本文選題：龜板　切入點：成骨分化　出處：《廣州中醫(yī)藥大學》2010年碩士論文　論文類型：學位論文

【摘要】： 一、研究目的 骨髓間充質(zhì)干細胞(mesenchymalstem cells, MSCs)是中胚層來源的具有多向分化能力的干細胞,在不同誘導條件下,具有向成骨細胞、軟骨細胞、脂肪細胞、神經(jīng)細胞及骨髓基質(zhì)等組織細胞分化的潛能。由于MSCs具有易分離、擴增以及體外操作簡便、體內(nèi)免疫反應(yīng)較弱等特點,在臨床上有廣泛的應(yīng)用前景。但是由于骨髓間充質(zhì)干細胞分化能力不強,誘導其高效率單一分化是我們亟需解決的問題。 龜板入肝腎經(jīng),具有滋補肝腎,益腎填精之效。研究表明龜板含藥血清在不同時間可以促進MSCs的增殖和分化,這種作用和BMP4激活不同的受體有關(guān)。中醫(yī)“腎主骨生髓”的理論為指導依據(jù),我們前期的實驗已發(fā)現(xiàn)MSCS在龜板含藥血清誘導作用下,可向成骨細胞方向分化,表現(xiàn)為堿性磷酸酶(ALP)活性升高。龜板的化學成分復雜,其中發(fā)現(xiàn)龜板的乙酸乙酯部位作為洗脫劑分離出來的成分含有與成骨誘導劑地塞米松類似的甾類成分,此成分與成骨分化有著密切的聯(lián)系。 核受體(nuclear receptor,NR)是配體依賴性轉(zhuǎn)錄因子超家族,與機體生長發(fā)育、細胞分化、體內(nèi)許多生理、代謝過程中的基因表達調(diào)控密切相關(guān)。針對不同的刺激信號,核受體與不同輔助調(diào)節(jié)因子的相互作用從而激活或抑制下游基因的表達。 BMP能誘導成骨細胞和軟骨細胞的分化成熟,作為下游基因,受到核受體的支配,當受到核受體信號后,并能在體內(nèi)誘導異位成骨。BMPs與骨形成蛋白受體BMPR結(jié)合,通過Smads和MAPKs途徑進行信號轉(zhuǎn)導,并通過下游轉(zhuǎn)錄因子Cbfal等與相應(yīng)的成骨細胞特異蛋白堿性磷酸酶、骨鈣素、OPN等基因啟動子鏈接,促進細胞向成骨方向分化。 本課題以核受體為突破口,探討龜板提取物對骨髓間充質(zhì)干細胞成骨分化過程中核受體的影響。本實驗?zāi)繕嗽谟诖_定龜板提取物誘導大鼠骨髓間充質(zhì)干細胞向成骨分化和對核受體中維生素D受體的影響及通路BMP的表達情況。 二、實驗方法和內(nèi)容 首先體外培養(yǎng)大鼠骨髓間充質(zhì)干細胞,分別用龜板提取物,成骨誘導液誘導MSCs向成骨分化,空白對照不加任何處理因素,誘導七天后,通過免疫化學染色、Western blot、原位雜交、RT-PCR等方法觀察龜板提取物對原代培養(yǎng)的MSCS向成骨分化的成骨分化標記物ALP、OPN及VDR、VDR mRNA的表達情況。 為了更進一步研究,本實驗采用EndoFectinTM-Plus試劑瞬時轉(zhuǎn)染VDR的表達質(zhì)粒,一方面,觀察轉(zhuǎn)染后Vonkoss染色結(jié)果；另一方面,提取轉(zhuǎn)染后的MSCs細胞蛋白,運用Western blotting觀察龜板提取物對MSCS向成骨分化的成骨分化標記物ALP、OPN等及BMP通路相關(guān)蛋白的表達情況。 用同樣的轉(zhuǎn)染方法,對VDR基因沉默。一方面，，觀察VDR基因沉默后Vonkoss染色結(jié)果；另一方面,提取VDR基因沉默后的MSCs細胞蛋白,運用Western blotting觀察龜板提取物對原代培養(yǎng)的MSCs向成骨分化的成骨分化標記物ALP、OPN等及BMP通路相關(guān)蛋白的表達情況。 三、實驗結(jié)果 未轉(zhuǎn)染VDR表達質(zhì)粒及未進行VDR基因沉默時,免疫組化染色顯示龜板組成骨分化標記物ALP、OPN和VDR的陽性百分比明顯高于對照組；Western blot結(jié)果也顯示龜板組的ALP、OPN和VDR的蛋白表達水平高于對照組；同時原位雜交、RT-PCR提示了龜板誘導MSCs向成骨分化過程可促進VDR mRNA的表達。 轉(zhuǎn)染VDR表達質(zhì)粒后,Vonkoss染色結(jié)果顯示細胞呈復層生長,并形成鈣化結(jié)節(jié),Western Blotting顯示VDR轉(zhuǎn)染后VDR的表達明顯增加,成骨標記物ALP、OPN、RunX2、CollagenⅠ明顯高于轉(zhuǎn)空載體組,BMP通路相關(guān)蛋白BMPRIB、BMPRIA、BMPR-ⅡBMP4、SMAD5、P-Smadl/5/8的表達高于轉(zhuǎn)空載體組。 VDR基因沉默后,Vonkoss染色很難找到鈣化結(jié)節(jié)存在,Western Blotting顯示VDR基因沉默后VDR的表達明顯降低,成骨標記物ALP、OPN、RunX2、CollagenⅠ明顯低于轉(zhuǎn)空載體組,BMPs通路相關(guān)蛋白BMPRIB、BMPRIA、BMPR-ⅡBMP4、SMAD5、P-Smad1/5/8的表達低于轉(zhuǎn)空載體組。 四、結(jié)論 龜板提取物可促MSCs向成骨分化,其過程可能與VDR的上調(diào)有關(guān)。 VDR過表達質(zhì)粒有協(xié)同龜板提取物促進骨髓間充質(zhì)干細胞向成骨分化的作用。 VDR基因沉默抑制了龜板提取物誘導的骨髓間充質(zhì)干細胞成骨分化的效應(yīng)。 得出結(jié)論：維生素D受體介導龜板誘導的骨髓間充質(zhì)干細胞向成骨分化。推測,核受體VDR協(xié)同龜板提取物在骨髓間充質(zhì)干細胞向成骨分化過程中起到的重要的作用,其可能與BMP通路密切相關(guān)。
[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.

【學位授予單位】：廣州中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R329;R285.5

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