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目的:体外研究刀豆蛋白A(Concanavalin A,Con A)联合淋巴细胞对人卵巢癌细胞株SKOV3细胞形态、活力、凋亡以及顺铂敏感性的影响。方法:应用密度梯度离心法分离健康成人新鲜血中的单个核细胞,培养3～5d待单核细胞完全贴壁后分离出悬浮的淋巴细胞,与SKOV3细胞混合培养,并设对照组(未处理的SKOV3)、实验组1(SKOV3培养体系中仅加入5μg/mL Con A)、实验组2(SKOV3和淋巴细胞混合培养)和实验组3(SKOV3和淋巴细胞混合培养体系中加入5μg/mL Con A)。培养7d后将SKOV3分离出继续培养,并进行以下实验,在光学显微镜下观察细胞形态变化;CCK8检测24和48h细胞活性;流式细胞术检测24和48h细胞凋亡情况;用浓度0和60μmol/L顺铂处理SKOV3细胞24和48h后,流式细胞术检测细胞凋亡率以判断细胞对顺铂的敏感性。结果:与对照组比较,实验组1和实验组2中细胞无明显改变,而实验组3中SKOV3与淋巴细胞混合培养后发生明显改变,如细胞突起减少,细胞变圆。与对照组比较,实验组1中24和48h细胞存活率分别为(105.68±7.12)%(z=-1.256,P=0.30)和(111.90±8.28)%(z=-1.766,P=0.13),实验组2分别为(101.05±5.92)%(z=-0.215,P=0.79)和(109.72±6.54)%(z=-1.766,P=0.13),差异均无统计学意义;而实验组3分别为(25.08±7.71)%(z=-2.087,P=0.004)和(14.6±4.41)%(z=-3.431,P=0.001),差异有统计学意义。与对照组比较,实验组1中24和48h细胞凋亡率分别为(4.43±1.49)%(z=-2.78,P=0.61)和(10.95±1.09)%(z=-1.982,P=0.08),实验组2分别为(4.79±0.83)%(z=-1.349,P=0.28)和(10.59±1.03)%(z=-1.349,P=0.28),差异均无统计学意义;实验组3分别为(13.88±1.00)%(z=-2.247,P=0.003)和(21.37±2.35)%(z=-2.137,P=0.006),差异有统计学意义。顺铂处理各组SKOV3细胞后,与对照组比较,实验组1中24和48h细胞凋亡率分别为(11.25±1.86)%(z=-0.361,P=0.59)和(33.66±4.96)%(z=-0.536,P=0.54),实验组2分别为(10.32±0.14)%(z=-0.225,P=0.65)和(31.56±6.21)%(z=-0.163,P=0.83),差异均无统计学意义;实验组3分别为(41.99±6.66)%(z=-2.14,P=0.03)和(66.5±2.55)%(z=-1.909,P=0.04),差异有统计学意义。结论:Con A联合淋巴细胞可显著改变SKOV3细胞形态,降低细胞活力,促进细胞凋亡,并增加细胞对顺铂的敏感性,而Con A或淋巴细胞单独处理并无此效应。
OBJECTIVE: To study the effect of Con A combined with lymphocyte on the morphology, viability, apoptosis and cisplatin sensitivity of human ovarian cancer cell line SKOV3 in vitro. Methods: Mononuclear cells from fresh blood of healthy adults were isolated by density gradient centrifugation. Suspension lymphocytes were isolated after mononuclear cells were completely adhered to the culture medium for 3 ~ 5 days, mixed with SKOV3 cells and cultured in control group (SKOV3 and lymphocyte mixed culture) and experimental group 3 (SKOV3 and lymphocyte mixed culture system added 5μg / mL Con A), experimental group 1 (SKOV3 only added 5μg / mL Con A) A). After cultured for 7 days, SKOV3 was isolated and cultured, and the following experiments were carried out to observe the morphological changes of cells under light microscope. The activity of cells was detected by CCK8 at 24 and 48 hours. Flow cytometry was used to detect the apoptosis of cells at 24 and 48 hours. / L Cisplatin treatment of SKOV3 cells 24 and 48h, flow cytometry to detect apoptosis rate to determine the sensitivity of cells to cisplatin. Results: Compared with the control group, there was no significant change in the cells in experimental group 1 and experimental group 2, but in experimental group 3, SKOV3 and lymphocytes changed significantly after mixed culture, such as decreased cell protrusion and rounded cells. Compared with the control group, the survival rates of cells in experimental group 1 at 24 and 48 hours were (105.68 ± 7.12)% (z = -1.256, P = 0.30) and (111.90 ± 8.28)% , Respectively. There was no significant difference between the two groups in experimental group 2 (101.05 ± 5.92)% (z = -0.215, P = 0.79) and (109.72 ± 6.54)% 3 were (25.08 ± 7.71)% (z = -2.087, P = 0.004) and (14.6 ± 4.41)% respectively (z = -3.431, P = 0.001) .The difference was statistically significant. Compared with the control group, the apoptotic rates in experimental group 1 at 24 and 48 hours were (4.43 ± 1.49)% (z = -2.78, P = 0.61) and (10.95 ± 1.09)% ) In experimental group 2 were (4.79 ± 0.83)% (z = -1.349, P = 0.28) and (10.59 ± 1.03)%, respectively 3 were (13.88 ± 1.00)% (z = -2.247, P = 0.003) and (21.37 ± 2.35)% respectively (z = -2.137, P = 0.006). The difference was statistically significant. Cisplatin treated SKOV3 cells in each group compared with the control group, the experimental group 1 24 and 48h apoptosis rates were (11.25 ± 1.86)% (z = -0.361, P = 0.59) and (33.66 ± 4.96)% (10.32 ± 0.14)% (z = -0.225, P = 0.65) and (31.56 ± 6.21)% (z = -0.163, P = 0.83) (41.99 ± 6.66)% (z = -2.14, P = 0.03) and (66.5 ± 2.55)% (z = -1.909, P = 0.04) in the experimental group 3, the difference was statistically significant significance. CONCLUSION: Con A combined with lymphocytes can significantly change the morphology of SKOV3 cells, reduce cell viability, promote apoptosis and increase cell sensitivity to cisplatin, whereas Con A or lymphocytes alone did not.