高表達(dá)SDF-1人臍血源基質(zhì)細(xì)胞經(jīng)PECAM-1介導(dǎo)調(diào)控巨核細(xì)胞增殖遷移的機(jī)制研究
發(fā)布時(shí)間：2018-09-05 15:33
【摘要】：臨床上,患者進(jìn)行造血干細(xì)胞移植及大劑量放化療后,體內(nèi)較長時(shí)期血小板減少且恢復(fù)較慢。其中,巨核細(xì)胞損傷導(dǎo)致的血小板減少,除輸注血小板外,尚缺乏有效的治療手段,并且隨著血小板輸注的增多,也增加了輸血相關(guān)感染性疾病和潛在性的移植物抗宿主病(graft versus host disease,GVHD)的發(fā)生。巨核細(xì)胞發(fā)育以及血小板的生成是一個(gè)復(fù)雜的生物學(xué)過程,包括造血干細(xì)胞發(fā)育為巨核祖細(xì)胞(megakaryocytic progenitors cell, MKPC),MKPC又進(jìn)一步分化和成熟為MK,并釋出血小板。研究者發(fā)現(xiàn)利用造血干/祖細(xì)胞進(jìn)行定向誘導(dǎo)分化、體外擴(kuò)增巨核細(xì)胞,再將擴(kuò)增的巨核細(xì)胞輸注給患者,可能有助于解決骨髓移植后血小板恢復(fù)較慢的臨床問題,減少血小板的輸注。 造血基質(zhì)細(xì)胞作為造血微環(huán)境(hematopoietic inductive microenvironment,HIM)的主要成分,可以分泌多種細(xì)胞因子,促進(jìn)巨核細(xì)胞增殖分化、成熟產(chǎn)板。因此,從修復(fù)或重建骨髓造血微環(huán)境正常功能入手治療巨核細(xì)胞損傷,是一個(gè)值得探索的領(lǐng)域。以往對(duì)基質(zhì)細(xì)胞的研究多集中在人骨髓基質(zhì)細(xì)胞(human bone marrow stromal cells,hBMSCs),但是由于hBMSCs的數(shù)量及增殖分化潛能隨年齡增加而下降、采集骨髓增加供者痛苦和風(fēng)險(xiǎn),另外由于自體移植中患者自身造血微環(huán)境異常,而異體移植又存在組織相容性等諸多問題,限制了hBMSCs在臨床上的廣泛運(yùn)用。人臍血中的造血干細(xì)胞較外周血和骨髓更原始,具有來源廣泛,采集方便,免疫原性弱和長期造血重建的特點(diǎn),已成為新的造血干細(xì)胞來源。 那么,在人臍血中是否存在著造血基質(zhì)細(xì)胞?以及其具體的生物學(xué)特點(diǎn)有待探究。課題組長期從事人臍血源基質(zhì)細(xì)胞(human umbilical cord blood-derived stromal cells,hUCBDSCs)及臍血造血微環(huán)境的研究,首次發(fā)現(xiàn)并證實(shí)人臍血中存在基質(zhì)細(xì)胞的前體細(xì)胞,能通過特定的細(xì)胞因子組合使hUCBDSCs得以有效擴(kuò)增;以hUCBDSCs為滋養(yǎng)層的體外擴(kuò)增體系對(duì)臍血CD34~+細(xì)胞具有明顯的擴(kuò)增作用,可促進(jìn)巨核細(xì)胞集落生成單位(CFU-Meg)的形成;體內(nèi)試驗(yàn)中,hUCBDSCs促進(jìn)小鼠輻照后CFU-Meg形成和血小板恢復(fù)的作用明顯優(yōu)于hBMSCs。對(duì)于上述hUCBDSCs能夠促進(jìn)巨核細(xì)胞發(fā)育這一生物學(xué)現(xiàn)象,本室在國內(nèi)外雜志上已經(jīng)做了詳盡的報(bào)道,但是對(duì)于這一生物現(xiàn)象的具體機(jī)制尚不清楚。 血小板生成素(thrombopoietin,TPO)是巨核細(xì)胞發(fā)育以及血小板成熟的重要誘導(dǎo)因子,有關(guān)TPO調(diào)控巨核細(xì)胞發(fā)育和血小板生成的研究也有系列報(bào)道,但有研究發(fā)現(xiàn)TPO治療后存在產(chǎn)生抗凝血抗體、加重出血等危險(xiǎn)。另有文獻(xiàn)報(bào)道,TPO-/-小鼠體內(nèi)巨核祖細(xì)胞雖然減少,但是殘留的巨核細(xì)胞和血小板在形態(tài)和功能上并沒有受到損害,并且基質(zhì)細(xì)胞衍生因子(stromal cell derived factor,SDF-1)仍然可以促進(jìn)殘余血小板的成熟和釋放。Dhanjal和Wu兩個(gè)實(shí)驗(yàn)室于2007年先后發(fā)現(xiàn),PECAM-1-/-小鼠體內(nèi)巨核細(xì)胞無法沿SDF-1濃度梯度遷移,最終導(dǎo)致其成熟障礙。