【摘要】：造血微環(huán)境(hematopoietic inductive microenvironment,HIM)是支持和調(diào)節(jié)造血干/祖細胞(haemopoietic stem/progenitor cell,HSPC)生長發(fā)育的內(nèi)環(huán)境,其結(jié)構(gòu)和功能的完整統(tǒng)一是維系造血功能正常進行的重要環(huán)節(jié)。作為造血微環(huán)境的重要組成部分,基質(zhì)細胞可能通過形成造血干細胞(hematopoietic stem cell,HSC)生長的“龕”,分泌造血生長因子(hematopoietic growth factor,HGF)和細胞外基質(zhì)(extracellular matrix,ECM)等參與造血細胞的自我更新、增殖分化和歸巢定位,在免疫調(diào)節(jié)方面也具有重要的作用。深入探討基質(zhì)細胞對骨髓造血功能的影響,從修復(fù)或重建骨髓微環(huán)境正常功能入手治療造血功能損傷具有重要的理論價值和實際意義。 基質(zhì)細胞由間充質(zhì)干細胞(mesenchymal stem cell,MSC)分化而來,是一個包括成纖維細胞、內(nèi)皮細胞、成骨細胞、脂肪細胞、巨噬細胞和網(wǎng)狀細胞等成分復(fù)雜的異質(zhì)細胞群。自1977年Dexter在體外培養(yǎng)出人骨髓基質(zhì)細胞(human bone marrow stromal cells,hBMSCs)獲得成功后,人們對hBMSCs進行了深入的研究。經(jīng)實驗研究和臨床實踐證實,hBMSCs體外培養(yǎng)擴增聯(lián)合HSC回輸是重建造血功能的有效方法。但hBMSCs來源受限,采集骨髓增加供者痛苦和風(fēng)險,且細胞數(shù)量及增殖分化潛能隨供者年齡增加而下降。自體移植中患者自身基質(zhì)細胞存在異常,而異體移植亦有可能帶來移植物抗宿主病(graft-versus-host disease,GVHD)等免疫相關(guān)問題,均限制了hBMSCs在臨床上的廣泛運用。 人臍血來源豐富,取材方便,具有未成熟的干/祖細胞比例高且免疫原性較低等特點,在多種造血系統(tǒng)惡性疾病臨床治療中具有潛在的應(yīng)用價值。本課題組長期從事人臍血源基質(zhì)細胞(human umbilical cord blood-derived stromal cells,hUCBDSCs)及臍血造血微環(huán)境的研究,前期研究通過特定的細胞因子使hUCBDSCs得以有效擴增,擴增后的hUCBDSCs在細胞成分和免疫表型上與hBMSCs相似,能夠分泌表達多種細胞因子,具備造血基質(zhì)細胞的基本特征。以hUCBDSCs為滋養(yǎng)層的培養(yǎng)體系能有效支持臍血CD34+細胞體外擴增,特別對于促巨核細胞集落形成單位(colony forming unit-megakaryocte,CFU-Mk)形成的作用明顯優(yōu)于hBMSCs,提示hUCBDSCs在促進巨核系增殖分化成熟方面可能具有重要的意義。深入探討hUCBDSCs移植在體內(nèi)支持和調(diào)控造血、重建造血微環(huán)境的作用可為造血功能損傷修復(fù)治療提供新的思路。基于上述分析,本課題首先建立兩種不同來源基質(zhì)細胞滋養(yǎng)層(hUCBDSCs和hBMSCs)培養(yǎng)體系,采用CCK-8法和Transwell法分別觀察兩種基質(zhì)細胞對臍血單個核細胞(human umbilical cord mononuclear cells,hUCB-MNCs)增殖、粘附和遷移的影響,并采用RT-PCR法檢測hUCBDSCs對歸巢相關(guān)分子mRNA的表達情況。在此基礎(chǔ)上建立BABL/c小鼠造血微環(huán)境輻照損傷模型,采用hUCB-MNCs單移植,或分別聯(lián)合兩種不同來源基質(zhì)細胞共移植的方法,比較觀察兩種基質(zhì)細胞促進造血細胞體內(nèi)歸巢與植入、重建造血微環(huán)境及支持造血重建的作用,為安全有效的臨床移植治療提供新的輔助措施和手段。 方法: 1.體外構(gòu)建hUCBDSCs和hBMSCs兩種不同來源基質(zhì)細胞滋養(yǎng)層培養(yǎng)體系。采用CCK-8法和Transwell法分別檢測兩種基質(zhì)細胞對hUCB-MNCs體外增殖、粘附和遷移的影響;采用RT-PCR法檢測hUCBDSCs對歸巢相關(guān)因子(SDF-1、CXCR-4、ICAM-1、VCAM-1、HCAM、PECAM-1、Fn)mRNA的表達情況。 2.以近交系BABL/c小鼠作為受體鼠,經(jīng)60COγ射線致死劑量8.5 Gy全身照射預(yù)處理后分別接受不同劑量hUCB-MNCs(2、4、6或8×10~6/只)單移植,或聯(lián)合hUCBDSCs(2×10~6/只)共移植,移植后第6周流式細胞儀檢測小鼠骨髓人源CD45+細胞植入率。 3.采用CM-DiI熒光染料預(yù)染hUCB-MNCs,輻照后BABL/c小鼠分別接受hUCB-MNCs(2×10~6/只)單移植,或聯(lián)合兩種不同來源基質(zhì)細胞(2×10~6/只)共移植。激光共聚焦顯微鏡追蹤觀察移植后熒光標(biāo)記hUCB-MNCs在小鼠體內(nèi)的遷移分布情況,比較各組造血細胞歸巢率。 4.輻照后小鼠分別接受hUCB-MNCs(2×10~6/只)單移植,或聯(lián)合兩種不同來源基質(zhì)細胞(2×10~6/只)共移植。