改构型酸性成纤维细胞生长因子对庆大霉素诱导的大鼠肾小管上皮细胞损伤的预防作用
|
摘要
目的：通过体外培养SD大鼠肾小管上皮细胞的方法,探讨改构型酸性成纤维细胞生长因子(MaFGF)是否对庆大霉素(GM)引起的肾小管上皮细胞损伤有预防作用。
方法：在无菌条件下分离SD大鼠原代肾小管上皮细胞,然后接种于50ml培养瓶、6孔板或96孔板内：(1)培养24小时后于细胞处于对数生长期时在96孔板内向实验组加入不同浓度的MaFGF,12小时后再加入一系列浓度的GM,再继续培养24小时后用MTT法检测每组细胞的光密度(OD)值并计算细胞生长抑制率、相应每组的GM的IC50。(2)将处于对数生长期的细胞分为三组：对照组、GM组、MaFGF+GM组。首先给予MaFGF+GM组一定量的MaFGF作用12小时,然后GM组和MaFGF+GM组同时给予相同剂量的GM继续培养24小时,从形态学上观察MaFGF对GM诱导的肾小管上皮细胞损伤的预防作用；通过检测对照组、GM组、MaFGF+GM组相应的生化指标(MDA、NO)和酶学指标(SOD、GSH-Px),观察MaFGF对GM诱导的肾小管上皮细胞损伤的预防作用。
结果：(1)MaFGF预处理12h能使GM对肾小管上皮细胞的半数抑制浓度(IC50)升高。(2)加入2.0g／L以上浓度的GM作用24h后可见细胞由原有的多边鹅卵石样变圆皱缩、形态异常,细胞边界较为模糊甚至消失,细胞胞浆稀薄,立体感较差,细胞富集度降低,多数细胞胞浆内出现较多的黑色颗粒和空泡呈明显的细胞老化现象,有些甚至脱落悬浮于培养基中；MaFGF预处理12h后再加GM继续培养24 h,可见细胞变圆皱缩程度减轻,细胞轮廓较清晰,细胞边界较为清晰且折光性尚好,细胞胞浆较丰富,立体感较强,细胞富集度提高,胞浆内少见黑色颗粒和空泡。(3)GM组与对照组比较,MDA、NO的含量升高而SOD、GSH-Px的活性降低,差异均有统计学意义(P<0.01); MaFGF+GM组与GM组比较,MDA、NO的含量有所下降而SOD、GSH-Px的活性有所升高,差异均有统计学意义(P<0.01);而MaFGF+GM组与对照组比较,各生化和酶学指标含量或活性均未回落至正常水平,差异仍有统计学意义(P＜0.01)。
结论：MaFGF对庆大霉素诱导的肾小管上皮细胞损伤具有一定的预防作用。MaFGF对庆大霉素肾毒性的预防作用与其直接或间接拮抗自由基的产生,加强抗氧化酶对自由基的清除和保护抗氧化酶的活性有关。
Objective:To explore the preventive role of MaFGF in the injury of gentamicin on renal tubular epithelial cells of SD rats.
Method:SD rat'primary renal tubular epithelial cells were sterilely colleccted, and then cultured in 6-well plates,96-well plates and culture bottles: (1) After the cells grow in logarithmic growth phase in 96-well plates, the cells were divided into five groups randomly. Control group were incubated for 36h normally; GM group were incubated with DMEM culture medium containing GM respectively and the final concentrations were followed by 1.0g/L,2.0 g/L,3.0 g/L,4.0 g/L and 5.0g/L at the latter 24 hours; MaFGF groups(Ⅰ,Ⅱ,Ⅲ) were incubated with DMEM culture medium containing MaFGF respectively and the final concentrations were followed by 5.20μg/L,6.24μg/L and 7.28μg/L at the former 12 hours, and then added GM as the above serious of drug concentrations at the latter 24 hours. Assay the activity of each group cells by MTT methord and calculate the inhibition rate and the corresponding IC50 values of GM. (2) The cells were divided into three groups randomly when they grow in logarithmic growth phase:Control group, GM group and MaFGF+GM group. Control group were incubated for 36h normally; GM group were incubated with DMEM culture medium containing 3.0g/L GM at the latter 24 hours; MaFGF+GM group were incubated with DMEM culture medium containing 7.28μg/L MaFGF at the former 12 hours, and then added 3.0 g/L GM at the latter 24 hours. And the preventive role of MaFGF in the injured renal epithelial cells was observed by using inverted phase contrast microscope and some biochemical and enzymological indexes.
