【摘要】：[研究背景及目的] 不同原因所致各種類(lèi)型骨缺損在臨床上十分常見(jiàn),頜骨缺損的修復(fù)問(wèn)題一直是臨床上難以解決的問(wèn)題。組織工程學(xué)的出現(xiàn)和發(fā)展為骨缺損修復(fù)帶來(lái)了機(jī)遇,其最終目標(biāo)是將功能細(xì)胞與可降解三維生物支架材料在體外聯(lián)合培養(yǎng),構(gòu)建成為有活性的組織或器官,然后植入體內(nèi),替代病損的組織,恢復(fù)其形態(tài)、結(jié)構(gòu)和功能。目前組織工程骨構(gòu)建方法多用單純種子細(xì)胞接種,雖然能夠成骨,但存在組織工程骨血管化緩慢,新骨生長(zhǎng)遲緩等問(wèn)題。 骨髓間充質(zhì)干細(xì)胞(bone marrow Mesenchymal stem cells, BMSCs)具有多向分化的潛能,在適當(dāng)條件下不僅可以分化為脂肪細(xì)胞、骨細(xì)胞、軟骨細(xì)胞、心肌細(xì)胞、神經(jīng)元細(xì)胞、成肌細(xì)胞、肌腱細(xì)胞及星形膠質(zhì)細(xì)胞等。目前關(guān)于誘導(dǎo)單純BMSCs向成骨細(xì)胞分化的研究取得了較大的進(jìn)展,然而誘導(dǎo)單純BMSCs向成骨細(xì)胞分化存在成骨周期長(zhǎng)、成骨效率低、細(xì)胞易老化等缺點(diǎn)。 近年來(lái),研究人員發(fā)現(xiàn)內(nèi)皮細(xì)胞可以分泌骨形態(tài)發(fā)生蛋白(Bone morphogenetic protein, BMP),促進(jìn)成骨分化的同時(shí)刺激成骨細(xì)胞及其前體細(xì)胞分泌血管內(nèi)皮生長(zhǎng)因子(vascular endothelial growth factor, VEGF),而VEGF在血管發(fā)生和形成過(guò)程中發(fā)揮著重要作用,可以促進(jìn)內(nèi)皮細(xì)胞增殖。然而目前血管內(nèi)皮細(xì)胞對(duì)骨髓間充質(zhì)干細(xì)胞成骨分化作用的具體機(jī)制還不清楚,尚缺乏從基因水平驗(yàn)證血管內(nèi)皮細(xì)胞對(duì)骨髓間充質(zhì)干細(xì)胞成骨分化的作用。 為此,本課題著重探討人臍靜脈內(nèi)皮細(xì)胞(human umbilical vein endothelial cells, HUVECs)在聯(lián)合培養(yǎng)體系中對(duì)人骨髓間充質(zhì)干細(xì)胞(human bone marrow Mesenchymal stem cells, hBMSCs)的形態(tài)、生長(zhǎng)、細(xì)胞分化及其Bmi-1基因和Runx2基因表達(dá)的影響,從基因水平驗(yàn)證血管內(nèi)皮細(xì)胞對(duì)骨髓間充質(zhì)干細(xì)胞成骨分化的作用。為臍靜脈內(nèi)皮細(xì)胞和骨髓間充質(zhì)干細(xì)胞聯(lián)合培養(yǎng)作為骨組織工程的種子細(xì)胞提供理論基礎(chǔ)。 [方法] (1)抽取志愿者骨髓液,使用密度梯度離心法分離骨髓單個(gè)核細(xì)胞,并借助MSCs黏附于塑料瓶底這一特性進(jìn)行純化,相差顯微鏡觀察形態(tài)變化。將MSCs傳代擴(kuò)增培養(yǎng)至第三代,流式細(xì)胞儀檢測(cè)CD34.CD29.CD44表面抗原表達(dá),鑒定MSCs; (2)將訂購(gòu)的HUVECs用ECM+10%新生胎牛血清擴(kuò)增至第三代后與第三代HUVECs按1：1比例建立以DMEM+10%胎牛血清為培養(yǎng)基的聯(lián)合培養(yǎng)體系。以單獨(dú)培養(yǎng)的hBMSCs組及hUVECs組作為陰性對(duì)照組,分別于第4、6、8、10天相差顯微鏡觀察形態(tài)變化,用計(jì)數(shù)板計(jì)數(shù)各組hBMSCs數(shù)量; (3)分別于第4、6、8、10天每組每個(gè)時(shí)間點(diǎn)取6孔檢測(cè)三組培養(yǎng)體系中堿性磷酸酶(Alkaline phosphatase,ALP)及骨鈣素(Osteocalin,OC)含量。用SPSS17.0軟件對(duì)各項(xiàng)檢測(cè)值進(jìn)行統(tǒng)計(jì)學(xué)分析； (4)采用實(shí)時(shí)熒光定量PCR(fluorescence quantitative PCR,FQ-PCR)檢測(cè)第4、6、8、10天單獨(dú)培養(yǎng)的hBMSCs組及聯(lián)合培養(yǎng)組中hBMSCs的Bmi-1和Runx2基因表達(dá)的情況,每組每個(gè)時(shí)間點(diǎn)取6孔。用SPSS17.0軟件對(duì)各項(xiàng)檢測(cè)值進(jìn)行統(tǒng)計(jì)學(xué)分析。 [結(jié)果] (1)采用Ficoll液密度梯度離心法分離、提純hBMSCs可達(dá)到較高的純度。用流式細(xì)胞儀對(duì)第3代hMSCs進(jìn)行細(xì)胞表型分析鑒定,CD34低表達(dá),CD29.CD44高表達(dá)； (2)新生胎牛血清分離培養(yǎng)的人骨髓間充質(zhì)干細(xì)胞成細(xì)長(zhǎng)梭形,細(xì)胞較細(xì)小,原代培養(yǎng)4至5天就可見(jiàn)成團(tuán)生長(zhǎng)的細(xì)胞；第三代骨髓間充質(zhì)干細(xì)胞形態(tài)單一,成梭形,呈旋渦狀分布,沒(méi)有細(xì)胞重疊現(xiàn)象。hBMSCs在4-6天呈對(duì)數(shù)生長(zhǎng),8-10天后生長(zhǎng)進(jìn)入平臺(tái)期。