發(fā)布時間：2018-04-08 11:35

  本文選題：血小板衍生因子　切入點：巨核細(xì)胞　出處：《南方醫(yī)科大學(xué)》2017年碩士論文

【摘要】：研究背景與研究目的血小板衍生生長因子(PDGF)是一種多肽生長因子,其儲存于人血小板α顆粒中。PDGF家族有5個不同的二聚體,PDGF-AA,AB,BB,CC和DD,其通過結(jié)合PDGFα受體和β受體發(fā)揮其生物學(xué)功能。PDGF受體(PDGFR)是一種跨膜糖蛋白,由α和β兩種酪氨酸蛋白激酶亞基構(gòu)成,具有酪氨酸蛋白激酶活性。研究發(fā)現(xiàn),PDGF-BB與兩種受體高親和力結(jié)合后,促增殖作用比其他亞型更強。PDGF-BB可以促進(jìn)多種結(jié)締組織細(xì)胞如成纖維細(xì)胞,內(nèi)皮細(xì)胞和平滑肌細(xì)胞等的生長和分化。除此以外,PDGF-BB對造血也具有重要的作用,如促進(jìn)紅系爆式集落形成單位(BFU-E)和粒系-紅系-單核-巨核細(xì)胞集落形成單位(CFU-GEMM)的形成。然而,PDGF-BB在造血中的具體機制尚不明確,可能與骨髓微環(huán)境中的骨髓間充質(zhì)細(xì)胞TPO的生成有關(guān)。因此,本研究旨在探索PDGF-BB對CFU-MK形成的作用,并將其與IL-3、IL-6、TPO和GM-CSF的作用進(jìn)行對比,以及加入抗IL-3、IL-6或TPO單克隆抗體后對CFU-MK生成的影響。進(jìn)一步探討PDGF-BB促進(jìn)巨核系造血的作用機制:PDGF作用于骨髓間充質(zhì)細(xì)胞表面的PDGF受體,激活下游相關(guān)的轉(zhuǎn)錄因子調(diào)控TPO產(chǎn)生,從而間接調(diào)節(jié)骨髓巨核系造血。研究內(nèi)容與研究方法第一部分PDGF-BB促進(jìn)巨核細(xì)胞生成一、PDGF-BB對小鼠CFU-MK集落形成的影響1.檢測不同濃度的 PDGF-BB(0ng/ml、5ng/ml、10ng/ml、20ng/ml、50ng/ml、100ng/ml)對小鼠CFU-MK形成的影響;2.檢測不同細(xì)胞因子(PDGF-BB、IL-3、IL-6、GM-CSF及TPO)及其不同組合對小鼠CFU-MK的作用;3.加入抗IL-3、IL-6、或TPO抗體后研究PDGF-BB對小鼠CFU-MK形成的影響。二、PDGF-BB對人CFU-MK集落形成的作用檢測不同濃度 PDGF-BB(0 ng/ml、5 ng/ml、20 ng/ml、50 ng/ml、100 ng/ml)對人類 CFU-MK的作用。第二部分PDGF作用于骨髓間充質(zhì)細(xì)胞調(diào)控TPO產(chǎn)生1.CCK-8法檢測PDGF對人MSCs的增殖作用;2.Annexin-V/PI染色法檢測PDGF對人MSCs凋亡的影響;3.流式細(xì)胞術(shù)檢測人MSCs細(xì)胞表面PDGFR-β的表達(dá);4.Western blot檢測PDGF-BB對骨髓間充質(zhì)細(xì)胞MAPK/ERK、STAT信號通路的影響;5.Q-PCR 檢測 PDGF-BB 對人 MSCs TPO mRNA 表達(dá)水平。研究結(jié)果第一部分PDGF-BB促進(jìn)巨核細(xì)胞生長一、PDGF-BB對小鼠CFU-MK集落形成的影響1.PDGF-BB以劑量依賴的方式促進(jìn)小鼠CFU-MK的形成,其中50 ng/ml作用最為明顯(n=5,P0.001);2.PDGF-BB 對 CFU-MK 的促增殖作用比 GM-CSF 強(n=5,P =0.0002),但低于 IL-3、IL-6 和 TPO(n=5,P0.05);3.PDGF-BB+IL-3 或 PDGF-BB+IL-6 與單獨使用 IL-3 或IL-6 無明顯差異(n=5,P0.05),但是IL-3+IL-6對CFU-MK的增殖具有明顯的協(xié)同作用,是單獨作用的2倍(n=5,P0.00001);4.PDGF-BB+IL-3 MoAb組與單用PDGF-BB組相比,CFU-MK計數(shù)無顯著差異(n=4,P=0.127);而 PDGF-BB+IL-6MoAb 組的 CFU-MK 數(shù)量減少了53.8%(n=4,P=0.003);PDGF-BB+TPOMoAb 組減少了 51.2%(n=4,P=0.002)。二、PDGF-BB對人CFU-MK形成的作用PDGF-BB以劑量依賴的方式促進(jìn)人CFU-MK的增殖,其中50 ng/ml作用最為明顯(n=4,P0.001)。第二部分PDGF作用于骨毮間充質(zhì)細(xì)胞調(diào)控TPO產(chǎn)生間接促進(jìn)骨髓造血生成1.PDGF-BB促進(jìn)人MSCs的增殖,最適濃度為50 ng/ml(n=8,P=0.0027);2.PDGF-BB能夠顯著減少MSCs的凋亡(n=5,P=0.00004);3.Q-PCR結(jié)果顯示,MSCs高表達(dá)PDGFR-β mRNA;流式結(jié)果顯示,MSCs表達(dá)PDGFR-β;4.Western blot結(jié)果顯示,PDGF-BB處理組的骨髓間充質(zhì)細(xì)胞P-ERK、P-STAT3蛋白表達(dá)增加;5.PDGF-BB 處理 MSCs 后,TPO mRNA 表達(dá)水平顯著增高(n=7,P=0.021)。研究結(jié)論1.PDGF-BB可以顯著促進(jìn)小鼠和人CFU-MK的形成;2.PDGF-BB對MSCs有促增殖和抗凋亡作用,MSCs高表達(dá)PDGFR-β,其與PDGF-BB結(jié)合后激活下游MAPK/ERK、STAT信號通路;3.PDGF-BB作用于骨髓微環(huán)境中的骨髓間充質(zhì)細(xì)胞,在轉(zhuǎn)錄水平促進(jìn)TPO產(chǎn)生,間接促進(jìn)巨核系造血。
