【作者】 尹强；

【导师】 钟德玝；

【作者基本信息】 中南大学 ， 外科学， 2011， 博士

【摘要】 本课题前期研究表明全虫蜈蚣提取液(Extract of centipedes, ECP)对肝细胞肝癌(HCC)有一定的治疗作用,并对其治疗肝细胞肝癌的机制作了初步的探讨。我们在此基础上,进一步探讨蜈蚣头足部分、全虫部分、躯干部分提取液对实验性肝癌的抑制作用,并对蜈蚣不同部分提取液(Extract from different parts of Centipedes, ECPs)进行系统的毒理研究,通过观察ECPs的毒理学反应,以期确定ECPs的毒性大小、毒性作用的靶器官,以及探讨ECPs对肝脏毒性作用的机制,为临床合理选择用药提供实验基础。第一章蜈蚣不同部分提取液对肝癌细胞Bel-7404移植瘤的抑制作用目的：研究ECPs在体内对肝癌细胞Bel-7404移植瘤的抑制作用。方法：建立裸鼠Bel-7404人异位肝癌移植模型,以ECPs予以灌服,观察肿瘤生长及肝癌转移情况。结果：①裸鼠Bel-7404人异位肝癌移植模型成功率100%；②ECPs对裸鼠Bel-7404移植瘤有明显抑制作用,移植瘤术后30天,荷瘤对照组肿瘤体积为1000.5+87.4mm3,而头足组、全虫组、躯干组肿瘤体积分别为：460.3+61.7、526.1±75.7、530.7±81.3mm3。移植瘤重量荷瘤对照组为1120.01±101.94mg,而头足组、全虫组、躯干组肿瘤分别为593.73±37.01、659.57+62.88、669.42±45.00mg,对照组与治疗组间均有明显统计学差异(P<0.05),计算头足组、全虫组、躯干组肿瘤抑制率分别达到46.99%、41.11%、40.23%。头足组、全虫组、躯干组组间比较,全虫组与躯干组平均肿瘤重量较为接近,无显著性差异；而头足组平均肿瘤重量低于全虫组与躯干组,有显著性差异(P<0.05)。结论：①成功建立人肝癌Bel-7404细胞株裸鼠移植瘤动物模型；②ECPs以10g生药/kg的剂量给药时,有抑制裸鼠Bel-7404移植瘤的作用,其中头足提取液抑瘤作用更显著。第二章蜈蚣不同部分提取液的毒性研究目的：通过急性毒性试验和长期毒性试验评价ECPs的一般毒性作用；通过体内微核试验、精子畸形试验、Ames试验评价ECPs的遗传毒性作用。方法：(1)急性毒性试验：用不同剂量ECPs灌胃昆明小鼠,观察小鼠活动、毒性反应及死亡情况。(2)长期毒性试验：用不同剂量ECPs灌胃SD大鼠,观察动物的反应及体重变化,分别于第4周和第13周取血作血常规及生化检查及行病理解剖,检查主要脏器的病理变化及计算脏器系数。留下的动物停药后继续观察2周(第15周)后再活杀,了解毒性反应的恢复情况和可能出现的延迟性毒性。(3)遗传毒性试验：①微核试验：用不同剂量ECPs灌胃昆明小鼠。给药后取股骨骨髓细胞制作涂片,观察含有微核的嗜多染红细胞数,以微核率表示。②精子畸变试验：用不同剂量ECPs灌胃昆明小鼠。给药后附睾滤液制作涂片,观察畸形精子数,计算其畸变率。③AMES试验：采用标准平板法,计数每皿上长出的回变菌落数。结果：(1)急性毒性试验：头足组的LD5o为39.18g生药/kg,95%平均可信区间32.89-45.01g生药/kg；全虫组的LD5o为51.20g生药/kg,95%平均可信区间41.81-59.97g生药/kg,躯干组LD5o为53.85g生药/kg,95%平均可信区间45.09-62.12g生药/kg。(2)长期毒性试验：①一般生理指标：实验初期ECPs可引起部分动物呕吐和大便性状改变,随后可缓解。实验过程中,与同期对照组相比,ECPs对SD大鼠摄食量无明显影响；ECPs可引起头足高剂量组大鼠体重增长减慢。②血液学指标：给药期间(第4周,第13周),与同期对照组相比,ECPs可引起RBC、HB降低,ALT、AST增高,BUN、CREA增高,PT、TT、APTT延长,并且有显著性差异(P<0.01,P<0.05)；到恢复期结束,除头足高剂量组ALT、AST外,其它指标基本恢复正常。同时期同处理组不同剂量水平组内比较：高、低剂量之间,以上指标存在统计学差异。同时期同剂量水平不同处理组组间比较：低剂量时,各组间以上指标无统计学差异；高剂量时,头足组与全虫组、头足组与躯干组比较,ALT、AST、BUN、CREA、PT、 APTT存在统计学差异(P<0.01,P<0.05),而全虫组与躯干组比较,无显著性差异(P>0.05)。③病理检查：处死大鼠行病理学检查,各组对脏器系数无明显影响。给药期间,头足中高剂量组、全虫高剂量组、躯干高剂量组可引起肝细胞肿胀改变,头足高剂量组甚至导致肝脏坏死改变,还可引起肾脏轻度水变性改变。停药后2周,肝肾损害程度减轻,肾脏基本恢复正常,部分肝脏遗留坏死改变。各组对心脏、脑、肺脏、脾脏组织无明显损害。(3)遗传毒性试验：①微核试验：蜈蚣提取液各剂量组微核率与阴性对照组之间无显著性差异(P>0.05),而环磷酰胺阳性对照组与阴性对照组间有显著性差异(P<0.01),说明该蜈蚣提取液无致小鼠骨髓嗜多染红细胞微核增生作用。②精子畸形试验：蜈蚣提取液对大鼠精子畸形发生率无明显影响,各剂量组与阴性对照组比较差异无显著性(P>0.05),而阳性对照组与阴性对照组有显著性差异(P<0.01),说明蜈蚣提取液无致小鼠精子畸形作用。③Ames试验：阳性对照组的回变菌落数超过白发回变对照组菌落数的2倍,而蜈蚣提取液各剂量组的回变菌落数均末超过自发回变菌对照组落数的2倍,结果为阴性。结论：①中药蜈蚣头足部分提取液为小毒,全虫部分、躯干部分提取液毒性接近,毒性更小。②相同剂量下,中药蜈蚣不同提取液的毒性大小依次为：头足部分>全虫部分三躯干部分。③ECPs可引起肝肾损害和凝血功能异常,肝脏、肾脏、血液系统可能为主要的毒性靶器官,其中肝脏损害较重,而肾脏和血液系统损害相对较轻。④ECPs在本实验条件下无明显遗传毒性作用。⑤临床治疗肝癌时,以蜈蚣全虫入药为宜,并且可适当加大蜈蚣的用量。第三章蜈蚣不同部分提取液对肝脏毒性损害机制研究目的：通过检测SD大鼠肝脏组织MDA、SOD、CytP450、TNF-α、IL-6水平,探讨ECPs对肝脏毒性损害的机制。方法：硫代巴比妥酸法检测肝组织MDA含量,黄嘌呤氧化酶法检测肝组织SOD活力,CO还原差示光谱法(Omura法)测定肝组织CytP450含量,双抗体夹心ABC-酶联免疫吸附试验(ELISA)检测肝组织TNF-a、IL-6。结果：与同期对照组相比,ECPs可引起MDA水平增高、SOD活力降低、CytP450含量降低、TNF-α、IL-6含量增高,差异显著(P<0.01,P<0.05)；其中头足高剂量组IL-6、TNF-α的差异到恢复期结束仍然存在(P<0.05)。同时期同处理组不同剂量水平组内比较：高、低剂量之间,以上指标存在统计学差异。同时期同剂量水平不同处理组组间比较：低剂量时,各组间以上指标无统计学差异；高剂量时,头足组与全虫组、头足组与躯干组比较,MDA、SOD、CytP450、TNF-α、IL-6存在统计学差异(P<0.01,P<0.05),而全虫组与躯干组比较,无显著性差异(P>0.05)。结论：①ECPs可引起肝组织MDA水平增高、SOD活力下降,通过脂质过氧化导致肝细胞化学性损伤。②ECPs可引起肝组织TNF、IL-6水平增高,通过诱导细胞因子、炎性因子等对肝细胞产生免疫性损伤。③ECPs可引起肝组织CytP450水平下降,提示中药蜈蚣可能通过影响动物体内组胺—HP450这一途径对肝脏造成损伤。更多还原