SDF-1主要由基質(zhì)細(xì)胞分泌產(chǎn)生,屬于趨化因子CXC亞家族,在造血干/祖細(xì)胞的增殖、分化、遷移和歸巢中發(fā)揮重要作用。那么在巨核細(xì)胞發(fā)育過程中是否存在著SDF-1/PECAM-1調(diào)控軸?其具體機(jī)制又是什么?基于此,我們提出“高表達(dá)SDF-1人臍血源基質(zhì)細(xì)胞在PECAM-1協(xié)同下促進(jìn)巨核細(xì)胞增殖、遷移”這一假設(shè)。本課題在構(gòu)建巨核細(xì)胞/hUCBDSCs共培養(yǎng)模型的基礎(chǔ)上,以SDF-1/PECAM-1為切入點(diǎn),觀察hUCBDSCs體外促進(jìn)巨核細(xì)胞增殖和遷移的作用;圍繞PECAM-1的上下游蛋白/信號(hào)通路,深入探討hUCBDSCs促進(jìn)巨核細(xì)胞發(fā)育的可能機(jī)制,有望為臨床上運(yùn)用hUCBDSCs治療巨核細(xì)胞損傷、促進(jìn)血小板恢復(fù)這一新的細(xì)胞治療手段提供理論依據(jù)和實(shí)驗(yàn)基礎(chǔ)。 方法: 1.人臍血源基質(zhì)細(xì)胞(hUCBCSCs)共培養(yǎng)影響巨核細(xì)胞PECAM-1的表達(dá)實(shí)驗(yàn)培養(yǎng)hUCBDSCs和HEL細(xì)胞;建立Transwell HEL細(xì)胞/hUCBDSCs共培養(yǎng)模型;ELISA檢測(cè)hUCBDSCs分泌SDF-1的情況;CCK-8檢測(cè)人臍血源基質(zhì)細(xì)胞hUCBDSCs對(duì)HEL細(xì)胞增殖的影響;細(xì)胞遷移實(shí)驗(yàn)檢測(cè)人臍血源基質(zhì)細(xì)胞hUCBDSCs對(duì)HEL細(xì)胞的遷移的影響;RT-PCR檢測(cè)HEL細(xì)胞PECAM-1在mRNA水平的表達(dá);免疫熒光組化和Western blot檢測(cè)HEL細(xì)胞PECAM-1的蛋白表達(dá)水平。 2. SDF-1/PECAM-1在巨核細(xì)胞發(fā)育中的機(jī)制探討 分兩部分:第一節(jié),SDF-1/PECAM-1慢病毒RNAi載體的構(gòu)建 siRNA的設(shè)計(jì),vshRNA載體的構(gòu)建,慢病毒包裝,慢病毒感染靶細(xì)胞,RNAi的效率檢測(cè)。 第二節(jié),SDF-1/PECAM-1在巨核細(xì)胞增殖遷移中的機(jī)制探討 SDF-1和PECAM-1分別敲低后,RT-PCR、Western blot檢測(cè)HEL細(xì)胞中PECAM-1的表達(dá)變化;SDF-1和PECAM-1分別敲低后,RT-PCR、免疫熒光組織化學(xué)檢測(cè)HEL細(xì)胞中CXCR4的表達(dá)變化;CCK-8檢測(cè)RNAi后HEL細(xì)胞的增殖變化情況;細(xì)胞遷移實(shí)驗(yàn)檢測(cè)RNAi后細(xì)胞遷移情況;Western blot檢測(cè)SHP-2蛋白(Src homology 2 domain-containing tyrosine phosphatase)的表達(dá);Western blot檢測(cè)PI3K/Akt,MAKP/ERK兩信號(hào)通路中Akt,ERK磷酸化蛋白的表達(dá)變化。 結(jié)果: 1.鏡下觀察人臍血源基質(zhì)細(xì)胞和HEL細(xì)胞。ELISA檢測(cè)到hUCBDSCs,較之hBMSCs,能表達(dá)較高量的SDF-1,特別是在第7天細(xì)胞融合時(shí)分泌量達(dá)到峰值,約3.5ng/ml;hUCBDSCs對(duì)HEL細(xì)胞的趨化作用強(qiáng)于hBMSCs(p0.05);在和hUCBDSCs共培養(yǎng)后HEL細(xì)胞增殖加快,在第4天和第7天時(shí),其增殖的OD值均高于對(duì)照組(p0.05);從RT-PCR、Western blot及免疫熒光染色實(shí)驗(yàn)結(jié)果得到,hUCBDSCs共培養(yǎng)促進(jìn)HEL細(xì)胞的PECAM-1的表達(dá)。 2.利用RNAi的理論和方法制備出靶向SDF-1和PECAM-1基因的兩個(gè)慢病毒載體,經(jīng)RT-PCR、Western blot檢測(cè)兩個(gè)載體工作正常,能有效敲低目的基因。 