【作者】 孙鹏；

【导师】 任先达；

【作者基本信息】 暨南大学 ， 药理学， 2003， 硕士

【摘要】 目的：研究眼镜蛇毒灌胃后的兔血清（the serum of rabbit taking orally cobra venom，SRCV）对肝癌的体内外抑瘤作用和诱导凋亡作用，并评价其安全性。 实验方法：根据血清药理学方法，制备SRCV，以人肝癌HepG-2细胞及正常人胚肺成纤维细胞为靶细胞，以小鼠肝癌移植瘤为动物模型，通过台盼蓝拒染实验、在体抑瘤实验、流式细胞仪分析、电镜观察、TUNEL测试等实验方法，观察该制剂对肝癌的体内外抑瘤及在体促凋亡作用。通过急性毒性实验和半数有效量测定评价其安全性。 实验结果：1)高、低剂量SRCV各组的细胞毒作用均显著强于同浓度正常血清（P＜0.01），其中5mg／ml剂量组的增殖抑制率均高于5μg／ml阿霉素（P＜0.01）；而高剂量SRCV的抑制率略高于同浓度低剂量SRCV，但无统计学显著性。同时，高剂量SRCV（5mg／ml）有显著的抗肿瘤细胞增殖作用，其作用与15μg／ml阿霉素相近。2)SRCV及正常兔血清各剂量组对人胚肺成纤维细胞有不同程度促进细胞增殖的作用，而前者促进细胞增殖的作用更强，其中1、2、5mg／ml剂量组与同浓度正常兔血清比较具统计学差异（P＜0.05）。3)急性毒性实验结果，SRCV的急性毒性最小，经酸热处理后蛇毒的毒性比眼镜蛇毒原毒也有所下降，其LD₅₀均有非常显著性差异（P＜0.01）。以三者LD₁的1／3量给药进行的抑瘤实验，均有显著抑瘤作用（P＜0.01），其中SRCV的抑瘤率最高，经酸热处理后蛇毒的抑瘤效果与原毒相似。SRCV的半数有效量测定结果及其相对毒性参数，说明它的相对毒性较低。4)流式细胞仪分析及TUNEL测试表明SRCV使小鼠肝癌移植瘤的凋亡率显著升高，而电镜下观察可见典型凋亡细胞，光镜下观察TUNEL处理的组织切片也可见凋亡细胞。根据上述实验结果。 结论：1)SRCV对肿瘤细胞具高度选择性抑制作用，且其细胞毒作用与抑制肿瘤细胞的增殖有关；而对正常增殖细胞，不但不抑制，还能促进细胞增殖。2)SRCV较原毒的毒性降低，但仍保留了抗癌活性。3)SRCV对小鼠肝癌移植瘤具在体促凋亡作用，与前期的体外促凋亡研究结果一致，提示SRCV的抑瘤作用与其诱导细胞凋亡有关。4)该种血清制剂仍然不够理想，有必要在今后研究中通过对有效组分的分离纯化，进一步提高其效价，降低其毒性。更多还原

【Abstract】 AIM To investigate the inhibitory and apoptosis-inducing effect of the serum of rabbit taking orally cobra venom (SRCV) on hepatocarcinoma.METHOD We used the human HepG-2 cells and human fetal lung fibroblast as target cells and established the animal model of mice transplanted hepatoma. The inhibitory effect was observed by means of trypan blue dye exclusion test in vitro and inhibitory rate test in vivo. The toxicity of SRCV was studied by using the acute toxicity and EDso test. In addition, the flow cytometry (FCM), transmission electron microscopy (TEM) and the method of TUNEL were applied to observe the apoptosis-inducing effect of SRCV in vivo.RESULT 1) The cytotoxicity action of SRCV was markedly stronger than that of the normal rabbit serum at the same dosage, and SRCV 5 mg/ml showed striking opposing-proliferation action on HepG-2 cells. Meanwhile, SRCV also displayed some auxo-action on proliferation of human fetal lung fibroblast. 2) The results of LDso and EDso tests showed that the toxicity of SRCV was the lowest among three cobra venom (CV) preparations. Comparing the inhibitory rate (IR) of three CV preparations in a dose of the 1/3 of their LDi, we found that IR of SRCV was the highest and IR of the other CV preparation which was denatured by the acid and heat was no more than that of the CV. 3) By FCM and TUNEL assay, we found that the apoptosis rate of tumor tissue was significantly increased by the SRCV. Under the TEM, the apoptosis cells could be observed. And we could, also see the apoptosis cells on the TUNEL slice.CONCLUSION 1) SRCV selectively inhibits the growth of HepG-2 cells but has no cytotoxicity on human fetal lung fibroblast. And this action is related to its suppressing proliferation of tumor cells. 2) After the CV is denatured or digested, its anticancer activity is still remained. And the toxicity of SRCV is lower than that of the others, but the tumor inhibitory effect more remarkable. 3) SRCV can induce apoptosis of tumor cells in vivo, which is identical with the results of apoptosis-inducing effect research in vitro, and hints that the tumor inhibitory effect of SRCV is related to its apoptosis-inducing effect. 4) The toxicity of SRCV is still remained to the extent, so it is necessary that SRCV would be separated and purified to reduce its toxicity.更多还原