鲜无蹼壁虎抗肿瘤活性成分脂质体研制及抗肿瘤实验研究
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摘要
目的:鲜无蹼壁虎抗肿瘤活性成分提取分离纯化,鲜无蹼壁虎抗肿瘤活性成分脂质体的研制,鲜无蹼壁虎抗肿瘤活性成分抗实验性恶性肿瘤的药效学研究,初步探讨鲜无蹼壁虎抗肿瘤活性成分抗癌作用机制,探索癌症治疗药物靶向给药的新思路、新方法和新技术。方法:捕捉地道鲜无蹼壁虎,并与蜥蜴相区别,MTT法体外药理,药效学指导下考察不同提取方法的有效性,筛选最佳提取方案,采用85%乙醇超声提取鲜无蹼壁虎抗肿瘤活性成分,利用逆相蒸发法、薄膜分散法、旋转薄膜-超声法制备抗肿瘤活性成分脂质体,以粒径及其分布、包封率、载药量等为指标,通过单因素考察类脂材料用量、药物用量、超声强度、超声时间和水浴温度等对脂质体的影响,制备包封率较高的药物脂质体。药效学体外实验用不同浓度的鲜无蹼壁虎抗肿瘤活性成分混悬液,分别处理CT-26和Heper-G2细胞株,MTT检测肿瘤细胞的生长活性,检测细胞凋亡率;体内动物实验采用4～6周龄,体重(22±2)g,♀,BALB/C小鼠动物造模。小鼠恒温(25℃),恒湿度(45%)常规饲养,各小鼠左后肢背侧皮下接种CT-26细胞悬液0.1ml(5.5×105·ml-1),筛选出造模成功小鼠,随机分组:即正常对照组;荷瘤小鼠对照组;环磷酰胺阳性对照组;鲜无蹼壁虎抗肿瘤活性成分治疗组。鲜无蹼壁虎抗肿瘤活性成分治疗组:于接种高致瘤性CT-26肿瘤细胞10d后开始腹腔注射(ip)鲜无蹼壁虎抗肿瘤活性成分混悬液及其脂质体,剂量分别为1mg·kg-1、5mg·kg-1、10 mg·kg-1;荷瘤小鼠对照组:腹腔注射等体积的生理盐水。阳性对照组:腹腔注射环磷酰胺100mg·kg-1。各组均qd,每天上午一次,共12天。于治疗结束后脱颈处死全部小鼠,将小鼠摘眼球取血,ELISA法测定TNF-α含量,剥离瘤块,称瘤重,计算抑瘤率。结果:体外药理实验显示,85%乙醇超声法提取物抑瘤率为46.95%。旋转薄膜-超声法制备抗肿瘤活性成分脂质体,优化得到的药物脂质体在透射电镜下呈类圆形,平均粒径为(355.6±51.2)nm,包封率为52.50%,载药量为4.16%,pH5.85,粒径符合靶向要求,达到预期目标。体外药效学实验表明:鲜无蹼壁虎抗肿瘤活性成分混悬液呈时间及剂量依赖性抑制肿瘤细胞的增殖,抑制率9.52%～62.30%。Heper-G2细胞经过1mg/ml,10mg/ml鲜无蹼壁虎抗肿瘤活性成分分别作用48h后,随着提取物浓度提高,早期凋亡率逐渐增加,高剂量浓度的药物(凋亡率为33.2%)与对照组比较均能明显诱导细胞凋亡(P<0.05),但低浓度抗肿瘤活性成分引起的凋亡率与对照组的差异无显著意义(P>0.05)。光镜观察结果显示,经鲜无蹼壁虎提取液处理的Heper-G2细胞可见凋亡小体形成等典型的细胞凋亡形态,对照组细胞形态正常。体内动物实验显示:鲜无蹼壁虎醇提混悬液抑制小鼠肿瘤生长,有一定剂量效应关系。造模给药后,给药组眼球血的TNF-α水平与对照组相比具有显著差异性(P<0.01),鲜无蹼壁虎抗肿瘤活性成分脂质体高剂量平均抑制率为52.17%,显示明显的抗瘤作用(P<0.01),中低剂量抑制小鼠肿瘤生长与阴性对照组比较,无显著性差异(P>0.05),高剂量组与壁虎散组比较有显著差异性(P<0.01)。同浓度鲜无蹼壁虎抗肿瘤活性成分脂质体比其混悬液抑瘤率显著增高。结论:85%乙醇超声法提取鲜无蹼壁虎抗肿瘤活性成分效果最佳,旋转薄膜-超声法制备抗肿瘤活性成分脂质体包封率较高;鲜无蹼壁虎抗肿瘤活性成分抗肿瘤活性成分能抑制多种肿瘤细胞的增殖,提取物混悬液与脂质体均有抗癌作用,且在相同剂量下,脂质体比药物混悬液抑瘤效果更显著(P<0.05)。本实验首次证实鲜无蹼壁虎抗肿瘤活性成分对BALB/C模型小鼠肿瘤细胞的调亡,可能通过调节体内的TNF-α水平而发挥抗癌作用,为无蹼壁虎抗肿瘤活性成分抗癌作用机制提供了实验依据。
Objective: To extract and purify fresh Gekko.swinhonis.Gunther anticancer active component. To manufacture fresh Gekko.swinhonis.Gunther antineoplasmic activity component extractive liposome and study antitumor pharmacodynamics for Gekko.swinhonis.Gunther anticancer active component, investigate its antitumor mechanism in vivo and vitro.METHODS:To capture fresh Gekko.swinhonis.Gunther and distinguished with lizard,to sieve out the best extrat schema by MTT,to hypersound extrat fresh Gekko.swinhonis.Gunther anticancer active component with 85% alcohol. To perpare antineoplasmic active component liposome with antiphase evaporation method,film dispersion method,spin film–hypersound method.Using particle diameter, particle size distribution, envelopment efficency, drug loading as target,the single factor was investigated,such as lipoid materials usage amount,drug usage, hypersound intensity,hypersound time and aqueous bath temperature. Anticancer active component liposome was prepared with turning membrane- hypersoun. MTT and was used to detect CT-26 and Heper-G2 growth activity according to different drug concentration in vitro.The experimental animal mould was BALB/C mouse which age was 4～6 weeks and weight was 22±2g ,♀.Athymic mouse was hypodermic inoculated CT-26 cell suspension in its left hind limb dorsi,it was routine rear of constant temperature(25℃) and constant humidity(45%).CT-26 and Heper-G2 tumor cells treated for 24-144h by various concentrations suspension of fresh Gekko.swinhonis.Gunther antineoplasmic activity component extraction (FGE) were assessed in vitro. Antiproliferation was obtained by MTT. The transplant tumor model of CT-26 in BALB/C mice was established. CT-26 tumor model mice were divided randomly into five groups ,the Cytoxan