Result:(1) MaFGF pretreatment renal tubular epithelial cells,which can increase IC50 value of GM. (2) Added 2.0g/L or higher concentrations of GM at the latter 24 hours, renal tubular epithelial cells became abnormal shape, which lost the original multilateral cobblestone-like shape and became rounded shrinkage. Cell borders became fuzzier or even disappear.Cytoplasm became thin, and three dimensional sense became worse, and cell concentration was low. More black particles and vacuoles existed in cytoplasm. Some cells even were sheded and suspended in culture medium. But the cells which were pretreated whith MaFGF at the former 12 hours and added 3.0g/L GM at the latter 24 hours also showed that the extent of rounded shrinkage was remission, and the cell shapes became clearer, and the refraction of cell boundary was better. Cytoplasm became abundant, and three dimensional sense became better, and cell concentration was higher. A litter of black particles and vacuoles existed in cytoplasm. (3) Compared GM group and Control group, the content of MDA and NO was increased while the activity of SOD and GSH-Px was decreased, the differences were significant statistically (P<0.01); Compared MaFGF+GM group and GM group, the content of MDA and NO was decreased while the activity of SOD and GSH-Px was increased, the differences were significant statistically (P<0.01); Compared MaFGF+GM group and Control group, the content of the four indexes were not restored to the normal levels, the differences were still significant difference statistically (P<0.01).
Conclusion:MaFGF could prevent the injury of renal tubular epithelial cells in some degree. The preventive effect of MaFGF on GM nephrotoxicity was related to antagonize free radicals directly or indirectly, enhance the activity of antioxidant enzymes which could scaven free radicals, and protect antioxidant enzymes.
方法：在无菌条件下分离SD大鼠原代肾小管上皮细胞,然后接种于50ml培养瓶、6孔板或96孔板内：(1)培养24小时后于细胞处于对数生长期时在96孔板内向实验组加入不同浓度的MaFGF,12小时后再加入一系列浓度的GM,再继续培养24小时后用MTT法检测每组细胞的光密度(OD)值并计算细胞生长抑制率、相应每组的GM的IC50。(2)将处于对数生长期的细胞分为三组：对照组、GM组、MaFGF+GM组。首先给予MaFGF+GM组一定量的MaFGF作用12小时,然后GM组和MaFGF+GM组同时给予相同剂量的GM继续培养24小时,从形态学上观察MaFGF对GM诱导的肾小管上皮细胞损伤的预防作用；通过检测对照组、GM组、MaFGF+GM组相应的生化指标(MDA、NO)和酶学指标(SOD、GSH-Px),观察MaFGF对GM诱导的肾小管上皮细胞损伤的预防作用。
结果：(1)MaFGF预处理12h能使GM对肾小管上皮细胞的半数抑制浓度(IC50)升高。(2)加入2.0g／L以上浓度的GM作用24h后可见细胞由原有的多边鹅卵石样变圆皱缩、形态异常,细胞边界较为模糊甚至消失,细胞胞浆稀薄,立体感较差,细胞富集度降低,多数细胞胞浆内出现较多的黑色颗粒和空泡呈明显的细胞老化现象,有些甚至脱落悬浮于培养基中；MaFGF预处理12h后再加GM继续培养24 h,可见细胞变圆皱缩程度减轻,细胞轮廓较清晰,细胞边界较为清晰且折光性尚好,细胞胞浆较丰富,立体感较强,细胞富集度提高,胞浆内少见黑色颗粒和空泡。(3)GM组与对照组比较,MDA、NO的含量升高而SOD、GSH-Px的活性降低,差异均有统计学意义(P<0.01); MaFGF+GM组与GM组比较,MDA、NO的含量有所下降而SOD、GSH-Px的活性有所升高,差异均有统计学意义(P<0.01);而MaFGF+GM组与对照组比较,各生化和酶学指标含量或活性均未回落至正常水平,差异仍有统计学意义(P＜0.01)。
结论：MaFGF对庆大霉素诱导的肾小管上皮细胞损伤具有一定的预防作用。MaFGF对庆大霉素肾毒性的预防作用与其直接或间接拮抗自由基的产生,加强抗氧化酶对自由基的清除和保护抗氧化酶的活性有关。
Objective:To explore the preventive role of MaFGF in the injury of gentamicin on renal tubular epithelial cells of SD rats.