HUVECs成單層生長(zhǎng),形態(tài)成多角形、鵝卵石狀鑲嵌排列,邊界清楚,胞漿豐富,胞核呈圓形或橢圓形,偶見(jiàn)雙核,第5d融合成片,第一代至第四代生長(zhǎng)速度較快,2-3天即可傳代； (3)各組堿性磷酸酶檢測(cè)量隨時(shí)間延長(zhǎng)先增高后降低,各時(shí)間聯(lián)合培養(yǎng)組ALP較高,8天時(shí)聯(lián)合培養(yǎng)組堿性磷酸酶最高；骨髓間充質(zhì)干細(xì)胞組和臍靜脈內(nèi)皮細(xì)胞組ALP基本沒(méi)有變化；聯(lián)合培養(yǎng)組和其它各組之間兩兩比較均有顯著統(tǒng)計(jì)學(xué)意義(P0.01)；各組骨鈣素檢測(cè)量隨時(shí)間延長(zhǎng)先增高后降低；8天時(shí)聯(lián)合培養(yǎng)組骨鈣素最高；聯(lián)合培養(yǎng)組和其它各組之間兩兩比較均有顯著統(tǒng)計(jì)學(xué)意義(P0.01)； (4)聯(lián)合培養(yǎng)組Bmi-1、Runx2基因檢測(cè)量隨時(shí)間延長(zhǎng)逐漸增高,各時(shí)間聯(lián)合培養(yǎng)組Runx2基因表達(dá)較高；骨髓間充質(zhì)干細(xì)胞組Bmi-1基因隨時(shí)間延長(zhǎng)略有增高,Runx2基因表達(dá)基本沒(méi)有變化；8th天時(shí)聯(lián)合培養(yǎng)組Bmi-1、Runx2基因檢測(cè)量最高；聯(lián)合培養(yǎng)組和骨髓間充質(zhì)干細(xì)胞組之間比較有顯著統(tǒng)計(jì)學(xué)意義(P0.01)。 [結(jié)論] (1)采用Ficoll液密度梯度離心法分離、提純的hBMSCs,經(jīng)流式細(xì)胞儀鑒定為較高純度的細(xì)胞； (2)骨髓間充質(zhì)干細(xì)胞與臍靜脈內(nèi)皮細(xì)胞聯(lián)合培養(yǎng)相容性良好,臍靜脈內(nèi)皮細(xì)胞對(duì)體外聯(lián)合培養(yǎng)體系中骨髓間充質(zhì)干細(xì)胞具有促進(jìn)增殖的作用； (3)在體外聯(lián)合培養(yǎng)體系中,臍靜脈內(nèi)皮細(xì)胞能促進(jìn)骨髓間充質(zhì)干細(xì)胞Bmi-1及Runx2基因的表達(dá),誘導(dǎo)其向成骨細(xì)胞方向分化。
[Abstract]:[Background and purpose of the study] The various types of bone defects due to different causes are very common in the clinic, and the problem of the repair of the defect of the jaw is always difficult to solve. Problems. The emergence and development of tissue engineering brings an opportunity for the repair of bone defects. The ultimate goal is to combine functional cells and degradable three-dimensional biological scaffold materials in vitro, to construct an active tissue or organ, Form, structure and The present tissue engineering bone construction method is multi-purpose simple seed cell inoculation, although it can be formed, the tissue engineering bone vascularization is slow, the growth of the new bone is slow, and the like. The bone marrow mesenchymal stem cells (BMSCs) have the potential of multi-directional differentiation, and can not only be differentiated into adipocytes, osteocytes, chondrocytes, cardiac muscle cells, neuron cells, myoblasts, tendon cells and star-like cells under appropriate conditions. At present, it has made great progress in the study of inducing the differentiation of only BMSCs to the osteoblast, but the induction of the differentiation of the BMSCs to the osteoblast is long, the osteogenic efficiency is low, and the cells are easy to use. In recent years, the researchers have found that endothelial cells can secrete bone morphogenetic protein (BMP), promote osteogenic differentiation, and stimulate the secretion of vascular endothelial growth factor (VEGF) in the osteoblast and its precursor cells. or, VEGF), and VEGF plays an important role in angiogenesis and formation, and can To promote the proliferation of the endothelial cells, the specific mechanism of the current vascular endothelial