觀察移植后各組小鼠存活情況,記錄生存率;動態(tài)檢測外周血血象恢復(fù)情況,骨髓組織病理切片觀察骨髓病理變化;不同時相點計數(shù)各組小鼠骨髓成纖維細胞集落形成單位(CFU-F)、脾集落形成單位(CFU-S)、粒/巨噬細胞集落形成單位(CFU-GM)和巨核細胞集落形成單位(CFU-Mk)產(chǎn)率。 結(jié)果: 1. hUCBDSCs對臍血單個核細胞體外增殖、粘附和遷移能力的影響 同hBMSCs共培養(yǎng)組和hUCB-MNCs單獨培養(yǎng)組相比較,hUCB-MNCs在hUCBDSCs共培養(yǎng)條件下增殖能力顯著增強。兩種基質(zhì)細胞均能促進hUCB-MNCs的粘附,且共培養(yǎng)后hUCB-MNCs遷移能力也顯著(P0.05)強于無基質(zhì)細胞支持對照。hUCBDSCs明顯表達與造血細胞歸巢植入密切相關(guān)的粘附分子、細胞因子及受體(SDF-1、CXCR-4、ICAM-1、VCAM-1、HCAM、PECAM-1、Fn)mRNA,揭示其對造血細胞在體內(nèi)的歸巢和植入具有重要作用。 2. hUCBDSCs聯(lián)合移植促進造血細胞歸巢和植入 輻照后小鼠分別接受不同劑量hUCB-MNCs(2、4、6或8×10~6/只)單移植,或聯(lián)合hUCBDSCs(2×10~6/只)共移植。采用不同劑量的hUCB-MNCs單移植后的植入率隨hUCB-MNCs輸注量增加而增長。hUCBDSCs聯(lián)合移植能不同程度提高小鼠骨髓中人CD45~+細胞植入比例,尤其當(dāng)輸注低劑量hUCB-MNC(s2×10~6/只)時,hUCBDSCs共移植較單移植的植入率提高最為顯著。激光共聚焦顯微鏡下觀察CM-DiI標(biāo)記的hUCB-MNCs在移植后2～3天主要歸巢至骨髓和脾臟。移植后48小時,hUCBDSCs共移植組歸巢率顯著(P0.05)高于hBMSCs共移植組和單移植組,顯示移植后早期hUCBDSCs能促進造血細胞向骨髓遷移歸巢。 3.hUCBDSCs聯(lián)合移植修復(fù)受損微環(huán)境,支持造血重建 兩種基質(zhì)細胞共移植組在移植后均能促進小鼠存活,血象恢復(fù)較快,在移植后28天內(nèi)恢復(fù)至照射前水平。其中,hUCBDSCs共移植組血小板受抑程度較輕,回升也較快,并于移植后21天恢復(fù)至照射前水平;h BMSCs共移植組白細胞于移植后10天迅速回升,在此后恢復(fù)趨勢高于hUCBDSCs共移植組;共移植兩組間血紅蛋白變化趨勢無顯著性差異。hUCBDSCs聯(lián)合移植移植后促進骨髓組織恢復(fù),重建受損微環(huán)境,提高CFUs(CFU-F,CFU-S,CFU-GM,CFU-Mk)產(chǎn)率,特別是CHU-Mk數(shù)量較h BMSCs聯(lián)合移植增多,提示hUCBDSCs對于促巨核系增殖分化具有重要作用。 結(jié)論: 1. hUCBDSCs能促進臍血單個核細胞增殖、粘附和遷移,且促增殖能力較h BMSCs強。人臍血源基質(zhì)細胞顯著表達一些歸巢相關(guān)因子的mRNA。 2. hUCBDSCs聯(lián)合移植能提高移植后小鼠造血植入率,特別當(dāng)造血細胞輸注為低劑量時,這種作用更加顯著。 3. hUCBDSCs聯(lián)合移植能促進造血細胞早期遷移歸巢至骨髓和脾臟,提高歸巢效率。 4. hUCBDSCs聯(lián)合移植能提高小鼠存活,促進移植后造血重建并修復(fù)受損微環(huán)境。
[Abstract]:Hematopoietic microenvironment (HIM) is an internal environment that supports and regulates the growth and development of haemopoietic stem/progenitor cell (HSPC). The integrity of its structure and function is an important link in maintaining normal hematopoietic function. Cells may participate in the self-renewal, proliferation, differentiation and homing of hematopoietic stem cells (HSCs), secrete hematopoietic growth factor (HGF) and extracellular matrix (ECM) through the formation of "niches" for the growth of hematopoietic stem cells (HSCs), and play an important role in immune regulation. To investigate the effect of stromal cells on hematopoietic function of bone marrow, it is of great theoretical value and practical significance to treat hematopoietic damage by repairing or reconstructing the normal function of bone marrow microenvironment.
