羊胎盘抗肿瘤活性蛋白多肽的制备分离

Preparation and Isolation of Anti-tumor Active Proteins and Polypeptides from Sheep Placenta

摘要:

以羊胎盘为原料，蛋白含量和MTT肿瘤抑制活性作为评价指标，筛选羊胎盘抗肿瘤活性蛋白多肽组分的提取和制备工艺。结果表明，混合磷酸盐缓冲溶液搅拌提取法可用于制备具有结肠癌抑制活性的羊胎盘提取物。其具体条件为：pH值为7.4的混合磷酸盐缓冲溶液（其中含0.9 wt.%的NaCl溶液，50 mmol/L常规PBS溶液，0.5~1 mmol/L苯甲基磺酰氟），提取温度4~10 ℃，提取2 h，总提取物得率为37.91%，蛋白含量为55.62%。抗肿瘤活性结果表明当提取物质量浓度为2 mg/mL，作用于HT-29细胞和HCT-116细胞抑制率分别56.88%和53.07%。总提取物经Sephadex-G-25凝胶柱层析分离纯化得到2个对结肠癌HT-29和HCT-116细胞株具有良好抑制活性的蛋白多肽组分F2和F3。抗肿瘤活性测试F2组分的IC50值分别为0.328 mg/mL和0.183 mg/mL，F3组分的IC50值分别为0.213 mg/mL和0.095 mg/mL。综上证实羊胎盘混合磷酸盐提取物经凝胶柱层析得到的活性蛋白多肽组分具有良好的抑制结肠癌细胞活性，可为羊胎盘废弃物的有效开发，综合利用提供理论与技术支撑。

Abstract:

Sheep placenta was used as the raw material to extract proteins and peptides. The protein content and MTT tumor inhibitory activity were used as the evaluation indexes to screen the extraction and fractions of sheep placenta with anti-tumor activity. The results showed that the stirred extraction method mixed phosphate buffer solution could be used to prepare sheep placenta extracts with inhibitory activity against a colon cancer cell lines. The specific conditions were as follows: mixed phosphate buffer solution (containing 0.15 mol/L NaCl solution, 50 mmol/L conventional PBS solution, and 0.5~1 mmol/L phenylmethylsulfonyl fluoride) at pH value 7.4, extraction temperature of 4~10 ℃, and extraction for 2 h. The total extract yield and the protein content were 37.91 and 55.62%, respectively. The results of antitumor activity showed that the inhibition rate was 56.88 and 53.07% in HT-29 and HCT-116 cells, respectively, when the concentration of the extract was 2 mg/mL. The total extract was isolated and purified using a Sephadex-G-25 gel column chromatography to obtain F2 and F3 fractions with good inhibitory activity against HT-29 and HCT-116 cells; the IC50 of F2 was 0.328 and 0.183 mg/mL, respectively, and the IC50 of F3 was 0.213 and 0.095 mg/mL, respectively. In conclusion, the active fractions obtained from the gel column chromatography of the mixed phosphate extract of sheep placenta had good inhibitory activity against colon cancer cell lines. The results provide theoretical and technical support for the effective development and comprehensive utilization of sheep placenta waste.