维生素D受体介导龟板诱导的骨髓间充质干细胞向成骨分化

发布时间：2018-03-22 09:19

本文选题：龟板　切入点：成骨分化　出处：《广州中医药大学》2010年硕士论文　论文类型：学位论文

【摘要】： 一、研究目的骨髓间充质干细胞(mesenchymalstem cells, MSCs)是中胚层来源的具有多向分化能力的干细胞,在不同诱导条件下,具有向成骨细胞、软骨细胞、脂肪细胞、神经细胞及骨髓基质等组织细胞分化的潜能。由于MSCs具有易分离、扩增以及体外操作简便、体内免疫反应较弱等特点,在临床上有广泛的应用前景。但是由于骨髓间充质干细胞分化能力不强,诱导其高效率单一分化是我们亟需解决的问题。龟板入肝肾经,具有滋补肝肾,益肾填精之效。研究表明龟板含药血清在不同时间可以促进MSCs的增殖和分化,这种作用和BMP4激活不同的受体有关。中医“肾主骨生髓”的理论为指导依据,我们前期的实验已发现MSCS在龟板含药血清诱导作用下,可向成骨细胞方向分化,表现为碱性磷酸酶(ALP)活性升高。龟板的化学成分复杂,其中发现龟板的乙酸乙酯部位作为洗脱剂分离出来的成分含有与成骨诱导剂地塞米松类似的甾类成分,此成分与成骨分化有着密切的联系。核受体(nuclear receptor,NR)是配体依赖性转录因子超家族,与机体生长发育、细胞分化、体内许多生理、代谢过程中的基因表达调控密切相关。针对不同的刺激信号,核受体与不同辅助调节因子的相互作用从而激活或抑制下游基因的表达。 BMP能诱导成骨细胞和软骨细胞的分化成熟,作为下游基因,受到核受体的支配,当受到核受体信号后,并能在体内诱导异位成骨。BMPs与骨形成蛋白受体BMPR结合,通过Smads和MAPKs途径进行信号转导,并通过下游转录因子Cbfal等与相应的成骨细胞特异蛋白碱性磷酸酶、骨钙素、OPN等基因启动子链接,促进细胞向成骨方向分化。本课题以核受体为突破口,探讨龟板提取物对骨髓间充质干细胞成骨分化过程中核受体的影响。本实验目标在于确定龟板提取物诱导大鼠骨髓间充质干细胞向成骨分化和对核受体中维生素D受体的影响及通路BMP的表达情况。二、实验方法和内容首先体外培养大鼠骨髓间充质干细胞,分别用龟板提取物,成骨诱导液诱导MSCs向成骨分化,空白对照不加任何处理因素,诱导七天后,通过免疫化学染色、Western blot、原位杂交、RT-PCR等方法观察龟板提取物对原代培养的MSCS向成骨分化的成骨分化标记物ALP、OPN及VDR、VDR mRNA的表达情况。为了更进一步研究,本实验采用EndoFectinTM-Plus试剂瞬时转染VDR的表达质粒,一方面,观察转染后Vonkoss染色结果；另一方面,提取转染后的MSCs细胞蛋白,运用Western blotting观察龟板提取物对MSCS向成骨分化的成骨分化标记物ALP、OPN等及BMP通路相关蛋白的表达情况。用同样的转染方法,对VDR基因沉默。一方面，，观察VDR基因沉默后Vonkoss染色结果；另一方面,提取VDR基因沉默后的MSCs细胞蛋白,运用Western blotting观察龟板提取物对原代培养的MSCs向成骨分化的成骨分化标记物ALP、OPN等及BMP通路相关蛋白的表达情况。三、实验结果未转染VDR表达质粒及未进行VDR基因沉默时,免疫组化染色显示龟板组成骨分化标记物ALP、OPN和VDR的阳性百分比明显高于对照组；Western blot结果也显示龟板组的ALP、OPN和VDR的蛋白表达水平高于对照组；同时原位杂交、RT-PCR提示了龟板诱导MSCs向成骨分化过程可促进VDR mRNA的表达。转染VDR表达质粒后,Vonkoss染色结果显示细胞呈复层生长,并形成钙化结节,Western Blotting显示VDR转染后VDR的表达明显增加,成骨标记物ALP、OPN、RunX2、CollagenⅠ明显高于转空载体组,BMP通路相关蛋白BMPRIB、BMPRIA、BMPR-ⅡBMP4、SMAD5、P-Smadl/5/8的表达高于转空载体组。 VDR基因沉默后,Vonkoss染色很难找到钙化结节存在,Western Blotting显示VDR基因沉默后VDR的表达明显降低,成骨标记物ALP、OPN、RunX2、CollagenⅠ明显低于转空载体组,BMPs通路相关蛋白BMPRIB、BMPRIA、BMPR-ⅡBMP4、SMAD5、P-Smad1/5/8的表达低于转空载体组。四、结论龟板提取物可促MSCs向成骨分化,其过程可能与VDR的上调有关。 VDR过表达质粒有协同龟板提取物促进骨髓间充质干细胞向成骨分化的作用。 VDR基因沉默抑制了龟板提取物诱导的骨髓间充质干细胞成骨分化的效应。得出结论：维生素D受体介导龟板诱导的骨髓间充质干细胞向成骨分化。推测,核受体VDR协同龟板提取物在骨髓间充质干细胞向成骨分化过程中起到的重要的作用,其可能与BMP通路密切相关。
[Abstract]:First, the purpose of the study
Bone marrow mesenchymal stem cells (mesenchymalstem, cells, MSCs) is a mesenchymal stem cells capable of multi-directional differentiation, under different induction conditions, has to osteoblasts, cartilage cells, fat cells, nerve cells and bone marrow stromal tissue cell differentiation potential. Because the MSCs is easily separated and amplified in vitro the operation is simple, the body weaker immune responses and other characteristics, has a wide application prospect in clinic. But the differentiation of bone marrow mesenchymal stem cells induced by its high efficiency ability is not strong, single differentiation we need to solve the problem.
Tortoise shell into the kidney, nourishing the liver and kidney, yishentianjing effect. The research showed that Seropharmacological Plastrum Testudinis can promote the proliferation and differentiation of MSCs in different time, the function and the activation of BMP4 receptor. Different TCM theory of kidney and bone marrow "as a guide, our previous experiments have found that MSCS the induction effect of Seropharmacological Plastrum Testudinis, can be induced to differentiate into osteoblasts, expression of alkaline phosphatase (ALP) activity increased. The chemical constituents of turtle shell complex, which found that ethyl acetate extract shells as eluent separated the ingredients contain osteogenic agent dexamethasone similar steroid components induced by this component is closely the contact and osteogenic differentiation.
The nuclear receptor (nuclear receptor NR) is a ligand dependent transcription factor superfamily, growth and cellular differentiation, many physiological and metabolic process, regulation of gene expression is closely related. According to different stimuli, and different expression of nuclear receptor coregulators interact to activate or inhibit downstream genes.
BMP can induce the differentiation of osteoblast and chondrocyte maturation as a downstream gene, dominated by the nuclear receptor, when the signal by the nuclear receptor, and the combination of the in vivo ectopic bone.BMPs and bone morphogenetic protein receptor BMPR signal transduction through Smads and MAPKs pathways, and the downstream transcription factor Cbfal and etc. bone cell specific protein alkaline phosphatase, corresponding osteocalcin, OPN gene promoter links, promote cell osteogenic differentiation.
