CAJ | 학술논문

背景:许旺细胞分泌多种神经营养因子在神经再生中发挥关键作用,但其分泌能力受诸多因素影响,寻找可行方法促进许旺细胞分泌神经生长因子是神经缺损后再生的重要环节.目的:观察不同浓度甲泼尼龙琥珀酸钠对许旺细胞分泌功能的影响.方法:酶消化法分离培养SD乳鼠许旺细胞,倒置相差显微镜下观察细胞生长情况;传代后,一部分进行S-100蛋白免疫鉴定许旺细胞的纯度;另一部分用细胞计数板调整细胞浓度为1×10~9 L~(-1),移入到6孔平底培养板(接种15个孔)继续培养.培养板中培养4 d后4个孔分别加入不同浓度的甲泼尼龙琥珀酸钠(10~(-3),10~(-4),10~(-6),10~(-8) mol/L),并设立1个孔为不加药物的空白对照组,分别作用于细胞24,48,72 h后行RT-PCR检测,观察许旺细胞24,48,72 h神经生长因子mRNA水平的变化.结果与结论:原代细胞培养至第7天数量明显增加,细胞铺满培养瓶底部80%以上;传代细胞形态为梭形,有2个细长的突起,荧光染色阳性,成纤维细胞为圆形或扁圆形,荧光染色阴性.RT-PCR检测结果显示,10~(-8) mol/L的甲基强地松龙作用72 h分泌的神经生长因子与空白对照组及其他浓度、时间组相比均表达增加(P < 0.05);10~(-3) mol/L甲基强地松龙在各时间段分泌的神经生长因子与空白对照组及其他浓度、时间组相比表达均减少(P < 0.05).提示高浓度的甲泼尼龙琥珀酸钠抑制许旺细胞分泌神经生长因子,长时间、低浓度的甲泼尼龙琥珀酸钠则促进许旺细胞分泌神经生长因子.
배경:허왕세포분비다충신경영양인자재신경재생중발휘관건작용,단기분비능력수제다인소영향,심조가행방법촉진허왕세포분비신경생장인자시신경결손후재생적중요배절.목적:관찰불동농도갑발니룡호박산납대허왕세포분비공능적영향.방법:매소화법분리배양SD유서허왕세포,도치상차현미경하관찰세포생장정황;전대후,일부분진행S-100단백면역감정허왕세포적순도;령일부분용세포계수판조정세포농도위1×10~9 L~(-1),이입도6공평저배양판(접충15개공)계속배양.배양판중배양4 d후4개공분별가입불동농도적갑발니룡호박산납(10~(-3),10~(-4),10~(-6),10~(-8) mol/L),병설립1개공위불가약물적공백대조조,분별작용우세포24,48,72 h후행RT-PCR검측,관찰허왕세포24,48,72 h신경생장인자mRNA수평적변화.결과여결론:원대세포배양지제7천수량명현증가,세포포만배양병저부80%이상;전대세포형태위사형,유2개세장적돌기,형광염색양성,성섬유세포위원형혹편원형,형광염색음성.RT-PCR검측결과현시,10~(-8) mol/L적갑기강지송룡작용72 h분비적신경생장인자여공백대조조급기타농도、시간조상비균표체증가(P < 0.05);10~(-3) mol/L갑기강지송룡재각시간단분비적신경생장인자여공백대조조급기타농도、시간조상비표체균감소(P < 0.05).제시고농도적갑발니룡호박산납억제허왕세포분비신경생장인자,장시간、저농도적갑발니룡호박산납칙촉진허왕세포분비신경생장인자.
BACKGROUND: Secretion of various neurotrophic factors by Schwann cells plays important roles in neural regeneration. However, the secretion capability is affected by many factors. To seek a feasible method for promoting nerve growth factor secretion by Schwann cells is a key of regeneraion following neurologic defect.OBJECTIVE: To explore the effects of methylprednisolone(solu-medrol) on the secreted function of Schwann cells of cultured rats.METHODS: Schwann cells were isolated and cultured by enzyme digestion method. Cell growth was observed under an inverted phase contrast microscope. Following passage, purity of some Schwann cells was identified using S-100 protein immunity. Other Schwann cells were regulated using cell counting plate into 1×10~9/L, and incubated in a 6-well culture plate (15 wells) for further incubation. Following 4 days of culture, different concentrations of solu-medrol (10~(-3), 10~(-4), 10~(-6), 10~(-8) mol/L) were administrated to the cell, while blank control group (1 well) was given no drug. 24, 48 and 72 hours after administration, reverse trancription-polymerase chain reaction (RT-PCR) was used in the detection of the levels of nerve growth factor mRNA.RESULTS AND CONCLUSION: Number of primarily cultured cells was significantly increased at day 7, and 80% cells were confluent. Subcultured cells were spindle-shaped, with 2 thin long processes, showing positive fluorescence staining. Fibroblasts were round or flat, showing negative reaction of fluorescence staining. Reserve transcription-polymerase chain reaction demonstrated that nerve growth factor number at 72 hours affected by 10~(-8) mol/L radiosone was increased compared with the blank control group and other concentrations and other time points (P < 0.05). Number of nerve growth factor was reduced following treatment of 10~(-3) mol/L radiosone compared with the blank control group and other concentrations (P < 0.05). These results suggested that high concentration of solu-medrol prohibits secreted function of Schwann's cells, but long time and low dosage solu-medrol promotes secreted function of Schwann's cells.