【作者】 张浪辉；

【导师】 陈志哲； 韩忠朝； 刘拥军；

【作者基本信息】 福建医科大学 ， 内科学， 2007， 博士

【摘要】 目的本研究旨在建立一种分离、培养扩增脐带源间充质干细胞(human umbilical cord derived mesenchymal stem cell,hUC-MSC)的方法,通过对其基本生物学特性以及在促进造血细胞归巢,提高造血干细胞植入与造血重建、调节移植免疫中作用的研究,在临床前水平验证hUC-MSC支持造血的功能,以期找到一条通过hUC-MSC与造血干细胞共移植治疗来提高造血干细胞移植成功率的有效途径,以满足临床上日益增多的移植需求。方法研究分四个部分:1.首先建立hUC-MSC分离和培养扩增的方法并通过细胞形态学,细胞增殖和传代培养生长曲线的绘制,成纤维样细胞集落形成实验,流式细胞仪检测细胞周期与免疫表型分析对其基本生物学特性进行研究。2.通过RT-PCR检测hUC-MSC支持造血、促进造血归巢与植入相关分子表达;通过造血干祖细胞集落培养了解hUC-MSC维系造血的能力;通过示踪技术检测移植后24h,hUC-MSCs在NOD/SCID小鼠的体内分布以及其引导造血细胞在小鼠体内归巢的情况。3.通过流式细胞仪检测hUC-MSC对脐带血T细胞及各亚型早期激活标志—CD69表达的影响和MTT法检测hUC-MSC对脐带血T细胞增殖的影响以了解其对T细胞免疫功能的调节作用。4.最后通过建立hUC-MSC与脐带血共移植NOD/SCID小鼠的动物模型,分析移植存活率和造血植入程度(人CD45+细胞嵌合率),在临床前动物实验水平了解hUC-MSC对造血干细胞移植的影响。结果通过本法分离、培养的原代hUC-MSC于接种后24-48h内贴壁, 6-10d后可见成纤维细胞样克隆形成(Unit-fibroblast like colony-forming,CFU-F),集落计数平均每459±76个脐带单个核细胞可形成1个CFU-F, 2-3w后即可传代。传代细胞增殖较快,对数生长期细胞倍增时间约为32h,经连续传代培养扩增29d后细胞总数可达(9.82±0.79)×109。细胞生长周期分析示超过80%的hUC-MSC处于G0/G1期。免疫表型分析显示hUC-MSC表达CD13、CD29、CD44和CD105,不表达CD34、CD38、CD45。对组织相容性表型的分析显示hUC-MSC不表达HLA-II抗原,不表达或低表达HLA-I抗原。RT-PCR检测结果提示hUC-MSC表达SCF, Flt3L, IL-3, IL-6, G-CSF, GM-CSF等造血生子因子以及SDF-1/CXCR-4、CD11a/ICAM-1、CD49d/VCAM-1等造血归巢与植入相关分子。造血前体细胞集落培养显示hUC-MSC可维持脐带血造血前体细胞长达6周以上的集落形成能力。示踪技术显示hUC-MSC在植入NOD/SCID小鼠体内24h后主要分布于骨髓中,肝、脾亦有少量分布,而在心脏、肺脏、肾脏、脑组织、外周血中未检出;在hUC-MSC共移植情况下,流式细胞仪检测和荧光显微镜下细胞计数均发现人脐带血MNC在小鼠骨髓中的含量与单独脐带血移植相比较有显著提高(145±59/1×105vs298±72/1×105;755±158/5×105vs1598±227/5×105个骨髓有核细胞;n=9,P<0.05)。流式细胞仪检测在hUC-MSC共培养情况下,经丝裂原刺激后,脐带血T淋巴细胞CD69表达与对照组(22. 6±5.2%)的阳性率相比较,下降至7.8±3.5%(p<0.01),而且这种抑制对不同亚型T细胞(CD4+/CD8+)均起作用。MTT检测提示在hUC-MSC共培养条件下,丝裂原刺激T细胞增殖受到抑制,抑制的程度与hUC-MSC的剂量正相关。hUC-MCS与脐带血共移植NOD/SCID小鼠动物模型研究显示hUC-MSC的共移植治疗可使小鼠存活率由单独脐带血单个核细胞移植的44.4%提高到88.9%(p<0.05);流式细胞仪检测提示hUC-MSC的造血共移植使得人CD45+细胞的小鼠嵌合率较单独脐带血单个核细胞移植的8.97±1.85%明显提高至35.72±15.41%(p<0.05)。结论1.成功建立了从人足月脐带中分离培养扩增hUC-MSC的方法,操作简单、易行,所得细胞完全能够满足临床治疗的需要;2.hUC-MSC具有间充质干细胞的基本生物学特性和表型特征,而其高增殖潜能的特点尤为突出。3.初步探讨了3种hUC-MSC保存的有效方法,为建立脐带间充质干细胞库提供可能的实施方案;4.hUC-MSC具有维持造血前体细胞长期存活,延缓分化,达到支持长期造血的能力;5.hUC-MSC具有促进造血干祖细胞有效归巢与植入,加速造血重建的作用;6.hUC-MSC对T淋巴细胞激活和增殖具有抑制作用,这种作用为非特异性的,呈剂量依赖性,提示了hUC-MSC的调节移植免疫的潜能,这为hUC-MSC应用于异基因造血干细胞移植预防和治疗移植排斥和移植物抗宿主病,提供了实验依据;7.共移植动物模型进一步证实了hUC-MSC促进造血植入,支持造血重建,提高移植存活率的能力。更多还原

【Abstract】 Objective To establish the methods of isolation and expansion of (human umbilical cord derived mesenchymal stem cell, hUC-MSC). By studying the basic biological characteristics of hUC-MSC and its effects on the homing, engraftment, reconstrution of hematopoiesis and its immunomodulatory function after cotransplantation with the hematopoietic stem/progenitor cells to confirm its capacity of hematpoietic-support which should open the horizons for hUC-MSC cotransplantation with hematopoietic stem/progenitor cells to improve the clinical outcome of transplatantions.Methods The study was divided into four parts: 1. hUC-MSCs were isolated and expanded from human umbilical cord after enzyme digested. The basic biological characteristics of hUC-MSC were investigated by the morphology investigation, growth curve drawing of proliferation and passage cultures, unit-fibroblast like colony-forming (CFU-F) assay. Cell cycle analysis and immunophenotype were also detected by flow cytometre (FCM). 