【作者】 张小贺；

【导师】 杜标炎；

【作者基本信息】 广州中医药大学 ， 中西医结合基础， 2008， 硕士

【摘要】 恶性黑素瘤(malignant melanoma)是来源于黑色素细胞的恶性肿瘤,发病率为全身恶性肿瘤的1%—2%,其恶性程度高,预后差。随着分子生物学的发展,基因治疗给恶性肿瘤的治疗带来了曙光,恶性黑素瘤是最早进行基因治疗的恶性肿瘤之一。肿瘤的基因治疗,特别是肿瘤的自杀基因疗法,已经成为继传统的手术切除、放疗、化疗等方法后的一种新的抗肿瘤治疗措施。但单纯自杀基因治疗由于目前载体转染效率较低、靶向性不够等问题没解决,仍有部分肿瘤细胞残余导致肿瘤复发,这是该疗法存在的重大缺陷。因而增强旁杀伤效应目前已成为提高肿瘤自杀基因治疗疗效的重要策略。由于缝隙连接机制是旁杀伤效应的重要机制,故改善肿瘤的细胞间缝隙连接功能(GJIC)是自杀基因疗法增效的重要途径之一。以往的研究证明:六味地黄丸能够提高自杀基因的旁杀伤效应,而缝隙连接机制是旁杀伤效应的重要机制之一,故推测六味地黄丸可能具有提高GJIC功能的作用。本研究应用中药复方血清药理学的方法,以小鼠恶性黑色素瘤B16细胞为研究对象,初步探讨六味地黄丸含药血清对小鼠恶性黑色素瘤增效作用的旁杀伤效应机制。通过研究以期为临床自杀基因疗法联合中药复方的中西医结合治疗方案的建立提供实验数据、科学依据及前期工作基础。目的:本实验旨在以往研究的基础上,应用中药复方血清药理学方法,研究六味地黄丸含药血清对小鼠恶性黑色素瘤细胞(B16细胞)旁杀伤效应增效作用的缝隙连接机制。方法:1六味地黄丸药物血清和对照血清的制备:SD大鼠(230～280g),分为对照组和六味地黄丸给药组(药物剂量为等效剂量的6～8倍)。灌胃八天。末次给药后1小时腹主动脉取血,-70℃冰箱保存备用。2六味地黄丸药物血清和对照血清添加量的确定:MTT法检测检测B16细胞对六味地黄丸含药血清和对照血清的敏感程度,确定用药浓度。3六味地黄丸含药血清对恶性黑色素瘤GJIC功能的影响:用流式细胞技术检测和SLDT法观察六味地黄丸含药血清对B16细胞的GJIC功能的影响。4六味地黄丸含药血清对恶性黑色素瘤细胞的缝隙连接蛋白Cx的影响:用western blot技术和间接免疫荧光技术检测六味地黄丸含药血清对恶性黑色素瘤细胞的缝隙连接蛋白Cx的影响。结果:1.血清的制备及含药血清的添加量本实验给药方案采用每天灌胃2次,间隔8小时;灌胃后1h取血。从MTT结果来看,对照血清及六味地黄丸含药血清在5%～10%浓度下对小鼠恶性黑色素瘤细胞B16的生长有一定的促进作用,但是随着血清浓度增高(＞20%)会对细胞的形态产生明显的影响,镜下观察细胞形态发生空泡状改变。但数量上还是有所增值的,故选择对B16细胞影响不大的浓度(＜10%)作为大鼠血清的工作浓度。但六味地黄丸含药血清与对照血清对细胞影响无统计学差异(P＞0.05)。2六味地黄丸含药血清对恶性黑色素瘤细胞GJIC功能的影响SLDT法和流式细胞仪技术检测的结果都是:对照血清和含药血清都使得B16细胞GJIC功能得到增强,至于大鼠血清为什么使得GJIC功能增强,具体机制有待进一步研究,但是单看六味地黄丸含药血清组可以看出有一个量效关系,随着浓度的增加,其GJIC功能得到增强,说明六味地黄丸含药血清能够提高小鼠恶性黑色素瘤细胞的GJIC功能。3六味地黄丸含药血清对恶性黑色素瘤细胞的缝隙连接蛋白的影响Western blot技术和FITC检测结果显示:对照血清和六味地黄丸含药血清都使得Cx蛋白的表达提高,至于对照血清为什么能够提高Cx的表达,具体机制不明,有待进一步研究。单看六味地黄丸含药血清也能使Cx提高。且有明显的量效关系。说明六味地黄丸含药血清能够使小鼠恶性黑色素瘤细胞的Cx的表达有所提高。结论:本研究在细胞水平探讨了六味地黄丸含药血清对小鼠恶性黑色素瘤细胞自杀基因疗法旁杀伤增效的作用机制。研究结果表明,六味地黄丸可能通过增强恶性黑色素瘤细胞Cx表达,从而增强缝隙连接功能,故旁杀伤效应的缝隙连接机制,可能是六味地黄丸对自杀基因疗法增效的机制之一。更多还原

