isolated ones, including the ability to form blood vessels in vivo. Our results eliminate the necessity of performing cell isolation procedures ahead of future clinical needs and suggest that EPCs derived from cryopreserved UCB may be suitable for EPC-related therapies.  相似文献   

15.
目的 观察人雪旺细胞(hSCs)对人脐带间充质干细胞(hUC-MSCs)在脊髓损伤(SCI)体内存活、分化的影响,通过分子生物学技术检测胶质纤维酸性蛋白(GFAP)、髓磷脂碱性蛋白(MBP)与高分子量神经丝蛋白(NF-H)的表达,描述其变化的时间特征.方法 分离、培养及纯化hUC-MSCs和hSCs.取8周龄雌性Wistar大鼠60只,以Impactor Model-Ⅱ型打击器制作脊髓胸10(T10)损伤模型.大鼠随机均分为4组:DMEM对照组(A)、hSCs移植组(B)、hUC-MSCs移植组(C)、hUC-MSCs与hSCs联合移植组(D),各组分别于移植后1、2、3、4周取材.结果 D组可见MBP与核因子(NF)-H染色阳性细胞,其他各组为阴性.A组3种神经标志物在蛋白与mRNA水平的表达量随时间延长均逐渐增高,且GFAP在移植后2周明显增高,MBP与NF-H在移植后3周明显增高[2周与1周GFAP条带灰度比值为1.34,而聚合酶链反应(PCR)所测比值为2.86;3周与1周MBP和NF-H条带灰度比值分别为3.43、4.12,而PCR所测比值分别为6.47、6.78].与对照组相应时间点比较,移植组MBP、NF-H的表达量较高而GFAP的表达量较低,其中D组的MBP、NF-H的表达量最高(D组与A组MBP3周条带灰度比值为2.11,而NF-H3周条带比值为2.11),差异有统计学意义(P<0.05),但D组GFAP各时间点的表达量差异(各周所测条带灰度比值依次为1.00:1.12:1.13:1.14)无统计学意义(P>0.05).结论 hSCs可促进hUC-MSCs向神经元与少突胶质细胞方向分化并增强其对胶质瘢痕的抑制作用.  相似文献   

16.
目的 体外分离培养人脐带静脉血内皮祖细胞(EPCs),观察EPCs对损伤脐动脉的修复作用.方法 采用磁珠分选法(MACS)从人脐血中分离培养EPCs,用流式细胞术、免疫细胞化学和免疫荧光检测进行鉴定;牵拉钳夹损伤法制备去内膜脐动脉段,与EPCs共孵育7 d后,通过病理切片、免疫组织化学和图像分析技术评价EPCs对动脉损伤的修复效果.结果 成功从人脐血中分离培养EPCs,流式细胞术分析结果 为培养7 d后CD133+细胞>90%;CD34、vWF因子相关抗原免疫染色均为阳性.EPCs移植组新生内膜厚度(43.5±5.5)ìm显著低于对照组内膜厚度(90.7±12.7)ìm,(t=-28.88,P<0.01);EPCs移植组的再内皮化程度(77.8±0.1)%明显高于对照组(52.2±0.1)%,(t=21.86,P<0.01).结论 成功从人脐血中培养出EPCs,人脐血EPCs可修复内皮损伤血管.  相似文献   

17.
目的 探讨人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,HUGMSCs)在受体内分化为血管内皮细胞、治疗皮瓣缺血再灌注损伤从而促进皮瓣成活的可能性.方法 体外增殖培养HUC-MSCs,并用流式细胞技术鉴定,以5-乙炔基-2’脱氧尿嘧啶核苷(EdU)标记HUC-MSCs后,移植于皮瓣缺血再灌注损伤的大鼠腹部皮瓣术区局部(治疗组),设PBS为对照组.术后每天观察皮瓣的颜色、皮纹、厚度、毛发生长、坏死范围及针刺出血情况等,并于术后7d处死大鼠,比较2组皮瓣的成活率,切取皮瓣组织行常规病理组织切片,分别进行免疫组织化学染色检测血管内皮生长因子(VEGF)的表达,EdU染色检测供体细胞在受体皮瓣组织内的分布或分化.结果 术后7d治疗组大鼠皮瓣成活率为(97.58±3.41)%,对照组为(54.37±8.78)%,治疗组高于对照组(P<0.05).治疗组大鼠VEGF的表达密度为138.27±8.67,明显高于对照组的56.17±14.13(P<0.05).在成活皮瓣的部分小血管内皮可见EdU阳性细胞连续分布.结论 HUC-MSCs在体内可以分化为血管内皮细胞,直接参与生成新血管,建立新的微循环,并能增加皮瓣局部VEGF的表达,促进血管形成,修复缺血再灌注损伤的皮瓣,减轻皮瓣坏死,促进皮瓣成活.  相似文献   

