CAJ | 학술논문

目的：采用人工合成的外源性C2-神经酰胺研究其对体外培养的间皮瘤细胞和体内移植瘤的移植作用，为是否能将 C2-神经酰胺作为治疗人恶性间皮瘤的新化疗药提供初步理论依据。方法：在体外实验中，C2-神经酰胺作用于人恶性间皮瘤细胞后，采用CCK-8法测定细胞增殖中活细胞数目并制作细胞剂量和时间的生长曲线。荧光激活流式细胞分离术（FACS）观察间皮瘤细胞周期的变化。DNA ladder对凋亡细胞进行分析。Fluorescent assay法检测间皮瘤细胞的Caspase-3活性。在体内实验中，建立荷瘤裸鼠动物模型。随机分为四组，C2-神经酰胺腹腔注射组、C2-神经酰胺皮下注射组、DMSO 组和生理盐水组。观察治疗后裸鼠自然状态、重量，计算肿瘤体积，21天时处死裸鼠，肉眼及镜下观察。免疫组织化学染色观察C2-ceram-ide对人间皮瘤细胞的增殖、凋亡和侵袭的影响。结果：体外实验中，在一定的时间与剂量范围内，C2-神经酰胺对间皮瘤细胞的抑制呈现剂量依从和时间依从性。FACS及DNA电泳细胞凋亡分析均表明，C2-神经酰胺在体外可诱导间皮瘤细胞的凋亡。FACS检测到G0/G1期细胞明显减少，S期细胞增多，细胞周期阻滞于G2/M期。Caspase-3活性测定发现C2-神经酰胺能增加间皮瘤细胞Caspase-3的活性。体内实验中，C2-神经酰胺组裸鼠肿瘤的重量、体积均小于未治疗组。C2-神经酰胺组肿瘤组织 Caspase-3蛋白表达明显强于未治疗组。结论：本实验证实C2-ceramide在体内、外均可抑制恶性间皮瘤细胞增殖、侵袭，诱导间皮瘤细胞凋亡，进一步了解间皮瘤细胞的生物学特性，为临床试验奠定了一定的基础。
목적：채용인공합성적외원성C2-신경선알연구기대체외배양적간피류세포화체내이식류적이식작용，위시부능장 C2-신경선알작위치료인악성간피류적신화료약제공초보이론의거。방법：재체외실험중，C2-신경선알작용우인악성간피류세포후，채용CCK-8법측정세포증식중활세포수목병제작세포제량화시간적생장곡선。형광격활류식세포분리술（FACS）관찰간피류세포주기적변화。DNA ladder대조망세포진행분석。Fluorescent assay법검측간피류세포적Caspase-3활성。재체내실험중，건립하류라서동물모형。수궤분위사조，C2-신경선알복강주사조、C2-신경선알피하주사조、DMSO 조화생리염수조。관찰치료후라서자연상태、중량，계산종류체적，21천시처사라서，육안급경하관찰。면역조직화학염색관찰C2-ceram-ide대인간피류세포적증식、조망화침습적영향。결과：체외실험중，재일정적시간여제량범위내，C2-신경선알대간피류세포적억제정현제량의종화시간의종성。FACS급DNA전영세포조망분석균표명，C2-신경선알재체외가유도간피류세포적조망。FACS검측도G0/G1기세포명현감소，S기세포증다，세포주기조체우G2/M기。Caspase-3활성측정발현C2-신경선알능증가간피류세포Caspase-3적활성。체내실험중，C2-신경선알조라서종류적중량、체적균소우미치료조。C2-신경선알조종류조직 Caspase-3단백표체명현강우미치료조。결론：본실험증실C2-ceramide재체내、외균가억제악성간피류세포증식、침습，유도간피류세포조망，진일보료해간피류세포적생물학특성，위림상시험전정료일정적기출。
Objective:The effects of exogenous C2 ceramide on the induction of apoptosis of mesothelioma cells in vitro and the cell growth of BALB/c (nu/nu)nude mice in vivo were investigated,in order to provide experimental data for the systemic treatment of this disease.Methods:In vitro,cell viability and apoptosis were analyzed by cell counting kit-8 assay,fluorescence activated cell sorter (FACS)analysis,DNA fragmentation analysis,Caspase -3 assay.In vivo,the BALB/c (nu/nu)nude mice models were developed,devided into four groups randomly and trea-ted with saline,vehicle (DMSO,1%),C2-ceramide direct injection and C2-ceramide intraperitoneal injection re-spectively.Tumor volume was calculated as length ×width2 ×0.5 with caliper.After the single tumor-bearing mouse was killed 21 days after treatment,samples of tissue were subjected to histological examination for the absence of mes-othelioma.Results:In vitro,C2 -ceramide demonstrated a dose -and time -dependent inhibition of cell prolifera-tion,induction of apoptosis,and cell-cycle arrest at G1/S phase in mesothelioma cells.The antiproliferative effect of 80μm C2-ceramide was paralleled by an increase in Caspase-3 activity in the two cell lines investigated.FACS a-nalysis showed that mesothelioma cells underwent apoptosis in a time-dependent manner.An increase in the portion of cells in the G0 -G1 phase and subG1 was observed in mesothelioma cells.The cells located in the subG1 portion were considered as being apoptotic.After cells transiently were transfected with C2 ceramide for 24h,C2 ceramide dramatically enhanced the level of DNA ladders in mesothelioma cells,whereas the DMSO resulted in undetectable laddering of the DNA.C2-ceramide reduced the volume of established tumors in mesothelioma xenograft mice.Con-clusion:Our results indicated that C2-ceramide induced apoptosis of malignant mesothelioma cells in vitro and re-duced growth of mesothelioma in vivo,the biological mechanism of mesothelioma cells were understood further.There-fore C2-ceramide might potentially provide clinical approach in the treatment of patients with mesothelioma.