【Abstract】 Malignant melanoma is the malignant tumor which comes from melanin cell. Which is a type of melanin cell malignant tumor which originates neural crest .The outbreak rate is 1% to2% of the whole body malignant tumor. Its malignant degree is high, differing after preparing. With the development of molecular biology , the suicide gene therapy of the tumor has represented the good application prospect as a kind of new approach. But the problem in this remedy is insufficient killing ability, low efficiency of transfection and uncertain safety. It’ s reported that increasing the bystander effect is a good approach to resolve this problem. Considering the most important mechanism of bystander effect conducted by the gap junction intercellular communication (GJIC) of cell, researchers today enhance the bystander effect by method of medical inducement to elevate GJIC. But there were not many medicines especially natural medicine with this effect had been found until today. Although some ingredients of TCM had been proved by the clinic or basic investigations on the effects of inducing apoptosis of tumor cells and increasing immunity of the patients, few articles were released about the investigation on increasing bystander effect and cooperating the tumor gene therapy with TCM or their ingredients. At the same time, research in the past proves that: Liu Wei Di Huang Bolus can be able to improve suicide gene bystander. The mechanism of gap junction is other one of bystander effect. Guess that Liu Wei Di Huang Bolus may be improving the GJIC function, which strengthen suicide gene bystander.This research applies the method that the serum pharmacology of the Chinese herbal medicine compound prescribe learns, With malignant melanoma of mou-se cell for research object, We study the function mechanism research of enhancingutility of serum pharmacology of Liu wei Di huang Bolus on "bystander effect"of suicide gene therapy of malignant melanoma. We provid experimentdata, science basis and work foundation to treatment project of suicide genetherapy consociatio-n serum pharmacology of the Chinese herbal medicinecompound prescribe .Objective:During the research, we hoped to explore the possible mechanism of adjusting cell GJIC and inducing of cell apoptosis with Serum Phamacology of LiuweiDiduang Bolus, then we could get the further comprehension about the mechanism of increasing bystander effect with Serum Phamacology of LiuweiDiduang Bolus. Methods:1. Preparation of the herbs serum of LiuweiDiduang Bolus and control serum:SD rat (230～280g), divided into two groups: one was control serum group, the other was Serum Phamacology of Liuwei Diduang Bolus (Administration: 6～8 times of isoeffect dosage). Getting blood from abdominal aorta after the Intragastric administration for one hour, Conservation of serum: -70°C frige.2. The suitable Medication level of the herbs serum of LiuweiDiduang Bolus and control serum. Using MTT assay to detect the sensitivity of herbs serum of Liu Wei Di Huang Bolus and control serum on B16 cell. with the results to decide the suitable medication level.3. The effects of the herbs serum of Liu Wei Di Huang Bolus on GJIC of malignant melanoma. the effects of herbs serum of Liu Wei Di Huang Bolus on the GJIC of B16 cell were detected by SLDT and FACS cell analyzer.4. The effects of the herbs serum of Liu Wei Di Huang Bolus on C_X of B16. the effects of herbs serum of Liu Wei Di Huang Bolus on C_X of B16 were detected by western blot and FITC.Results:1. The preparation of rat serum and the addition of the herbs serum of Liuwei Diduang BolusThe control serum and herbs serum of Liuwei Diduang Bolus showed a promotion effect on B16 cell when the final concentration was 5%～15% but showed inhibiting effect when higher than 20%(micro. Observation: the cells transformed into vacuoles). There was no statistical difference comparing the influences between the herbs serum of Liuwei Diduang Bolus and control serum on CBRH7919. (P>0. 05) 2. The effects of the herbs serum of Liu Wei Di Huang Bolus on GJIC of malignant melanomaThe result of SLDT and FACS cell analyzer showed that the ability of GJIC function of B16 was up-regulated by herbs serum of Liu Wei Di Huang Bolus and control serum. But why control serum can strengthen the ability of GJIC function of B16, the concrete mechanism pending further studies. Along with the density of herbs serum of Liu Wei Di Huang Bolus increase, its GJIC function obtains the enhancement, This indicated the ability of GJIC function of B16 was up-regulated by herbs serum of Liuwei Diduang Bolus.3. The effects of the herbs serum of Liu Wei Di Huang Bolus on C_X protein of B16.The result of western blot and FITC showed that C_X was up-regulated by herbs serum of Liu Wei Di Huang Bolus and control serum. But why control serum can strengthen C_X, the concrete mechanism pending further studies. Along with the density of herbs serum of Liuwei Diduang Bolus increase, The connexion expression has the enhancement, This indicated the expression of C_X was up-regulated by herbs serum of Liu Wei Di Huang Bolus.Conclusion:This research discussed The Mechanism Research of herbs serum of LiuWei Di Huang Bolus on GJIC of malig-nant melanoma .The analysis result indicated Liu Wei Di Huang Bolus strengthens the express of C_X of malignant melanoma, Which can strengthen the function of gap junction. The mechanism of gap junction of bystander effect may be one of the Liu Wei Di Huang Bolus mechanism to suicide gene treatment synergy.更多还原