The nuclear receptor as a breakthrough of Plastrum Testudinis extracts on bone marrow mesenchymal stem cells differentiation process of nuclear receptor. The goal is to determine the expression of Plastrum Testudinis extracts of rat bone marrow mesenchymal stem cells to osteoblast differentiation and influence on vitamin D receptor nuclear receptors and pathways of BMP.
Two, experimental methods and contents
The first in vitro culture of rat bone marrow mesenchymal stem cells with osteogenic induction medium of Plastrum Testudinis extracts, MSCs induced osteogenic differentiation and blank control without any treatment, after seven days of induction, by immunohistochemical staining, in situ hybridization, Western blot, RT-PCR and other methods of observation of Plastrum Testudinis extracts on cultured MSCS osteogenic differentiation marker ALP osteoblast differentiation, OPN and VDR, the expression of VDR mRNA.
In order to further study the expression plasmid of EndoFectinTM-Plus transfected VDR reagent on the one hand, the transfected Vonkoss staining results; on the other hand, MSCs cell protein extraction after transfection, using Western blotting MSCS to observe Plastrum Testudinis extracts on bone differentiation markers ALP into osteogenic differentiation, the expression of OPN and etc. BMP pathway related proteins.
The same transfection, the VDR gene silencing. On the one hand, Vonkoss was observed after VDR gene silencing; on the other hand, extraction of protein in MSCs cells after VDR gene silencing, using the Western blotting observation of Plastrum Testudinis extracts on cultured MSCs osteogenic differentiation marker ALP into osteogenic differentiation, the expression of OPN and the BMP pathway related proteins.
Three, experimental results
Non transfected VDR expression plasmid and VDR gene silencing, immunohistochemical staining showed that the osteogenic differentiation markers of ALP shells, positive percentage of OPN and VDR were significantly higher than the control group; Western blot results also show that the tortoise platron group ALP, the expression level of OPN and VDR protein was higher than the control group; and in situ hybridization, RT-PCR the tortoise shell induced during the osteoblast differentiation of MSCs could promote the expression of VDR mRNA.
Transfection of VDR expression plasmid, Vonkoss staining showed that the cells were double layer growth, and the formation of calcified nodules, Western Blotting showed that VDR increased the expression of VDR after transfection, osteogenic markers ALP, OPN, RunX2, Collagen I was significantly higher than that in turn empty vector group, BMP pathway related protein BMPRIB, BMPRIA, BMPR- II BMP4 SMAD5, P-Smadl/5/8, was higher than that of empty vector group.
After VDR gene silencing, Vonkoss staining is very difficult to find the calcified nodules, Western Blotting showed that VDR gene silencing VDR expression was significantly decreased, bone markers ALP, OPN, RunX2, Collagen I was significantly lower than that in turn empty vector group, BMPs pathway related protein BMPRIB, BMPRIA, BMPR- II BMP4, SMAD5, P-Smad1/5/8 was lower than empty vector group.
Four. Conclusion
Tortoise shell extract could promote MSCs osteogenic differentiation, the process may be related to the upregulation of VDR.
VDR over expression plasmid coordinate with PTE to promote bone marrow mesenchymal stem cells to osteoblast differentiation.
VDR gene silencing PTE inhibited the induction of bone marrow mesenchymal stem cells into osteoblasts effect.
Conclusion: vitamin D receptor mediated Testudinis induced bone marrow mesenchymal stem cells into osteoblasts. Speculated that the nuclear receptor VDR synergy of Plastrum Testudinis extracts in bone marrow mesenchymal stem cells during osteogenic differentiation has played an important role, which may be closely related to the BMP pathway.

【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329;R285.5

【参考文献】