Method:SD rat'primary renal tubular epithelial cells were sterilely colleccted, and then cultured in 6-well plates,96-well plates and culture bottles: (1) After the cells grow in logarithmic growth phase in 96-well plates, the cells were divided into five groups randomly. Control group were incubated for 36h normally; GM group were incubated with DMEM culture medium containing GM respectively and the final concentrations were followed by 1.0g/L,2.0 g/L,3.0 g/L,4.0 g/L and 5.0g/L at the latter 24 hours; MaFGF groups(Ⅰ,Ⅱ,Ⅲ) were incubated with DMEM culture medium containing MaFGF respectively and the final concentrations were followed by 5.20μg/L,6.24μg/L and 7.28μg/L at the former 12 hours, and then added GM as the above serious of drug concentrations at the latter 24 hours. Assay the activity of each group cells by MTT methord and calculate the inhibition rate and the corresponding IC50 values of GM. (2) The cells were divided into three groups randomly when they grow in logarithmic growth phase:Control group, GM group and MaFGF+GM group. Control group were incubated for 36h normally; GM group were incubated with DMEM culture medium containing 3.0g/L GM at the latter 24 hours; MaFGF+GM group were incubated with DMEM culture medium containing 7.28μg/L MaFGF at the former 12 hours, and then added 3.0 g/L GM at the latter 24 hours. And the preventive role of MaFGF in the injured renal epithelial cells was observed by using inverted phase contrast microscope and some biochemical and enzymological indexes.
Result:(1) MaFGF pretreatment renal tubular epithelial cells,which can increase IC50 value of GM. (2) Added 2.0g/L or higher concentrations of GM at the latter 24 hours, renal tubular epithelial cells became abnormal shape, which lost the original multilateral cobblestone-like shape and became rounded shrinkage. Cell borders became fuzzier or even disappear.Cytoplasm became thin, and three dimensional sense became worse, and cell concentration was low. More black particles and vacuoles existed in cytoplasm. Some cells even were sheded and suspended in culture medium. But the cells which were pretreated whith MaFGF at the former 12 hours and added 3.0g/L GM at the latter 24 hours also showed that the extent of rounded shrinkage was remission, and the cell shapes became clearer, and the refraction of cell boundary was better. Cytoplasm became abundant, and three dimensional sense became better, and cell concentration was higher. A litter of black particles and vacuoles existed in cytoplasm. (3) Compared GM group and Control group, the content of MDA and NO was increased while the activity of SOD and GSH-Px was decreased, the differences were significant statistically (P<0.01); Compared MaFGF+GM group and GM group, the content of MDA and NO was decreased while the activity of SOD and GSH-Px was increased, the differences were significant statistically (P<0.01); Compared MaFGF+GM group and Control group, the content of the four indexes were not restored to the normal levels, the differences were still significant difference statistically (P<0.01).
Conclusion:MaFGF could prevent the injury of renal tubular epithelial cells in some degree. The preventive effect of MaFGF on GM nephrotoxicity was related to antagonize free radicals directly or indirectly, enhance the activity of antioxidant enzymes which could scaven free radicals, and protect antioxidant enzymes.
引文
1.叶任高,陆再英.内科学[M].北京：人民卫生出版社,2004,536.
2. Kintzel PE.Anticancer drugs-induced kidney disorders[J].Drug Safety,2001, 24(1):19-38.
3. Nagai J,Takano M.Molecular aspects of renal handling of aminoglycosides and strategies for preventing the nephrotoxicity[J].Drug Metab Pharma-cokinet,2004,19(3):159-170.
4.黄巨恩,黄全勇,陈维平,等.碱性成纤维细胞生长因子对庆大霉素肾损害保护作用的体外实验研究[J].中国药理学通报,2005,21(2)：232-235.
5.胡文娟,黄巨恩,李校堃,等.酸性成纤维细胞生长因子对大鼠肾缺血再灌注损伤的保护作用[J].解剖学研究,2006,28(3)：172-175.
6.胡文娟,黄巨恩,李校堃,等.改构型酸性成纤维细胞生长因子对大鼠肾缺血／再灌注损伤的保护作用[J].中国药理学通报,2007,23(2)：166-169.