positive group,the FGE group and the negative control group,they were treated respectively with intraperitoneal injection of Cytoxan 100 mg·kg-1, FGE 10mg·kg-1,5mg·kg-1and 1mg·kg-1 respectively,and equal volume of saline, intragastric administration of Gekko pulvis 100 mg·kg-1. The medication was given for 12 times totally, and mice were to take in food and water freely , after putting to death by dislocation to measure mice body weight, to dissect subcutaneouly tumor.The anti-tumor activity was evaluated by tumor tissue weighing. Antiproliferation rate of tumor cell was detected in vivo and in vitro. The light microscop results manifested that typical apoptosis cell morphology change in administer experiment group ,cell membrane integrated, cell volume became smaller,synapse became shorter,cell shrinked to round, cell outline became clear. RESULTS: The pharmaco-essay manifested that Antiproliferation rate of extractive with 85% alcohol hypersound method was 46.95 % . The liposome of fresh Gekko.Swinhonis.Gunther antineoplasmic activity component was prepared by turnning film - hypersound.The similar round was seen with transmission electron microscope, which mean diameter was 355.6±51.2nm,EE was 52.50%,DL was 4.16%, pH 5.85,the liposome diameter consisitent with target desire. The pharmacodynamics experiment in vitro manifested that fresh Gekko.Swinhonis.Gunther antineoplasmic activity component suspension could obviously inhibit cell proliferate in a time-and dose-dependet manner in vitro and vivo,the ratio of inhibiting was 9.52%～62.30%,Heper-G2 cell was expose to FGE of 1 mg/ml,10.0mg/ml for 48h,Apoptosis ratio was enhanced gradually according to increasing FGE concentration .Higher dosage groups (apoptosis ratio: 33.2%) hadn’t significant difference compared with positive control group, apoptosis ratio of lower dosage groups hadn’t significant difference compared with control group(P>0.05) .The animal experimental results manifested that FGE could inhibit mouse cancer growth with some dosage-efficency relationship. The content of TNF-αin eyeball blood and NO secreted by mouse macrophage had significant difference compared with control group(P<0.05). The mean inhibiting ratio of high dosage group of FGE liposome was 52.17%,which had significant difference compared with‘Tianlongsan’group(P<0.01), Antiproliferation rate of liposome had significant enhance compared with FGE in same concentration. CONCLUSION: Our test results showed that fresh Gekko.swinhonis.Gunther antineoplasmic activity component suspension could effectively decrease CT-26, Heper-G2 cell proliferation both in vitro and vivo,suggesting that antineoplasmic activity component extraction of fresh Gekko.Swinhonis.Gunther might be a potential anticancer drugs. The extract antineoplasmic activity component effective was best with 85% alcohol-hypersound,EE was higher with turning membrane-hypersound to prepare liposome. The extraction can inhibit several varieties of tumor cell. In the limited scope of drug concentration, the ratio of anti-cancer rises with the drug concentration rising.This study first confirmed that fresh Gekko.swinhonis.Gunther antineoplasmic activity component suspension induced BALB/C model mouse tumour cell apoptosis might be by enhancing TNF-αlevel to bring into full playing antitumouse effect,it provided experimental evidence for fresh Gekko.swinhonis.Gunther antineoplasmic activity component suspension anticancer mechanism .
