【作者】 高蕾；

【导师】 陈幸华；

【作者基本信息】 第三军医大学 ， 内科学， 2008， 博士

【摘要】 造血功能障碍是恶性肿瘤化学药物治疗和大剂量放射线治疗、放射源意外泄漏和核战争条件下急性放射损伤的主要并发症之一。其中,巨核细胞损伤导致的血小板减少除输注血小板外,尚缺乏有效的治疗手段。血小板生成素(thrombopoietin,TPO)作为刺激巨核细胞增殖和血小板生成的重要细胞因子仍存在治疗后产生抗凝血抗体、加重出血的危险性。如何安全有效地促进血小板数量和功能的恢复,尚需探索。造血微环境(hematopoietic inductive microenvironment,HIM)是支持和调节造血细胞生长发育的内环境,具有促进巨核细胞增殖分化、成熟产板的作用。因此,从修复或重建骨髓造血微环境正常功能入手治疗巨核细胞损伤,是一个值得探索的领域。骨髓基质细胞(human bone marrow stromal cells,hBMSCs)是造血微环境的主要成分,自1977年Dexter在体外培养出骨髓基质细胞获得成功后,人们对BMSCs进行了深入的研究。目前,骨髓基质细胞体外培养扩增联合造血干细胞回输经实验和临床验证是重建造血功能损伤的有效方法。由于自体移植中患者自身造血微环境异常,而异体移植又存在组织相容性的问题,限制了骨髓基质细胞在临床上的广泛运用。人脐血中的细胞成分丰富且较骨髓和外周血更原始,具有来源广泛,采集方便,免疫原性弱和长期造血重建的特点,已成为新的造血干细胞来源。本课题组长期从事人脐血源基质细胞(human umbilical cord blood-derived stromal cells,hUCBDSCs)及脐血造血微环境的研究,前期研究经体外培养扩增获得hUCBDSCs,证明其具备造血基质细胞的基本特征和造血调控功能的物质基础,以hUCBDSCs为滋养层的体外扩增体系促进巨核细胞集落(colony forming unit-megakaryocte,CFU-Meg)形成的作用明显优于hBMSCs,提示hUCBDSCs在促进巨核细胞增殖分化方面可能具有特殊的意义,对其作用特点和机制的深入研究可为巨核细胞损伤修复治疗提供新的思路。鉴于此,本课题在构建巨核细胞/hUCBDSCs共培养模型和裸鼠造血微环境损伤模型的基础上,观察hUCBDSCs体外促进巨核细胞增殖和体内重建造血微环境、促进巨核细胞生成和血小板数量恢复的作用。从TPO途径、SDF-1途径和间隙连接细胞间通`讯三个方面深入探讨hUCBDSCs促进巨核细胞增殖和移植裸鼠血小板恢复的可能机制,为安全有效促进血小板功能和数量恢复提供新的辅助治疗措施。方法:1.观察人脐血源基质细胞促进巨核细胞系HEL增殖的体外研究实验分组:①HEL细胞悬浮培养组;②HEL细胞/hBMSCs共培养组;③HEL细胞/hUCBDSCs共培养组。倒置显微镜和扫描电镜观察HEL细胞和基质细胞的位象关系;CCK8法绘制HEL细胞生长曲线;流式细胞仪检测HEL细胞的细胞周期和凋亡坏死情况,透射电镜观察HEL细胞超微结构。2.观察人脐血源基质细胞移植重建造血微环境促进巨核细胞生成的在体研究对裸鼠实施不同辐照剂量照射,观察裸鼠辐照后不同时相点血象、骨髓象、CFU-F等指标变化,探索适宜本研究的造血微环境损伤动物模型的辐照剂量。根据上述实验结果,选定5.0Gy作为造血微环境损伤动物模型的辐照剂量;实验分组:①生理盐水组;②hBMSCs组;③hUCBDSCs组。观察不同移植组造血微环境重建和巨核细胞、血小板数量恢复情况。动态观察CM-DiI标记hUCBDSCs裸鼠体内迁移情况。3.人脐血源基质细胞促进巨核细胞增殖的机制研究3.1 TPO途径在人脐血源基质细胞促进巨核细胞增殖中的作用ELISA法动态检测hBMSCs和hUCBDSCs培养上清TPO浓度;激光共聚焦、流式细胞仪检测不同培养条件下HEL细胞C-mpl蛋白表达,RT-PCR检测HEL细胞C-mpl mRNA表达水平。3.2 SDF-1途径在人脐血源基质细胞促进巨核细胞增殖中的作用ELISA法动态检测hBMSCs和hUCBDSCs培养上清SDF-1浓度;激光共聚焦、流式细胞仪检测不同培养条件下HEL细胞CXCR4蛋白表达,RT-PCR检测HEL细胞CXCR4 mRNA表达水平。3.3间隙连接细胞间通讯在人脐血源基质细胞促进巨核细胞增殖中的作用荧光漂白恢复(FRAP)技术检测细胞间通讯功能;激光共聚焦显微镜观察共培养条件下HEL细胞和hUCBDSCs Cx43蛋白表达;RT-PCR法检测共培养条件下HEL细胞和hUCBDSCs Cx43 mRNA水平表达情况。透射电镜观察共培养条件下HEL细胞和hUCBDSCs细胞连接处超微结构。结果:1.倒置显微镜观察,HEL细胞粘附在hUCBDSCs表面,呈球形,随培养时间的延长,HEL细胞数量逐渐增多;扫描电镜观察发现HEL细胞可通过伪足粘附于hUCBDSCs表层,或龛于hUCBDSCs融合所形成的“网眼”中,甚至移行到hUCBDSCs层下,部分hUCBDSCs胞膜突起与多个HEL细胞粘附,形成“放射状”排列。HEL细胞生长曲线显示HEL/hUCBDSCs组细胞生长速度最快;细胞周期结果显示HEL/hUCBDSCs组S+G2/M期细胞比例最高(P<0.05),突显出hUCBDSCs在巨核细胞增殖中的特殊促进作用。凋亡率结果显示三组之间无显著性差异(p>0.05)。2.裸鼠接受不同剂量辐照所表现出来的血象变化程度不同,5.0Gy组裸鼠有明显骨髓抑制和CFU-F计数降低,且在照射后自行恢复,是裸鼠造血微环境损伤的理想辐照剂量。移植裸鼠实验结果显示:基质细胞有明显的促进血小板恢复的作用,其中hUCBDSCs作用最强。采用CM-DiI标记hUCSDCs移植裸鼠,激光共聚焦显微镜观察发现:hUCSDCs在肝脏、脾脏和肺脏组织间隙短暂“停留”后,迅速“归巢”至骨髓重建损伤造血微环境。3.人脐血源基质细胞促进巨核细胞增殖的机制研究3.1 ELISA检测结果显示hUCBDSCs分泌表达TPO水平明显高于hBMSCs,分泌高峰稍迟于hBMSCs,传代后hUCBDSCs培养上清中的TPO浓度仍高于hBMSCs。流式细胞仪和激光共聚焦检测均显示其C-mpl蛋白的表达明显增强;RT-PCR检测不同培养条件下HEL细胞C-mpl mRNA表达情况,结果显示无显著性差异;3.2 ELISA结果显示:体外液体培养前6天,hUCBDSCs和hBMSCs SDF-1分泌水平相当,6天后hUCBDSCs SDF-1分泌水平明显高于hBMSCs,传代后hUCBDSCs培养上清中的SDF-1浓度仍高于hBMSCs;流式细胞仪和激光共聚焦检测均显示与hUCBDSCs和hBMSCs共培养的HEL细胞CXCR4蛋白表达明显减弱,且可在部分HEL细胞胞浆中发现红色点状荧光;RT-PCR检测不同培养条件下HEL细胞CXCR4 mRNA表达情况,结果显示无显著性差异;3.3在本试验条件下,荧光漂白恢复技术检测结果提示hUCBDSCs和HEL细胞之间无明显荧光物质交换,而相互粘连的hUCBDSCs的荧光强度在淬灭后荧光迅速恢复;激光共聚焦显微镜观察共培养条件下HEL细胞Cx43表达较弱,呈均匀分布,hUCBDSCs Cx43表达较强,可见呈点状群聚分布的高亮斑块;RT-PCR检测在HEL/hUCBDSCs共培养体系中,HEL细胞Cx43 mRNA的表达明显低于hUCBDSCs。结论:1.人脐血源基质细胞具有促进巨核细胞增殖的作用:①生长曲线――细胞扩增速度加快;②细胞周期――S+G2/M期细胞比例明显增多;③扫描电镜――可见hUCBDSCs粘附、龛合、包裹HEL细胞;④透射电镜――可见大量增殖旺盛的HEL细胞和核分裂相;2.人脐血源基质细胞具有重建移植裸鼠造血微环境和促进巨核细胞和血小板数量和功能恢复的作用;3. hUCBDSCs通过分泌高水平TPO,上调巨核细胞C-mpl蛋白的表达,促进巨核细胞增殖、分化、产板;4. hUCBDSCs通过分泌高水平SDF-1,促进巨核细胞的迁移和增殖,在迁移的过程中巨核细胞表面CXCR4表达降低,形成“内吞小泡”,调控巨核细胞的迁移;5.本试验条件下,hUCBDSCs和HEL细胞间未检测到间隙连接细胞间通讯,提示GJIC功能对hUCBDSCs促进巨核细胞增殖的作用不明显。更多还原

