本文選題：間充質(zhì)干細胞 + 細胞培養(yǎng)�。� 參考：《華中科技大學(xué)》2008年博士論文

【摘要】： 第一部分人骨髓間充質(zhì)干細胞的體外培養(yǎng)及鑒定 目的體外分離培養(yǎng)人骨髓間充質(zhì)干細胞(hMSCs)并進行鑒定,為組織工程提供適宜的種子細胞。 方法抽取健康成人骨髓,采用人淋巴細胞分離液分離純化獲取hMSCs,觀察細胞形態(tài)特征、生長狀況,從表面抗原表達及成骨分化能力兩個方面進行鑒定。 結(jié)果分離細胞貼壁生長,呈集落樣增殖,放射狀、渦漩狀分布排列。免疫細胞化學(xué)檢測細胞表達CD44和CD71,CD34、CD45呈陰性表達,細胞表面標志表達與間充質(zhì)干細胞的特征一致。分離的細胞經(jīng)過成骨條件培養(yǎng)基誘導(dǎo)分化后2周,細胞堿性磷酸酶染色陽性,4周后可以形成鈣結(jié)節(jié),證明所培養(yǎng)細胞有向成骨細胞分化的潛能。表明分離細胞為間充質(zhì)干細胞。 結(jié)論采用人淋巴細胞分離液分離細胞,能獲得較為純化的hMSCs,可為培養(yǎng)hMSCs提供穩(wěn)定的分離方法。 第二部分低氧環(huán)境對人骨髓間充質(zhì)干細胞生物學(xué)行為的影響 目的建立人骨髓間充質(zhì)干細胞(human mesenchymal stem cells ,hMSCs)體外低氧培養(yǎng)及向成骨細胞分化的生物模型,研究其生物學(xué)特征,為骨組織工程提供實驗依據(jù)。 方法采用人淋巴細胞分離液分離健康成人骨髓中的間充質(zhì)干細胞。取第3代細胞,根據(jù)培養(yǎng)氧濃度及培養(yǎng)基類型分為4組:正常氧組(20%O2加DMEM-LG)、低氧組(1%O2加DMEM-LG)、正常氧成骨誘導(dǎo)組(20%O2加條件培養(yǎng)基)及低氧成骨誘導(dǎo)組(1%O2加條件培養(yǎng)基),通過細胞計數(shù)、細胞增殖測定(MTT法)、增殖細胞核抗原(PCNA)檢測、凋亡蛋白及集落形成檢測,觀察低氧對hMSCs增殖凋亡的影響;RT-PCR檢測、ALP活性檢測及茜素紅染色,研究低氧環(huán)境對hMSCs成骨分化的影響;檢測Fibronectin、Laminin和CollagenⅠ蛋白表達,觀察低氧對hMSCs細胞外基質(zhì)分泌的影響。 結(jié)果與正常氧組相比,低氧組中的hMSCs有較高的增殖速度,生長差異顯著(P0.05);低氧組中PCNA染色細胞增多,高于正常氧組(P0.05);低氧組中凋亡細胞減少,抗凋亡蛋白bcl-2表達增多(P0.05);低氧組中的hMSCs生成的集落逐漸增多,明顯高于正常氧組。低氧成骨誘導(dǎo)組培養(yǎng)的hMSC堿性磷酸酶活性逐漸增高,但明顯低于正常氧成骨誘導(dǎo)組,兩者有明顯的差異(P0.01);定量RT-PCR檢測,正常氧成骨誘導(dǎo)組ALP、OC、COL1A2及BSP mRNA表達量明顯增加,ALP、OC、COL1A2及BSP mRNA明顯增高(P0.01);培養(yǎng)4周后,與其它三組相比,正常氧成骨誘導(dǎo)組可見到明顯的鈣鹽沉積和染成紅色的鈣結(jié)節(jié)。免疫組織化學(xué)和Western blot檢測,低氧組中FN和LA分泌增多(P0.05),CollagenⅠ蛋白表達無顯著差異(P0.05)。 結(jié)論低氧使hMSCs增殖率增加,集落形成能力增強,抑制成骨細胞分化,保持干細胞特征,促進hMSCs分泌細胞外基質(zhì)。 第三部分慢性低氧影響HIF-1α、SDF-1及VEGF165在hMSCs中的表達 目的建立體外hMSCs低氧模型,觀察慢性低氧對hMSCs表達HIF-1α(低氧誘導(dǎo)因子-1α)、SDF-1(基質(zhì)細胞衍生因子-1)及VEGF165(血管內(nèi)皮細胞生長因子)的影響,為hMSCs治療缺氧性疾病提供實驗依據(jù)。通過比較HIF-1α、SDF-1及VEGF165,探討低氧抑制hMSCs成骨分化的機制。 方法采用人淋巴細胞分離液分離健康成人骨髓中的間充質(zhì)干細胞。取第3代細胞,根據(jù)培養(yǎng)氧濃度及培養(yǎng)基類型分為4組:正常氧(n組)(20%O2加DMEM-LG)、低氧組(h組)(1%O2加DMEM-LG)、正常氧成骨誘導(dǎo)組(nos組)(20%O2加條件培養(yǎng)基)及低氧成骨誘導(dǎo)組(hos組)(1%O2加條件培養(yǎng)基),運用免疫組織化學(xué)檢測HIF-1α、SDF-1及VEGF蛋白表達, western blot檢測HIF-1α及SDF-1蛋白表達,定量RT-PCR檢測HIF-1α、SDF-1及VEGF165 mRNA表達。 結(jié)果n組和nos組hMSCs細胞質(zhì)內(nèi)HIF-1α染色陽性,h組和hos組hMSCs胞質(zhì)及胞核內(nèi)均強陽性染色。低氧條件下,SDF-1染色細胞數(shù)目明顯增多,VEGF染色呈陽性。hMSCs在慢性低氧的條件下,h組和hos組HIF-1α、SDF-1及VEGF165 mRNA表達水平明顯增高,與n組相比,兩者有顯著差異(P0.05) ,而n組和nos組,HIF-1αmRNA表達始終處于較低的水平(P0.05),nos組SDF-1 mRNA表達量持續(xù)增高,14天時達到峰值,隨后開始降低(P0.05),n組、h組、nos組及hos組隨著時間的延長,VEGF165 mRNA表達均逐漸增多,但h組及hos組VEGF mRNA表達始終高于n組和nos組,nos組14天時VEGF1165 mRNA表達量快速增加達到峰值,隨后開始降低(P0.05)。western blot檢測,h組和hos組可見HIF-1α及SDF-1蛋白表達增多,明顯高于n組和nos組(P0.05),nos組7天后SDF-1蛋白表達量明顯增加(P0.05),14天后減少。 結(jié)論SDF-1和VEGF在正常氧條件下有協(xié)同成骨的作用,低氧條件下HIF-1雖然刺激SDF-1和VEGF分泌增多,但抑制SDF-1和VEGF協(xié)同成骨的作用,SDF-1和VEGF分泌還存在著除HIF-1調(diào)控以外的途徑。
[Abstract]:Part one: culture and identification of human bone marrow mesenchymal stem cells in vitro
Objective to isolate and culture human bone marrow mesenchymal stem cells (hMSCs) in vitro and identify them, so as to provide suitable seed cells for tissue engineering.
Methods the bone marrow of healthy adults was extracted, and hMSCs was obtained by separation and purification by human lymphocyte separation solution. The morphological characteristics and growth status of the cells were observed. The expression of surface antigen and the ability of osteogenic differentiation were identified in two aspects.