【Abstract】 The previous research of this programme initially clarified that the extract of Centipedes(ECP) has some therapeutic effect on Hepatocellular carcinoma(HCC), and made an initial probe into the mechanism of ECP for the treatment of hepatocellular carcinoma. On the basis of the previous research, we plan to further approach inhibitive effect of extract from different parts of Centipedes(ECPs) on Bel-7404hepatic carcinoma xenografts and make a systematic toxicological study on ECPs-through observing the different toxicological reactions caused by ECPs try to determine the latter’s toxicity and the target organs of the toxicity, and to probe into the mechanism of the toxicity on the target organs, so that an experimental basis can be provided for reasonable choice of clinical medication.Chapter One:Inhibitive Effect of ECPs on Bel-7404hepatic carcinoma xenograftsObjective:To investigate the inhibitive effect of ECPs on Bel-7404hepatic carcinoma xenografts.Method:To set up model of Bel-7404hepatic carcinoma xenografts in nude mouse,and observe its tumor growth and metastasis by intragastric administration of ECPs.Result:①The achievement ratio for Bel-7404hepatic carcinoma xenografts in nude mouse is100%.②ECPs had distinctive inhibitive effect on Bel-7404hepatic carcinoma xenografts in nude mouse. At the30st day postgraft of nude mouse,the average cancer volume of the contrast group was1000.5±87.4mm3, and that of the treatment groups are460.3±61.7、526.1±75.7、530.7±81.3mmj respectively; the average grafting carcinoma weight of the contrast group was1120.01±101.94mg, but that of the treatment groups were593.73±37.01、659.57±62.88、669.42±45.00mg respectively; all these differences are all distinctive (P<0.05). The inhibitive ratio of the head&foot group, the holo-centipede group and the truck group reach46.990%、41.11%、40.23%respectively. The average weight of the grafting carcinoma in the holo-centipede group and the truck group are of no significant difference; but that of the head&foot gorup is less than the other two groups, the difference is significant (P<0.05).Conclusion:①The model of Bel-7404hepatic carcinoma xenografts in nude mouse is set up successfully;②ECPs(10g/kg) can inhibit Bel-7404hepatic carcinoma xenografts in nude mouse; Extract from the head&foot of centipede has the most significant inhibitive effect. Chapter Two:Toxicity study on extract from different parts of centipedesObjective:To evaluate the general toxicity of the ECPs through acute toxicity test and long-term toxicity test. To evaluate the genetic toxicity of the ECPs through Micronucleus test, Sperm Malformation Test and Ames Test.Method:(1)Acute toxicity test:The KM mice were lavaged with the different dose of ECPs, and their activities, reactions and death were observed.(2)Long-term toxicity test:The SD rats were lavaged with the different dose of ECPs in successive4weeks and13weeks, and their reactions and weight changes were observed. The blood routine test and biochemical test were detected. Then pathological anatomy was done on the rats, and the organ coefficient was calculated.(3)Genetic toxicity test:①Micronucleus test:The different dose of ECPs were lavaged to the rats, Then1000polychromatic erythrocytes (PCE) per rat were counted on each smear and get the micronucleus rates.