3. SDF-1/PECAM-1在巨核細(xì)胞增殖遷移中的機(jī)制探討 3.1 HEL細(xì)胞和hUCBDSCs共培養(yǎng)后,其PECAM-1在mRNA水平和蛋白水平均表達(dá)上調(diào),當(dāng)hUCBDSCs敲低SDF-1后,其作為滋養(yǎng)層細(xì)胞再和HEL細(xì)胞共培養(yǎng),導(dǎo)致HEL細(xì)胞PECAM-1表達(dá)下調(diào);另一面,當(dāng)HEL細(xì)胞的PECAM-1敲低后,再和正常的hUCBDSCs共培養(yǎng),其表面的PECAM-1表達(dá)仍然下降; 3.2不論是mRNA水平還是蛋白水平,HEL細(xì)胞的PECAM-1的敲低并不會(huì)影響其CXCR4的表達(dá)(p0.05); 3.3 HEL細(xì)胞和hUCBDSCs共培養(yǎng)后,hUCBDSCs對(duì)HEL細(xì)胞的增殖和遷移作用均增強(qiáng)(p0.01)。而當(dāng)hUCBDSCs的SDF-1敲低后,共培養(yǎng)后hUCBDSCs對(duì)HEL細(xì)胞的增殖和遷移均受到抑制(p0.01);另一方面,當(dāng)HEL細(xì)胞的PECAM-1敲低后,再進(jìn)行共培養(yǎng)后,HEL細(xì)胞的增殖和遷移也同樣受到了抑制(p0.01); 3.4 HUCBDSCs共培養(yǎng)后增強(qiáng)HEL細(xì)胞SHP-2的表達(dá);而SDF-1和PECAM-1的敲低抑制HEL細(xì)胞SHP-2蛋白的表達(dá); 3.5 HUCBDSCs共培養(yǎng)后增強(qiáng)HEL細(xì)胞Akt和ERK的磷酸化;而SDF-1和PECAM-1的敲低抑制HEL細(xì)胞Akt和ERK的磷酸化。 結(jié)論: 1. HUCBDSCs,較之hBMSCs,能分泌較高水平的SDF-1,促進(jìn)巨核細(xì)胞PECAM-1的表達(dá); 2.在巨核細(xì)胞/人臍血源基質(zhì)細(xì)胞共培養(yǎng)體系中,存在著SDF-1/PECAM-1聯(lián)合信號(hào)調(diào)控,從而促進(jìn)巨核細(xì)胞的增殖和遷移; 3. SDF-1/PECAM-1聯(lián)合通過激活pI3K/Akt,MAKP/ERK信號(hào)通路,促進(jìn)巨核細(xì)胞的增殖和遷移。
[Abstract]:In clinic, thrombocytopenia and recovery of platelets in patients after hematopoietic stem cell transplantation and high-dose radiotherapy and chemotherapy are slow. Among them, thrombocytopenia caused by megakaryocyte injury is lack of effective treatment besides platelet transfusion, and with the increase of platelet transfusion, it also increases the incidence of transfusion-related infectious diseases and The development of megakaryocytes and platelet formation is a complex biological process, including the development of hematopoietic stem cells into megakaryocytic progenitors cells (MKPC), the further differentiation and maturation of MKPC into MK and the release of hemorrhagic platelets. It was found that directional induction of differentiation by hematopoietic stem/progenitor cells, expansion of megakaryocytes in vitro, and infusion of expanded megakaryocytes into patients may help to solve the clinical problem of slow platelet recovery after bone marrow transplantation and reduce platelet transfusion.
