本文關(guān)鍵詞：BMP-2因素對血管內(nèi)皮細(xì)胞共培養(yǎng)干細(xì)胞成骨分化以及相關(guān)基因表達(dá)的影響研究　出處：《昆明醫(yī)科大學(xué)》2012年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 血管內(nèi)皮細(xì)胞 骨髓間充質(zhì)干細(xì)胞 Bmi-1 Runx2 聯(lián)合培養(yǎng)BMP-2 RNA干擾

【摘要】：[研究背景及目的] 由各類先天或后天原因造成的骨缺損是臨床上的常見疾病之一,而此類骨缺損的修復(fù)一直是較難解決的問題。運(yùn)用組織工程技術(shù)體外成骨后移植是這類骨缺損除手術(shù)外新的修復(fù)方法。骨髓間充質(zhì)干細(xì)胞(bone marrow Mesenchymal stem cells, BMSCs)作為體內(nèi)一種較易獲得的干細(xì)胞,其具有分化為多種不同類型細(xì)胞的潛能。BMSCs在適當(dāng)誘導(dǎo)條件下能夠分化為骨細(xì)胞、軟骨細(xì)胞、脂肪細(xì)胞等各類相關(guān)細(xì)胞。目前關(guān)于體外培養(yǎng)單一種類的骨髓間充質(zhì)干細(xì)胞并誘導(dǎo)其成骨分化的研究取得了很多進(jìn)展,但仍然存在細(xì)胞增殖慢、成骨效率低、成骨周期長等缺點(diǎn)。 目前有研究發(fā)現(xiàn)血管內(nèi)皮細(xì)胞(vein endothelial cells, VECs)在與BMSCs共培養(yǎng)可以通過骨形態(tài)發(fā)生蛋白(Bone morphogenetic protein, BMP),促進(jìn)成骨分化的同時刺激成骨細(xì)胞及成骨前細(xì)胞釋放血管內(nèi)皮生長因子(vascular endothelial growth factor, VEGF),而VEGF在促進(jìn)內(nèi)皮細(xì)胞的增殖的同時,也在血管發(fā)生和形成過程中發(fā)揮著重要作用。但目前從基因水平研究血管內(nèi)皮細(xì)胞對骨髓間充質(zhì)干細(xì)胞成骨分化作用仍未見報(bào)道。 本研究采用RNAi技術(shù)原理靜默hUVECs的BMP-2基因,分別采用正常hUVECs和BMP-2基因靜默的hUVECs與hBMSCs構(gòu)建聯(lián)合培養(yǎng)體系,利用熒光定量PCR技術(shù)定量檢測培養(yǎng)體系中各組hUVECs對hBMSCs的Bmi-1和Runx2基因的影響；驗(yàn)證hUVECs對hBMSCs內(nèi)增殖和成骨誘導(dǎo)促進(jìn)作用；探討hUVECs的BMP-2因子對hBMSCs的Bmi-1和Runx2基因的影響,明確BMP-2是否是hUVECs調(diào)控hBMSCs的Bmi-1和Runx2基因表達(dá)的主要因素。本研究為hUVECs共培養(yǎng)hBMSCs在骨組織工程中的種子細(xì)胞體外培養(yǎng)分化提供理論依據(jù),并期望研究成果能夠探索出新的方法從而解決組織工程種子細(xì)胞在支架材料上增殖、黏附和成骨分化的難題。 [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核細(xì)胞,并借助hBMSCs(?)壁生長的特性進(jìn)行純化,在相差顯微鏡觀察干細(xì)胞形態(tài)變化。將hBMSCs傳代擴(kuò)增培養(yǎng)至第三代,流式細(xì)胞儀檢測CD34、CD29、CD44表面抗原表達(dá),鑒定hBMSCs; (2)取hUVECs細(xì)胞進(jìn)行體外培養(yǎng),并運(yùn)用免疫組化染色方法鑒定hUVECs細(xì)胞,培養(yǎng)至第三代后用Western Blotting蛋白檢測方法分別檢測第4、6、8、10天BMP-2蛋白的表達(dá)情況。 (3)設(shè)計(jì)四組BMP-2基因的shRNA干擾序列,將所設(shè)計(jì)好的shRNA序列插入質(zhì)粒載體,運(yùn)用脂質(zhì)體轉(zhuǎn)染法將構(gòu)建的干擾基因序列轉(zhuǎn)染到血管內(nèi)皮細(xì)胞內(nèi),并采用熒光顯微鏡觀察質(zhì)粒轉(zhuǎn)染的效果。Western Blotting檢測血管內(nèi)皮細(xì)胞BMP-2蛋白的表達(dá),鑒定對hUVECs細(xì)胞BMP-2的RNA靜默的效果。 (4)將hUVECs細(xì)胞用ECM+10%新生胎牛血清擴(kuò)增至第三代后與第三代hBMSCs細(xì)胞按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)組。