本文選題：成骨細胞 + 神經(jīng)細胞��； 參考：《成都中醫(yī)藥大學(xué)》2006年碩士論文

【摘要】：目的 考察大鼠骨膜中的成骨細胞經(jīng)漏蘆總黃酮體外誘導(dǎo)后向神經(jīng)細胞轉(zhuǎn)化的可行性，為臨床中樞和外周神經(jīng)損傷的修復(fù)治療提供新的選擇。 方法 從大鼠脛骨內(nèi)側(cè)獲取骨膜，組織塊培養(yǎng)法得到成骨細胞，經(jīng)鑒定確認后體外純化擴增培養(yǎng)，然后借助MTT(噻唑藍)比色法了解漏蘆總黃酮對體外培養(yǎng)成骨細胞生長的影響，選出合適的誘導(dǎo)濃度，分別加入0.0613％漏蘆總黃酮和2％二甲基亞砜(DMSO)進行誘導(dǎo)，于誘導(dǎo)24h、48h、72h時相差顯微鏡下觀察細胞形態(tài)變化，并于72h時通過逆轉(zhuǎn)錄聚合酶鏈反應(yīng)技術(shù)(RT-PCR法)檢測經(jīng)誘導(dǎo)后的細胞中是否具有神經(jīng)細胞特有的神經(jīng)元烯醇化酶(NSE)和特異性膠質(zhì)纖維酸性蛋白(GFAP)的mRNA表達，以此確定神經(jīng)細胞和神經(jīng)膠質(zhì)細胞的存在。 結(jié)果 細胞毒實驗中，在0.09％到1.92％的濃度范圍內(nèi)，與空白組比較，各濃度漏蘆總黃酮組活細胞吸光度值(optical density，OD)明顯減小(P＜0.01)。折線圖顯示隨著漏蘆總黃酮濃度加大，OD值呈現(xiàn)持續(xù)上升狀態(tài)，對細胞的抑制率呈現(xiàn)下降趨勢。篩選合適誘導(dǎo)濃度(0.0613％)誘導(dǎo)72h后，漏蘆總黃酮組和二甲基亞砜組細胞形態(tài)均發(fā)生了改變，由原來的不規(guī)則三角形變?yōu)橥挥|樣生長；RT-PCR法顯示經(jīng)誘導(dǎo)后的細胞NSE、GFAP的mRNA表達陽性，，且在NSE的mRNA表達上，漏蘆總黃酮組強于二甲基亞砜組，而在GFAP的mRNA表達上，二甲基亞砜組強于漏蘆總黃酮組；空白組細胞在形態(tài)上無明顯變化，NSE和GFAP的mRNA有極微弱表達。 結(jié)論 在0.09％到1.92％的濃度范圍內(nèi)，漏蘆總黃酮對體外培養(yǎng)成骨細胞的生長有一定抑制作用，但呈現(xiàn)濃度越大抑制率越小的趨勢，考慮不僅與漏蘆總黃酮對細胞的毒性作用有關(guān)，也與其對細胞的誘導(dǎo)作用有關(guān)。成骨細胞經(jīng)0.0613％漏蘆總黃酮體外誘導(dǎo)后有向神經(jīng)細胞和神經(jīng)膠質(zhì)細胞轉(zhuǎn)化的可行性，且更傾向于向神經(jīng)細胞轉(zhuǎn)化，因此有望成為神經(jīng)種子細胞的另外一個來源。
[Abstract]:Objective to investigate the feasibility of osteoblast in rat periosteum induced by total flavone in vitro and to provide a new choice for the repair and treatment of central and peripheral nerve injury. Methods Osteoblasts were obtained from the medial tibia of rats, and osteoblasts were obtained by tissue mass culture. After identification, the osteoblasts were purified and cultured in vitro. Then the effect of total flavonoids on the growth of osteoblasts cultured in vitro was investigated by MTT colorimetry. The suitable induction concentration was selected and 0.0613% total flavone and 2% DMSO were added respectively. The morphological changes of the cells were observed under a phase-contrast microscope at 24 h, 48 h or 72 h after induction. At 72 h, the mRNA expression of neuronal enolase (NSE) and specific glial fibrillary acidic protein (GFAP) were detected by reverse transcriptase polymerase chain reaction (RT-PCR). This determines the existence of nerve cells and glial cells. Results in the cytotoxicity test, in the range of 0.09% to 1.92%, compared with the blank group, the absorbance of living cells in the total flavone group decreased significantly (P < 0.01). The curve diagram showed that the OD value increased with the increase of total flavonoids concentration, and the inhibition rate of the cells decreased. After 72 hours of induction, the morphology of the cells in the total flavone group and the dimethyl sulfoxide group changed from irregular triangle to synaptic growth. RT-PCR showed that the mRNA expression of NSEG FAP was positive in the induced cells. In the mRNA expression of NSE, the total flavone group was stronger than that in the dimethyl sulfoxide group, while the mRNA expression in the GFAP group was stronger than that in the GFAP group, and the mRNA expression in the blank group had no obvious change in morphology. Conclusion in the range of 0.09% to 1.92%, the total flavone of Leymus chinensis has a certain inhibitory effect on the growth of osteoblasts cultured in vitro, but the higher the concentration is, the smaller the inhibition rate is, which is related not only to the toxicity of the total flavonoids to the cells, but also to the growth of osteoblasts in vitro. It is also related to its induction to cells. Osteoblasts induced by 0.0613% total flavonoids have the feasibility of transforming into nerve cells and glial cells, and are more inclined to convert to nerve cells, so it is expected to be another source of neural seed cells.
【學(xué)位授予單位】：成都中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R329

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