cells to the osteogenic differentiation of the bone marrow-derived mesenchymal stem cells is not clear. To study the effects of human umbilical vein endothelial cells (HUVECs) on human bone marrow mesenchymal stem cells (hBMSCs), the morphology, growth, cell differentiation and Bmi-1 gene and R of human bone marrow-derived mesenchymal stem cells (hBMSCs) in human umbilical vein endothelial cells (HUVECs) were discussed in this paper. The effect of unx 2 gene expression on the expression of bone marrow from vascular endothelial cells from the gene level The role of the osteogenic differentiation of the mesenchymal stem cells is the combination of the umbilical vein endothelial cells and the bone marrow mesenchymal stem cells as bone tissue engineering. fine seed The cell provides a theoretical basis.[Method] (1) The bone marrow liquid of the volunteers is extracted, and the bone marrow mononuclear cells are separated by using a density gradient centrifugation method, and the bone marrow mononuclear cells are adhered to the bottom of the plastic bottle by means of MSCs. The culture of MSCs was cultured to the third generation, and the CD34. CD29 was detected by flow cytometry. The expression of the surface antigen of CD44 was identified and the MSCs were identified; (2) the newly-ordered HUVECs was expanded to the third generation with the ECM + 10% of the new fetal calf serum, and the second generation HUVECs was set up in a 1:1 ratio with the third generation HUVECs for DME The combined culture system of M + 10% fetal calf serum was used as the negative control group for the cultured hBMSCs group and the hUVECs group. the number of hBMSCs in each group is counted by using a counting plate, and (3) the number of each group of hBMSCs is detected by a counting plate; and (3) six holes are taken at each time point of each group in groups 4,6,8 and 10 respectively to detect the alkaline in three groups of culture systems Phosphatase, AL P) and the content of Osteocin (OC). (4) Bmi of hBMSCs and hBMSCs in group 4,6,8 and 10 were detected by real-time fluorescence quantitative PCR (FQ-PCR). -1 and Runx2 gene expression,6 for each group of time points per group Hole. S The PSS17.0 software performs a statistical analysis of the detection values.[Results] (1) Use F The purification of hBMSCs can achieve higher purity by the method of density gradient centrifugation, and the third generation hMSCs were determined by flow cytometry. The analysis and identification of the cell phenotype, the low expression of CD34, the high expression of CD29. CD44, and (2) the isolation and culture of the new fetal bovine serum. the mesenchymal stem cells are in an