SD大鼠颌下腺放射性损伤细胞模型的建立及中药乌梅、甘草配伍的修复作用
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摘要
【目的】建立SD大鼠颌下腺(SMG)放射性损伤细胞模型,观察中药乌梅、甘草配伍对SMG模型细胞增殖的影响。【方法】原代培养SMG细胞经。经免疫荧光鉴定、绘制生长曲线后,分别给予SMG细胞不同剂量(0.1、0.2、1、2、4 Gy)的X电子射线一次性照射,再分别给予浓度梯度为500、50、5、0.5μg/m L的乌梅甘草溶液培养,采取四甲基偶氮唑蓝(MTT)法检测不同剂量X电子射线照射后SMG细胞存活率及不同浓度药物作用24 h后模型细胞的增殖活力。【结果】随着X电子射线剂量的增加,SMG细胞存活率逐渐降低。当中药乌梅甘草溶液浓度介于50~0.5μg/m L时,随着给药浓度升高,细胞增殖率呈上升趋势,具有剂量依赖性;但当给药浓度为500μg/m L时,细胞增殖率下降。【结论】一次性照射剂量为0.1 Gy,能量为9 Mev,剂量率为3 Gy/min,跳数为12 MU,源皮距(SSD)=100 cm的造模条件适用于建立放射性损伤SMG细胞模型;按照3∶1配伍的乌梅和甘草溶液浓度为50μg/m L时,对模型细胞具有明显的修复作用。
Objective To establish a submandibular gland(SMG)cell model of radiation injury,and to observe the effect of compatibility of Fructus Mume(Wumei)and Radix Glycyrrhizae(Gancao)on the proliferation of the modeled cells. Methods SMG cells were primarily cultured in vitro for the experiment. After immunofluorescence identification and growth curve analysis,SMG cells were given one-time exposure to X electron-ray at dose of0.1,0.2,1,2,4 Gy respectively,and then were cultured with Wumei-Gancao solution at the concentration of500,50,5,0.5 μg/m L separately. Methyl thiazolyl tetrazolium(MTT)was used to determine the survival rate of SMG cells exposed to different doses of X electron-ray and proliferation vitality of SMG cells treated with different concentrations of Wumei-Gancao solution for 24 h. Results The survival rate of SMG cells was decreased gradually with the increase of X electron-ray dose. When the concentrations of Wumei-Gancao solution were in the range of 50 ~ 0.5 μg/m L,the cell proliferation rate showed an increasing trend with the increase of drug concentration, the effect being dose-dependent. When the solution concentration arrived to 500 μ g/m L,the cell proliferation rate was declined. Conclusion The SMG cell model of radiation injury has been established successfully under the conditions of 0.1 Gy of one-time exposure, 9 Mev of energy at dose rate 3 Gy/min,hop count at 12 MU and source-skin distance being 100 cm. The concentration of the solution with the compatibitity ratio of Wumei to Gancao being 3 ∶ 1 has certain effects on repairing the modeled cells.
Objective To establish a submandibular gland(SMG)cell model of radiation injury,and to observe the effect of compatibility of Fructus Mume(Wumei)and Radix Glycyrrhizae(Gancao)on the proliferation of the modeled cells. Methods SMG cells were primarily cultured in vitro for the experiment. After immunofluorescence identification and growth curve analysis,SMG cells were given one-time exposure to X electron-ray at dose of0.1,0.2,1,2,4 Gy respectively,and then were cultured with Wumei-Gancao solution at the concentration of500,50,5,0.5 μg/m L separately. Methyl thiazolyl tetrazolium(MTT)was used to determine the survival rate of SMG cells exposed to different doses of X electron-ray and proliferation vitality of SMG cells treated with different concentrations of Wumei-Gancao solution for 24 h. Results The survival rate of SMG cells was decreased gradually with the increase of X electron-ray dose. When the concentrations of Wumei-Gancao solution were in the range of 50 ~ 0.5 μg/m L,the cell proliferation rate showed an increasing trend with the increase of drug concentration, the effect being dose-dependent. When the solution concentration arrived to 500 μ g/m L,the cell proliferation rate was declined. Conclusion The SMG cell model of radiation injury has been established successfully under the conditions of 0.1 Gy of one-time exposure, 9 Mev of energy at dose rate 3 Gy/min,hop count at 12 MU and source-skin distance being 100 cm. The concentration of the solution with the compatibitity ratio of Wumei to Gancao being 3 ∶ 1 has certain effects on repairing the modeled cells.
