本文選題：雪旺細(xì)胞高葡萄糖　切入點：S-100細(xì)胞培養(yǎng)　出處：《昆明醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：第一部分雪旺細(xì)胞培養(yǎng)方法建立及高糖的作用 目的：建立原代雪旺細(xì)胞培養(yǎng)模型,并觀察高糖狀態(tài)下雪旺細(xì)胞的生長規(guī)律,測量雪旺細(xì)胞面積,突起長度和細(xì)胞增殖活力。為高糖環(huán)境對雪旺細(xì)胞影響分子機制研究提供可用的模型。 方法：取新生1-3天的SPF級SD大鼠10只,斷頭處死后無菌切取雙側(cè)坐骨神經(jīng),剝離神經(jīng)外膜,取神經(jīng)剪碎,用低濃度雙酶消化法消化神經(jīng)制成細(xì)胞懸液,接種于6孔培養(yǎng)板。2小時差速貼壁后取上清轉(zhuǎn)入另一培養(yǎng)板,加阿糖胞苷抑制成纖維細(xì)胞生長,傳代培養(yǎng),第三代后將細(xì)胞分為兩組,一組應(yīng)用5.5mmol/L的葡萄糖,另一組應(yīng)用25mmol/L的葡萄糖分別進行培養(yǎng)。觀察兩組雪旺細(xì)胞的細(xì)胞突起,細(xì)胞面積及增殖能力。用雪旺細(xì)胞標(biāo)記物S-100抗體鑒定細(xì)胞。 結(jié)果：1、應(yīng)用差速貼壁法、酶消化法和化學(xué)法可以得到高純度的雪旺細(xì)胞。建立了雪旺細(xì)胞原代培養(yǎng)模型。 2、分析不同糖濃度對雪旺細(xì)胞胞生長的影響,結(jié)果顯示,與低糖培養(yǎng)組相比,在高糖培養(yǎng)條件下原代的雪旺細(xì)胞胞體面積減小,突起長度變長,增殖活性變大。 結(jié)論：1、應(yīng)用雙酶消化,差速貼壁及阿糖胞苷法可以得到純度比較高的雪旺細(xì)胞。 2、雪旺細(xì)胞在高糖培養(yǎng)環(huán)境下,細(xì)胞突起變長,細(xì)胞面積減小,增殖活性變大。說明高糖明顯影響體外雪旺細(xì)胞生長。本細(xì)胞模型可用于高糖環(huán)境對雪旺細(xì)胞影響的分子機制研究。 第二部分高糖對培養(yǎng)雪旺細(xì)胞POU3F3基因的影響及其對DLL1分子的調(diào)控 目的：分析高糖對培養(yǎng)雪旺細(xì)胞POU3F3基因的影響,觀察其在高糖作用的SC中的表達(dá)變化規(guī)律,探討POU3F3對DLL1的調(diào)控及其對細(xì)胞的影響。 方法：高糖條件培養(yǎng)SC48小時,獲取高糖組(25mmol/L葡萄糖)和對照組(5.5mmol/L葡萄糖)細(xì)胞,利用RT-PCR技術(shù)擴增各組BDNF, CNTF, EGF, GMF, IGF-1, EGR-2, MMP9, NF-KB,NGF,NRG-1, POU3F3, SLC2al,SOX-10,TrkA,TrkB基因。挑出其中有統(tǒng)計學(xué)差異的基因進行功能研究。本實驗確定POU3F3為目的基因,化學(xué)合成該基因siRNA干擾片段,用脂質(zhì)體轉(zhuǎn)染法將POU3F3siRNA干擾片段導(dǎo)入雪旺細(xì)胞株RSC96,觀察干擾前、后RSC96細(xì)胞株的細(xì)胞突起,大小。 結(jié)果： 1、RT-PCR結(jié)果顯示在被檢測的15個基因中,與對照組(5.5mmol/L葡萄糖)相比,僅POU3F3基因在高糖(25mmol/L葡萄糖)環(huán)境下培養(yǎng)48h時,其mRNA表達(dá)明顯升高,P0.05,差異有統(tǒng)計學(xué)意義,其余因子均未見明顯變化,差異無統(tǒng)計學(xué)意義,P0.05；所有因子高糖(25mmol/L葡萄糖)培養(yǎng)96小時后mRNA表達(dá)與對照組(5.5mmol/L葡萄糖)相比P0.05,差異無統(tǒng)計學(xué)意義。 2、利用生物信息學(xué)查詢發(fā)現(xiàn)POU3F3與DLL1密切相關(guān),查詢資料發(fā)現(xiàn)POU3F3可能激活DLL1轉(zhuǎn)錄。 3、利用RT-PCR擴增DLL1基因,觀察其mRNA的表達(dá),結(jié)果顯示SC在高糖條件下培養(yǎng)48小時,與對照組(5.5mmol/L葡萄糖)相比,DLL1mRNA表達(dá)增加3.4倍,P0.05,差異有統(tǒng)計學(xué)意義；培養(yǎng)96小時表達(dá)增加1.5倍,P0.05,差異無統(tǒng)計學(xué)意義。 4、化學(xué)合成POU3F3siRNA干擾片段,利用脂質(zhì)體轉(zhuǎn)染的方式將片段導(dǎo)入雪旺細(xì)胞株RSC96,結(jié)果顯示,與陰性對照組相比,將POU3F3基因干擾24小時對細(xì)胞突起長度,細(xì)胞面積均無影響,P0.05；干擾48小時后,細(xì)胞突起長度無統(tǒng)計學(xué)差異,P0.05,而干擾組細(xì)胞面積明顯減小,P0.05。陰性對照組及只加了轉(zhuǎn)染試劑組細(xì)胞面積比正常組面積減小,P0.05。 5、POU3F3與DLL1表達(dá)正相關(guān),相關(guān)系數(shù)r=0.999,P0.05。 結(jié)論： 1、高糖培養(yǎng)SC,檢測了15個因子的mRNA,結(jié)果顯示僅POU3F3mRNA升高。提示該基因在高糖狀態(tài)下的雪旺細(xì)胞中起著十分重要的作用。 2、化學(xué)合成POU3F3的siRNA干擾片段,用脂質(zhì)體轉(zhuǎn)染的方式將片段導(dǎo)入雪旺細(xì)胞株RSC96后發(fā)現(xiàn)細(xì)胞突起長度無變化,而細(xì)胞面積減小。表明POU3F3可能因影響雪旺細(xì)胞的體積而影響雪旺細(xì)胞形成髓鞘。 3、POU3F3可以調(diào)控DLL1的表達(dá)。在高糖環(huán)境下,POU3F3的升高導(dǎo)致DLL1伴隨升高。
[Abstract]:The first part of the culture of Schwann cells and the effect of high sugar