Objective: To extract and purify fresh Gekko.swinhonis.Gunther anticancer active component. To manufacture fresh Gekko.swinhonis.Gunther antineoplasmic activity component extractive liposome and study antitumor pharmacodynamics for Gekko.swinhonis.Gunther anticancer active component, investigate its antitumor mechanism in vivo and vitro.METHODS:To capture fresh Gekko.swinhonis.Gunther and distinguished with lizard,to sieve out the best extrat schema by MTT,to hypersound extrat fresh Gekko.swinhonis.Gunther anticancer active component with 85% alcohol. To perpare antineoplasmic active component liposome with antiphase evaporation method,film dispersion method,spin film–hypersound method.Using particle diameter, particle size distribution, envelopment efficency, drug loading as target,the single factor was investigated,such as lipoid materials usage amount,drug usage, hypersound intensity,hypersound time and aqueous bath temperature. Anticancer active component liposome was prepared with turning membrane- hypersoun. MTT and was used to detect CT-26 and Heper-G2 growth activity according to different drug concentration in vitro.The experimental animal mould was BALB/C mouse which age was 4～6 weeks and weight was 22±2g ,♀.Athymic mouse was hypodermic inoculated CT-26 cell suspension in its left hind limb dorsi,it was routine rear of constant temperature(25℃) and constant humidity(45%).CT-26 and Heper-G2 tumor cells treated for 24-144h by various concentrations suspension of fresh Gekko.swinhonis.Gunther antineoplasmic activity component extraction (FGE) were assessed in vitro. Antiproliferation was obtained by MTT. The transplant tumor model of CT-26 in BALB/C mice was established. CT-26 tumor model mice were divided randomly into five groups ,the Cytoxan positive group,the FGE group and the negative control group,they were treated respectively with intraperitoneal injection of Cytoxan 100 mg·kg-1, FGE 10mg·kg-1,5mg·kg-1and 1mg·kg-1 respectively,and equal volume of saline, intragastric administration of Gekko pulvis 100 mg·kg-1. The medication was given for 12 times totally, and mice were to take in food and water freely , after putting to death by dislocation to measure mice body weight, to dissect subcutaneouly tumor.The anti-tumor activity was evaluated by tumor tissue weighing. Antiproliferation rate of tumor cell was detected in vivo and in vitro. The light microscop results manifested that typical apoptosis cell morphology change in administer experiment group ,cell membrane integrated, cell volume became smaller,synapse became shorter,cell shrinked to round, cell outline became clear. RESULTS: The pharmaco-essay manifested that Antiproliferation rate of extractive with 85% alcohol hypersound method was 46.95 % . The liposome of fresh Gekko.Swinhonis.Gunther antineoplasmic activity component was prepared by turnning film - hypersound.The similar round was seen with transmission electron microscope, which mean diameter was 355.6±51.2nm,EE was 52.50%,DL was 4.16%, pH 5.85,the liposome diameter consisitent with target desire. The pharmacodynamics experiment in vitro manifested that fresh Gekko.Swinhonis.Gunther antineoplasmic activity component suspension could obviously inhibit cell proliferate in a time-and dose-dependet manner in vitro and vivo,the ratio of inhibiting was 9.52%～62.30%,Heper-G2 cell was expose to FGE of 1 mg/ml,10.0mg/ml for 48h,Apoptosis ratio was enhanced gradually according to increasing FGE concentration .Higher dosage groups (apoptosis ratio: 33.2%) hadn’t significant difference compared with positive control group, apoptosis ratio of lower dosage groups hadn’t significant difference compared with control group(P>0.05) .The animal experimental results manifested that FGE could inhibit mouse cancer growth with some dosage-efficency relationship. The content of TNF-αin eyeball blood and NO secreted by mouse macrophage had significant difference compared with control group(P<0.05). The mean inhibiting ratio of high dosage group of FGE liposome was 52.17%,which had significant difference compared with‘Tianlongsan’group(P<0.01), Antiproliferation rate of liposome had significant enhance compared with FGE in same concentration. CONCLUSION: Our test results showed that fresh Gekko.swinhonis.Gunther antineoplasmic activity component suspension could effectively decrease CT-26, Heper-G2 cell proliferation both in vitro and vivo,suggesting that antineoplasmic activity component extraction of fresh Gekko.Swinhonis.Gunther might be a potential anticancer drugs. The extract antineoplasmic activity component effective was best with 85% alcohol-hypersound,EE was higher with turning membrane-hypersound to prepare liposome. The extraction can inhibit several varieties of tumor cell. In the limited scope of drug concentration, the ratio of anti-cancer rises with the drug concentration rising.This study first confirmed that fresh Gekko.swinhonis.Gunther antineoplasmic activity component suspension induced BALB/C model mouse tumour cell apoptosis might be by enhancing TNF-αlevel to bring into full playing antitumouse effect,it provided experimental evidence for fresh Gekko.swinhonis.Gunther antineoplasmic activity component suspension anticancer mechanism .
引文
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