【作者】 李洪伟；

【导师】 孙俊英；

【作者基本信息】 苏州大学 ， 外科学， 2011， 博士

【摘要】 第一部分人骨髓间充质干细胞的分离、培养及鉴定目的从人骨髓中分离培养hMSCs并对其细胞生物学特性进行观察。方法无菌条件下在健康志愿者髂棘处穿刺抽取骨髓,运用密度梯度离心法分离骨髓单个核细胞,培养在含10%胎牛血清的DMEM培养基中,再利用贴壁筛选法纯化获得人骨髓间充质干细胞,对其进行细胞生长和形态学的观察,流式细胞仪检测第3代hMSCs细胞表面标志并分析细胞周期。结果所分离培养出的细胞形态呈长梭形,漩涡状生长,纯度随传代次数增加而提高,8代以后细胞扩增速度变慢,细胞形态也发生改变。P3代骨髓间充质干细胞的表面标志CD90、CD105表达强阳性,而CD34、CD11b阴性。第1、3、5代hMSCs生长曲线呈S形,无显著性差异,均经历潜伏期、对数增殖期和停滞期三个时期。培养的第3代骨髓间充质干细胞中处于G0/G1期的细胞比例约为77.42%,处于S+G2/M期的细胞比例为22.58%左右。结论采用密度梯度离心结合贴壁筛选培养法可获得纯度较高且增殖活性强的人骨髓间充质干细胞,是一种简单、经济、有效的分离纯化骨髓间充质干细胞的方法。第二部分硅酸二钙涂层离子溶液对人骨髓间充质干细胞增殖及凋亡的影响目的通过硅酸二钙涂层离子溶液与人骨髓间充质干细胞复合培养,试图阐明硅酸二钙涂层离子溶液对人骨髓间充质干细胞增殖、细胞周期及凋亡的影响。方法将等离子喷涂的硅酸二钙涂层钛片浸泡在DMEM培养基中,37℃下静置72小时,电感耦合等离子体原子发射光谱仪(ICP)测定培养基中Si、Ca、P离子浓度的变化。骨髓间充质干细胞分别培养在四种培养基(OS-DS-、OS-DS+、OS+DS-、OS+DS+)中,MTT法测定细胞增殖情况,流式细胞仪检测hMSCs位于G0/G1、S、G2/M期的细胞数及增殖指数、细胞凋亡率。结果与OS-DS-培养基相比,OS-DS+、OS+DS+培养基中Si离子浓度显著升高(P<0.05);OS-DS+、OS+DS-和OS+DS+培养基中Ca离子浓度略有下降,但没有统计学差异;OS+DS-、OS+DS+培养基中P离子浓度明显增高。在培养的1~4天,OS-DS+和OS+DS+组细胞增殖率高于OS-DS-组,并分别在第2、3天时差异具有统计学意义;OS+DS-组的细胞数始终低于OS-DS-组,在第7天时有显著性差异(P<0.05)。流式细胞仪测定结果显示,在第4天时,OS-DS+和OS+DS+组处于G0/G1期的细胞比例明显低于OS-DS-组,G2/M期细胞比例明显高于OS-DS-组(P<0.05);OS+DS-组处于S期的细胞数在第4天时比OS-DS-组明显减少,而OS+DS+和OS-DS+组S期的细胞比例分别在第4、14天明显高于OS-DS-组。和OS-DS-组比较,OS+DS-和OS+DS+组细胞凋亡率在第7、14天显著增高,而OS-DS+组细胞凋亡率在第14天明显高于OS-DS-组(P<0.05)。结论硅酸二钙涂层离子溶液能促进人骨髓间充质干细胞的早期增殖,其促细胞增殖的能力可能与培养基中较高的硅离子浓度有关,通过加速骨髓间充质干细胞周期的转换而达到的。第三部分硅酸二钙涂层离子溶液促进人骨髓间充质干细胞成骨分化的研究目的通过检测骨髓间充质干细胞成骨分化过程中特异性基因的表达,探讨硅酸二钙涂层离子溶液对骨髓间充质干细胞成骨分化的影响。方法人骨髓间充质干细胞分别培养在四种培养基(OS-DS-、OS-DS+、OS+DS-、OS+DS+)中,采用对硝基苯磷酸盐法测定细胞内碱性磷酸酶(ALP)活性,茜素红S染色方法观察钙化结节的形成,以半定量RT-PCR检测转录因子Runx2、I型胶原、骨结合素及骨钙素的基因表达情况。结果在整个培养过程中OS+DS-、OS+DS+组ALP活性显著高于OS-DS-和OS-DS+组;在第7、14和21天时,OS+DS+组细胞内ALP活性明显高于OS+DS-组;从第7天起,OS-DS+组ALP活性高于OS-DS-组,并于第14天时差异具有统计学意义。在培养的第14天,OS-DS+、OS+DS-和OS+DS+组钙沉积的OD值均高于OS-DS-组,有显著性差异(P<0.05);与OS+DS-组相比,OS+DS+组钙沉积的OD值明显增高,差异具有统计学意义(P<0.05)。培养28天后,OS+DS+组钙化结节的数量和着色程度均较OS+DS-组明显增加,其他两组未见钙化结节形成。在第4、7、14天时,成骨诱导组hMSCs成骨分化相关标志基因的表达量显著高于非成骨诱导组;OS-DS+组Runx2与骨结合素mRNA表达水平分别在第7、14天明显高于OS-DS-组;在第14天,OS+DS+组各特异性基因的表达量均显著高于OS+DS-组。结论硅酸二钙涂层离子溶液自身能够在一定程度上诱导骨髓间充质干细胞向成骨细胞分化;和成骨诱导剂协同,则促进钙沉积及骨髓间充质干细胞的成骨分化。第四部分硅酸二钙涂层离子溶液促进人骨髓间充质干细胞成骨分化机制的研究目的探讨硅酸二钙涂层离子溶液促进人骨髓间充质干细胞成骨分化的可能分子机制。方法骨髓间充质干细胞分别培养在三组培养基(OS+DS-、OS+DS+、OS+DS+/PD)中,以Western blot方法检测细胞内ERK的磷酸化表达情况;加入ERK特异性抑制剂PD98059后,分别采用对硝基苯磷酸盐法、茜素红S染色方法测定碱性磷酸酶活性与钙沉积变化。结果与OS+DS-组相比,在第7、14及21天,OS+DS+组细胞内碱性磷酸酶活性显著增加(P<0.05),其作用可被ERK途径抑制剂PD98059阻断(P<0.01)。OS+DS+组钙沉积在第14天时明显高于OS+DS-组(P<0.05),其作用也可被PD98059抑制,差异具有统计学意义。hMSCs受到OS+DS+培养基作用后10 min,细胞中磷酸化的ERK表达显著增强(P<0.01),并维持在较高水平至60 min,随后磷酸化的ERK表达逐渐降低到基线水平。比较三组培养基作用10 min后ERK相对表达量,发现PD98059显著抑制ERK磷酸化的水平,有统计学差异(P<0.01)。结论硅酸二钙涂层离子溶液能够促进人骨髓间充质干细胞的成骨分化,ERK信号通路在此过程中发挥了调控作用。更多还原

【Abstract】 PartⅠIsolation, culture and identification of human mesenchymal stem cellsObjective To isolate and culture human mesenchymal stem cells (hMSCs) from bone marrow in vitro and observe their biological characteristics. Methods Bone marrow aspirates were obtained from the posterior iliac crest of healthy human donors under aseptic conditions. hMSCs were isolated from the marrow by Percoll density gradient centrifugation method. The nucleated cells were plated in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). hMSCs were highly purified and expanded through adherence filtration. Cell morphology and proliferation status were observed by phase contrast microscope. Standard flow cytometry techniques were used to determine the cell surface epitope profile and