CD147分子在巨核细胞系HEL细胞分化发育中的作用研究

发布时间：2018-04-26 01:31

本文选题：CD147 + 巨核细胞　；参考：《中国人民解放军军事医学科学院》2008年硕士论文

【摘要】： 金属基质蛋白酶诱导剂(matrix metalloproteinase inducer)CD147作为一种粘附分子,参与肿瘤、造血等多种病理生理过程。CD147广泛表达于造血及非造血细胞,包括上皮细胞,内皮细胞,粒细胞,以及在白细胞中也有弱表达。最近研究表明CD147参与血细胞的发育过程并与基质金属蛋白酶MMP密切相关。基质金属蛋白酶(extracellular matrix metalloproteinase,MMP)是一族锌离子依赖性酶。大量研究表明CD147在多种组织中高表达,是通过诱导分泌MMPs而发挥作用。MMPs通过重塑骨髓造血微环境ECM参与造血生理和病理过程,如造血、造血干细胞动员和移植、血液肿瘤等。 Coste等报道:在红细胞发育过程的不同阶段,表达在其细胞膜表而的粘附分子的种类和数量是有差异的,有些粘附分子随着红细胞的脱核而消失。而CD147在红细胞发育的所有阶段,包括成熟红细胞均表达,用CD147特异性抗体处理可引起红细胞在脾脏的破坏和EPO介导的红细胞生成。说明CD147在红细胞的发育中发挥重要作用。大量研究表明血小板的分化途径和红细胞是密切相关的:如红系和巨核细胞系细胞表达共同的转录因子;促红细胞生成素(Erythropoietin,EPO)是红细胞分化的主要调节因子,也与巨核细胞生成有关;而巨核细胞特异性生长因子血小板生成素Thrombopoietin (TPO)可增强红系祖细胞的增殖;EPO和TPO与他们各自的细胞表面受体结合后,激活相同的信号转导途径;长期大剂量使用EPO可引起小鼠血小板减少。而急性血小板的减少则引起血小板生成增加和红细胞生成降低;大量的人白血病细胞系均同时表达红系和巨核细胞系特异性蛋白标志,改变培养条件可使白血病细胞向红系或巨核细胞系分化等。因此我们推测CD147在血小板的发育中可能也发挥重要作用。我们发现成熟血小板表面表达CD147分子并且与血小板的活化状态和功能有关。活化血小板CD147的表达较静息血小板增高、MMP分泌活性增强。加入CD147抗体后,血小板达到最大聚集率时间延长,即CD147抗体抑制了血小板的聚集功能。血小板是从骨髓成熟的巨核细胞浆脱落下来的具有生物活性的小块胞质。由于血小板没有细胞核,并不是一个完整的细胞单位,胞浆中有少量RNA,它所表达的粘附分子和产生的细胞因子大部分在巨核细胞阶段合成。巨核细胞作为血小板的前体细胞,CD147分子在其分化中的作用尚无报道,因此我们首先选取巨核细胞系HEL细胞作为模型,检测了HEL细胞表面CD147的表达和MMP的分泌活性,发现HEL细胞CD147和MMP表达水平均很低。为了研究巨核细胞在分化为血小板的过程中CD147及MMP的变化,以及CD147和MMP在分化中的作用,我们克隆了CD147基因,构建了含CD147基因的慢病毒载体并包装病毒;感染HEL细胞,得到了高表达CD147的巨核细胞系HEL细胞,然后用分化诱导剂TPA进行诱导分化。使用光学显微镜观察分化过程中细胞形态,应用RT-PCR在转录水平、流式细胞术在蛋白水平检测CD147和巨核细胞分化标志CD41的表达情况、应用明胶酶谱分析MMPs的表达与活性,并考察了MMP抑制剂对分化的影响。结果表明,巨核细胞系HEL细胞经TPA诱导后,细胞形态和表面标志CD41的表达均发生了变化。野生型HEL细胞经TPA诱导分化后,流式结果显示随着HEL细胞分化标志CD41的表达增加,CD147的表达也增加,同时明胶酶谱实验结果显示MMP分泌和活性增强,而MMP抑制剂抑制了CD41的表达。慢病毒介导的CD147表达增加促进了TPA诱导的HEL细胞的分化,CD41表达增加,MMP抑制剂同样抑制HEL分化。这说明,CD147分子在巨核细胞分化过程中表达逐渐增加,其作用可能与MMP活性相关。结论:1.发现CD147在血小板表达并与血小板状态聚集功能相关。活化血小板CD147表达较静息血小板表达高,而血小板的前体巨核细胞系HEL细胞CD147的表达却低于成熟血小板,MMP的分泌也表现了相似的规律。2.通过慢病毒载体感染获得了高表达CD147的巨核细胞系HEL细胞。采用Western blot方法检测CD147蛋白,表明获取高表达CD147的巨核细胞系HEL细胞,成功建立了实验细胞模型。3.我们建立了诱导巨核细胞系HEL细胞分化的方法,观察细胞形态和巨核细胞分化的表面标志,结果表明TPA确实诱导了巨核细胞系HEL细胞的分化。通过检测分化过程中CD147的表达和MMP的分泌与活性,发现CD147在巨核细胞分化中可能发挥了作用。加入MMP抑制剂后,分化被抑制,提示CD147的作用可能与MMP密切相关。其具体机制仍有待进一步研究。下一步研究计划:研究人脐血造血干细胞分化发育为血小板过程中CD147、MMP的表达情况和作用,进一步探讨CD147在血小板发育中的作用及二者的关系。