18.
目的分析以脐血细胞作为第三方细胞辅助输注对小鼠单倍型造血干细胞移植后造血重建的影响。 方法以CB6/F1雄性小鼠为供鼠,以BALB/C小鼠为受鼠。受鼠经Co60全身照射清髓后回输供鼠干细胞,建立单倍型造血干细胞移植模型。将40只受鼠分为对照组和实验组,每组各20只,对照组受鼠回输供鼠干细胞,实验组受鼠回输供鼠干细胞+脐血单个核细胞。于移植后+7 d、+14 d、+21 d、+28 d和+50 d对存活受鼠进行断尾采血,检测白细胞、血红蛋白和血小板,分析造血重建情况。处死小鼠时取外周血1 mL,采用荧光原位杂交技术检测Y染色体比例,计算嵌合率。两组受鼠各时间点白细胞、血红蛋白及血小板检测结果采用成组t检验进行比较,嵌合率采用卡方检验进行比较。P<0.05为差异有统计学意义。 结果移植后+14 d实验组和对照组白细胞数量分别为(8.4±2.6)×109/L和(3.8±1.0)×109/L,差异有统计学意义(t=6.968,P<0.05);其他时间点两组白细胞数量差异无统计学意义。移植后两组受鼠各时间点血红蛋白检测结果差异均无统计学意义。移植后+7 d实验组和对照组血小板数量分别为(125±40)×109/L和(64±15)×109/L,移植后+14 d分别为(282±47)×109/L和(163±41)×109/L,差异均有统计学意义(t=6.366和8.093,P均<0.05);其他时间点两组血小板数量差异无统计学意义。 结论使用脐血细胞作为第三方细胞辅助输注后,可促进单倍型造血干细胞移植受鼠早期白细胞和血小板较快恢复。  相似文献   

19.
脐血间充质干细胞是-类从脐带血中分离和培养的成体干细胞,具有高度自我更新和多向分化的潜能.脐血间充质干细胞的这些特性,吸引了众多国内外学者的目光,目前,脐血间充质干细胞移植的临床治疗取得了一定进展.本文就脐血间充质干细胞的采集分离、纯化及生物学特性及研究前景作一简要综述.  相似文献   

20.
目的 探讨氯化锂体外诱导人脐血间充质干细胞(MSCs)向神经细胞分化的可行性.方法 无菌条件下收集正常足月儿的脐带血,肝素抗凝,密度梯度离心法分离人脐血单个核细胞,贴壁法纯化,用含15%FBS的低糖DMEM培养基进行扩增培养,流式细胞仪检测表面抗原.取3代的脐血MSCs进行诱导,A组:用含15%FBS和20 ng/ml bFGF的DMEM完全培养基预诱导24 h,3 mol/L LiCI的DMEM培养基继续诱导6 d:B组:含3 mol/L LiCI的DMEM培养基诱导7 d;C组:含15%FBS的DMEM培养基正常培养7 d.光镜下观察细胞形态,用免疫组化技术检测细胞NSE、MAP2及GFAP的表达.结果 A组与B组诱导3 d后细胞即出现形态学上的改变,细胞变成不规则形,立体感增强,从胞体伸出突起.免疫组织化学和免疫荧光方法鉴定显示,诱导后的细胞能表达神经元特异性标志NSE和MAP2,阳性表达率A组[分别为(73.6±7.8)%,(75.5±8.5)%]明显高于B组[分别为(31.0±4.3)%、(33.5±5.O)%],而星形胶质细胞特异性标志GFAP阳性细胞较少,A、B、C三组阳性表达率分别为(4.7 ±3.3)%、(5.1±4.6)%、(8.5±3.2)%.结论 LiCI联合生长因子bFGF体外可诱导人脐血MSCs分化为神经元样细胞.  相似文献   

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