將經(jīng)RNAi處理的hUVECs細(xì)胞和第三代hBMSCs細(xì)胞按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)干擾組。設(shè)置單獨(dú)hBMSCs培養(yǎng)組、單獨(dú)hUVECs培養(yǎng)組為陰性對照組。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；用SPSS17.0軟件對各項(xiàng)檢測值進(jìn)行統(tǒng)計(jì)學(xué)分析。 (5)分別于第4、6、8、10天每組每個時間點(diǎn)取6孔檢測單獨(dú)hBMSCs組、聯(lián)合培養(yǎng)組和聯(lián)合培養(yǎng)干擾組中堿性磷酸酶(Alkaline phosphatase, ALP)及骨鈣素(Osteocalin, OC)含量。用SPSS17.0軟件對各項(xiàng)檢測值進(jìn)行統(tǒng)計(jì)學(xué)分析。 (6)分別于第4、6、8、10天每組每個時間點(diǎn)取6孔應(yīng)用Western Blotting方法檢測單獨(dú)hUVECs組、聯(lián)合培養(yǎng)組和聯(lián)合培養(yǎng)干擾組中hUVECs的BMP-2蛋白表達(dá)情況。 (7)采用熒光定量PCR法(fluorescence quantitative PCR, FQ-PCR)檢測第4、6、8、10天單獨(dú)hBMSCs組、聯(lián)合培養(yǎng)組和聯(lián)合培養(yǎng)干擾組中hBMSCs的Bmi-1和Runx2基因表達(dá)的情況,每組每個時間點(diǎn)取6孔。用SPSS17.0軟件對各項(xiàng)檢測值進(jìn)行統(tǒng)計(jì)學(xué)分析。 [結(jié)果] (1)采用密度梯度離心法結(jié)合貼壁培養(yǎng)法分離、提純hBMSCs,所獲得細(xì)胞的純度較高,達(dá)到實(shí)驗(yàn)要求。用流式細(xì)胞儀對提取純化的第3代hBMSCs進(jìn)行細(xì)胞表型分析鑒定,CD34低表達(dá),CD29、CD44高表達(dá),符合hBMSCs的表型特征。 (2)對所培養(yǎng)hUVECs行免疫組化染色鑒定,符合該細(xì)胞表型,Western Blotting檢測結(jié)果顯示hUVECs能正常表達(dá)BMP-2蛋白。 (3)2號質(zhì)粒轉(zhuǎn)染后的細(xì)胞BMP-2蛋白表達(dá)量明顯降低,本研究設(shè)計(jì)合成的shRNA干擾質(zhì)粒對hUVECs細(xì)胞BMP-2蛋白表達(dá)的靜默是有效的。質(zhì)粒3ug、脂質(zhì)體10ul,轉(zhuǎn)染6小時是最佳轉(zhuǎn)染條件,轉(zhuǎn)染效率約為60%。 (4)培養(yǎng)體系中hBMSCs的形態(tài)變化結(jié)果顯示各時間點(diǎn)聯(lián)合培養(yǎng)組hBMSCs呈現(xiàn)出一定的成骨分化表現(xiàn),聯(lián)合培養(yǎng)干擾組hBMSCs成骨分化的形態(tài)表現(xiàn)弱于聯(lián)合培養(yǎng)組,單獨(dú)hBMSCs組未見成骨分化表現(xiàn)。 (5)各組別細(xì)胞數(shù)目隨時間延長先增高后降低,各時間點(diǎn)聯(lián)合培養(yǎng)組細(xì)胞數(shù)目最高,聯(lián)合培養(yǎng)干擾組次之,單獨(dú)hBMSCs最低。各組之間兩兩比較,差異均有顯著統(tǒng)計(jì)學(xué)意義(P0.01)。 (6)Western Blotting檢測結(jié)果顯示聯(lián)合培養(yǎng)組hUVECs對BMP-2的表達(dá)較單獨(dú)hUVECs組增高。聯(lián)合培養(yǎng)干擾組對BMP-2的表達(dá)顯著降低,達(dá)到實(shí)驗(yàn)對RNA干擾的要求。 (7)聯(lián)合培養(yǎng)組和聯(lián)合培養(yǎng)干擾組內(nèi)堿性磷酸酶檢測量隨時間延長先增高后降低,單獨(dú)hBMSCs組的堿性磷酸酶含量在各時間點(diǎn)變化較小。各時間點(diǎn)聯(lián)合培養(yǎng)組ALP最高,聯(lián)合培養(yǎng)干擾組次之,單獨(dú)hBMSCs最低。各組之間兩兩比較,差異均有顯著統(tǒng)計(jì)學(xué)意義(P0.01)。 (8)聯(lián)合培養(yǎng)組和聯(lián)合培養(yǎng)干擾組內(nèi)骨鈣素檢測量隨時間延長先增高后降低；單獨(dú)hBMSCs組的骨鈣素含量在各時間點(diǎn)變化較�。