elongated shuttle shape, the cells are small, and the cells growing in a cluster can be seen in the primary culture for 4 to 5 days; The third generation of the bone marrow-derived mesenchymal stem cells has a single form, a spindle shape, a vortex-like distribution, no cells, the hBMSCs are logarithmic growth on 4-6 days and grow into the platform stage after 8-10 days; the HUVECs are single-layer growth, the shape is polygonal, the cobblestone is embedded and arranged, the boundary is clear, the cytoplasm is rich, the nucleus is circular or elliptical, 2-3 days after the first-generation to fourth-generation growth, and 2-3 days of passage; (3) the amount of alkaline phosphatase in each group was decreased with the increase of time, the ALP of the combined culture group was higher, and the combination of 8 days was combined. The alkaline phosphatase of the culture group was the highest, and the ALP of the bone marrow mesenchymal stem cell group and the umbilical vein endothelial cell group did not change basically, and there was a significant difference between the combined culture group and the other groups (P0 .01). The amount of bone-bone detection in each group was decreased with the increase of time, and the combined culture group was cultured on 8 days. There was a significant difference between the two groups in the combined culture group and the other groups (P0.01), and (4) the combined culture group Bmi- 1. The amount of the Runx2 gene was increased with the time, and the expression of the Runx2 gene in the combined culture group was high; the Bmi-1 gene of the bone marrow-derived mesenchymal stem cell group increased slightly with the time, and the expression of the Runx2 gene was basically unchanged; the Bmi-1 and Runx2 groups in the combined culture group at the 8th day due to the amount of detection There was a significant difference between the combination culture group and the bone marrow mesenchymal stem cell group (P0.01).[Conclusion] (Conclusion] ( 1) Separation and purification of hBMSC by Ficoll density gradient centrifugation (2) bone marrow mesenchymal stem cells and umbilicus; the combined culture of the vein endothelial cells is good, the umbilical vein endothelial cells have the effect of promoting the proliferation of the bone marrow mesenchymal stem cells in the in vitro combined culture system, and (3) in vitro combination
【學(xué)位授予單位】：昆明醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R329

【共引文獻(xiàn)】

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10 竇慧慧;郭文君;于麗;馬麗;孫麗;;人臍帶間充質(zhì)干細(xì)胞分離培養(yǎng)方法的研究[J];中國(guó)組織化學(xué)與細(xì)胞化學(xué)雜志;2010年02期

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