Hematopoietic stromal cells (HSCs), as the main component of hematopoietic microenvironment (HIM), can secrete a variety of cytokines, promote the proliferation and differentiation of megakaryocytes and mature platelets. Previous studies on stromal cells have focused on human bone marrow stromal cells (hBMSCs). However, the number and proliferation and differentiation potential of hBMSCs decrease with age. Bone marrow collection increases donor pain and risk. In addition, the patient's own hematopoietic microenvironment is abnormal in autologous transplantation, and allogeneic transplantation exists. Human umbilical cord blood hematopoietic stem cells (HBMSCs) are more primitive than peripheral blood and bone marrow, and have the characteristics of extensive sources, convenient collection, weak immunogenicity and long-term hematopoietic reconstruction. HBMSCs have become a new source of hematopoietic stem cells.
So, whether there are hematopoietic stromal cells in human umbilical cord blood and its specific biological characteristics need to be explored. Somatic cells can effectively amplify hUCBDSCs by specific cytokine combinations; the in vitro amplification system with hUCBDSCs as trophoblast has obvious effect on the proliferation of cord blood CD34~+ cells and promotes the formation of megakaryocyte colony-forming unit (CFU-Meg); in vivo experiments, hUCBDSCs can promote the formation of CFU-Meg and platelets in irradiated mice. Recovery is obviously superior to hBMSCs. For the biological phenomenon that hUCBDSCs can promote megakaryocyte development, our laboratory has done a detailed report in domestic and foreign journals, but the specific mechanism of this biological phenomenon is still unclear.
Thrombopoietin (TPO) is an important inducer of megakaryocyte development and platelet maturation. There are a series of reports on the regulation of TPO on megakaryocyte development and platelet formation, but some studies have found that there is a risk of anti-coagulation antibody and aggravation of bleeding after TPO treatment. Although megakaryocyte progenitor cells were reduced, the morphology and function of residual megakaryocytes and platelets were not impaired, and stromal cell derived factor (SDF-1) could still promote the maturation and release of residual platelets. SDF-1 is produced mainly by stromal cells and belongs to the CXC subfamily. It plays an important role in the proliferation, differentiation, migration and homing of hematopoietic stem/progenitor cells. Based on this, we proposed the hypothesis that human umbilical cord blood stromal cells with high expression of SDF-1 can promote the proliferation and migration of megakaryocytes in synergy with PECAM-1. The possible mechanism of hUCBDSCs promoting megakaryocyte development around the upstream and downstream protein/signaling pathway of PECAM-1 will provide theoretical and experimental basis for the clinical application of hUCBDSCs in the treatment of megakaryocyte injury and platelet recovery.
Method:
1. Human umbilical cord blood stromal cells (hUCBCSCs) were co-cultured to influence the expression of megakaryocyte PECAM-1. hUCBDSCs and HEL cells were cultured in vitro. Transwell HEL cells / hUCBDSCs co-cultured model was established; SDF-1 secreted by hUCBDSCs was detected by ELISA; proliferation of HEL cells was detected by human umbilical cord blood stromal cells hUCBDSCs was detected by CCK-8; The migration of HEL cells was influenced by hUCBDSCs, the expression of PECAM-1 in HEL cells was detected by RT-PCR, and the expression of PECAM-1 in HEL cells was detected by immunofluorescence histochemistry and Western blot.