[Abstract]:Metal matrix protease inducer (matrix metalloproteinase inducer) CD147, as a adhesion molecule, participates in a variety of pathophysiological processes, such as tumor and hematopoiesis,.CD147 is widely expressed in hematopoiesis and non hematopoietic cells, including epithelial cells, endothelial cells, granulocytes, and weak expressions in white blood cells. Recent studies have shown that CD147 is involved in blood. The process of cell development is closely related to matrix metalloproteinase MMP. Matrix metalloproteinase (extracellular matrix metalloproteinase, MMP) is a family of zinc dependent enzymes. A large number of studies have shown that CD147 is highly expressed in many tissues. It plays a role in the secretion of MMPs by inducing.MMPs to participate in the remodeling of bone marrow hematopoietic microenvironment ECM participation. Hematopoietic physiological and pathological processes, such as hematopoiesis, hematopoietic stem cell mobilization and transplantation, hematological malignancies, and so on.
Coste and other reports: there are different types and numbers of adhesion molecules expressed in the cell membrane surface at different stages of the erythrocyte development, and some of the adhesion molecules disappear with the denucleation of red cells. And CD147 is expressed in all stages of the development of red blood cells, including the mature red cells, and the CD147 specific antibody can cause red blood to cause red blood. The destruction of the spleen and the formation of red blood cells mediated by EPO indicate that CD147 plays an important role in the development of red blood cells. A large number of studies have shown that the differentiation pathways of platelets are closely related to red cells: the red and megakaryocyte cells express the common transcription factors; the erythropoietin (Erythropoietin, EPO) is the red cell division. The major regulatory factor is also associated with megakaryocyte formation, and megakaryocyte specific growth factor thrombopoietin Thrombopoietin (TPO) can enhance the proliferation of erythroid progenitor cells; EPO and TPO activate the same signal transduction pathway after the combination of their respective cell surface receptors; long term large dose use of EPO can cause a small amount of blood in mice. The reduction of platelets and acute thrombocytopenia cause increased platelet formation and erythropoiesis, and a large number of human leukemia cell lines simultaneously express the specific protein markers of the red and megakaryocyte lines, and the change of culture conditions can cause leukemia cells to differentiate into the red or megakaryocyte lines. Therefore, we speculate that CD147 is in the platelets. It may also play an important role in development.
