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  本文選題：成骨生長肽(10-14) + 骨髓間充質干細胞�。� 參考：《蘭州大學》2007年博士論文

【摘要】： 以往的實驗證據(jù)表明，骨髓間充質干細胞(MSCs)具有多向分化潛能，，在一定條件下可定向分化為成骨細胞和脂肪細胞，此已分化的成骨細胞、脂肪細胞可以去分化再分化，并且二者之間存在“此消彼長”的關系。研究發(fā)現(xiàn)，在各種類型的骨質疏松病人中，其骨髓基質中脂肪細胞的增多并伴隨松質骨骨量低下。成骨生長肽(OGP)可促進骨形成、刺激骨折愈合、增加小梁骨骨密度；過度表達OGP基因的轉基因小鼠，尤其是雌性鼠，因其骨形成增加而有著較高的骨量峰值；在成骨性細胞及起源于人及其它哺乳動物的骨髓基質細胞培養(yǎng)中，OGP調節(jié)細胞的增殖、堿性磷酸酶活性及基質礦化。正常生理狀態(tài)下，在嚙齒類動物和人類血液中80～90％的OGP是以與成骨生長肽結合蛋白(OGPBP)相結合的形式存在的。OGP從OGP-OGPBP復合物中分離出來后，在蛋白水解酶作用下，產生OGP的C末端五肽——OGP(10-14)。OGP(10-14)是具有OGP全部生物活性的最小片段，它通過激活靶細胞內Gi蛋白-促有絲分裂素激動蛋白(MAP)激酶信號途徑發(fā)揮其生物學作用。以上實驗結果促使我們對OGP(10-14)預防和治療骨質疏松的機理進行探討。OGP(10-14)是否通過調節(jié)MSCs的成骨、成脂分化來影響骨的形成尚未可知。 在本研究中，OGP(10-14)采用Boc策略和Fmoc策略的固相多肽合成法手工合成，經(jīng)分析型HPLC鑒定純度＞95％，質譜鑒定分子量與理論值相符。生物活性鑒定結果顯示，OGP(10-14)對NIH3T3細胞具有促增殖作用，劑量反應曲線呈一“近似鐘形”，發(fā)揮最大生物活性的濃度為10~(-10)M。用貼壁篩選法分離MSCs，傳3代后再進行成脂、成骨誘導，通過相差顯微鏡下觀察細胞生長形態(tài)學變化，并行油紅O染色、堿性磷酸酶(ALP)染色、Von Kossa鈣染色、細胞內甘油三酯(TG)和ALP活性的測定，半定量RT-PCR分析成脂相關性基因PPARγ2,、LPL,、aP_2、C/EBP及成骨相關性基因cbfal、ALP、OC的表達情況，進而觀察OGP(10-14)在大鼠MSCs成骨、成脂分化中的作用。 本實驗結果顯示，不管是對rMSCs用10~(-10)M OGP(10-14)預處理后再成脂誘導還是在成脂誘導培養(yǎng)體系中直接加入10~(-10)M OGP(10-14)，其成脂分化與對照組相比明顯受抑，表現(xiàn)為所形成的脂肪細胞數(shù)量減少、細胞內甘油三酯水平降低。同樣，對rMSCs用10~(-10)M OGP(10-14)預處理后再成骨誘導或在成骨誘導培養(yǎng)體系中直接加入10~(-10)MOGP(10-14)，均可促進其成骨分化，ALP陽性細胞及所形成的骨樣小結明顯增多，細胞內ALP活性也明顯高，且OGP(10-14)促進rMSCs成骨分化可不依賴于地塞米松的存在，但地塞米松(10~(-8)M)與OGP(10-14)(10~(-10)M)聯(lián)合作用時，其效應大于單一因素的作用，呈現(xiàn)出顯著的協(xié)同作用。不管是OGP(10-14)處理組還是對照組，均可探及cbfal和PPARγ2基因的表達；至于ALP，OC，LPL，aP_2和C/EBP，其表達水平非常低或未探及表達；與對照組比較，對rMSCs用10~(-10)M OGP(10-14)預處理7天可顯著提高cbfal基因的表達水平，同時抑制PPARγ2基因的表達。 本實驗證實成骨細胞與脂肪細胞均可由MSCs分化而來；OGP(10-14)可以通過抑制MSCs向脂肪細胞的分化并促進其分化為更多的成骨細胞，從而有效地促進骨形成；OGP(10-14)對rMSCs成脂、成骨分化的影響可能在細胞定向分化階段而非細胞成熟階段；OGP(10-14)具有預防和治療骨質疏松的潛在臨床應用價值。
[Abstract]:Previous experimental evidence suggests that bone marrow mesenchymal stem cells (MSCs) have multiple differentiation potential and can differentiate into osteoblasts and adipocytes under certain conditions. The differentiated osteoblasts, adipocytes can dedifferentiated and redifferentiated, and the relationship between the two is "this elimination" relationship. Studies have found that various types of bone are found. In the patients with osteoporosis, the increase of fat cells in the bone marrow stroma and the low bone mass of the cancellous bone. Osteogenic growth peptide (OGP) promotes bone formation, stimulates fracture healing and increases the bone mineral density of the trabecular bone; the transgenic mice that overexpress the OGP gene, especially the female mice, have a higher bone peak value because of the increase of bone formation; and in osteogenesis. In the culture of cells and bone marrow stromal cells derived from human and other mammals, OGP regulates cell proliferation, alkaline phosphatase activity and matrix mineralization. Under normal physiological conditions, 80 to 90% of OGP in rodents and human blood are in the form of.OGP from OGP-OGPBP in the form of a combination of osteogenic growth peptide binding protein (OGPBP). After the separation, the C terminal five peptide of OGP, OGP (10-14).OGP (10-14), is the smallest fragment of all OGP bioactivity under the action of protein hydrolase. It plays its biological role by activating the Gi protein in the target cells - the signal pathway of the mitogen activator kinkinin (MAP) kinase (MAP) kinase. The results of the above results urge us on O GP (10-14) the mechanism of prevention and treatment of osteoporosis is discussed..OGP (10-14) is not yet known whether the formation of bone can be affected by adipogenic differentiation by regulating MSCs osteogenesis.