Results the cell proliferation, radiate and whirlpool distribution were arranged in the isolated cells. The expression of CD44 and CD71, CD34, CD45 in immunocytochemical detection cells were negative, and the expression of the cell surface markers was consistent with the characteristics of mesenchymal stem cells. The isolated cells were induced 2 weeks after the differentiation of bone conditioned medium, and the alkaline phosphatase of the cells was induced. Staining positive, calcium nodules can be formed after 4 weeks, indicating that the cultured cells have the potential to differentiate into osteoblasts, indicating that the isolated cells are mesenchymal stem cells.
Conclusion the purified hMSCs can be obtained by separating the cells from human lymphocytes, which can provide a stable separation method for hMSCs culture.
The second part is the effect of hypoxic environment on the biological behavior of human bone marrow mesenchymal stem cells.
Objective to establish a biological model of human bone marrow mesenchymal stem cells (human mesenchymal stem cells (hMSCs) in vitro hypoxia culture and osteoblast differentiation, and to study the biological characteristics of bone marrow mesenchymal stem cells, and to provide experimental basis for bone tissue engineering.
Methods human mesenchymal stem cells were separated from healthy adult bone marrow by human lymphocyte separation solution. Third generation cells were divided into 4 groups according to the oxygen concentration and medium type: normal oxygen group (20%O2 plus DMEM-LG), hypoxia group (1%O2 plus DMEM-LG), normal oxygen induction group (20% O2 plus conditioned medium) and hypoxia induction group (1%O2 plus conditioned culture). Yang Ji), by cell count, cell proliferation assay (MTT), proliferating cell nuclear antigen (PCNA) detection, apoptotic protein and colony formation detection, the effect of hypoxia on hMSCs proliferation and apoptosis was observed. RT-PCR detection, ALP activity detection and alizarin red staining were used to study the effect of hypoxia on the osteogenesis of hMSCs, and Fibronectin, Laminin and Collagen I were detected. Protein expression was observed to observe the effect of hypoxia on the secretion of extracellular matrix in hMSCs cells.
Results compared with the normal oxygen group, the hMSCs in the hypoxia group had a higher proliferation rate and significant difference in growth (P0.05), and the number of PCNA stained cells in the hypoxia group was higher than that in the normal oxygen group (P0.05), the apoptotic cells decreased and the expression of anti apoptotic protein Bcl-2 increased (P0.05) in the hypoxia group, and the colony of hMSCs generated in the hypoxia group increased gradually, obviously higher than that in the normal oxygen group. Oxygen group. The activity of hMSC alkaline phosphatase in the hypoxia induction group increased gradually, but it was significantly lower than that of the normal oxygen induction group (P0.01). Quantitative RT-PCR detection, the expression of ALP, OC, COL1A2 and BSP mRNA in the normal oxygen induction group increased obviously, ALP, OC, COL1A2 and BSP. In the other three groups, there were obvious calcium salts and red calcium nodules in the normal oxygen induction group. Immunohistochemistry and Western blot detection, the increase of FN and LA secretion in the hypoxia group (P0.05), and no significant difference in the expression of Collagen I protein (P0.05).
