【作者】 岳文玲；

【导师】 钟廷机；

【作者基本信息】 广州中医药大学 ， 中西医结合基础， 2006， 硕士

【摘要】 基因治疗就是将外源基因导入到正常或病变细胞中,利用其转录或翻译产物发挥治疗作用的一种方法,是分子生物学技术在临床医学中的一项重要应用。近年来基因治疗技术发展迅速,有希望成为肿瘤的有效治疗方法。但由于目前基因治疗研究中目的基因的表达水平很低,而且还缺乏调节目的的基因表达组织的特异性、表达时序性的有效手段,已成为影响基因治疗效果的严重障碍。在这种情况下,引入“自杀基因”,设计病变细胞的自杀机制,对于抗肿瘤具有重要的实际应用价值。 目前应用最多的是HSV-tk基因,将HSV-tk基因导入肿瘤细胞,配合以GCV治疗,使该基因的表达产物将GCV转化为GCV-TP,进而杀伤肿瘤细胞,而正常细胞则不被伤害。该疗法的作用不仅仅引起被导入“自杀基因”的细胞死亡,而且可以通过“旁观者”效应(by stander effect)杀伤未导入基因的邻近分裂细胞,显著扩大其杀伤效应,这对恶性肿瘤的治疗显得尤为重要。 缝隙连接(GJ)是介导HSV-tk基因治疗“旁杀伤效应”的重要途径。GJ除一些终末分化细胞如骨胳肌细胞,红细胞和循环淋巴细胞外几乎在所有动物中存在。GJ含有通道,它们位于两细胞之间,可非选择性通过小分子物质,但不允许蛋白和核酸大分子通过。GCV的代谢产物通过细胞间的直接接触进人tk~-细胞,而GCV的磷酸化产物不能自由通过细胞膜,故推测细胞间的缝隙连接是GCV的磷酸产物的通道。进一步的实验也证明了缝隙连接在肿瘤局部旁观者效应中的作用。 目的:本实验旨在以往研究的基础上,将tk基因导入H22细胞,应用HSV-tk/GCV系统进行小鼠肝癌自杀基因治疗的体内实验,并联合运用丹参注射液,观察联合治疗的疗效,并运用病理学技术及免疫组化等技术初步探讨其机制,另外对大鼠的含药(丹参注射液)血清和丹参注射液直接作用的H22细胞的CX-43的检测,进一步探讨其机制,为建立中西医结合肿瘤基因治疗方案提供必要条件。 方法:在已建立能稳定产病毒的PT67/tk细胞株的基础上,用NIH 3T3测定病毒滴度。将此病毒感染小鼠肝癌细胞株H22,G418筛选出抗性克隆命名为H22/tk。并将H22细胞与H22/tk细胞以4∶1的比例接种Balb/c小鼠,建立移植瘤模型进行体内实验,应用前药GCV及腹腔注射丹参注射液,16日后处死测肿瘤质量,通过病理切片观察组织的病理变化,应用免疫组化技术观察肿瘤细胞CX-43的表达,应用细胞化学法和流式细胞术体外检测药物作用过的H22细胞的CX-43含量的表达。 结论:体内实验中,自杀基因系统联合丹参注射液显示出明显的协同抗肿瘤效应;丹参注射液增效作用的机制与增强CX-43的表达而增强了旁观者效应密切相关;联合组肿瘤细胞CX-43的含量表达增多。体外实验中,细胞化学法和流式细胞术检测到肿瘤细胞的CX-43的含量表达同样增多。实验证实丹参注射液对自杀基因疗法具有增效作用,通过进一步实验证实后,可望成为肿瘤基因治疗的新方案。更多还原

【Abstract】 Gene therapy plays an important role in clinic coming from molecular biology technique. It could make effect with the translation outcome by taking exterior gene into normal or sick cells. During these years, it has made great development in gene therapeutic technique, which could be efficient therapy for tumor. It is great obstruction to because of lack of available measures for that lower level expression in the situation, introduced suicide gene and designed the suicide mechanism of the diseased cell, it is an important practise value for the anticancer.At present, HSV-TK is the most used, which is transfixed in tumour cells, cooperating GCV therapy , the expression of gene will turn GCV into GCV-TP , then kill tumour cells , but normal cells are not hurt .This use of therapy not only educe the death of cells transfecteded suicide gene , but also kill adjacent divided cells not transfecteded suicide gene by stander effect, and extend the effect of killing .It is important for the therapy of cancer .GJ is almost existed all of animals , dispose of no differentiate cells , eg. Skeletal muscle cells , red blood cells and circulate lympH cells . GJ has the passages , which are placed between cells , may pass small molecule matter by no choice , but not allowed pass large molecule and nucleic acid molecule .The expression product of GCV by direct touch of cells ,get in tk" cells .however , the pHospHoric acid product of GCV don’ t get by a cell membrane .Thus , it is inferred that GJ of cells are thoroughfare of the pHospHoric acid product of GCV. The GJ of tumor pratial stander effect is tested by more experiments.Objective: To construct a new cell stain by transfect tk gene to H22 cell stain and name it H22/tk. Then use it for treating tumor in vivo and observethe curative effect of treatment by combining tan shin and GCV. And study the mechanism by immunohistochemical and TUNEL method.Through examination of the rat’ s having tan shin serum and the CX-43 H22 cells, the mechanism is furthermore studied. Provide requirement for gene therapy of tumor by combining traditional Chinese and western medicine.Methods: Based on PT67/tk cell stain has been construction by DNA recombinant technique, then determining vival titer by infecting NIH 3T3 cells. H22 was infected with the recombinant retrovirus. The positive clones were obtained after G418 selection and named H22/tk. The morpHological changes after GCV treatment were observed with microscope. The liver tumor model on the KM mice were made in vivo by inoculate the cell mix which comprise 80% H22 and 20% H22/tk. Give a treatment by injecting pro-drug GCV, and combined with tan shin. Observe the weight of tumor after 16 days and histopathological expression of the tumors were observed in every group. Observe the expression of CX-43 of cells by immunohistochemical technic. Using cell Chemistry method and streamline cell skill to examine the CX-43 of cells, which are acted by tanshin.Conclusion: In vivo, transfer of HSV-tk gene plus GCV combined with tan shin have synergistic antitumor effects, and the mechanism of which relate with the promotion of stander effect and the expression of CX-43. In vitro.the expression of CX-43 is a higher level using the cell Chemistry method and streamline cell skill. The combined therapy might have therapeutic potentials for human cancer.更多还原