2. RT-PCR technique was used to detect the gene expression of hematopoietic growth factors, chemokine and adhesion molecules associated with homing and engraftment of hUC-MSC. Colony-forming assay of hematopoietic stem/progenitor cell was also performed to test the capability of hUC-MSC to maintain hematpoiesis. A cell tracing technique was used to detect the spatial localization of transplanted hUC-MSCs in nonobese diabetic/severe combined immundeficicency (NOD/SCID) mouse afer 24h transplantation. The homing of human hematopoietic cells mediated by hUC-MSC after 24h cotransplantation was also detected through tracing technique. 3. In order to study the immunomodulatory capacity of hUC-MSC, FCM technique was used to detect the CD69 expression, which serves as an ideal indicator of a T-cell early activation, on PHA+PMA activated T cells after 8 hours hUC-MSCs coculture. The effect of hUC-MSC on T-lymphocyte proliferation induced by PHA were also detected by MTT. 4. Lastly a NOD/SCID mouse model of hUC-MSC cotransplantation with human umbilical cord blood was developed to detect the survival rate of NOD/SCID mouse after cotransplantation and the extent of engraftment of umbilical cord blood cells (chimerism of human CD45+ cells) in the bone marrow of NOD/SCID mouse were also measured by FCM.Results The primary hUC-MSCs isolated and cultured by such method adhered to culture flask within 24-48h. Unit-fibroblast like colony-forming (CFU-F) can be seen after 6-10d, and about 459±76 mononuclear cells digested from umbilical cord can form a fibroblast-like colone. After a 2-3 weeks primary expansion, hUC-MSCs can be passaged. The passaged hUC-MSCs proliferated quite rapidly and the number of hUC-MSCs in logarithemic growth period can be doubled within 32h. After 29d sequential passages about 9.82±0.79)×109 hUC-MSCs can be hearvested. Cell cycle analysis of hUC-MSCs showed more than 80 % cells were in G0-G1 phase. The immunophenotypic analysis showed that hUC-MSCs express CD13, CD29, CD44 and CD105, but negative expression of CD34, CD38 and CD45. The MHC phenotypic analysis indicated negative expression of HLA class II and negative or less expression of HLA class I. RT-PCR showed hUC-MSCs express hematopoietic growth factors such as SCF, Flt3L, IL-3, IL-6, G-CSF, GM-CSF, and also express chemokine receptor CXCR-4 and its ligend, stromal derived factor (SDF-1) and other adhesion molecules, CD11a/ICAM-1, CD49d /VCAM-1 associated with homing