②Sperm Malformation Test: The different dose of ECPs were the same to the micronucleus test.1000sperm cells per rat were observed through the light microscope and the aberration rate was got.③Ames test:Standard plate method was used. The numbers of the strains in each culture dish were scored.Result:(1)Acute toxicity test:Through the acute toxicity test, the LD50of the head&food group was39.18g/kg, with the average95%confidence interval (CI)32.89～45.01g/kg; the LD50of the holo-centipede group was51.20g/kg, with the average95%CI41.81～59.97g/kg; and the LD50of the trunk group was53.85g/kg, with the average95%CI45.09～62.12g/kg.(2)Long-term toxicity test:①General physiological indices:Early in the experiment, ECPs caused some animals vomit and changes of stool trait, then the symptom could be relieved. Compared with the contrast group of the same period, no influence on the SD rats’food intake was found in any group during the experiment.But the group of rats lavaged with high dose of extract from the head&food of centipede were found conspicuous hold-back in their weight growth.②Hematological indices: During the period of administration (in the4th and the13th week), compared with the contrast group of the same period, it was found that, ECPs caused a drop in RBC and Hb, a raise in ALT, AST, BUN, and CREA, and extended PT、TT、APTT, and was found significant difference (P<0.01, P<0.05). When the convalescence is over (in the15th week), all the other indicators mentioned above, except ALT and AST, basically recovered to their normal level. Comparison among the groups of the same period with the same treatment but different dose level:significant differences were found between the high dose group and the low dose group.Comparison among the groups of the same period with the same dose level but different treatment:In the level of low dose, no differences was found having statistical sense; In the level of high dose, Significant differences were found in ALT、AST、BUN、CREA、PT、APTT between the head&foot group and the holo-centipede group, and between the head&foot group and the trunk group(P<0.01, P<0.05);but no difference was found between the holo-centipede group and the trunk group(P>0.05).③Pathological examination:After the experiment, the rats were killed and pathological examination was made on them. The results show that the groups of any dose level have no significant influence on organ coefficient. During the administration, high dose of head&foot extract, holo-centipede extract, and trunk extract caused swelling in the liver cells, especially the high dose of head&foot extract even caused liver necrosis, and mild cellular edema in kidney. When the convalescence was over, the damage on liver and kidney was reduced, the kidney basically recovered, hepatic necrosis remained. None group made significant damage on the rats’ heart, brain, lung or spleen.(3)Genetic toxicity test:①Micronucleus test:No significant deviation was found in micronucleus rate between all the groups lavaged with ECPs and their negative contrast groups (P>0.05); while high significant deviation was found between cyclophosphamide positive contrast group and negative contrast group (P<0.01). This shows that the ECPs have no effect on proliferation of mice polychromatic erythrocytes.