7.黄巨恩,王慧杰,刘华刚,等.酸性成纤维细胞生长因子对庆大霉素诱导的肾小管上皮细胞损伤的保护作用[J].中国药理学通报,2007,23(11)：1455-1457.
8.刘华钢,黄巨恩,刘丽敏,等.酸性成纤维细胞生长因子对庆大霉素和顺铂所致肾小管上皮细胞损伤的保护作用[J].现代生物医学进展,2007,7(12)：1765-1768.
9.刘丽敏,刘华钢,黄巨恩,等.改构型酸性成纤维细胞生长因子对顺铂肾损伤保护作用的研究[J].中国生物工程杂志,2007；27(6)：10-14.
10.章静波.组织和细胞培养技术[M].北京：人民卫生出版社,2002：3-6.
11.司徒镇强,吴军正.细胞培养[M].西安：世界图书出版西安公司,2004：250-252.
12.顾光,支媛,殷召雪,等.大鼠肾细胞分离制备及培养方法的比较[J].卫生研
究,2005,34(5)：574-576.
13.王林,卓丽玲,顾建红,等.SD大鼠近端肾小管上皮细胞的原代培养及鉴定[J].中国兽医学报,2008,28(12)：1445-1448.
14.卢永科,李秋娟,宫德正,等.利用原代培养细胞进行药物短期毒性筛选方法[J].卫生毒理杂志,2003,17(2)：112-114.
15.赵斌,葛金芳,朱娟娟,等.小议在MTT法测细胞增殖抑制率中IC50的计算方法[J].安徽医药,2007,11(9)：834-835.
16.周一平.用SPSS软件计算新药的LD50[J].药学进展,2003,27(5)：314-316.
17.季建平.实验方法学(南京建成生物工程研究所).
18.熊祖应,梅俏,徐叔云.肾组织上皮细胞的培养及其应用进展[J].中国药理学通报,1999，15(4):303-306.
19.廖晓星,唐洪林,孙庭,等.肾小管上皮细胞原代培养及鉴定[J].中国现代医药杂志,2004(6)：12-14.
20.马世兴,陈志龙.成纤维细胞清除的时机与方法选择[J].细胞与分子免疫学杂志,1996,12(1)：54-55.
21.方开云,石明隽,肖瑛,等.原代肾小管上皮细胞的培养和鉴定[J].贵阳医学院学报,2008,33(2)：136-138.
22.王林,卓丽玲,顾建红.SD大鼠近端肾小管上皮细胞的原代培养及鉴定[J].中国兽医学报,2008,28(12)：1445-1448.
23.何娅妮,廖立生,蒋建新.庆大霉素肾毒性对大鼠肾皮质线粒体、溶酶体损伤机理的研究[J].第三军医大学学报,1996,18(3)：243-245.
24. Decorti QMalusa N,Furlan G,et al. Endocytosis of gentamicin in a proximal tubular renal cell line[J].Life Sci,1999,65(11):1115-1118.
25.李校堃,黄巨恩,梁刚,等.BFGF对庆大霉素及缺血性肾损伤的保护作用[J].中国药科大学学报,2002,24(2)：110-112.
26.曹阳,祁俊生,王远亮.自由基介导细胞质凋亡的机制[J].自然科学研究研 究,2004,3(20)：118-122.
27. Rougie F,Claude D,Maurin M.Aminoglycoside nephro toxicity [J].CurrDrug Targets Infect Disord,2004,4(2):153-162.
28.黄云剑,廖立生,袁发焕.近端肾小管上皮细胞原代培养及庆大霉素对其的影响[J].解放军医学杂志.1998,23(5)：453-455.
29.钟波,张璟.酸性成纤维细胞生长因子诱导大鼠肾小管上皮细胞转分化的作用及机制[J].第三军医大学学报,2002,24(7)：778-781.
30.刘华伟,赵勇,李贵生,等.FGF-1改构体对小鼠脾淋巴细胞增值与凋亡的影响[J].中国生物工程杂志,2005,25(6)：45-50.
31.海广范,詹合琴,许华,等.nmhaFGF对肾脏缺血再灌注损伤大鼠不同时间血尿素氮、肌酐和尿蛋白含量的影响[J].新乡医学院学报,2007,24(5)：450-452.
