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  本文關鍵詞： IGF-1 內皮細胞 雪旺細胞 神經元 共培養(yǎng)　出處：《重慶醫(yī)科大學》2011年碩士論文　論文類型：學位論文

【摘要】：目的：觀察胰島素樣生長因子-1 (Insulin-like Growth Factor-1, IGF-1)對受損血管內皮細胞(Vascular Endothelial Cell, VEC)增殖及存活的影響,并對其機制進行初步探討。建立雪旺細胞、神經元、內皮細胞的共培養(yǎng)模型,為外周神經損傷研究打基礎。 方法：用不同濃度(10ng/ml、20ng/ml、40ng/ml、80ng/ml、160ng/ml)的IGF-1作用于血管內皮細胞48h后,通過MTT法檢測細胞的生長情況。采用劃痕實驗觀察IGF-1對受損內皮細胞增殖的影響,并用PI染色觀察細胞情況,ELISA法進行VEGF含量測定。采用組織塊貼壁法、機械消化法分別培養(yǎng)雪旺細胞及神經元原代細胞,內皮細胞采用細胞株,并用S-100、β-tubulin-Ⅲ、vWF分別對其進行免疫學鑒定。用NB、DMEM/F-12混合培養(yǎng)基,根據(jù)生長周期不同先后將雪旺細胞、神經元、內皮細胞接種在培養(yǎng)皿里,37℃培養(yǎng)7d后進行免疫熒光鑒定。 結果：濃度為Ong/ml、20ng/ml、40ng/ml、80ng/ml的IGF-1對血管內皮細胞增殖都有不同程度的促進作用,濃度為40ng/ml、80ng/ml實驗組與未加IGF-1的對照組比較,差異有統(tǒng)計學意義(P 0.05),160ng/ml的IGF-1對細胞的增殖作用下降,差異無統(tǒng)計學意義(P 0.05)。取40ng/ml的IGF-1作用于受損血管內皮細胞48h后,細胞增殖速度較對照組明顯加快,PI染色顯示實驗組死亡細胞明顯減少,實驗組培養(yǎng)液上清VEGF含量較對照組高,差異均有統(tǒng)計學意義(P0.05)。激光共聚焦顯微鏡下可見雪旺細胞呈長梭形,胞核呈卵圓形或長圓形,“海魚狀”排列生長。神經元胞體較小,從胞體長出許多長而細的突起,細胞間突起可連接成網狀。內皮細胞胞體多呈梭形、三角形,團簇存在,胞核清晰,胞漿豐富,細胞生長活躍,成“鋪路石”排列生長。 結論：MTT法觀察細胞增殖程度時發(fā)現(xiàn),10ng/ml、20ng/ml、40ng/ml、80ng/ml的IGF-1對血管內皮細胞的增殖都有不同程度的促進作用(P0.05),160ng/ml的IGF-1對細胞增殖作用下降,差異無統(tǒng)計學意義(P0.05),提示IGF-1在低劑量時對內皮細胞的增殖有促進作用,且表現(xiàn)出一定的量效關系,40ng/ml作用明顯,但高劑量(160ng/ml) IGF-1卻對細胞增殖有抑制作用,具體機制有待進一步探討。根據(jù)IGF-1細胞增殖研究結果,進一步觀察IGF-1對受損內皮細胞存活和增殖的影響。以40ng/ml作為實驗組,在倒置顯微鏡下觀察到同時相點實驗組劃痕空白寬度比對照組明顯縮窄；DAPI/PI雙標記觀察死亡細胞數(shù)目,實驗組死亡細胞數(shù)較對照組少；對其分泌的細胞生長因子VEGF測定,觀察到實驗組經過IGF-1作用后內皮細胞分泌VEGF明顯高于對照組,提示IGF-1可能促進受損內皮細胞的存活、增殖和遷移,以修復內皮細胞的損傷,其機制與其抑制細胞死亡和分泌營養(yǎng)因子VEGF有關,至于該作用具體的信號通路機制尚需以后深入研究。激光共聚焦顯微鏡下顯示雪旺細胞、神經元、內皮細胞模型清晰可見,雪旺細胞為藍色染色細胞,細胞呈長梭形,胞核呈卵圓形或長圓形,海魚狀排列生長。神經元細胞為綠色染色,胞體較小,從胞體長出許多長而細的突起,細胞間突起可連接成網狀。內皮細胞為紅色染色細胞,胞體細胞多呈梭形、三角形,胞核清晰,胞漿豐富,細胞生長活躍,成“鋪路石”排列生長,表明共培養(yǎng)模型構建成功。
[Abstract]:Objective: To observe the effect of insulin-like growth factor -1 (Insulin-like Growth Factor-1, IGF-1) on the damage of vascular endothelial cells (Vascular Endothelial Cell, VEC) proliferation and survival, and to investigate its possible mechanism. The establishment of Schwann cells, neurons, endothelial cell co culture model, laying the foundation for the study of peripheral nerve injury.
Methods: with different concentrations (10ng/ml, 20ng/ml, 40ng/ml, 80ng/ml, 160ng/ml) IGF-1 effects on vascular endothelial cells after 48h, the growth of MTT cells detected by scratch test. The effect of IGF-1 on endothelial cell proliferation, and PI staining cells, ELISA method for determination of VEGF. Using tissue explant method, mechanical digestion method cultured Schwann cells and neurons in primary cells, endothelial cells and cell lines by using S-100, -tubulin-, beta III, vWF respectively on the immunological identification. With NB, DMEM/F-12 mixed medium, according to the different growth periods will have Schwann cells, neurons, endothelial cells inoculated in a culture dish, at 37 7d after culture were identified by immunofluorescence.