桓鲿r間點(diǎn)聯(lián)合培養(yǎng)組骨鈣素檢測量最高,聯(lián)合培養(yǎng)干擾組次之,單獨(dú)hBMSCs組最低。各組之間兩兩比較,差異有顯著統(tǒng)計(jì)學(xué)意義(P0.01)。 (9)干細(xì)胞Bmi-1基因表達(dá)量在聯(lián)合培養(yǎng)組和聯(lián)合培養(yǎng)干擾組內(nèi)按時間增長逐漸上升；單獨(dú)hBMSCs組的Bmi-1基因表達(dá)量在各時間點(diǎn)變化較小；各時間點(diǎn)聯(lián)合培養(yǎng)組和聯(lián)合培養(yǎng)干擾組Bmi-1基因表達(dá)較高,單獨(dú)hBMSCs組最低；相同時間點(diǎn)聯(lián)合培養(yǎng)組和聯(lián)合培養(yǎng)干擾組之間比較,差異無統(tǒng)計(jì)學(xué)意義(P0.05)；單獨(dú)hBMSCs組與聯(lián)合培養(yǎng)組和聯(lián)合培養(yǎng)干擾組之間比較,差異有顯著統(tǒng)計(jì)學(xué)意義(P0.01)。 (10)聯(lián)合培養(yǎng)組和聯(lián)合培養(yǎng)干擾組內(nèi)Runx2基因表達(dá)量隨時間延長逐漸增高；單獨(dú)hBMSCs組的Runx2基因表達(dá)量在各時間點(diǎn)變化較��；各時間點(diǎn)聯(lián)合培養(yǎng)組Runx2基因表達(dá)量最高,聯(lián)合培養(yǎng)干擾組次之,單獨(dú)hBMSCs組最低；各組之間兩兩比較,差異均有顯著統(tǒng)計(jì)學(xué)意義(P0.01)。 [結(jié)論] (1)采用密度梯度離心法分離以及貼壁法純化的hBMSCs,經(jīng)流式細(xì)胞儀表型鑒定為骨髓來源的干細(xì)胞,可在后期聯(lián)合培養(yǎng)中應(yīng)用。 (2)臍靜脈血管內(nèi)皮細(xì)胞能正常表達(dá)BMP-2蛋白。實(shí)驗(yàn)中構(gòu)建的質(zhì)粒序列正確有效,能夠達(dá)到實(shí)驗(yàn)對BMP-2的RNA干擾要求。 (3)聯(lián)合培養(yǎng)體系中骨髓間充質(zhì)干細(xì)胞和臍靜脈血管內(nèi)皮細(xì)胞有相互促進(jìn)增殖的作用。兩種細(xì)胞聯(lián)合培養(yǎng)相容性好,未見抑制現(xiàn)象。 (4)聯(lián)合培養(yǎng)體系中骨髓間充質(zhì)干細(xì)胞分泌堿性磷酸酶和骨鈣素增加,向成骨細(xì)胞方向分化速度顯著加快。 (5)血管內(nèi)皮細(xì)胞能提高聯(lián)合培養(yǎng)體系中骨髓間充質(zhì)干細(xì)胞的Bmi-1表達(dá),這與骨髓間充質(zhì)干細(xì)胞的增殖密切相關(guān)。 (6)聯(lián)合培養(yǎng)體系對骨髓間充質(zhì)干細(xì)胞Bmi-1的表達(dá)有促進(jìn)作用,但此信號通路上游的調(diào)控并不是通過BMP-2而完成。 (7)骨髓間充質(zhì)干細(xì)胞在聯(lián)合血管內(nèi)皮細(xì)胞培養(yǎng)時成骨分化速度加快,這與其Runx2基因表達(dá)增加重要相關(guān)。 (8)骨髓間充質(zhì)干細(xì)胞Runx2基因的表達(dá)與血管內(nèi)皮細(xì)胞分泌的BMP-2密切相關(guān),這證明在聯(lián)合培養(yǎng)體系中存在BMP-2/Runx2通路。但BMP-2不是聯(lián)合培養(yǎng)體系中Runx2通路上游唯一的影響因子。
[Abstract]:[research background and purpose]
Bone defect caused by various types of congenital or acquired causes is one of the common clinical diseases, and repair such bone defects has been a problem difficult to solve. Using tissue engineering technology in vitro osteogenesis after transplantation is the bone defect except for surgery a new repair method. Bone marrow mesenchymal stem cells (bone marrow Mesenchymal stem