[Abstract]:The research background and research purpose of platelet derived growth factor (PDGF) is a polypeptide growth factor, which is stored in the.PDGF granule family has 5 different two mer, PDGF-AA, AB, BB, CC and DD, the PDGF by binding receptor alpha and beta receptor (.PDGF receptor exerts its biological functions PDGFR) is a transmembrane glycoprotein composed of alpha and beta two subunit protein tyrosine kinase, a protein tyrosine kinase activity. The study found that high affinity binding of PDGF-BB and two kinds of receptors, proliferation is stronger than the other subtypes of.PDGF-BB can promote a variety of connective tissue cells such as fibroblasts, endothelial cells and smooth muscle cell growth and differentiation. In addition, PDGF-BB also plays an important role in hematopoiesis, such as promoting the erythroid burst forming unit (BFU-E) and myeloid erythroid - monocyte megakaryocyte colony forming unit (CFU-GEMM) of the form . however, the specific mechanism of PDGF-BB in hematopoiesis is not clear, and may be generated in the microenvironment of bone marrow mesenchymal stem cells TPO. Therefore, this study aims to explore the role of PDGF-BB on the formation of CFU-MK, and IL-3, IL-6, TPO and GM-CSF were compared and the effect, adding anti IL-3, CFU-MK, IL-6 or TPO effects on the formation of monoclonal antibody. After further explore the mechanism of PDGF-BB in promoting megakaryocytopoiesis: the effect of PDGF on bone marrow mesenchymal cell surface PDGF receptor, activation of downstream related transcription factor TPO, thus indirectly regulate bone marrow megakaryocytopoiesis. The research contents and methods the first part of PDGF-BB promoting megakaryocytopoiesis, PDGF-BB on mouse CFU-MK colony forming PDGF-BB 1. detection of different concentrations (0ng/ml, 5ng/ml, 10ng/ml, 20ng/ml, 50ng/ml, 100ng/ml) influence on the formation of mouse CFU-MK; 2. Detection of different cytokines (PDGF-BB, IL-3, IL-6, GM-CSF and TPO) on mice of different combinations of CFU-MK and IL-6; 3. joined the anti IL-3 TPO antibody, or after PDGF-BB on mice CFU-MK formation. The effects of two PDGF-BB on CFU-MK colony formation test of different concentrations of PDGF-BB (0 ng/ml, 5 ng/ml, 20 ng/ml, 50 ng/ml, 100 ng/ml) of human CFU-MK. The second part of the PDGF effect on bone marrow mesenchymal cells to produce 1.CCK-8 TPO control method was used to detect PDGF on MSCs proliferation; effect of detection of PDGF 2.Annexin-V/PI staining on MSCs apoptosis; detect the expression of human MSCs cell surface PDGFR- beta 