Results: the concentration of Ong/ml, 20ng/ml, 40ng/ml, 80ng/ml and IGF-1 have different effect on the proliferation of vascular endothelial cells, the concentration of 40ng/ml, compared to control group, 80ng/ml group and without IGF-1, the difference was statistically significant (P 0.05), decreased the proliferation of 160ng/ml IGF-1 cells, no significant difference the significance of 40ng/ml (P 0.05). The effects of IGF-1 on damaged vascular endothelial cells after 48h cell proliferation rate compared with the control group increased significantly, PI staining showed that the experimental group significantly reduced cell death in the experimental group, the supernatant VEGF content was higher than the control group, the differences were statistically significant (P0.05). Laser scanning confocal microscope the Schwann cells were fusiform, nuclei were ovoid or oblong, "fish shape arrangement growth. Neurons were small, growing from the cell bodies of many long and thin neurites, cell processes could be connected into the network. The cells of the skin cell are mostly spindle, triangular, cluster, clear, rich in cytoplasm and active in cell growth, and grow up as "pave stone".
Conclusion: MTT was used to observe the proliferation of 10ng/ml, 20ng/ml, 40ng/ml, 80ng/ml, IGF-1 on the proliferation of vascular endothelial cells have different degrees of promotion (P0.05), decreased effect of 160ng/ml IGF-1 on cell proliferation, there was no statistically significant difference (P0.05), suggesting that IGF-1 at low doses on endothelial cells the proliferation promoting effect, and showed a dose-response relationship, the 40ng/ml effect is obvious, but the high dose (160ng/ml) IGF-1 has inhibitory effect on cell proliferation, the specific mechanism needs to be further discussed. According to the result of IGF-1 cell proliferation, further observe the effect of IGF-1 on endothelial cell survival and proliferation. Using 40ng/ml as the experimental group in, observed under the inverted microscope at the same time point blank width of the experimental group was shorter than the control group; DAPI/PI double marker to observe the number of dead cells, the number of dead cells in experimental group than in the control The group is less; the secretion of cell growth factor VEGF were observed in the experimental group after VEGF of endothelial cells after IGF-1 treatment was significantly higher than the control group, suggesting that IGF-1 may promote the survival of injured endothelial cells, proliferation and migration, in order to repair the injury of endothelial cells, and its mechanism is related to the inhibition of cell death and the secretion of neurotrophic factor VEGF as for the future, the role of specific signal transduction mechanism remains to be further studied. Laser confocal microscopy showed that Schwann cells, neurons, endothelial cell model is clearly visible, Schwann cells for blue stained cells, cells were fusiform, nuclei were ovoid or oblong, arranged in the form of fish growth. Neurons for green staining. Smaller cell body, growing from the cell bodies of many long and thin neurites, cell protrusions can be connected into the network. Endothelial cells were red stained cells, somatic cells were spindle shaped, triangular, cell The nucleus is clear, the cytoplasm is rich, the cell growth is active, and the "paving stone" is arranged and grown, which indicates that the co culture model is constructed successfully.

【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R363

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