【Abstract】 Dysfunction of hematogenesis is a main complication under the condition of chemotherapy, radiotherapy, unexpected leakage of radioactive material and nuclear warfare. Thrombocytopenia caused by the injury of megakaryocyte is to be a formidable puzzle to solve till recently. Many risks, such as bleeding caused by the anticoagulated antibody, might be caused by the application of thrombopoietin (TPO), which is used as a tool to deal with the thrombocytopenia. So, the methods to restore the quantity and function of platelets efficiently and securely are remained to explore.Hematopoietic inductive microenvironment (HIM), the internal environment for sustaining and regulating the growth and development of hematopoietic cell, acts as an important role in the proliferation, differentiation and maturation of megakaryocytes. So, it is worth finding some methods to cope with the injury of megakaryocytes start with the reconstruction of HIM.Bone marrow stromal cells(BMSCs) is the main component of hematopoietic inductive microenvironment, BMSCs was researched systematically since it was cultured in vitro by Dexter in 1977. Infusion of the suspension containing BMSCs expanded in vitro and hemapoietic stem cells is thought to be an effective method to restore the normal function of hematopoiesis previously. But, it can’t be applied extensively in clinical practices for some reasons, such as the dysfunction of HIM in autotransplantation and histocompatibility in allograft. Based on some advantages, such as easy-to-obtain, convenient to collect and low immunogenicity, cord blood is now used as a new source of haemopoietic stem cells. Our group also has studied the human umbilical cord blood-derived stromal cells (hUCBDSCs) and the cord blood associated HIM for many years. After the successful acquirement of hUCBDSCs in vitro in our preliminary experiments, bio-characteristics and hematopoietic supporting-capacity of these cells were observed. Interestingly, it was found that many stromal cell specific features was possessed by hUCBDSCs. Moreover, as compared with the alternative expansion system, which human bone marrow stromal cells (hBMSCs) were used as the trophoblastic cells, the colony forming unit-megakaryocte rate was much higher in the co-culture system with hUCBDSCs to be trophoblastic cells. It is hinted that hUCBDSCs might act as a special role for the proliferation of megakaryocytes. Thus, some therapeutic improvements on the megakaryocytic injury might be made if the further understanding of this phenomenon could be reached.Accordingly, based on the construction of megakaryocyte/hUCBDSCs co-culture model and nude mice HIM injury model, the mechanisms of hUCBDSCs to promote the proliferation of megakaryocytes and to reconstruct the HIM in vivo, especially, to restore the quantity of platelet were explored in this