32.海广范,杨宇平,许华,等.非促分裂型人酸性成纤维细胞生长因子对急性肾功能衰竭大鼠血尿素氮、肌酐和尿蛋白含量的影响[J].新乡医学院学报,2008,25(3)：232-234.
33. Lozano RM,Pineda-Lucena A,Gonzalez C,et al.1H NMRstructural charact-erization of a nonmitogenic vasodilatory,ischemia-protector and neuromo-dulatory acid fibroblast growth factor[J]. Biochemistry,2000,39:4982-4993.
34.卢永科,仲来福.一氧化氮在顺铂致大鼠肾损害过程中的作用[J].卫生毒理学杂志,2000,14(3)：152-155.
1.曹定国,李校坤,付小兵.酸性成纤维细胞生长因子非促分裂活性的研究进展[J].中国危重病急救医学,2004,16(8)：502-504.
2.吴喆,黄巨恩.成纤维细胞生长因子对海马神经元损伤保护作用的研究进展[J].医学综述,2009,15(4)：496-498.
3.钟波,张璟.酸性成纤维细胞生长因子诱导大鼠肾小管上皮细胞转分化的作用及机制[J].第三军医大学学报,2002,24(7)：778-781.
4. La Rosa S,Uccella S,Erba s,et al.Immunohistochemical detection of fibroblast growth factor receptors in normal endocrine cells and related tumors of the digestive system[J].Appl Immunohistochem Mol Morphol,2001,9(4):319-328.
5. Lozano RM,Pineda-Lucena A,Gonzalez C,et al.1H-NMR structural charac-terization of a nonmitogenic,vasodilatory,ischemia-protector and neuromo-dulatory acidic fibroblast growth factor[J].Biochemistry,2000,39(17):4982-4993.
6.胡文娟,黄巨恩,李校堃,等.改构型酸性成纤维细胞生长因子对大鼠肾缺血／再灌注损伤的保护作用[J].中国药理学通报,2007,23(2)：163-169.
7.翁立新,付小兵,李秀霞,等.改构型和野生型aFGF对肠缺血再灌注损伤后肝肾功能的影响[J].中国危重病急救医学,2004,16(1)：19-21.
8.黄巨恩,杨志平,张绍峰,等.碱性成纤维细胞生长因子对庆大霉素诱导的急性肾功能衰竭疗效的探讨[J].广西医科大学学报,2002,19(1)：24-26.
9.石丽娟,汤浩.丹参注射液拮抗链霉素肾毒性损伤的实验研究[J].中国病理生理杂志,2007,23(4)：789-793.
10.刘晓华,黎七雄.川芎嗪对顺铂肾损伤大鼠肾细胞凋亡及凋亡相关蛋白表达的影响[J].中国药理学与毒理学杂志,2005,19(5)：352-356.
11.朱少铭,刘铭球,刘久波,等.银杏叶对关木通致大鼠急性肾损伤的保护作用[J].医药导报,2003,22(11)：760-763.
12.肖瑛,任进.马兜铃酸致小鼠急性肾毒性的早期研究[J].毒理学杂志,2007,21(4)：337.
13.唐帆,彭超,郑楠,等.小鼠急性肾损伤后细胞再生的动态观察[J].上海交通大学学报,2007,27(6)：670-672.
14.吴红赤,杨谦,于海涛,等.单次大剂量镉致急性肾损伤的实验研究[J].中国伤残医学,2007,15(6)：1-3.
15.海广范,杨宇平,许华,等.非促分裂型人酸性成纤维细胞生长因子对急性肾功能衰竭大鼠血尿素氮、肌酐和尿蛋白含量的影响[J].新乡医学院学报,2008,25(3)：232-234.
16.吴广礼,温进坤,韩梅,等.山莨菪碱对过度训练大鼠急性肾损伤的保护作用[J].中国微循环,2006,10(4)：241-244.
17.陈洪,周春华.高强度军事训练致急性肾损伤动物模型的研究[J].解放军医学杂志,2007,32(12)：1304-1306.
18.卢永科,李秋娟,宫德正,等.利用原代培养细胞进行药物短期毒性筛选方法[J].卫生毒理杂志,2003,17(2)：112-114.
19.黄巨恩,黄全勇,陈维平,等.碱性成纤维细胞生长因子对庆大霉素肾损害保护作用的体外实验研究[J].中国药理学通报,2005,21(2)：232-235.