cells, BMSCs) as a body is easy to obtain stem cells, which can differentiate into various types of cells.BMSCs can differentiate into bone cells, cartilage cells under appropriate induction conditions, fat cells and other related cells. At present about the single type of cultured bone marrow mesenchymal stem cells and its osteogenic differentiation have made great progress, but there are still slow cell proliferation, osteogenic bone defects of low efficiency, long cycle and so on.
Studies have found that vascular endothelial cells (vein endothelial cells, VECs) in BMSCs co cultured with the bone morphogenetic protein (Bone morphogenetic, protein, BMP), promote osteoblast differentiation and stimulate osteoblasts and osteogenic cells before release of vascular endothelial growth Naga Innoko (vascular endothelial growth factor, VEGF, and VEGF) in promoting the proliferation of endothelial cells and plays an important role in angiogenesis and the formation process. But the research from the gene level of vascular endothelial cells on bone marrow mesenchymal stem cells into osteoblasts has not been reported.
This study adopts BMP-2 RNAi technology principle of hUVECs gene silence, respectively using hUVECs and hBMSCs normal hUVECs and BMP-2 gene silence in the construction of joint training system, detected by fluorescence quantitative PCR and quantitative culture influence system were hUVECs to hBMSCs Bmi-1 and Runx2 gene; verification of hUVECs in hBMSCs proliferation and osteogenic effect; to investigate the effects of Bmi-1 and Runx2 gene BMP-2 factor hUVECs of hBMSCs, to determine whether BMP-2 is a major factor in the expression of Bmi-1 and Runx2 gene regulation of hUVECs hBMSCs. This study was hUVECs hBMSCs co cultured seed cells in vitro in bone tissue engineering provides a theoretical basis for the cultivation and differentiation, and expect the research results to explore new methods in order to solve the seed cells of tissue engineering scaffolds in proliferation, adhesion and osteogenic differentiation of the problem.