3. flow cytometry FCM; 4.Western blot detection of PDGF-BB of bone marrow mesenchymal stem cells MAPK/ERK, STAT signaling pathway; the level of 5.Q-PCR to detect the expression of PDGF-BB on MSCs TPO mRNA. The research results of the first part of PDGF-BB to promote megakaryocyte growth, P DGF-BB set 1.PDGF-BB in a dose dependent manner of mice CFU-MK promote the formation of CFU-MK of mice, of which 50 ng/ml the most obvious function (n=5, P0.001); the proliferation effect of 2.PDGF-BB on CFU-MK GM-CSF (n=5, P, =0.0002) but lower than IL-3, IL-6 and TPO (n=5, P0.05); 3.PDGF-BB+IL-3 or PDGF-BB+IL-6 and IL-3 alone or IL-6 showed no significant difference (n=5, P0.05), but IL-3+IL-6 on the proliferation of CFU-MK have obvious synergistic effect, is 2 times the individual effect (n=5, P0.00001); 4.PDGF-BB+IL-3 MoAb group compared with PDGF-BB group, there was no significant difference between CFU-MK count (n=4, P=0.127); and the number of CFU-MK in PDGF-BB+IL-6MoAb group was reduced by 53.8% (n=4, P=0.003); group PDGF-BB+TPOMoAb decreased 51.2% (n=4, P=0.002). Two effects of PDGF-BB PDGF-BB on CFU-MK formation in a dose-dependent manner promote the proliferation of CFU-MK, of which 5 0 ng/ml the most obvious function (n=4, P0.001). In the second part, the effect of PDGF on the regulation of mesenchymal cells produce TPO bone between Sha indirectly promote bone marrow hematopoietic 1.PDGF-BB promote the proliferation of MSCs, the optimal concentration is 50 ng/ml (n=8, P=0.0027); 2.PDGF-BB can significantly reduce the apoptosis of MSCs (n=5, P=0.00004); 3.Q-PCR the results showed that MSCs high expression of PDGFR- beta mRNA; flow cytometry showed that MSCs expression of PDGFR- beta 4.Western; blot results showed that PDGF-BB treatment group and bone marrow mesenchymal stem cells P-ERK, P-STAT3 protein expression was increased; 5.PDGF-BB after MSCs treatment, the expression level of TPO mRNA increased significantly (n=7, P=0.021). Conclusion 1.PDGF-BB can significantly promote the the formation of mouse and human CFU-MK; 2.PDGF-BB proliferation and anti apoptosis effect of MSCs, MSCs high expression of PDGFR- beta and its combination with PDGF-BB after activation of downstream MAPK/ERK, STAT signal pathway; the effect of 3.PDGF-BB on bone marrow microenvironment in Bone marrow mesenchymal cells, at the transcriptional level, promote TPO production and indirectly promote megakaryocyte hematopoiesis.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R331

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本文編號：1721425