cell cycle of hMSCs. Results The cells exhibited a fusiform shape and swirled when they assembled together. The amplification speed of hMSCs became slow and cell morphology changed after passage 8. The adherent cells did not express hematopoietic lineage markers CD34 and CD11b, while hMSCs collected from passage 3 were strongly positive for CD90 and CD105 markers. The growth curves of hMSCs from the first, third and fifth passage were S-shaped. The cell cycle analysis showed that the percentage of G0/G1 phase of cells from passage 3 was 77.42% and the percentage of S+G2/M phase was 22.58%. Conclusion The density gradient centrifugation technique in combination with adherence filtration is a simple, economical and efficient method to isolate and purify hMSCs. PartⅡEffect of ionic dissolution products of dicalcium silicate coating on human mesenchymal stem cells proliferation and apoptosisObjective To explore the influence of ionic dissolution products of dicalcium silicate coating on proliferation, cell cycle and apoptosis in human mesenchymal stem cells. Methods Ten pieces of plasma sprayed dicalcium silicate (DS) coatings were soaked in 200 ml DMEM for 72 h at 37℃in static conditions, then inductively coupled plasma atomic emission spectroscopy (ICP-AES) was employed to measure the concentrations of silicon (Si), calcium (Ca) and phosphorus (P). hMSCs were cultured in four media (OS-DS-, OS-DS+, OS+DS-, OS+DS+). The proliferation of hMSCs was determined using MTT assay. Cell cycle and apoptosis were analyzed by flow cytometry on days 4, 7 and 14. Results The concentration of Si in OS-DS+ and OS+DS+ was significantly higher than OS-DS-, Ca concentration decreased slightly, and P content of osteogenic medium (OS+DS-, OS+DS+) was higher than OS-DS- medium. Cell proliferation process in OS-DS+ and OS+DS+ was consistently higher than that in OS-DS- in the first 4 days of culture, and significant difference was seen at 2 and 3 days, respectively. Throughout the assay period, the number of hMSCs exposed to OS+DS- was generally lower than that in OS-DS-, and significant decrease did not become apparent until day 7 of culture. The percentage of G0/G1 phase of cells cultured in OS-DS+ and OS+DS+ was lower than that in OS-DS-, while the percentage of G2/M phase was higher on day 4. Smaller percentage of S phase was detected in hMSCs exposed to OS+DS- at 4 days, whereas bigger percentage of S phase was observed in OS+DS+ and OS-DS+ at 4 and 14 days, respectively. Apoptosis was markedly increased in OS+DS- and OS+DS+ on days 7 and 14, and in OS-DS+ on day 14 when compared to OS-DS-. Conclusion The ionic dissolution products of dicalcium silicate coating can promote the early proliferation of hMSCs by accelerating cell cycle, which may be attributed to the high concentration of silicon ion. PartⅢStudy of ionic dissolution products of dicalcium silicate coating on osteogenic differentiation in human mesenchymal stem cellsObjective To investigate the effect of ionic dissolution products of dicalcium silicate coating on human mesenchymal stem cells osteogenic differentiation. Methods hMSCs were cultured in four media (OS-DS-, OS-DS+, OS+DS-, OS+DS+). Alkaline phosphatase (ALP) activity was assessed at the protein level. Calcium deposition was analyzed by Alizarin Red S staining. Reverse transcription-polymerase chain reaction (RT-PCR) was used for analysis of mRNA levels of