[method]
(1) extraction of volunteer bone marrow, bone marrow mononuclear cells were isolated by density gradient centrifugation, and with the help of hBMSCs (?) the characteristics of wall growth were purified in phase contrast microscopy stem cell morphological changes. HBMSCs cells were cultured to the third generation, flow cytometric detection of CD34, CD29, expression of CD44 surface antigen identification of hBMSCs;
(2) hUVECs cells were cultured in vitro, and hUVECs cells were identified by immunohistochemical staining. After third generations, the expression of BMP-2 protein on 4,6,8,10 day was detected by Western Blotting protein assay.
(3) four groups of shRNA interference sequence of BMP-2 gene, the shRNA sequence design good insertion plasmid using liposome transfection method to construct the interference gene sequence was transfected into vascular endothelial cells, the expression of.Western Blotting protein effect detection of BMP-2 vascular endothelial cells were observed by fluorescence microscope. The transfection and identification of hUVECs cell BMP-2 RNA silencing effect.
(4) hUVECs cells of neonatal ECM+10% fetal bovine serum amplification to the third generation and the third generation of hBMSCs cells at the ratio of 1:1 to establish DMEM+10% fetal bovine serum for co culture medium group. The hUVECs cells and third RNAi treated hBMSCs cells according to the ratio of 1:1 to DMEM+10% fetal bovine serum for co culture interference group. Set up a separate hBMSCs culture group, hUVECs alone group was negative control group respectively. On day 4,6,8,10 is to observe the morphological changes by microscope, count the number of plates were counted hBMSCs; statistical analysis on the test value with SPSS17.0 software.
(5) were in the 4,6,8,10 days each time point for 6 hole detection alone hBMSCs group, co culture group and co culture group (Alkaline alkaline phosphatase phosphatase interference, ALP) and Osteocalcin (Osteocalin, OC). The content of the statistical analysis on the test value with SPSS17.0 software.
(6) on the day of 4,6,8,10, 6 holes were collected from each group at each time point. Western Blotting was used to detect the expression of BMP-2 protein in the hUVECs group, the co culture group and the co culture interference group respectively.
(7) using fluorescence quantitative PCR (fluorescence quantitative PCR, FQ-PCR) detection day 4,6,8,10 hBMSCs alone group, co culture of expression of Bmi-1 and Runx2 gene hBMSCs interference group in the group and joint training, each group of each time point for 6 holes. Statistical analysis of the measured value by SPSS17.0 software.
[results]
(1) by density gradient centrifugation and adherent cell culture separation, purification of hBMSCs, the cells with high purity, meet the requirement of experiment. The purification of the extract by flow cytometry third generation hBMSCs were analyzed to identify the cell phenotype, the low expression of CD34, CD29, high expression of CD44, hBMSCs. The phenotypic characteristics of character
(2) the immuno histochemical staining of the cultured hUVECs was identified, which conformed to the phenotype of the cell. The results of Western Blotting detection showed that hUVECs could express the BMP-2 protein normally.
(3) the expression of BMP-2 protein decreased significantly after transfection of plasmid 2. The shRNA interference plasmid designed in this study is effective for the silence of BMP-2 protein expression in hUVECs cells. Plasmid 3UG, liposome 10ul and transfection for 6 hours are optimal transfection conditions, and the transfection efficiency is about 60%..
(4) the morphological changes of hBMSCs in the culture system showed that hBMSCs showed a certain osteogenic differentiation at different time points. In the co culture interference group, the osteogenic differentiation of hBMSCs was weaker than that of the co culture group, and there was no osteogenic differentiation in the hBMSCs group alone.