Objective: to establish a primary Schwann cell culture model, observe the growth rule of Schwann cells in high glucose state, measure the area of Schwann cells, the length of neurite and cell proliferation activity, and provide an available model for studying the molecular mechanism of Schwann cell influence in high glucose environment.
Methods: the newborn 1-3 days in SPF SD 10 rats were decapitated after aseptic cut bilateral sciatic nerve, nerve membrane peeling, the nerve was cut, with a low concentration of double enzyme digestion of nerve cell suspension were seeded in 6 well plates.2 hours after differential adhesion to the supernatant in another culture plate, ara2c inhibited fibroblast growth, subculture, after third generations of cells were divided into two groups, one group with 5.5mmol/L glucose, another group using 25mmol/L glucose were cultured. Cell protrusions Schwann cells were observed in the two groups, the area and the cell proliferation ability. With antibody identification cells of Schwann cell marker S-100.
Results: 1, high purity Schwann cells could be obtained by differential adherence method, enzyme digestion and chemical method. The primary culture model of Schwann cells was established.
2, the effects of different sugar concentrations on the growth of Schwann cells were analyzed. The results showed that compared with the low sugar culture group, the cell area of primary Schwann cells decreased, the length of neurite grew longer and the proliferative activity increased.
Conclusion: 1, the high purity Schwann cells can be obtained by double enzyme digestion, differential adherence and cytarabine method.
2, Schwann cells in the high glucose environment, the cell protuberance increased, the cell area decreased, and the proliferative activity increased. This indicates that high glucose significantly affects the growth of Schwann cells in vitro. This cell model can be used to study the molecular mechanism of Schwann cells in high glucose environment.
The effect of high sugar on the culture of POU3F3 gene of Schwann cells in the second part and its regulation on DLL1
Objective: to analyze the effect of high glucose on the expression of POU3F3 gene in Schwann cells, observe its expression and change rule in high glucose SC, and explore the regulation of POU3F3 on DLL1 and its effect on cells.
Methods: high glucose cultured SC48 hours, to obtain the high glucose group (25mmol/L glucose) and control group (5.5mmol/L glucose) cells, BDNF was amplified by RT-PCR technology, CNTF, EGF, GMF, IGF-1, EGR-2, MMP9, NF-KB, NGF, NRG-1, POU3F3, SLC2al, SOX-10, TrkA, TrkB gene. Pick one function study on difference gene. POU3F3 gene was identified in this experiment, the chemical synthesis of the gene siRNA interference fragment by liposome transfection POU3F3siRNA interference fragment into Schwann cell line RSC96, observe the interference, cell processes, RSC96 cell line size.
Result錛，

本文編號：1640993