runt-related transcription factor 2 (Runx2), collagen type I (Col-I), osteonectin (ON) and osteocalcin (OC). Results Throughout the cultivation period, hMSCs exposed to osteogenic medium (OS+) had significantly higher ALP activity than did cells exposed to growth medium (OS-). ALP activity of hMSCs cultured in OS+DS+ was significantly higher than that of cells cultured in OS+DS- on days 7, 14 and 21. The level of ALP activity was appreciably higher in hMSCs exposed to OS-DS+ when compared to OS-DS- on day 14. The optical density (OD) value in hMSCs exposed to OS-DS+ was higher than that of cells exposed to OS-DS- at 14 days. Treatment in OS+DS- resulted in a significant increase in OD, and hMSCs in OS+DS+ further increased the OD value. The hMSCs grown in OS-DS- or OS-DS+ medium failed to form any detectable calcium nodules after 28 days of culture. Cells cultured in OS+DS- medium exhibited some small mineralized nodules, while a greater extent of mineralization with more detectable bone nodules was observed in hMSCs exposed to OS+DS+ medium. The mRNA levels of osteogenic markers investigated were higher in hMSCs exposed to osteogenic medium (OS+) than cells exposed to growth medium (OS-) throughout the assay period. hMSCs grown in OS-DS+ expressed higher levels of Runx2 and ON mRNA than did cells exposed to OS-DS- on days 7 and 14, respectively. The osteogenic markers were expressed at higher level in the (OS+DS+)-group at 14 days when compared to the (OS+DS-)-group. Conclusion The ionic dissolution products of dicalcium silicate coating alone partly induce hMSCs differentiation towards the osteoblastic lineage. The cooperation between ionic dissolution products of dicalcium silicate coating and osteogenic supplements can fully induce osteoblast-specific gene expression and significantly promote osteogenic activity of hMSCs.PartⅣStudy of the mechanisms of dicalcium silicate coating ionic dissolution products on human mesenchymal stem cells osteogenic differentiationObjective To explore the possible mechanisms of ionic dissolution products released from dicalcium silicate coating in promoting osteogenic differentiation of hMSCs. Methods hMSCs were cultured in OS+DS- medium, OS+DS+ medium or OS+DS+ medium supplemented with PD98059. The levels of extracellular signal-regulated kinase (ERK) phosphorylation were examined by Western blot. PD98059, a specific inhibitor of ERK, was added into OS+DS+ medium to investigate the changes in alkaline phosphatase activity and calcium deposition. Results ALP activity of hMSCs cultured in OS+DS+ was significantly higher than that of cells cultured in OS+DS- on days 7, 14 and 21, but the increase was inhibited by PD98059. Calcium deposition in OS+DS+ was greater than that in OS+DS- at 14 days, and the increase was also suppressed by PD98059. ERK was activated at 10 min after treatment in OS+DS+ medium. The ERK activation was maintained at higher level to 60 min, and then declined to the baseline. hMSCs in OS+DS- medium expressed a moderate level of ERK phosphorylation, and treatment with ionic dissolution products of dicalcium silicate coating further increased the level of ERK phosphorylation, but the increase was blocked by pre-treating cells with PD98059. Conclusion The ionic dissolution products from the dicalcium silicate coating may promote osteogenic differentiation of hMSCs mainly through ERK signal transduction pathway.更多还原