The mechanism of 2. SDF-1/PECAM-1 in megakaryocyte development
It is divided into two parts: Section 1, construction of SDF-1/PECAM-1 lentiviral RNAi vector.
SiRNA design, vshRNA vector construction, lentivirus packaging, lentivirus infection target cells, RNAi efficiency detection.
The second section, the mechanism of SDF-1/PECAM-1 in the proliferation and migration of megakaryocytes.
After SDF-1 and PECAM-1 were knocked down respectively, the expression of PECAM-1 in HEL cells was detected by RT-PCR and Western blot; after SDF-1 and PECAM-1 were knocked down, the expression of CXCR4 in HEL cells was detected by RT-PCR and immunofluorescence histochemistry; the proliferation of HEL cells was detected by CCK-8 after RNAi; and the migration of HEL cells after RNAi was detected by cell migration test. The expression of SHP-2 (Src homology 2 domain-containing tyrosine phosphatase) was detected by blot, and the expression of Akt and ERK phosphorylated proteins in PI3K/Akt and MAKP/ERK signaling pathways were detected by Western blot.
Result:
1. Microscopic observation of human umbilical cord blood stromal cells and HEL cells. ELISA detection of hUCBDSCs, compared with hBMSCs, can express a higher amount of SDF-1, especially on the 7th day of cell fusion secretion reached a peak, about 3.5 ng/ml; hUCBDSCs chemotaxis on HEL cells stronger than hBMSCs (p0.05); after co-culture with hUCBDSCs, HEL cells proliferation accelerated, and on the 4th day and On the 7th day, the proliferative OD value was higher than that of the control group (p0.05). The results of RT-PCR, Western blot and immunofluorescence staining showed that the co-culture of hUCBDSCs promoted the expression of PECAM-1 in HEL cells.
2. Two lentiviral vectors targeting SDF-1 and PECAM-1 genes were prepared by using the theory and method of RNAi. RT-PCR and Western blot showed that the two vectors worked well and knocked down the target gene effectively.
The mechanism of 3. SDF-1/PECAM-1 in the proliferation and migration of megakaryocytes
3.1 After co-culture of HEL cells and hUCBDSCs, the expression of PECAM-1 was up-regulated at both mRNA and protein levels. When hUCBDSCs knocked down SDF-1, it was co-cultured with HEL cells as trophoblast cells, resulting in the down-regulation of PECAM-1 expression in HEL cells. On the other hand, when HEL cells were knocked down by PECAM-1, PECAM-1 was co-cultured with normal hUCBDSCs and the PECAM-1 surface of HEL cells was observed. Da still fell.
3.2 The expression of CXCR4 was not affected by the knockdown of PECAM-1 in HEL cells at both mRNA and protein levels (p0.05).
3.3 After co-culture of HEL cells and hUCBDSCs, the proliferation and migration of HEL cells were enhanced by hUCBDSCs (p0.01). When the SDF-1 of hUCBDSCs was knocked down, the proliferation and migration of HEL cells were inhibited by hUCBDSCs after co-culture (p0.01); on the other hand, when the PECAM-1 of HEL cells was knocked down, the proliferation and migration of HEL cells were inhibited by co-culture. It was also inhibited (P0.01).
3.4 HUCBDSCs co-cultured HEL cells enhanced the expression of SHP-2, while SDF-1 and PECAM-1 knockdown inhibited the expression of SHP-2.
3.5 HUCBDSCs co-cultured HEL cells enhanced the phosphorylation of Akt and ERK, while SDF-1 and PECAM-1 knockdown inhibited the phosphorylation of Akt and ERK.
Conclusion:
1. HUCBDSCs, compared with hBMSCs, can secrete a higher level of SDF-1 and promote the expression of PECAM-1 in megakaryocytes.
2. In the megakaryocyte/human umbilical cord blood-derived stromal cells co-culture system, SDF-1/PECAM-1 combined with signal regulation can promote the proliferation and migration of megakaryocytes.