Conclusion hypoxia can increase the proliferation rate of hMSCs, enhance colony forming ability, inhibit osteoblast differentiation, maintain the characteristics of stem cells, and promote the secretion of extracellular matrix by hMSCs.
The third part of chronic hypoxia affects the expression of HIF-1, SDF-1 and VEGF165 in hMSCs.
Objective to establish an in vitro hMSCs hypoxia model and observe the effect of chronic hypoxia on hMSCs expression of HIF-1 alpha (hypoxia inducible factor -1 alpha), SDF-1 (matrix cell derived factor -1) and VEGF165 (vascular endothelial cell growth factor), and provide experimental basis for the treatment of hypoxia disease by hMSCs. Through comparison of HIF-1 alpha, SDF-1 and VEGF165, hypoxia inhibits hMSCs osteogenesis. The mechanism of differentiation.
Methods human mesenchymal stem cells were separated from healthy adult bone marrow by human lymphocyte separation solution. The third generation cells were divided into 4 groups according to the oxygen concentration and medium type: normal oxygen (group n) (20%O2 plus DMEM-LG), hypoxia group (H Group) (1%O2 plus DMEM-LG), normal oxygen induction group (NOS group) and hypoxia induction group. In group HOS) (1%O2 plus conditioned medium), the expression of HIF-1 alpha, SDF-1 and VEGF protein was detected by immunohistochemistry. The expression of HIF-1 A and SDF-1 protein was detected by Western blot, and HIF-1 alpha, SDF-1 and expression were detected by RT-PCR.
Results in group n and group NOS, HIF-1 alpha staining was positive in hMSCs cytoplasm, and both in group H and hos group were strongly positive in cytoplasm and nucleus of hMSCs. The number of SDF-1 stained cells increased obviously in hypoxia condition, VEGF staining showed positive.HMSCs in chronic hypoxia, H group and hos group were increased obviously, two There were significant differences (P0.05), while the expression of HIF-1 alpha mRNA in group n and NOS was always at a lower level (P0.05). The mRNA expression of SDF-1 in NOS Group continued to increase, reaching the peak at 14 days, then began to decrease (P0.05), N group, H group, group and group gradually increased with time. Higher than group n and group NOS, VEGF1165 mRNA expression increased rapidly at 14 days in group NOS, and then decreased (P0.05).Western blot detection. H group and hos group showed increased expression of HIF-1 alpha and SDF-1 protein, which was significantly higher than that in group and group. After 7 days, the expression of protein was obviously increased, and decreased after 14 days.
Conclusion SDF-1 and VEGF have synergistic osteogenesis in normal oxygen condition, while HIF-1 stimulates the increase of SDF-1 and VEGF secretion in hypoxic conditions, but inhibits the synergistic effect of SDF-1 and VEGF on osteogenesis, and there are other pathways other than HIF-1 regulation in SDF-1 and VEGF secretion.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R329

【引證文獻】

相關(guān)期刊論文 前1條

1 羅元一;殷明;殷嫦嫦;;序貫酶消化法分離培養(yǎng)大鼠髓核細胞[J];九江學(xué)院學(xué)報(自然科學(xué)版);2012年03期

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本文編號：2021645