study, which were mainly focused on the following three aspects: TPO pathway, SDF-1 pathway and gap junction communication. Some experimental data might be obtained for the improvement of megakaryocytic injury therapy.Methods:1. To observe the influence of hUCBDSCs on the proliferation of megakaryocytic cell line HEL.Groups: HEL suspended culture group; HEL/hBMSCs co-culture group; HEL/ hUCBDSCs co-culture group. The spatial relationship between the HEL cells and stromal cells was observed under the inverted microscope and scanning electron microscope, then, CCK8 assay was used to determine the proliferation of HEL cells. After this, the cell cycle distribution and the rate of apoptosis/necrosis of these cells were assessed by flow cytometry. At last, the ultrastructure of HEL cells was revealed under the transmission electron microscope.2. To observe the reconstruction of HIM, especially the recovery of megakaryocytes caused by the implantation of hUCBDSCs into nude mice.To observe the changes of hemogram, bone marrow biopsy and CFU-F in nude mice at different phases after irradiation in order to explore the proper irradiation dose to apply in this study. At last, 5.0 Gy was used as the experimental dose in the following experiments.Groups: Isotonic Na chloride group; hBMSCs group; hUCBDSCs group. After ionizing radiation, the reconstruction of HIM and the recovery of megakaryocytes in ` different groups were determined in the nude mice with transplantation.3. Possible mechanisms for hUCBDSCs to promote the proliferation of megakaryocytes.3.1 Possible role performed by the TPO pathway.The concentration of TPO in the culture supernatant of hBMSCs and hUCBDSCs was detected by ELISA assay. The expression of C-mp1 at mRNA and protein levels in HEL cells was determined by RT-PCR, confocal microscopy and flow cytometry under the different culture conditions.3.2 Possible role performed by the SDF-1 pathway.The concentration of SDF-1 in the culture supernatant of hBMSCs and hUCBDSCs was detected by ELISA assay. The expression of CXCR4 at mRNA and protein levels in HEL cells was determined by RT-PCR, laser confocal microscopy and flow cytometry under the different culture conditions.3.3 Possible role performed by the gap conjunction communication.The gap conjunction communication between HEL cells and hUCBDSCs was assessed by fluorescence recovery after photobleaching assay (FRAP). The expression of Connexin-43 (Cx43) at mRNA and protein levels in HEL cells and hUCBDSCs under co-culture condition was determined by RT-PCR and laser confocal microscopy, respectively.Results:1. It was revealed under the inverted microscopy that many HEL cells attached on the hUCBDSCs. Under the inverted microscopy, some of them adhered to the hUCBDSCs surface by pseudopodia, the others inserted into the meshes formed by several fused hUCBDSCs. The quantity of attached-HEL cells increased significantly as the culturing time prolonged. As compared with the other groups, HEL cells in the HEL/hUCBDSCs co-culture group grew faster. But, the rate of apoptosis/necrosis in different groups was the same. It is indicated that some special roles might be performed by hUCBDSCs