酸性神经磷脂酶及神经酰胺在吉西他滨诱导胰腺癌PANC-1细胞耐药中的作用

目的 建立对吉西他滨耐药的胰腺癌PANC-1/Gem细胞株,检测诱导耐药前后该细胞株生物学特性的变化.探讨吉西他滨诱导胰腺癌耐约的可能机制.方法 通过逐渐增加培养基中吉西他滨的浓度,建屯对吉西他滨耐药的胰腺癌PANC-1/Gem细胞株,TUNEL染色检测细胞株凋亡,MTT方法 检测胰腺癌PANC-1和胰腺癌PANC-1/Gem细胞的半数抑制浓度(IC_(50))和耐药指数(RI),Western印迹法检测酸性神经磷脂酶表达变化、二酰基甘油激酶(diacylglycerol kinase,DAGK)法检测神经酰胺含量变化.结果 经过24周成功诱导出对吉西他滨耐药的胰腺癌PANC-1/Gem细胞株,吉西他滨对PANC-1和PANC-1/Gem细胞的IC_(50)值分别为:亲本(8.13±0.85)μg/ml,24周(285.40±34.83)μg/ml,与亲本细胞相比耐药倍数为35.10倍,且凋亡率降低.Western印迹检测发现吉西他滨诱导24周的胰腺癌细胞PANC-1/Gem酸性神经磷脂酶的表达低于亲本细胞.两组细胞的神经酰胺含量分别为:亲本(364.95±46.11)pmol/mg protein,24周(120.61±20.07)pmol/mgprotein.结论 酸性神经磷脂酶的表达降低,导致神经酰胺含量减少可能是胰腺癌PANC-1细胞株对吉西他滨产生耐药的机制之一.

Significance of acid sphingomyelinase and ceramide in inducing gemcitabine-resistant pancreatic cancer cell line PANC-1

Objective To establish gemcitabine-resistant pancreatic cancer cell line PANC-1/ gem and discuss its biological characters and drug resistance mechanism. Methods gemcitabine-resist-ant pancreatic cancer cell line was obtained by culture of pancreatic cancer cell line PANC-1 in vitro with intermittently increasing the concentration of gemcitabine in the culture medium for 24 weeks. After that, TUNEL technique was employed to detect the apoptosis. Meanwhile, its drug sensitivity as well as the expression of sphingomyelinase, contents of C2-ceramide were determined respectively. Results Drug-resistant PANC-1/Gem cell was successfully induced with gemeitabine for 24 weeks. The IC_(50) increased from (8.13±0.85)μg/ml in PANC-1 to (285.40±34.83)μg/ml in PANC-1/Gem. Compared with the normal pancreatic cancer cell line PANC-1, the drug resistance indexes of PANC-1/gem to gemcitabine was 35.1 and the apoptosis rate was reduced from 6.21 to 2.74%. The ex-pression of sphingomyelinase was lower than that of pancreatic cancer cell line PANC-1, and the con-tents of ceramide was reduced from (364.95±46.11 pmol/mg protein) to (120.61±20.07 pmol/mg protein). Conclusion The durg resistance of gemcitabine to pancreatic cancer cell line is positively rel-evant to the low expression of acid sphingomyelinase as well as ceramide deficiency.