20.刘丽敏,刘华钢,黄巨恩,等.酸性成纤维细胞生长因子对顺铂肾损害保护作用的实验研究[J].解剖学研究,2006,28(4)：243-246.
21.刘丽敏,刘华钢,黄巨恩,等.改构型酸性成纤维细胞生长因子对顺铂肾损伤保护作用的研究[J].中国生物工程杂志,2007,27(6)：10-14.
22.裴秀丛,徐兆发.镉对肾小管上皮细胞钙黏蛋白的影响[J].中国工业医学杂志,2005,18(5)：266-267.
23.周桂凤,蒋云生,胡白英,等.铅抑制NF-κB、 Bcl-2蛋白表达诱导人肾小管
上皮细胞凋亡[J].实用预防医学,2008,15(5)：1330-1332.
24.孙同柱,付小兵,陈伟,等.改构型酸性成纤维细胞生长因子对肠缺血-再灌注的保护作用[J].中国危重病急救医学,2005,17(1)：46-48.
25.金伯泉,主编.细胞和分子免疫学[M].第2版.北京：科学出版社,2001,643-645.
26.梁钢,黄巨恩,陈纡,等.碱性成纤维细胞生长因子对大鼠肾缺血再灌注损伤的保护作用[J].广西医科大学学报,2002,19(4)：465-467.
27.曹定国,李校坤,付小兵,等.改构酸性成纤维细胞生长因子对小鼠胸腺细胞调亡的影响[J].中国生物工程杂志,2003,23(9)：80-83.
28.袁海平,陈佩杰,史仍飞,等.运动性蛋白尿与肾脏细胞凋亡及氧自由基代谢关系的研究[J].中国运动医学杂志,2003,22(3)：254-257.
2. Kintzel PE.Anticancer drugs-induced kidney disorders[J].Drug Safety,2001, 24(1):19-38.
3. Nagai J,Takano M.Molecular aspects of renal handling of aminoglycosides and strategies for preventing the nephrotoxicity[J].Drug Metab Pharma-cokinet,2004,19(3):159-170.
4.黄巨恩,黄全勇,陈维平,等.碱性成纤维细胞生长因子对庆大霉素肾损害保护作用的体外实验研究[J].中国药理学通报,2005,21(2)：232-235.
5.胡文娟,黄巨恩,李校堃,等.酸性成纤维细胞生长因子对大鼠肾缺血再灌注损伤的保护作用[J].解剖学研究,2006,28(3)：172-175.
6.胡文娟,黄巨恩,李校堃,等.改构型酸性成纤维细胞生长因子对大鼠肾缺血／再灌注损伤的保护作用[J].中国药理学通报,2007,23(2)：166-169.
7.黄巨恩,王慧杰,刘华刚,等.酸性成纤维细胞生长因子对庆大霉素诱导的肾小管上皮细胞损伤的保护作用[J].中国药理学通报,2007,23(11)：1455-1457.
8.刘华钢,黄巨恩,刘丽敏,等.酸性成纤维细胞生长因子对庆大霉素和顺铂所致肾小管上皮细胞损伤的保护作用[J].现代生物医学进展,2007,7(12)：1765-1768.
9.刘丽敏,刘华钢,黄巨恩,等.改构型酸性成纤维细胞生长因子对顺铂肾损伤保护作用的研究[J].中国生物工程杂志,2007；27(6)：10-14.
10.章静波.组织和细胞培养技术[M].北京：人民卫生出版社,2002：3-6.
11.司徒镇强,吴军正.细胞培养[M].西安：世界图书出版西安公司,2004：250-252.
12.顾光,支媛,殷召雪,等.大鼠肾细胞分离制备及培养方法的比较[J].卫生研
究,2005,34(5)：574-576.
13.王林,卓丽玲,顾建红,等.SD大鼠近端肾小管上皮细胞的原代培养及鉴定[J].中国兽医学报,2008,28(12)：1445-1448.
14.卢永科,李秋娟,宫德正,等.利用原代培养细胞进行药物短期毒性筛选方法[J].卫生毒理杂志,2003,17(2)：112-114.
15.赵斌,葛金芳,朱娟娟,等.小议在MTT法测细胞增殖抑制率中IC50的计算方法[J].安徽医药,2007,11(9)：834-835.
16.周一平.用SPSS软件计算新药的LD50[J].药学进展,2003,27(5)：314-316.