引文
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[13]赵琼,徐世军,李秀亮,等.加味人参乌梅汤对Caco-2细胞脱水模型AQP4基因表达的影响[J].中药与临床,2010,1(3):36.
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[15]刘言广.水通道蛋白AQP1/AQP5在双峰驼唾液腺的表达研究[D].兰州:兰州大学,2016.
[16]李志民.唾液腺辐射损伤发生分子机制与预防的研究[D].长春:吉林大学,2004.
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[18]刘燕.津血源增加口干症模型大鼠唾液分泌机制的实验研究[D].南京:南京中医药大学,2014.
[2]傅辰春,王晓萍.放射性口腔干燥症的防治及进展[J].中国肿瘤临床与康复,2008,15(5):475.
[3]曹兴午.颌下腺—睾丸轴[J].中国男科学杂志,2008,22(9):62.
[4]成梦.大鼠颌下腺上皮细胞中MRN复合体在不同剂量60Coγ电子射线照射后表达的变化[D].南宁:广西医科大学,2014.
[5]高洋,柯学平,杨建荣.大鼠颌下腺放射性损伤的细胞学模型的建立[J].口腔生物医学,2010,1(3):140.
[6]韩石.兔气管黏膜上皮细胞和骨髓间充质干细胞的体外培养与鉴定[D].扬州:扬州大学,2012.
[7]Berndt-Weis M L,Kauri L M,Williams A,et al.Global transcriptional characterization of a mouse pulmonary epithelial cell line for use in genetic toxicology[J].Toxicol in Vitro,2009,23(5):816.
[8]陈宝安,王骏,薛萌,等.MTT比色法的改良及其在LAK细胞活性检测中的应用[J].东南大学学报(医学版),2001,20(3):150.
[9]吴婷婷.基于单抗敲除技术的葛根素与葛根改善SH-SY5Y细胞糖氧剥夺损伤炎症因子作用的量效关系研究[D].北京:北京中医药大学,2014.
[10]Dozic I,Todorovi c T,Dozic B,et al.Immunohistochemical identification of cytokeratins in the rat submandibular salivary glands during ontogenesis[J].Acta Veterinaria,2009,59(1):69.
[11]陈浩,陈湘君.酸甘化阴法治疗干燥综合征[J].上海中医药大学学报,2005,19(3):22.
[12]曹裕杰,蔡传书,魏斌,等.酸甘化阴法治疗鼻咽癌放疗后口干症疗效观察[J].中国医药导报,2009,6(14):50.
[13]赵琼,徐世军,李秀亮,等.加味人参乌梅汤对Caco-2细胞脱水模型AQP4基因表达的影响[J].中药与临床,2010,1(3):36.
[14]赵琼,黄勤挽,代渊,等.加味人参乌梅汤对腹泻大鼠AQP4及其相关调控因子的影响[J].中国实验方剂学杂志,2015,21(7):99.
[15]刘言广.水通道蛋白AQP1/AQP5在双峰驼唾液腺的表达研究[D].兰州:兰州大学,2016.
[16]李志民.唾液腺辐射损伤发生分子机制与预防的研究[D].长春:吉林大学,2004.
[17]Nielsen S,King L S,Christensen B M,et al.Aquaporins in complex tissues.II.Subcellular distribution in respiratory and glandular tissues of rat[J].Am J Physiol,1997.273(5 Pt 1):C1549.
[18]刘燕.津血源增加口干症模型大鼠唾液分泌机制的实验研究[D].南京:南京中医药大学,2014.