in the proliferation of HEL cells.2. Different degrees of myelosuppression and CFU-F decreasing could be found in the mice, which were administrated with different dose of radiation. The 5.0 Gy was selected as the proper dose in the following studies. It was revealed by the in vivo assay that the two kinds of stromal cells, especially hUCBDSCs, possessed the capacity of promoting the proliferation of platelets. After processed a transient retention at liver, spleen and lung, hUCSDCs homing quickly to bone marrow to reconstruct the impaired HIM.3. The mechanisms of hUCBDSCs to promote the proliferation of megakaryocytes3.1 It was revealed by ELISA assay that the concentration of TPO secreted by hUCBDSCs was higher than that of the hBMSCs did, even though the passage was done. But, the appearance of secretion peak in hUCBDSCs group was late. The expression of C-mpl protein was enhanced as determined by flow cytometry and laser confocal microscopy. But, there was no significant differences of the C-mpl mRNA level between different groups.3.2 The concentration of SDF-1 in different groups was the same at the early stage of culturing. But, 6 days after seeding, it increased significantly in the hUCBDSCs group, even though the passage was done. The expression of CXCR4 protein was weaken as determined by flow cytometry and laser confocal microscopy. Red punctiform fluorescence was detected in endochylema by laser confocal microscopy after HEL cell line co-cultured with hUCBDSCs. But, there was no significant differences of the CXCR4 mRNA level between different groups.3.3 Between the hUCBDSCs and hUCBDSCs, it was revealed by FRAP assay that the fluorescence intensity was recovered quickly in the photobleached cells. But, this phenomenon was not found between the hUCBDSCs and HEL cells. Moreover, under the co-culture condition, the expression level of Cx43 was much weaker in HEL cells than that in hUCBDSCs. The spatial distribution of this protein was also quite different in these two cells. Under laser confocal microscopy, fluorescence displayed as a homogeneous distribution in HEL cells but a plaquelike distribution in hUCBDSCs.Conclusion:1. The proliferation of megakaryocytes can be enhanced by the hUCBDSCs in vitro.2. The impaired HIM, especially the decreasing and aberrant platelets, can be restored after the hUCBDSCs has been implanted into the nude mice.3. High level of TPO is secreted by hUCBDSC. Meanwhile, the expression of C-mpl protein, the receptor of TPO on megakaryocyte also can be upregulated by hUCBDSC. This might be one of the reasons for the hUCBDSC to promote the proliferation, differentiation and maturation of megakaryocytes.4. High level of SDF-1 is secreted by hUCBDSCs to promote the proliferation and migration of megakaryocyte. With migration, the expression of CXCR4 protein on megakaryocyte was decreased and formed“internalized vesic”, that regulate the migration of megakaryocyte.5. There is no obvious gap junction communication can be found between the hUCBDSCs and HEL cells. It is indicated that this kind of interaction might not be the main cause of the hUCBDSCs to promote the proliferation of megakaryocytes.更多还原