摘要
背景嗜神经侵袭是泪腺腺样囊性癌（ACC）有别于其他泪腺肿瘤最重要的生物学特征，胶质源性神经营养因子（GDNF）在涎腺ACC嗜神经侵袭中发挥了重要的作用，但GDNF对ACC细胞生物学功能的调控作用尚需进一步探讨。目的研究GDNF对体外培养的人ACC-2细胞增生和迁移能力的影响，为进一步明确ACC的神经侵袭机制提供实验依据。方法将ACC-2细胞系在含质量分数10％胎牛血清（FBS）、100u／mI（商品单位）青霉素、0．1g／L链霉素的RPMI-1640培养液中进行常规培养和传代，取对数生长期的ACC-2细胞制成单细胞悬液以2x10^4个／ml的密度接种于96孔板中，实验组加入终质量浓度分别为20、60、80、100、120μg／L的GDNF，对照组为仅含10％FBS的RPMI-1640培养液。细胞培养48h后，用免疫组织化学法检测ACC-2细胞中GDNF蛋白的表达，MTT法检测不同质量浓度GDNF作用不同时间后人ACC-2细胞的吸光度（A。）值；用Transwell小室法检测不同质量浓度GDNF作用不同时间后迁移的ACC-2细胞数目。结果免疫组织化学法检测表明，GDNF可在ACC-2细胞的细胞质中表达。标准培养48h后，随着GDNF质量浓度的提高，ACC-2细胞A”D值明显增加，各组间的差异有统计学意义（F=3．336，P=0．026）。与对照组比较，各质量浓度GDNF组ACC-2细胞A。值均明显升高，差异有统计学意义（P〈0．05）。80μg／LGDNF作用后，随着培养时间的延长，ACC-2细胞A570值逐渐增加，差异有统计学意义（F时间=39．979，P=0．000），各时间点GDNF组间ACC-2细胞A570值均明显高于对照组，组间总体比较的差异有统计学意义（F组别=13．212，P=0．003）。GDNF作用细胞30h后，随着GDNF质量浓度的增加，ACC-2细胞迁移数量明显增加，差异有统计学意义（F=144．886，P=0．000）。用100μg／LGDNF培养24、30、40h，随着培养时间的延长，ACC-2细胞系迁移数量明显增加，且各时间点GDNF组细胞迁移数均明显多于对照组，差异均有统计学意义（F目目=46．747，P=0．000；F组别=63．786，P=0．000）。结论GDNF可以促进体外培养的人ACC-2细胞的增生和迁移能力，并且分别具有剂量和时间依赖性。
Background Perineural invasion is an important biological character for adenoid cystic carcinoma （ACC） of lacrimal gland, which is different from those of other lacrimal gland tumors. As the important part of neurotrophic factors,glial-derived neurotrophic factor （GDNF） plays an important role in perineural invasion for ACC of salivary gland. GDNF regulation in the ACC cell biology function needs to be further explored. Objective This study was to investigate the effect of GDNF on proliferation and migration of ACC cells, and to explore the mechanism of neural invasion in ACC of lacrimal gland. Methods ACC-2 cell line was cultured and passaged in RPMI 1640 medium with 10% fetal bovine serum, 100 U/ml penicillin and 0.1 g/L streptomycin. Single-cell suspension was prepared with the density of 2x 104/ml using logarithmic phase of ceils and then incubated to 96-well plate. GDNF with the final concentration of 20,60,80,100 and 120 μg/L was added into the medium respectively in the experimental groups, and the cells were cultured in the medium without GDNF as the control group. The expression of GDNF in ACC-2 cells was detected by immunohistochemistry. MTT assay was employed to assay the absorbance value at the wavelength of 570 nm （As7o） for the evaluation of proliferation of ACC-2 cells after cultured by different concentrations of GDNF for different time points. Meanwhile,transwe]l chamber was used to examine the cell migrated number. Results Immunochemistry assay exhibited that ACC-2 cells showed the positive response for GDNF with the brown staining in the cytoplasm. In 48 hours after culture,the Asv0 value was elevated with the increase of GDNFconcentration,showing a significant difference among various groups （ F = 3. 336, P = 0. 026 ） , and the A570 value in various eoncentrations of GDNF groups was higher than that of 0 l, zg/L GDNF group （ all P〈0.05）. After action of 80 Ixg/E GDNF,the A570 value nf the eells was gradually increased with the prolong of culture time （F,~mo = 39. 979, P= 0. 000）. In 30 minutes after GDNF cultured, the number of migrated cells increased with the increase of GDNF concentration （F= 144. 886,P=0. 000）. ACC-2 cells were cultured by 100 I.μg/L GDNF for 24,30 and 40 hours,the number of migrated cells were more as the time lapse, and more migrated eeUs were seen in GDNF group at various time points （ Ftime = 46. 747, P = 0. 000 ; Fgroup = 63. 786, P = 0. 000 ）. Conclusions GDNF can stimulate the proliferation and migration of ACC-2 cells in a dose- and time-dependent manner.
出处
《中华实验眼科杂志》
CAS
CSCD
北大核心
2013年第3期243-247,共5页
Chinese Journal Of Experimental Ophthalmology
基金
天津医科大学科学基金面上项目（2011YK21）
关键词
胶质源性神经营养因子
腺样囊性癌
细胞增生
细胞迁移
Glial-derived neurotrophic factor
Adenoid cystic carcinoma
Cell proliferation
Cell migration