Stromal cells, derived from mesenchymal stem cells (MSCs), are a complex heterogeneous group of cells including fibroblasts, endothelial cells, osteoblasts, adipocytes, macrophages and reticular cells. Human bone marrow stromal cells (hBMSCs) have been cultured in vitro by Dexter since 1977. After successful transplantation, hBMSCs were studied in depth. Experiments and clinical practice proved that hBMSCs in vitro culture and amplification combined with HSC reinfusion is an effective method for reconstructing hematopoietic function. However, the source of hBMSCs is limited. Bone marrow collection increases donor pain and risk, and cell number and proliferation and differentiation potential decrease with the increase of donor age. Autologous transplantation has abnormal stromal cells, and allograft-versus-host disease (GVHD) and other immune-related problems may occur in autologous transplantation, which limits the wide use of hBMSCs in clinical practice.
Human umbilical cord blood-derived stromal cells (hUCBDSCs) and umbilical cord blood have long been engaged in the clinical treatment of malignant hematopoietic diseases. Previous studies have shown that specific cytokines can effectively amplify hUCBDSCs. The amplified hUCBDSCs are similar to hBMSCs in cell composition and immunophenotype. They can secrete and express many cytokines and possess the basic characteristics of hematopoietic stromal cells. The culture system with hUCBDSCs as trophoblast can effectively support umbilical cord blood. CD34+ cell proliferation in vitro, especially for colony forming unit-megakaryocte (CFU-Mk) formation, is significantly superior to that of hBMSCs, suggesting that hUCBDSCs may play an important role in promoting the proliferation, differentiation and maturation of megakaryocytes. Based on the above analysis, two different culture systems of stromal cell trophoblasts (hUCBDSCs and hBMSCs) were established to observe the effects of two kinds of stromal cells on human umbilical cord mononuclear cells (HUCBDSCs) by CCK-8 and Transwell methods. The effects of proliferation, adhesion and migration of R cells, hUCB-MNCs, and the expression of homing-related molecules mRNA in hUCBDSCs were detected by RT-PCR. On this basis, the model of hematopoietic microenvironment irradiation injury in BABL/c mice was established. The two methods were compared and observed by single transplantation of hUCB-MNCs or co-transplantation of two different stromal cells. Stromal cells promote the homing and implantation of hematopoietic cells in vivo, reconstruct the hematopoietic microenvironment and support the hematopoietic reconstruction.