17.季建平.实验方法学(南京建成生物工程研究所).
18.熊祖应,梅俏,徐叔云.肾组织上皮细胞的培养及其应用进展[J].中国药理学通报,1999，15(4):303-306.
19.廖晓星,唐洪林,孙庭,等.肾小管上皮细胞原代培养及鉴定[J].中国现代医药杂志,2004(6)：12-14.
20.马世兴,陈志龙.成纤维细胞清除的时机与方法选择[J].细胞与分子免疫学杂志,1996,12(1)：54-55.
21.方开云,石明隽,肖瑛,等.原代肾小管上皮细胞的培养和鉴定[J].贵阳医学院学报,2008,33(2)：136-138.
22.王林,卓丽玲,顾建红.SD大鼠近端肾小管上皮细胞的原代培养及鉴定[J].中国兽医学报,2008,28(12)：1445-1448.
23.何娅妮,廖立生,蒋建新.庆大霉素肾毒性对大鼠肾皮质线粒体、溶酶体损伤机理的研究[J].第三军医大学学报,1996,18(3)：243-245.
24. Decorti QMalusa N,Furlan G,et al. Endocytosis of gentamicin in a proximal tubular renal cell line[J].Life Sci,1999,65(11):1115-1118.
25.李校堃,黄巨恩,梁刚,等.BFGF对庆大霉素及缺血性肾损伤的保护作用[J].中国药科大学学报,2002,24(2)：110-112.
26.曹阳,祁俊生,王远亮.自由基介导细胞质凋亡的机制[J].自然科学研究研 究,2004,3(20)：118-122.
27. Rougie F,Claude D,Maurin M.Aminoglycoside nephro toxicity [J].CurrDrug Targets Infect Disord,2004,4(2):153-162.
28.黄云剑,廖立生,袁发焕.近端肾小管上皮细胞原代培养及庆大霉素对其的影响[J].解放军医学杂志.1998,23(5)：453-455.
29.钟波,张璟.酸性成纤维细胞生长因子诱导大鼠肾小管上皮细胞转分化的作用及机制[J].第三军医大学学报,2002,24(7)：778-781.
30.刘华伟,赵勇,李贵生,等.FGF-1改构体对小鼠脾淋巴细胞增值与凋亡的影响[J].中国生物工程杂志,2005,25(6)：45-50.
31.海广范,詹合琴,许华,等.nmhaFGF对肾脏缺血再灌注损伤大鼠不同时间血尿素氮、肌酐和尿蛋白含量的影响[J].新乡医学院学报,2007,24(5)：450-452.
32.海广范,杨宇平,许华,等.非促分裂型人酸性成纤维细胞生长因子对急性肾功能衰竭大鼠血尿素氮、肌酐和尿蛋白含量的影响[J].新乡医学院学报,2008,25(3)：232-234.
33. Lozano RM,Pineda-Lucena A,Gonzalez C,et al.1H NMRstructural charact-erization of a nonmitogenic vasodilatory,ischemia-protector and neuromo-dulatory acid fibroblast growth factor[J]. Biochemistry,2000,39:4982-4993.
34.卢永科,仲来福.一氧化氮在顺铂致大鼠肾损害过程中的作用[J].卫生毒理学杂志,2000,14(3)：152-155.
1.曹定国,李校坤,付小兵.酸性成纤维细胞生长因子非促分裂活性的研究进展[J].中国危重病急救医学,2004,16(8)：502-504.
2.吴喆,黄巨恩.成纤维细胞生长因子对海马神经元损伤保护作用的研究进展[J].医学综述,2009,15(4)：496-498.
3.钟波,张璟.酸性成纤维细胞生长因子诱导大鼠肾小管上皮细胞转分化的作用及机制[J].第三军医大学学报,2002,24(7)：778-781.
4. La Rosa S,Uccella S,Erba s,et al.Immunohistochemical detection of fibroblast growth factor receptors in normal endocrine cells and related tumors of the digestive system[J].Appl Immunohistochem Mol Morphol,2001,9(4):319-328.
5. Lozano RM,Pineda-Lucena A,Gonzalez C,et al.1H-NMR structural charac-terization of a nonmitogenic,vasodilatory,ischemia-protector and neuromo-dulatory acidic fibroblast growth factor[J].Biochemistry,2000,39(17):4982-4993.