②Sperm Malformation Test:No manifest effect by the ECPs was found on the sperm malformation of the mice; no significant deviation was found in any group (at different dose level) compared with their negative contrast group (P>0.05); and significant deviation was found in the positive control group compared with the negative contrast group (P<0.01). This shows that the ECPs have no effect on sperm malformation.③Ames test: The number of colony with back mutation in the positive contrast group exceeded the twice number of that in the negative contrast group; the number of colony with back mutation in varied strains of Salmonella typhimurium at different doses did not exceed the twice number of those with spontaneous mutation. The result of the test was negative.Conclusion:①Extract from head&foot of centipedes had low toxicity, while the toxicity of extract from holo-centipede is similar to that of the trunk.②When at the same dose level, toxicity of ECPs is:head&foot extract>holo-centipede extract>trunk extract.③ECPs can cause dysfunction in liver, kidney, and coagulopathy; the main target organs may be liver, kidney, etc., while more damage will be caused on liver, and less on kidney and hematological system.④No genetic toxicity of ECPs was found under our experiment condition.⑤It is advisable to use holo-centipede, and to increase the dose appropriately in the clinical treatment of HCC. Chapter Three:Experimental study on mechanisms of hepatotoxical damage in rats caused by ECPsObjective:To probe into the toxicological mechanism of ECPs on liver damage through detecting the level of MDA, SOD, CytP450, TNF-α, and IL-6in the livers of SD rats.Method:The MDA contents in livers were measured by the method of thiobarbituric acid reaction (TBA); the SOD activity of the livers was measured by Xanthine oxidase; the CytP450contents in livers were measured by Omura; TNF-a and IL-6in livers were measured by ELISA.Result:Compared with the contrast group of the same period, it was found that, during the period of administration, ECPs caused a drop in SOD and CytP450, a raise in MDA, IL-6, TNF-α (P<0.01, P<0.05).When the convalescence is over, all the other indicators mentioned above, except IL-6and TNF-α, basically recovered to their normal level. Comparison among the groups of the same period with the same treatment but different dose level:significant differences were found between the high dose group and the low dose group.Comparison among the groups of the same period with the same dose level but different treatment:Significant differences in MDA. SOD. CytP450、TNF-α、IL-6were found between the head&foot group and the holo-centipede group, and between the head&foot group and the trunk group(P<0.01, P<0.05),but no significant difference was found between the holo-centipede group and the trunk group (P>0.05).Conclusion:①ECPs can cause a raise in MDA contents and a drop in SOD activity, and may cause a chemical injury in liver cells through lipid peroxidation.②ECPs can cause a raise in TNF、IL-6contents and immunological injury in liver cells through inducing cytokine and inflammatory factors (such as TNF, IL-6,etc.).③ECPs can cause a drop in CytP450contents in liver, and the damage may be caused through affecting Histamine-HP450in the animals’body.更多还原