3. SDF-1/PECAM-1 promotes the proliferation and migration of megakaryocytes by activating pI3K/Akt, MAKP/ERK signaling pathway.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363
本文編號(hào)：2224700
[Abstract]:In clinic, thrombocytopenia and recovery of platelets in patients after hematopoietic stem cell transplantation and high-dose radiotherapy and chemotherapy are slow. Among them, thrombocytopenia caused by megakaryocyte injury is lack of effective treatment besides platelet transfusion, and with the increase of platelet transfusion, it also increases the incidence of transfusion-related infectious diseases and The development of megakaryocytes and platelet formation is a complex biological process, including the development of hematopoietic stem cells into megakaryocytic progenitors cells (MKPC), the further differentiation and maturation of MKPC into MK and the release of hemorrhagic platelets. It was found that directional induction of differentiation by hematopoietic stem/progenitor cells, expansion of megakaryocytes in vitro, and infusion of expanded megakaryocytes into patients may help to solve the clinical problem of slow platelet recovery after bone marrow transplantation and reduce platelet transfusion.
Hematopoietic stromal cells (HSCs), as the main component of hematopoietic microenvironment (HIM), can secrete a variety of cytokines, promote the proliferation and differentiation of megakaryocytes and mature platelets. Previous studies on stromal cells have focused on human bone marrow stromal cells (hBMSCs). However, the number and proliferation and differentiation potential of hBMSCs decrease with age. Bone marrow collection increases donor pain and risk. In addition, the patient's own hematopoietic microenvironment is abnormal in autologous transplantation, and allogeneic transplantation exists. Human umbilical cord blood hematopoietic stem cells (HBMSCs) are more primitive than peripheral blood and bone marrow, and have the characteristics of extensive sources, convenient collection, weak immunogenicity and long-term hematopoietic reconstruction. HBMSCs have become a new source of hematopoietic stem cells.
So, whether there are hematopoietic stromal cells in human umbilical cord blood and its specific biological characteristics need to be explored. Somatic cells can effectively amplify hUCBDSCs by specific cytokine combinations; the in vitro amplification system with hUCBDSCs as trophoblast has obvious effect on the proliferation of cord blood CD34~+ cells and promotes the formation of megakaryocyte colony-forming unit (CFU-Meg); in vivo experiments, hUCBDSCs can promote the formation of CFU-Meg and platelets in irradiated mice. Recovery is obviously superior to hBMSCs. For the biological phenomenon that hUCBDSCs can promote megakaryocyte development, our laboratory has done a detailed report in domestic and foreign journals, but the specific mechanism of this biological phenomenon is still unclear.
Thrombopoietin (TPO) is an important inducer of megakaryocyte development and platelet maturation. There are a series of reports on the regulation of TPO on megakaryocyte development and platelet formation, but some studies have found that there is a risk of anti-coagulation antibody and aggravation of bleeding after TPO treatment. Although megakaryocyte progenitor cells were reduced, the morphology and function of residual megakaryocytes and platelets were not impaired, and stromal cell derived factor (SDF-1) could still promote the maturation and release of residual platelets. SDF-1 is produced mainly by stromal cells and belongs to the CXC subfamily. It plays an important role in the proliferation, differentiation, migration and homing of hematopoietic stem/progenitor cells. Based on this, we proposed the hypothesis that human umbilical cord blood stromal cells with high expression of SDF-1 can promote the proliferation and migration of megakaryocytes in synergy with PECAM-1. The possible mechanism of hUCBDSCs promoting megakaryocyte development around the upstream and downstream protein/signaling pathway of PECAM-1 will provide theoretical and experimental basis for the clinical application of hUCBDSCs in the treatment of megakaryocyte injury and platelet recovery.
Method:
1. Human umbilical cord blood stromal cells (hUCBCSCs) were co-cultured to influence the expression of megakaryocyte PECAM-1. hUCBDSCs and HEL cells were cultured in vitro. Transwell HEL cells / hUCBDSCs co-cultured model was established; SDF-1 secreted by hUCBDSCs was detected by ELISA; proliferation of HEL cells was detected by human umbilical cord blood stromal cells hUCBDSCs was detected by CCK-8; The migration of HEL cells was influenced by hUCBDSCs, the expression of PECAM-1 in HEL cells was detected by RT-PCR, and the expression of PECAM-1 in HEL cells was detected by immunofluorescence histochemistry and Western blot.