Method:
1. To construct the trophoblast culture system of hUCBDSCs and hBMSCs in vitro. CCK-8 method and Transwell method were used to detect the effects of two kinds of stromal cells on the proliferation, adhesion and migration of hUCB-MNCs in vitro; RT-PCR was used to detect the expression of homing-related factors (SDF-1, CXCR-4, ICAM-1, VCAM-1, HCAM, PECAM-1, Fn) mRNA. Situation.
2. Inbred BABL/c mice were subjected to different doses of hUCB-MNCs (2,4,6 or 8 65507
3. HUCB-MNCs were pre-stained with CM-DiI fluorescent dye. After irradiation, BABL/c mice received either single transplantation of hUCB-MNCs (2 x106/mouse) or co-transplantation of two different stromal cells (2 x106/mouse). Laser confocal microscopy was used to observe the migration and distribution of fluorescent labeled hUCB-MNCs in mice, and to compare the hematopoietic cells of each group. Nesting rate.
4. After irradiation, mice received either single transplantation of hUCB-MNCs (2 *10~6/mouse) or co-transplantation of two different stromal cells (2 *10~6/mouse). Survival was observed and survival rate was recorded. The recovery of peripheral blood picture was dynamically detected, and pathological changes of bone marrow tissue were observed. Rat bone marrow fibroblast colony forming unit (CFU-F), spleen colony forming unit (CFU-S), granulocyte/macrophage colony forming unit (CFU-GM) and megakaryocyte colony forming unit (CFU-Mk) yields.
Result:
Effect of 1. hUCBDSCs on proliferation, adhesion and migration of cord blood mononuclear cells in vitro
Compared with hUCB-MNCs co-culture group and hUCB-MNCs single culture group, hUCB-MNCs proliferation ability was significantly enhanced under the co-culture condition of hUCB-MNCs. Both stromal cells could promote the adhesion of hUCB-MNCs, and the migration ability of hUCB-MNCs after co-culture was also significantly stronger (P 0.05) than that of control without stromal cell support. Implantation of closely related adhesion molecules, cytokines and receptors (SDF-1, CXCR-4, ICAM-1, VCAM-1, HCAM, PECAM-1, Fn) mRNA reveals their important role in the homing and implantation of hematopoietic cells in vivo.
2. hUCBDSCs combined transplantation promotes homing and implantation of hematopoietic cells
After irradiation, mice received different doses of hUCB-MNCs (2,4,6 or 8 65507 When low-dose hUCB-MNC was infused, the implantation rate of co-transplantation of hUCBDSCs was significantly higher than that of single transplantation. And single transplantation group showed that hUCBDSCs could promote hematopoietic cells homing to bone marrow in early stage after transplantation.
3.hUCBDSCs combined transplantation to repair damaged microenvironment and support hematopoietic reconstitution
In the co-transplantation group, the platelet inhibition was mild and the platelet recovery was rapid, and the leukocytes in the co-transplantation group returned to the pre-irradiation level 21 days after transplantation. There was no significant difference in hemoglobin between the two groups. hUCBDSCs combined transplantation promoted bone marrow tissue recovery, reconstructed the damaged microenvironment, and increased the CFUs (CFU-F, CFU-S, CFU-GM, CFU-Mk) production rate, especially the number of CHU-Mk increased compared with the hUCBDSCs combined transplantation. S plays an important role in promoting proliferation and differentiation of megakaryocytes.
Conclusion:
1. hUCBDSCs can promote the proliferation, adhesion and migration of umbilical cord blood mononuclear cells, and the proliferation-promoting ability is stronger than that of H BMSCs. Human umbilical cord blood stromal cells significantly express the mRNA of some homing-related factors.
2. Combined transplantation of hUCBDSCs can increase the rate of hematopoietic implantation in mice after transplantation, especially when the dosage of hematopoietic cells is low.
3. hUCBDSCs combined transplantation can promote early migration of hematopoietic cells, homing to bone marrow and spleen, and improving homing efficiency.
4. hUCBDSCs combined transplantation can improve the survival of mice, promote hematopoietic reconstitution after transplantation and repair the damaged microenvironment.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

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