6.胡文娟,黄巨恩,李校堃,等.改构型酸性成纤维细胞生长因子对大鼠肾缺血／再灌注损伤的保护作用[J].中国药理学通报,2007,23(2)：163-169.
7.翁立新,付小兵,李秀霞,等.改构型和野生型aFGF对肠缺血再灌注损伤后肝肾功能的影响[J].中国危重病急救医学,2004,16(1)：19-21.
8.黄巨恩,杨志平,张绍峰,等.碱性成纤维细胞生长因子对庆大霉素诱导的急性肾功能衰竭疗效的探讨[J].广西医科大学学报,2002,19(1)：24-26.
9.石丽娟,汤浩.丹参注射液拮抗链霉素肾毒性损伤的实验研究[J].中国病理生理杂志,2007,23(4)：789-793.
10.刘晓华,黎七雄.川芎嗪对顺铂肾损伤大鼠肾细胞凋亡及凋亡相关蛋白表达的影响[J].中国药理学与毒理学杂志,2005,19(5)：352-356.
11.朱少铭,刘铭球,刘久波,等.银杏叶对关木通致大鼠急性肾损伤的保护作用[J].医药导报,2003,22(11)：760-763.
12.肖瑛,任进.马兜铃酸致小鼠急性肾毒性的早期研究[J].毒理学杂志,2007,21(4)：337.
13.唐帆,彭超,郑楠,等.小鼠急性肾损伤后细胞再生的动态观察[J].上海交通大学学报,2007,27(6)：670-672.
14.吴红赤,杨谦,于海涛,等.单次大剂量镉致急性肾损伤的实验研究[J].中国伤残医学,2007,15(6)：1-3.
15.海广范,杨宇平,许华,等.非促分裂型人酸性成纤维细胞生长因子对急性肾功能衰竭大鼠血尿素氮、肌酐和尿蛋白含量的影响[J].新乡医学院学报,2008,25(3)：232-234.
16.吴广礼,温进坤,韩梅,等.山莨菪碱对过度训练大鼠急性肾损伤的保护作用[J].中国微循环,2006,10(4)：241-244.
17.陈洪,周春华.高强度军事训练致急性肾损伤动物模型的研究[J].解放军医学杂志,2007,32(12)：1304-1306.
18.卢永科,李秋娟,宫德正,等.利用原代培养细胞进行药物短期毒性筛选方法[J].卫生毒理杂志,2003,17(2)：112-114.
19.黄巨恩,黄全勇,陈维平,等.碱性成纤维细胞生长因子对庆大霉素肾损害保护作用的体外实验研究[J].中国药理学通报,2005,21(2)：232-235.
20.刘丽敏,刘华钢,黄巨恩,等.酸性成纤维细胞生长因子对顺铂肾损害保护作用的实验研究[J].解剖学研究,2006,28(4)：243-246.
21.刘丽敏,刘华钢,黄巨恩,等.改构型酸性成纤维细胞生长因子对顺铂肾损伤保护作用的研究[J].中国生物工程杂志,2007,27(6)：10-14.
22.裴秀丛,徐兆发.镉对肾小管上皮细胞钙黏蛋白的影响[J].中国工业医学杂志,2005,18(5)：266-267.
23.周桂凤,蒋云生,胡白英,等.铅抑制NF-κB、 Bcl-2蛋白表达诱导人肾小管
上皮细胞凋亡[J].实用预防医学,2008,15(5)：1330-1332.
24.孙同柱,付小兵,陈伟,等.改构型酸性成纤维细胞生长因子对肠缺血-再灌注的保护作用[J].中国危重病急救医学,2005,17(1)：46-48.
25.金伯泉,主编.细胞和分子免疫学[M].第2版.北京：科学出版社,2001,643-645.
26.梁钢,黄巨恩,陈纡,等.碱性成纤维细胞生长因子对大鼠肾缺血再灌注损伤的保护作用[J].广西医科大学学报,2002,19(4)：465-467.
27.曹定国,李校坤,付小兵,等.改构酸性成纤维细胞生长因子对小鼠胸腺细胞调亡的影响[J].中国生物工程杂志,2003,23(9)：80-83.
28.袁海平,陈佩杰,史仍飞,等.运动性蛋白尿与肾脏细胞凋亡及氧自由基代谢关系的研究[J].中国运动医学杂志,2003,22(3)：254-257.