The mechanism of 2. SDF-1/PECAM-1 in megakaryocyte development
It is divided into two parts: Section 1, construction of SDF-1/PECAM-1 lentiviral RNAi vector.
SiRNA design, vshRNA vector construction, lentivirus packaging, lentivirus infection target cells, RNAi efficiency detection.
The second section, the mechanism of SDF-1/PECAM-1 in the proliferation and migration of megakaryocytes.
After SDF-1 and PECAM-1 were knocked down respectively, the expression of PECAM-1 in HEL cells was detected by RT-PCR and Western blot; after SDF-1 and PECAM-1 were knocked down, the expression of CXCR4 in HEL cells was detected by RT-PCR and immunofluorescence histochemistry; the proliferation of HEL cells was detected by CCK-8 after RNAi; and the migration of HEL cells after RNAi was detected by cell migration test. The expression of SHP-2 (Src homology 2 domain-containing tyrosine phosphatase) was detected by blot, and the expression of Akt and ERK phosphorylated proteins in PI3K/Akt and MAKP/ERK signaling pathways were detected by Western blot.
Result:
1. Microscopic observation of human umbilical cord blood stromal cells and HEL cells. ELISA detection of hUCBDSCs, compared with hBMSCs, can express a higher amount of SDF-1, especially on the 7th day of cell fusion secretion reached a peak, about 3.5 ng/ml; hUCBDSCs chemotaxis on HEL cells stronger than hBMSCs (p0.05); after co-culture with hUCBDSCs, HEL cells proliferation accelerated, and on the 4th day and On the 7th day, the proliferative OD value was higher than that of the control group (p0.05). The results of RT-PCR, Western blot and immunofluorescence staining showed that the co-culture of hUCBDSCs promoted the expression of PECAM-1 in HEL cells.
2. Two lentiviral vectors targeting SDF-1 and PECAM-1 genes were prepared by using the theory and method of RNAi. RT-PCR and Western blot showed that the two vectors worked well and knocked down the target gene effectively.
The mechanism of 3. SDF-1/PECAM-1 in the proliferation and migration of megakaryocytes
3.1 After co-culture of HEL cells and hUCBDSCs, the expression of PECAM-1 was up-regulated at both mRNA and protein levels. When hUCBDSCs knocked down SDF-1, it was co-cultured with HEL cells as trophoblast cells, resulting in the down-regulation of PECAM-1 expression in HEL cells. On the other hand, when HEL cells were knocked down by PECAM-1, PECAM-1 was co-cultured with normal hUCBDSCs and the PECAM-1 surface of HEL cells was observed. Da still fell.
3.2 The expression of CXCR4 was not affected by the knockdown of PECAM-1 in HEL cells at both mRNA and protein levels (p0.05).
3.3 After co-culture of HEL cells and hUCBDSCs, the proliferation and migration of HEL cells were enhanced by hUCBDSCs (p0.01). When the SDF-1 of hUCBDSCs was knocked down, the proliferation and migration of HEL cells were inhibited by hUCBDSCs after co-culture (p0.01); on the other hand, when the PECAM-1 of HEL cells was knocked down, the proliferation and migration of HEL cells were inhibited by co-culture. It was also inhibited (P0.01).
3.4 HUCBDSCs co-cultured HEL cells enhanced the expression of SHP-2, while SDF-1 and PECAM-1 knockdown inhibited the expression of SHP-2.
3.5 HUCBDSCs co-cultured HEL cells enhanced the phosphorylation of Akt and ERK, while SDF-1 and PECAM-1 knockdown inhibited the phosphorylation of Akt and ERK.
Conclusion:
1. HUCBDSCs, compared with hBMSCs, can secrete a higher level of SDF-1 and promote the expression of PECAM-1 in megakaryocytes.
2. In the megakaryocyte/human umbilical cord blood-derived stromal cells co-culture system, SDF-1/PECAM-1 combined with signal regulation can promote the proliferation and migration of megakaryocytes.
3. SDF-1/PECAM-1 promotes the proliferation and migration of megakaryocytes by activating pI3K/Akt, MAKP/ERK signaling pathway.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363
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