We found that the expression of CD147 molecules on the surface of mature platelets is related to the activation state and function of platelets. The expression of activated platelet CD147 is higher than that of resting platelets, and the activity of MMP secretion is enhanced. After adding CD147 antibody, the maximum aggregation rate of platelet is prolonged, that is, CD147 antibody inhibits platelet aggregation function. Platelets are A small cell with biological activity from the megakaryocyte pulp that is mature in the bone marrow. Because the platelets have no nuclei, they are not a complete cell unit and a small amount of RNA in the cytoplasm. The adhesion molecules and the resulting cytokines are mostly synthesized in the megakaryocyte stage. Megakaryocyte is the precursor of the platelets. The role of CD147 molecules in its differentiation has not yet been reported. Therefore, we first selected megakaryocyte line HEL cells as a model to detect the expression of CD147 on the surface of HEL cells and the secretory activity of MMP, and found that the expression level of CD147 and MMP in HEL cells was very low. In order to study the changes of CD147 and MMP in the process of megakaryocyte differentiation into platelets, As well as the role of CD147 and MMP in the differentiation, we cloned the CD147 gene, constructed the lentivirus carrier containing the CD147 gene and packaged the virus, infected the HEL cells, and obtained the megakaryocyte HEL cells with high expression of CD147, and then used the differentiation inducer TPA to induce differentiation. The morphology of the cells during the differentiation process was observed with the light microscope, and RT was used to use RT. -PCR at the transcriptional level, flow cytometry at the protein level to detect the expression of CD147 and megakaryocyte differentiation marker CD41, the expression and activity of MMPs were analyzed by gelatinase spectrum, and the effect of MMP inhibitor on the differentiation was investigated. The results showed that the expression of cell morphology and surface marker CD41 in megakaryocyte line HEL cells was induced by TPA. There was a change. After TPA induced differentiation of wild type HEL cells, the flow pattern showed that the expression of CD147 increased with the increase of the expression of HEL cell differentiation marker CD41. Meanwhile, the results of gelase spectrum experiment showed that the secretion and activity of MMP were enhanced, while the MMP inhibitor inhibited the expression of CD41. The increase of CD147 expression promoted the HE of TPA induced HE. The differentiation of L cells, the increase of CD41 expression, and the inhibition of HEL differentiation by MMP inhibitors also suggest that the expression of CD147 molecules is gradually increased during the differentiation of megakaryocyte, and its effect may be related to the activity of MMP.
Conclusion: 1. the expression of CD147 in platelets was found to be related to platelet aggregation. The expression of activated platelets CD147 expression is higher than that of resting platelets, while the expression of CD147 in the HEL cells of the megakaryocyte line of the platelets is lower than that of the mature platelets, and the secretion of MMP is also similar to that of.2. through the infection of the lentivirus carrier. The megakaryocyte HEL cells of CD147 were expressed. The CD147 protein was detected by Western blot method. It showed that the megakaryocyte HEL cells with high expression of CD147 were obtained. The experimental cell model.3. was successfully established. We established the method of inducing the differentiation of the megakaryocyte HEL cell line, and observed the surface markers of the cell form and the megakaryocyte differentiation. The results showed that the cell differentiation of the megakaryocyte lines was marked. TPA did induce the differentiation of the megakaryocyte line HEL cells. By detecting the expression of CD147 and the secretion and activity of MMP during the differentiation, it was found that CD147 may play a role in the differentiation of megakaryocytes. The differentiation was inhibited after adding MMP inhibitors, suggesting that the role of CD147 may be related to the dense cutting of MMP. The specific mechanism of which is still to be further studied.
The next step of the study is to study the expression and role of CD147, MMP in the differentiation and development of human umbilical cord blood hematopoietic stem cells in the process of platelets, and further explore the role of CD147 in the development of platelets and the relationship between the two.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

【共引文献】