本文關(guān)鍵詞： IGF-1 內(nèi)皮細(xì)胞 雪旺細(xì)胞 神經(jīng)元 共培養(yǎng)　出處：《重慶醫(yī)科大學(xué)》2011年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的：觀察胰島素樣生長(zhǎng)因子-1 (Insulin-like Growth Factor-1, IGF-1)對(duì)受損血管內(nèi)皮細(xì)胞(Vascular Endothelial Cell, VEC)增殖及存活的影響,并對(duì)其機(jī)制進(jìn)行初步探討。建立雪旺細(xì)胞、神經(jīng)元、內(nèi)皮細(xì)胞的共培養(yǎng)模型,為外周神經(jīng)損傷研究打基礎(chǔ)。 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(nèi)皮細(xì)胞48h后,通過(guò)MTT法檢測(cè)細(xì)胞的生長(zhǎng)情況。采用劃痕實(shí)驗(yàn)觀察IGF-1對(duì)受損內(nèi)皮細(xì)胞增殖的影響,并用PI染色觀察細(xì)胞情況,ELISA法進(jìn)行VEGF含量測(cè)定。采用組織塊貼壁法、機(jī)械消化法分別培養(yǎng)雪旺細(xì)胞及神經(jīng)元原代細(xì)胞,內(nèi)皮細(xì)胞采用細(xì)胞株,并用S-100、β-tubulin-Ⅲ、vWF分別對(duì)其進(jìn)行免疫學(xué)鑒定。用NB、DMEM/F-12混合培養(yǎng)基,根據(jù)生長(zhǎng)周期不同先后將雪旺細(xì)胞、神經(jīng)元、內(nèi)皮細(xì)胞接種在培養(yǎng)皿里,37℃培養(yǎng)7d后進(jìn)行免疫熒光鑒定。 結(jié)果：濃度為Ong/ml、20ng/ml、40ng/ml、80ng/ml的IGF-1對(duì)血管內(nèi)皮細(xì)胞增殖都有不同程度的促進(jìn)作用,濃度為40ng/ml、80ng/ml實(shí)驗(yàn)組與未加IGF-1的對(duì)照組比較,差異有統(tǒng)計(jì)學(xué)意義(P 0.05),160ng/ml的IGF-1對(duì)細(xì)胞的增殖作用下降,差異無(wú)統(tǒng)計(jì)學(xué)意義(P 0.05)。取40ng/ml的IGF-1作用于受損血管內(nèi)皮細(xì)胞48h后,細(xì)胞增殖速度較對(duì)照組明顯加快,PI染色顯示實(shí)驗(yàn)組死亡細(xì)胞明顯減少,實(shí)驗(yàn)組培養(yǎng)液上清VEGF含量較對(duì)照組高,差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。激光共聚焦顯微鏡下可見(jiàn)雪旺細(xì)胞呈長(zhǎng)梭形,胞核呈卵圓形或長(zhǎng)圓形,“海魚(yú)狀”排列生長(zhǎng)。神經(jīng)元胞體較小,從胞體長(zhǎng)出許多長(zhǎng)而細(xì)的突起,細(xì)胞間突起可連接成網(wǎng)狀。內(nèi)皮細(xì)胞胞體多呈梭形、三角形,團(tuán)簇存在,胞核清晰,胞漿豐富,細(xì)胞生長(zhǎng)活躍,成“鋪路石”排列生長(zhǎng)。 結(jié)論：MTT法觀察細(xì)胞增殖程度時(shí)發(fā)現(xiàn),10ng/ml、20ng/ml、40ng/ml、80ng/ml的IGF-1對(duì)血管內(nèi)皮細(xì)胞的增殖都有不同程度的促進(jìn)作用(P0.05),160ng/ml的IGF-1對(duì)細(xì)胞增殖作用下降,差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05),提示IGF-1在低劑量時(shí)對(duì)內(nèi)皮細(xì)胞的增殖有促進(jìn)作用,且表現(xiàn)出一定的量效關(guān)系,40ng/ml作用明顯,但高劑量(160ng/ml) IGF-1卻對(duì)細(xì)胞增殖有抑制作用,具體機(jī)制有待進(jìn)一步探討。根據(jù)IGF-1細(xì)胞增殖研究結(jié)果,進(jìn)一步觀察IGF-1對(duì)受損內(nèi)皮細(xì)胞存活和增殖的影響。以40ng/ml作為實(shí)驗(yàn)組,在倒置顯微鏡下觀察到同時(shí)相點(diǎn)實(shí)驗(yàn)組劃痕空白寬度比對(duì)照組明顯縮窄；DAPI/PI雙標(biāo)記觀察死亡細(xì)胞數(shù)目,實(shí)驗(yàn)組死亡細(xì)胞數(shù)較對(duì)照組少；對(duì)其分泌的細(xì)胞生長(zhǎng)因子VEGF測(cè)定,觀察到實(shí)驗(yàn)組經(jīng)過(guò)IGF-1作用后內(nèi)皮細(xì)胞分泌VEGF明顯高于對(duì)照組,提示IGF-1可能促進(jìn)受損內(nèi)皮細(xì)胞的存活、增殖和遷移,以修復(fù)內(nèi)皮細(xì)胞的損傷,其機(jī)制與其抑制細(xì)胞死亡和分泌營(yíng)養(yǎng)因子VEGF有關(guān),至于該作用具體的信號(hào)通路機(jī)制尚需以后深入研究。激光共聚焦顯微鏡下顯示雪旺細(xì)胞、神經(jīng)元、內(nèi)皮細(xì)胞模型清晰可見(jiàn),雪旺細(xì)胞為藍(lán)色染色細(xì)胞,細(xì)胞呈長(zhǎng)梭形,胞核呈卵圓形或長(zhǎng)圓形,海魚(yú)狀排列生長(zhǎng)。神經(jīng)元細(xì)胞為綠色染色,胞體較小,從胞體長(zhǎng)出許多長(zhǎng)而細(xì)的突起,細(xì)胞間突起可連接成網(wǎng)狀。內(nèi)皮細(xì)胞為紅色染色細(xì)胞,胞體細(xì)胞多呈梭形、三角形,胞核清晰,胞漿豐富,細(xì)胞生長(zhǎng)活躍,成“鋪路石”排列生長(zhǎng),表明共培養(yǎng)模型構(gòu)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(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R363

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 陳曉東;神經(jīng)再生與胰島素樣生長(zhǎng)因子[J];國(guó)外醫(yī)學(xué)(創(chuàng)傷與外科基本問(wèn)題分冊(cè));1998年01期

2 瞿海龍;陳曉春;;大鼠胸主動(dòng)脈內(nèi)皮細(xì)胞培養(yǎng)方法的改進(jìn)[J];內(nèi)蒙古醫(yī)學(xué)院學(xué)報(bào);2007年03期

3 李紅;陳曉蓉;趙正梅;思盼盼;張金秋;湯俊峰;;胚鼠大腦皮層神經(jīng)元培養(yǎng)方法的改進(jìn)[J];四川解剖學(xué)雜志;2009年02期

4 白慧云;穆祥;丁庫(kù)克;李莉;郭洋;董小黎;;大鼠腦微血管內(nèi)皮細(xì)胞的培養(yǎng)[J];微循環(huán)學(xué)雜志;2011年01期

，

本文編號(hào)：1552291