卵巢癌细胞诱导T淋巴细胞凋亡的实验研究

Experimental study on apoptosis in Tcells induced by ovarian carcinoma cells

目的 探讨卵巢癌细胞诱导T淋巴细胞凋亡及一氧化氮与细胞内游离钙离子在T淋巴细胞凋亡过程中的作用。方法 分别采用电子显微镜、流式细胞仪观察 3个卵巢癌细胞株 (OVCAR3、CAOV3和SKOV3 )的培养上清液诱导的CD+ 8T淋巴细胞的凋亡 ;逆转录 聚合酶链反应 (RT PCR)技术检测诱导型一氧化氮合成酶 (iNOS)mRNA的表达 ;Fura 2荧光负荷技术和酶联免疫 (EIA)法分别检测细胞内游离钙离子浓度 ( [Ca2 + ]i)和环鸟苷酸 (cGMP)浓度。结果 ( 1)分别经卵巢癌细胞株OVCAR3、CAOV3和SKOV3细胞培养的上清液作用后 ,CD+ 8T淋巴细胞出现典型的凋亡改变 ,即染色质固缩和片段化 ;流式细胞仪上见凋亡峰 ;细胞凋亡率 [分别为 ( 2 3 8± 3 5) % ,( 2 3 1± 2 9) %、( 2 4 2± 4 9) % ]较对照 [( 4 6± 0 5) % ]明显增高 (P <0 0 1) ;( 2 )CD+ 8T淋巴细胞凋亡过程中iNOSmRNA的表达 (分别为 1 14± 0 0 3 ,1 0 5± 0 0 4,1 16± 0 0 2 )较对照 ( 0 60± 0 0 3 )显著增高 (P <0 0 1) ;细胞内游离 [Ca2 + ]i[分别为 ( 3 80± 3 8)、( 3 66± 13 )、( 3 56± 2 0 )nmol L]和cGMP浓度 [分别为 ( 0 65± 0 0 9)、( 0 62± 0 16)、( 0 57± 0 12 )pmol ml]均较对照 [分别为 ( 184± 11)nmol L、( 0 2 9± 0 0

Objective To investigate apoptosis in T cells induced by ovarian carcinoma cells and analyze the role of nitric oxide and intracellular free calcium in this process Methods Apoptosis induced by ovarian carcinoma cell lines supernatants was investigated by electron microscopy and flow cytometry Reverse trancscription polymerase chain reaction (RT PCR )was used to detect the expression of inducible nitric oxide synthase (iNOS) mRNA Calcium ions ([Ca 2+ ]i ) and cycle guanosine monophosphate (cGMP) were determined by Fura 2 fluorescein load technique and enzyme imunoassay (EIA),respectively Results Cluster of differentiation (CD)8 + T cells cocultured with ovarian carcinoma cell lines supernatants expressed typical change of apoptosis, such as condensation and fragmentation of chromosomes, apoptosis peak in flow cytometry and higher apoptotic ratios[(23 8±3 5)%,(23 1±2 9)%,(24 2 ±4 9) % vs (4 6±0 5)%, P <0 01] The expression of iNOS mRNA and cGMP level in the process of CD + 8 T cellular apoptosis were significantly enhanced as compared to that of control group [1 14±0 03,1 05±0 04,1 16±0 02 vs 0 60±0 03, P <0 01;(0 65±0 09),(0 62±0 16),(0 57±0 12)pmol/Lvs (0 29±0 04) pmol/ml, P <0 01],and [Ca 2+ ]i was significantly higher [(380±38),(366±13),(356±20) nmol/L vs (184±11) nmol/L, P <0 01) Conclusion Nitric oxide and intracellular free calcium may play a key role in apoptotic T cell induced by ovarian carcinoma