【摘要】 胎盘免疫调节因子(placenta immunoregulating factor,PF)是从胎盘提取的小分子多肽,对病毒性疾病、免疫缺陷疾病及恶性肿瘤等具有潜在的临床应用价值。为检测牛(Bos taurus)和绵羊(Ovis aries)胎盘免疫调节因对人(Homo sapiens)胰腺癌细胞(pancreatic cancer cells-Ⅰ,PANC-Ⅰ)增殖与迁移的影响,本研究对从屠宰场获得的牛和绵羊胎盘进行分离,利用双酶水解法制备PF,将质量浓度为100~2 000 ng/m L的牛、羊PF作用于体外培养的PANC-Ⅰ细胞,以3-(4,5-二甲基噻唑-2-基)-5-(3-羧甲酯基)-2-(4-磺苯基)-2H-四唑(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,MTS)法观察细胞增殖抑制率,应用流式细胞技术检测其对PANC-Ⅰ细胞周期的影响。并利用Transwell迁移实验检测添加PF对PANC-Ⅰ细胞迁移能力的影响,同时通过实时荧光定量PCR法检测肿瘤抑制基因(tumor suppressor gene,P53)和细胞周期依赖性蛋白激酶抑制因子1A基因(cyclin-dependent kinase inhibitor 1A,P21)的表达情况。结果显示,17.11 g绵羊胎盘制得0.588 mg PF,6.61 g牛胎盘制得0.312 mg PF,胎盘质量与所得PF的质量比约为20 000∶1。在体外培养的PANC-Ⅰ细胞培养液中添加PF,从形态上观察,牛羊PF对PANC-Ⅰ细胞没有明显的影响。绵羊PF处理后PANC-Ⅰ细胞增殖抑制率为9.75%~22.89%,牛PF处理后PANC-Ⅰ细胞增殖抑制率为-3.81%~23.56%,增殖抑制率最显著的绵羊PF浓度为500 ng/m L,牛PF浓度为1 000 ng/m L。绵羊PF在浓度为500 ng/m L时,PANC-Ⅰ细胞的PI和SPF最低,而牛PF各浓度下的胰腺癌细胞增殖指数(proliferation index,PI)和增殖活性(S-phase fraction,SPF)没有明显的变化。同时,牛羊PF对PANC-Ⅰ细胞的迁移能力无显著影响。牛羊PF使PANC-I细胞P53表达上调而P21表达下调。上述结果表明,在一定浓度下,PF对体外培养的PANC-Ⅰ细胞增殖有负调控作用,且牛羊胎盘免疫调节因子对胰腺癌细胞的影响不同,可能因为其存在的活性成分有区别。本研究为胰腺癌的治疗提供了新的思路。更多还原

【Abstract】 Placenta immunoregulating factor(PF) is composed of small polypeptides, which is extracted from the placenta. PF is valuable candidate for treatment of viral disease, immune deficiency disorder and malignant tumor. To detect the effects of sheep(Ovis aries) and cow(Bos taurus) placenta immunoregulating factor on the proliferation and migration of the human(Homo sapiens) pancreatic cancer cells- Ⅰ(PANC- Ⅰ)in vitro. PF was prepared from placenta by bienzyme hydrolysis. PANC-Ⅰ cells cultured in vitro were exposed to PF at the mass concentrations of 100~2 000 ng/m L. The effect of PF on the PANC- Ⅰ cell cycle was detected by flow cytometric method. The migration ability of PANC-Ⅰ cell was investigated with Transwell assay. The inhibition to proliferation rate of PANC-Ⅰ cells were evaluated by 3-(4, 5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)- 2-(4- sulfophenyl)- 2H- tetrazolium(MTS) assay. The m RNA expression levels of tumor suppressor gene(P53) and cyclin-dependent kinase inhibitor 1A(P21) were analyzed by using real-time PCR. The relative expression was determined by using the comparative 2-??Ctmethod. The results showed that0.588 mg PF derived from 17.11 g sheep placenta and 0.312 mg PF derived from 0.616 g cow placenta, and the quality ratio was 20 000∶1. The morphology of the PANC-Ⅰ cells was not affect by adding the PF. The cell inhibition rate of PANC-Ⅰ cells was from 9.75% ~22.89% with different concentration of sheep PF and from-3.81%~23.56% with different concentration of cow PF. The most remarkable concentration of sheep PF and cow PF were 500 ng/m L and 1 000 ng/m L, respectively. At the same time, the PI and SPF of PANC-Ⅰcells was the lowest with 500 ng/m L of sheep PF, but the proliferation index(PI) and S-phase fraction(SPF) were not affected by cow PF. The migration ability of PANC-Ⅰcells was not affected by PF. The expression levels of P53 and P21 of PANC- Ⅰ cells were increased and decreased respectively after the treatment of PF. The results showed that the PF had a negative effect on the proliferation of PANC-Ⅰ cells in vitro, and the different effects of sheep and cow PF on PANC-Ⅰcells may related to the composition of the PF. This study provides new ideas for the treatment of pancreatic cancer.更多还原