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目的构建天蚕素 B-黄体生成素释放激素结合部位(CB-LHRH’)重组基因,探讨 CB-LHRH’蛋白成为新型抗肿瘤靶向药物的可能性。方法设计并人工合成 CB-LHRH′重组基因序列,应用昆虫细胞-杆状病毒表达系统表达目的蛋白并通过蛋白斑点印迹法鉴定,采用二甲氧唑黄(XTT)比色法检测不同剂量的目的蛋白对卵巢上皮性癌细胞株 SKOV3(LHRH 受体阴性)和子宫内膜癌细胞株 HEC-1B(LHRH 受体阳性)细胞的生长抑制率,并进行镜下形态观察。结果蛋白斑点印迹法检测结果显示,重组杆状病毒感染的 Sf9细胞裂解液呈现棕色,证实 CB-LHRH’蛋白已表达。重组杆状病毒感染的昆虫细胞系 Sf9细胞裂解液对 SKOV3及 HEC-1B 细胞的生长抑制率,随作用时间的延长和剂量的增加而增加,SKOV3细胞的生长抑制率从(5.03±0.08)%增加至(53.24±1.22)%,HEC-1B细胞的生长抑制率从(5.13±0.37)%增加至(56.16±1.08)%。相同剂量的重组杆状病毒感染的 Sf9细胞裂解液,作用相同时间,其对 HEC-1B 细胞的生长抑制率高于 SKOV3细胞,差异有统计学意义(P<0.01)。经重组杆状病毒感染的 St9细胞裂解液作用后,镜下可见癌细胞出现明显改变甚至成片坏死崩解为无定形的颗粒状物质。结论 CB-LHRH’蛋白可有效杀伤 LHRH 受体阳性的HEC-1B 细胞,有望成为治疗表达 LHRH 受体的肿瘤的抗肿瘤靶向药物。
Objective To construct recombinant gene of cecropin B-LHZ release hormone binding site (CB-LHRH’) and to explore the possibility of CB-LHRH’ protein becoming a novel antitumor drug targeting drug. Methods The CB-LHRH′ recombinant gene sequence was designed and synthesized. The target protein was expressed by the insect cell-baculovirus expression system and identified by protein dot blotting. The purpose of different doses was determined by the colorimetric method of dimethoxazole yellow (XTT). Proteins were observed for growth inhibition of epithelial ovarian cancer cell line SKOV3 (LHRH receptor negative) and endometrial cancer cell line HEC-1B (LHRH receptor positive), and were observed under microscope. Results Protein dot blot analysis showed that recombinant baculovirus-infected Sf9 cell lysates showed brown color, confirming that CB-LHRH’ protein has been expressed. The growth inhibition rate of SKOV3 and HEC-1B cells elicited by recombinant baculovirus-infected insect cell line Sf9 cells increased with the prolonged action time and dose, and the growth inhibition rate of SKOV3 cells was (5.03±0.08)%. Increasing to (53.24±1.22)%, the growth inhibition rate of HEC-1B cells increased from (5.13±0.37)% to (56.16±1.08)%. The same dose of recombinant baculovirus-infected Sf9 cell lysate, the effect of the same time, its growth inhibition rate of HEC-1B cells was higher than SKOV3 cells, the difference was statistically significant (P<0.01). After being lysed by recombinant baculovirus-infected St9 cell lysate, the cancerous cells apparently changed under microscope and even became necrotic and disintegrated into amorphous granular material. Conclusion The CB-LHRH’ protein can effectively kill LHRH receptor-positive HEC-1B cells, and it is expected to become an anti-tumor drug targeting tumors expressing LHRH receptors.