and engrftment. Colony-forming assay of hematopoietic stem/progenitor cell showed that hUC-MSC can maintain capability of hematpoietic colony-forming more than 6 weeks. Tracing technique showed the spatial localization of transplanted hUC-MSCs in NOD/SCID mouse afer 24h transplantation were mainly in marrow , less in liver and spleen, but no in peripheral blood, lung, kidney, heart and brain. The data showed that cotransplantation of hUC-MSCs together with umbilical cord blood (UCB) resulted in a significant increse of human UCB mononuclear cells (MNCs) in NOD/SCID mouse bone marrow nucleated cells as compared with the infusion of UCB alone through FCM technique and fluorescent microscope (145±59 / 1×105 vs 298±72 / 1×105; 755±158/ 5×105 vs 1598±227/ 5×105; n=9, p<0.05). FCM showed hUC-MSC could inhibit CD69 expression on PHA and PMA activated T cells from 22. 6±5.2% to 7.8±3.5% (p<0.01), further more such inhibitation affected both CD4+ and CD8+ T-cell subsets. A dose dependence of inhibitory effect of hUC-MSC on T-lymphocyte proliferation induced by PHA was also found by MTT. The NOD/SCID mouse model of cotransplantation showed the survival rate of NOD/SCID mouse after cotransplantation was improved from 44.4% to 88.9% as compared with MNCs transplantation alone, and the extent of engraftment of umbilical cord blood cells (chimerism of human CD45+ cells) in the bone marrow of NOD/SCID mouse were also increased from 8.97±1.85% to 35.72±15.41% measured by FCM.Conclusion1. A simple method to isolate and culture-expand hUC-MSCs from human umbilical cord was established successfully. The number of hearvested hUC-MSCs was appropriate for therapeutic purpose.2. hUC-MSC had the basic biological and phenotypic characteristics of MSC, and its capacity of high proliferation was quite outstanding.3. Three methods to preserve hUC-MSCs were primally explored which might do favor to establishing bank of hUC-MSC in the future.4. hUC-MSC had the capacity of hematpoietic-support in a long term through maintaining suvival and preventing differentiation of hematpoietic stem/progenitor cells.5. hUC-MSC could augment the homing and engraftment of hematpoietic stem/progenitor cells which would accelerate the reconstrution of hematopoiesis.6. hUC-MSC had a dose dependent inhibitory effect on T lymphocyte activation and proliferation, and such effect was not selective. These results indicated the immunomodulatory capacity of hUC-MSC which should open the horizons for hUC-MSCs used in much more allogeneic hemotopoietic stem cell transplantations to prevent and treat engraftment rejection and GVHD.7. The NOD/SCID mouse model of cotransplantation confirmed the capacity of hUC-MSC to augment the engraftment and reconstrution of hematopoiesis following a significant improvement of transplantation outcome.更多还原