In this study, OGP (10-14) was manually synthesized by the Boc strategy and the solid-phase polypeptide synthesis of Fmoc strategy. The purity of the analytical HPLC was more than 95%, and the molecular weight identified by the mass spectrometry was in accordance with the theoretical value. The results of bioactivity identification showed that OGP (10-14) had a proliferation promoting effect on NIH3T3 cells, and the dose response curve showed a "approximate bell shape" and played the most important role. The concentration of large biological activity was 10~ (-10) M. to separate MSCs with adherent screening method. After 3 generations, lipid formation and osteogenesis were induced. The morphological changes of cell growth were observed under phase contrast microscope, oil red O staining, alkaline phosphatase (ALP) staining, Von Kossa calcium staining, determination of intracellular triglyceride (TG) and ALP activity, semi quantitative RT-PCR analysis. Lipid related gene PPAR gamma 2, LPL, aP_2, C/EBP and the expression of cbfal, ALP, OC in bone related genes, and then observe the role of OGP (10-14) in rat MSCs osteogenesis and lipid differentiation.
The results showed that the lipid differentiation of rMSCs 10~ (-10) M OGP (10-14) was induced or 10~ (-10) M OGP (10-14) was directly added to the lipid induced culture system, and its lipid differentiation was obviously inhibited compared with the control group, which showed that the number of adipocytes formed and the level of triglyceride in the cells decreased. The same, rMSCs. 10~ (-10) M OGP (10-14) was pretreated with bone induction or direct addition of 10~ (-10) MOGP (10-14) into the osteogenic induction culture system, which could promote osteogenesis. ALP positive cells and bone like nodules were significantly increased, and the intracellular ALP activity was significantly higher. OGP (10-14) promoted rMSCs osteogenesis to be dependent on the survival of dexamethasone. At the same time, the effect of dexamethasone (10~ (-8) M) and OGP (10-14) (10~ (-10) M) was greater than that of a single factor, showing a significant synergistic effect. Both OGP (10-14) treatment group and control group could detect the expression of cbfal and PPAR gamma 2 genes. Compared with the control group, the pretreatment of rMSCs with 10~ (-10) M OGP (10-14) for 7 days could significantly increase the expression level of cbfal gene and inhibit the expression of PPAR gamma 2 gene.
It is verified that osteoblasts and adipocytes can be differentiated by MSCs; OGP (10-14) can effectively promote bone formation by inhibiting the differentiation of MSCs into adipocytes and promoting its differentiation into more osteoblasts, and the effect of OGP (10-14) on rMSCs formation and osteogenic differentiation may be in the phase of differentiation of cells instead of cell maturity. OGP (10-14) has potential clinical value in the prevention and treatment of osteoporosis.

【學位授予單位】：蘭州大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R329

【引證文獻】

相關碩士學位論文 前1條

1 吳銳;OGP對兔骨髓間充質干細胞體外成骨分化干預的實驗研究[D];昆明醫(yī)科大學;2013年

本文編號：1866359