(5) the number of cells in each group increased first and then decreased with the time prolonging, each time point of maximum number of cells co culture group, co culture interference group, alone hBMSCs minimum. 22 comparison between groups, the difference was statistically significant (P0.01).
(6) Western Blotting detection showed that the expression of BMP-2 in hUVECs group was higher than that in single hUVECs group. The expression of BMP-2 in the co culture interference group was significantly reduced, which reached the requirement of RNA interference.
(7) co culture group and co culture group in alkaline phosphatase detection interference amount increased firstly and then decreased with time prolonging, alkaline phosphatase hBMSCs alone group at each time point were relatively small. Each time point of joint training group had the highest ALP, co culture interference group, alone hBMSCs minimum. 22 comparison between the groups was statistically significant. There was significant difference (P0.01).
(8) co culture group and co culture group osteocalcin detection interference first increased and then decreased with time prolonging; osteocalcin content of hBMSCs only groups at each time point changes little; each time point of the co culture group was the highest detection of osteocalcin co culture, interference group and single hBMSCs group is the lowest. 22 comparison between the groups. The difference was statistically significant (P0.01).
(9) stem cell Bmi-1 gene expression increased gradually in the co culture group and co culture group interference according to the time of growth; Bmi-1 gene expression of hBMSCs alone group at each time point changes little; each time point of joint training and joint training group the expression of Bmi-1 gene interference group was higher, hBMSCs alone was the lowest; at the same time comparison between co culture group and the co cultured interference group, the difference was not statistically significant (P0.05); hBMSCs alone group and co culture group and co culture between interference group comparison, the difference was statistically significant (P0.01).
(10) joint training group and the co cultured interference group Runx2 expression increased with time gradually; the Runx2 gene alone group hBMSCs expression at each time point changes little; each time point of the co culture group the expression of Runx2 gene was the highest, joint training of interference group, hBMSCs alone group was the lowest; comparison between groups 22, the difference was statistically significant (P0.01).
[Conclusion]
(1) hBMSCs purified by density gradient centrifugation and adherent method was identified as bone marrow derived stem cells by flow cytometry.
(2) the umbilical vein endothelial cells can express the BMP-2 protein normally. The plasmid sequence constructed in the experiment is correct and effective, and can meet the RNA interference requirements of the experimental BMP-2.
(3) in the combined culture system, bone marrow mesenchymal stem cells and umbilical vein endothelial cells promote each other's proliferation. The two kinds of cells have good compatibility and no inhibition phenomenon.
(4) bone marrow mesenchymal stem cells (MSCs) secreted alkaline phosphatase and osteocalcin in the combined culture system, and the velocity of osteoblast differentiation was accelerated significantly.
(5) vascular endothelial cells can improve the Bmi-1 expression of bone marrow mesenchymal stem cells in the joint culture system, which is closely related to the proliferation of bone marrow mesenchymal stem cells.
(6) the combined culture system can promote the expression of Bmi-1 in bone marrow mesenchymal stem cells, but the upstream regulation of this signal pathway is not accomplished through BMP-2.
(7) bone marrow mesenchymal stem cells (MSCs) have accelerated osteogenic differentiation at the combined culture of vascular endothelial cells, which are closely related to the increase in the expression of Runx2 gene.
(8) the expression of Runx2 gene in bone marrow mesenchymal stem cells is closely related to the BMP-2 secreted by vascular endothelial cells. This proves that there is BMP-2/Runx2 pathway in the co culture system, but BMP-2 is not the only upstream factor in the Runx2 pathway of co culture system.

【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R329

【參考文獻(xiàn)】

中國期刊全文數(shù)據(jù)庫 前4條

1 劉流;王�？�;趙德萍;何曉光;鐘玲;代曉明;李逸松;;聯(lián)合培養(yǎng)VECs與ADSCs與部分脫蛋白生物骨體外構(gòu)建組織工程骨的實(shí)驗(yàn)研究[J];中國生物美容;2010年01期

2 王芳;王e，

本文編號：1437820