摘要
目的:应用异硫氰酸荧光素标记天花粉蛋白,在激光共聚焦显微镜下直接观察天花粉蛋白进入黑色素瘤B16细胞的动态过程并分析其对黑色瘤B16细胞的损伤作用。方法:实验于2003-08/2004-08在南通大学神经再生重点实验室完成。将常规传代培养的黑色素瘤B16细胞用胰酶消化,以5×109个/L的浓度种植于24孔培养板。将天花粉蛋白、异硫氰酸荧光素-天花粉蛋白、异硫氰酸荧光素-牛血清白蛋白分别加入培养细胞中,终浓度为50mg/L,同时加入等量生理盐水作为正常对照组,每组再分别设3,6,12h不同孵育时间组。异硫氰酸荧光素标记天花粉蛋白后,利用激光共聚焦显微镜观察异硫氰酸荧光素-天花粉蛋白进入细胞的过程及其荧光强度,CCK-8细胞毒性实验评估各组细胞存活率、并用单细胞凝胶电泳及hoechst33258染核观察天花粉蛋白对黑色素瘤B16细胞致DNA损伤作用。结果:①终浓度50mg/L的异硫氰酸荧光素-天花粉蛋白孵育3h时已经进入黑色素瘤细胞,6h时进入细胞量达到最高,12h时已轻度下降,各时间点比较差异有统计学意义(P<0.01)。异硫氰酸荧光素-天花粉蛋白组与异硫氰酸荧光素-牛血清白蛋白组荧光强度差异有显著性(P<0.01)。②终浓度50mg/L异硫氰酸荧光素-天花粉蛋白、天花粉蛋白孵育黑色素瘤细胞3,6h后利用单细胞电泳和Hoechst33258染色未观察到核形态的变化;但孵育6h后CCK-8细胞毒性实验显示活细胞数量明显下降;孵育12h后出现DNA损伤的特征性彗星尾现象,并观察到明显的核浓缩和边集现象,出现特征性凋亡小体。结论:孵育6h时天花粉蛋白进入细胞量最多,但并没有明显的细胞DNA损伤的作用,而孵育12h时产生明显的细胞毒作用和凋亡的发生。
AIM： To observe the dynamic access of fiuorescein isothiocyanate （FITC）-Iabeled trichosanthin （TCS） into melanoma B-16 cells under laser confocal microscopy, and investigate the effect of trichosanthin on melanoma B-16 cell injury. METHODS： The experiment was carried out in the Key Laboratory of Nerve Regeneration, Nantong University from August 2003 to August 2004. Primarily cultured melanoma B-16 cells were digested by pancreatin, and seeded onto a 24-well culture plate at concentration of 5×10^9 cells/L TCS, FITC labeled TCS, FITC-bovine serum albumin （BSA） were added into the cultured cells, respectively at a final concentration of 50 mg/L; meanwhile, matching normal saline was added as the control group. Moreover, each group was subdivided into 3, 6 and 12 hours incubation groups. With FITC labeled TCS, the process of FITC-TCS entering the cell and its fluorescence intensity were observed by laser confocal microscopy. Cell survival was evaluated by CCK-8 cytotoxicity assay, and the effect of TCS on the melanoma B-16 injury was observed by single cell gel electrophoresis （SCGE） and hoechst33258 staining method. RESULTS： ①50 mg/L FITC-TCS entered the cell within 3 hours, and the endocytosis amount achieved the peak at 6 hours, followed by a slight decrease within 12 hours; differences of each time point had statistical significance （P 〈 0.01 ）. Fluorescence intensity in the FITC-TCS group was remarkably different from the FITC-BSA （P 〈 0.01）. ②With TCS （50 mg/L） exposure for 3 hours and 6 hours, no changes in cell nucleus were found in SCGE and hoechst33258 staining methods. CCK-8 cytotoxicity assay, however, suggested a distinct decrease in the number of living cells at 6 hours; after 12 hours incubation, comet tail, the specific phenomena of DNA injury, chromatin condensation even apoptotic body were observed. CONCLUSION： The amount of trichosanthin entering the cells reaches the peak after 6 hours incubation, but no obvious cell DNA injury is found. Strong cytotoxicity and apoptosis appear after 12 hours incubation.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第29期5792-5796,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
