新城疫病毒疫苗及白细胞介素-15对卵巢癌细胞株体外作用的实验研究

Experimental Study on the Effect of Newcastle Disease Virus Vaccine and Interleukin-15 to the Human Ovarian Carcinoma Cell Strain in Vitro

目的研究新城疫病毒疫苗(ATV-NDV)和白细胞介素-15(IL-15)对人卵巢癌细胞株体外生长的作用。方法将正常人卵巢上皮细胞和人卵巢癌细胞株3AO体外培养;分离正常人外周血淋巴细胞;制备人卵巢癌细胞株3AOATV-NDV,(1-2)×107细胞/0.5ml;将ATV-NDV、IL-15、ATV-NDV+IL-15分别与正常人外周血淋巴细胞共培养24、48、72、96小时;用正常人外周血淋巴细胞作对照,用MTT比色法测定不同浓度ATV-NDV、IL-15作用不同时间对淋巴细胞增殖程度的影响,将ATV-NDV(50μl)、rhIL-15(50ng/ml)及ATV-NDV(50μl)+IL-15(50ng/ml)与人外周血淋巴细胞共培养48小时,按效靶比分别为50∶1、25∶1、10∶1加入正常人卵巢上皮细胞和人卵巢癌细胞株3AO,继续培养24、48、72、96小时,MTT法测定细胞生长抑制率;HE染色法观察效靶比为50∶1、继续培养24-96小时后细胞形态学变化。结果1.淋巴细胞增殖改变:ATV-NDV、IL-15具有对淋巴细胞的增殖活性,随浓度的增加和时间的延长而增强(P<0.05),与对照组相比差异有显著性(P<0.01)。IL-15与ATV-NDV具有协同作用,并且这种协同作用与IL-15、ATV-NDV的单独作用相比差异有显著性(P<0.01);IL-15、ATV-NDV对正常人外周血淋巴细胞增殖作用的影响与对照组相比差异有显著性(P<0.01);IL-15与ATV-NDV作用相比差异无显著性(P=0.35)。2.ATV-NDV、IL-15对肿瘤细胞生长抑制作用:活化的淋巴细胞与靶细胞共培养可杀死肿瘤细胞,并具有时效性(P<0.05)。3.细胞形态学变化:活化的淋巴细胞作用48小时后肿瘤细胞即已出现凋亡形态,作用96小时时即有坏死细胞形态出现。结论ATV-NDV和IL-15对正常人外周血淋巴细胞增殖有影响,经ATV-NDV、IL-15及ATV-NDV+IL-15共培养的正常人外周血淋巴细胞可有效地抑制细胞株3AO生长,具有时间、浓度依赖性。

Objective To investigate the effect of autologous tumor cell vaccines-Newcastle disease virus-infected（ATV-NDV） and interleukin-15 （IL-15） on the growth of human ovarian carcinoma cell strains in vitro. Methods Normal human ovarian epithelium cell and human ovarian carcinoma cell strain 3AO were cultured in vitro. Peripheral blood lymphocyte was separated from normal individual and ATVNDV with human ovarian carcinoma cell strain 3AO was prepared（2-4 × 10^7 cells/ml）. ATV-NDV, IL-15 and ATV-NDV＋IL-15 were cultured for 24-96 hours together with human peripheral blood lymphocyte （the controls） respectively. MTT colorimetric assay was used to analyze the effect of ATV-NDV and IL-15 with different concentration and different acting time on the proliferation degree of lymphocyte. ATVNDV（50/A）, rhIL-15（50ng/ml） and ATV-NDV（50μl）＋rhIL-15（50ng/ml） were cultured together withhuman peripheral blood lymphocyte for 48 hours, according to the ratio of effect and target（E ： T） which were 50 ： 1, 25 ： 1 and 10 ： 1, added into normal human ovarian epithelial cells and human ovarian carcinoma cell strain 3AOrespectively, continued to culture for 24-96 hours, the cell growth inhibiting rate was determined using the MTT assay and the change of cell modality was observed by hematoxylin-eosin （HE） staining cultured for 24-96 hours whose E ： T was 50 ： 1. Results 1. Lymphocyte proliferation influence： ATV-NDV and IL-15 showed proliferation effect on lymphocyte, and the effect went up with the increasing of dosage and acting time（P〈0.05）. The difference was significant compared with the controls（P~ 0.01）. IL-15 had synergism with ATV-NDV, and compared with the effect of each alone, the difference was significant（P〈0.01） ; The effect of IL-15 and ATV-NDV on human peripheral blood lymphocytes had significant difference compared with the controls（P〈0.01）; There was no significant difference between the effect of IL-15 and ATV-NDV（P=0. 35）. 2. The inhibitory effect of ATV-NDV and IL-15 on tumor ceils： The activated lymphocytes could kill carcinoma ceils if they were cultured together with target cells, and the effect was promoted as the time prolonged（P〈0.05）. 3. Cell modality changing： Tumor cells displayed the modality of apoptosis when the activated lymphocytes acted on 48 hours and the necrosis cell form arose when acted on 96 hours. Conelnsion ATV-NDV and IL-15 are proved to have effect on the proliferation of normal human peripheral blood lymphocyte, and the human peripheral blood lymphocyte can effectively inhibit the growth of the ovarian carcinoma cell strain 3AO after cultured together with ATV-NDV, IL-15 and ATV-NDV＋IL-15. The effect has the time and concentration dependence.