特异性启动子控制双自杀基因系统靶向治疗多药耐药胶质瘤的研究
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摘要
目的:
采用逐渐增加培养基中药物浓度的方法联合短期暴露于高浓度的抗癌药物的方法,在体外建立脑胶质瘤C6耐阿霉素细胞株(C6/ADR)MDR细胞系,观察C6及C6/ADR细胞的形态学改变,并对其多药耐药特性进行鉴定,为下一步的实验打下基础。
研究方法:
(1)取对数生长期C6细胞在37℃,5%CO_2全湿度培养箱内培养于低浓度的阿霉素(0.001,0.01,0.1,0.25,0.5μg/ml),每种药物浓度维持四周后,然后暴露在高药物浓度中进行培养36小时,利用MTT法,计算不同药物(终浓度0.001,0.01,0.1,1,10,100μg/ml)对C6细胞的抑制率,来确定阿霉素药物终浓度为1μg/ml。
(2)取对数生长期的C6胶质瘤细胞培养基中加入阿霉素,使其终浓度达1μg/ml,将逐渐增加培养液中药物浓度的方法和反复短期暴露法在细胞传代过程中互相结合,4个月后C6细胞可生长于含ADM 0.5μg/ml RPMI-1640完全培养液中,每2～3天传代一次,耐药细胞系(命名C6/ADR)在无药培养液中培养2月。用光学显微镜及电子显微镜观察活细胞的形态结构及生长特点或经HE染色后观察。
(3)利用MTT法,以540nm为测试波长,630nm为参考波长的酶标仪上读取C6及C6/ADR细胞的光密度值,以OD值代表细胞数为纵坐标,以时间为横坐标,绘制生长曲线,来观察细胞增殖情况,并计算细胞倍增时间。并利用克隆形成试验测定C6及C6/ADR单个细胞增殖能力,将培养板倒置并叠加一张带网络的透明胶片,在显微镜下计数大于50个细胞的克隆数,来计算克隆形成率。
(4) RT-PCR法来检测C6及C6/ADR细胞MDR1基因mRNA水平变化:用UNIQ-10柱离心式Trizol总RNA抽提试剂盒提取细胞总RNA,利用非特异性引物进行逆转录反应,然后用MDR1检测引物进行PCR扩增,取PCR扩增产物在1%琼脂糖凝胶中电泳30 min,紫外透射分析仪下观察结果并拍照。同时,用Western blot法分析C6及C6/ADR细胞中MDR1蛋白的表达情况。利用流式细胞免疫学方法对多药耐药基因MDR1编码蛋白P-gp的进行定量研究。
(5)收集并整理实验数据,进行统计学分析,P＜0.05有统计学差异。
结果:
(1)形态学观察:在光学显微镜下,C6/ADR细胞与C6细胞形态相似,细胞生长较快,二者形态相同,均为成纤维样增殖,透明性强,折光性弱,细胞生长状态良好,细胞内无颗粒。HE染色显示:有突起,核大,圆形,不均质,多为单核,可见双核,可见有丝分裂像,胞浆少;在电子显微镜下,C6/ADR细胞较C6细胞相比,细胞外形不规则,有较多的突起或微绒毛,核大形态不规则,畸形核可见,内质网及线粒体丰富。
(2)利用MTT法测定细胞增殖情况,绘制生长曲线,结果显示:C6细胞倍增时间59.52h,C6/ADR细胞倍增时间64.58h。这说明C6/ADR细胞在产生耐药性后,其增殖能力未受到明显的影响。利用克隆形成试验测定C6及C6/ADR单个细胞增殖能力,结果显示:通过与C6细胞的细胞克隆形成率相比,C6/ADR细胞产生耐药后,其单个细胞增殖能力没有显著差异(P＞0.05),说明其增殖能力并未受到影响。
(3) RT-PCR检测MDR1表达结果:电泳观察C6/ADR细胞出现约170bp的阳性条带,证实其高表达MDR1 mRNA;而C6细胞则不表达。Western blot检测C6及C6/ADR细胞MDR1蛋白的表达结果显示MDR1蛋白在C6/ADR细胞中表达,而在C6细胞则不表达。采用流式细胞术进行定量分析,其阳性细胞P-gp蛋白表达率42.51±7.82%,证明该药诱导的耐药细胞其耐药基因为MDR1基因,蛋白表达P-gp。
结论:
(1) C6及C6/ADR细胞在光镜下两者差别不大,但透射电镜发现C6/ADR细胞和C6细胞的超微结构明显不同,特别是内质网及线粒体丰富,这提示随着耐药性的增加,酶类的合成也在增加,进一步提示了耐药的复杂性。
(2) C6/ADR细胞产生耐药后,其细胞倍增时间以及单个细胞的克隆能力与C6细胞没有太大差异,这说明C6/ADR细胞的增殖能力以及单个细胞的克隆能力并没有受到明显的影响。
(3)多药耐药蛋白基因能够在C6/ADR细胞中能够高表达P-gp,而在C6细胞中基本不表达。
目的:
从耐药胶质瘤C6/ADR细胞中克隆多药耐药基因启动子(MDR1P),构建MDR1P控制的双自杀基因表达载体,并初步研究其在C6/ADR中的表达情况。
研究方法:
(1)提取C6/ADR基因组DNA,PCR扩增MDR1P,克隆入T载体,NdeⅠ和HindⅢ双酶切MDR1P,获得相应粘性末端的MDR1P片段,纯化并进行加A反应,将其连接入通用载体pMD 18Simple-T中,形成MDR1P-T载体,转化JM109感受态菌后小量提取质粒,PCR及测序鉴定。
(2) NdeⅠ/HindⅢ双酶切pcDNA3-TK质粒和MDR1P-T载体,利用T4 DNA连接酶用MDR1P置换pcDNA3-TK中的CMV启动子,获得pcDNA3-MDR1P-TK,进行PCR及测序鉴定。
(3)利用BamHⅠ酶切pcDNA3-MDR1P-TK及pcDNA3-CD-TK,将pcDNA3-CD-TK中的CD基因插入到pcDNA3-MDR1P-TK中TK基因的上游,从而获得pcDNA3-MDR1P-CD-TK,进行PCR及测序鉴定。
(4)在脂质体介导下将pcDNA3-MDR1P-CD-TK转染C6和C6/ADR细胞,G418筛选获得稳定转染细胞株,用PCR鉴定自杀基因在两种细胞中的整合情况,RT-PCR鉴定CD、TK在在两种转染细胞中的表达情况。
结果:
(1)从C6/ADR基因组DNA中提取的MDR1P片段,PCR成功扩增出260bp的MDR1P并克隆入T载体后,PCR产物测序结果,与基因bank的序列一致,说明MDR1P已成功克隆。
(2)经分子克隆技术将MDR1P成功插入pcDNA3-TK质粒中,形成pcDNA3-MDR1P-TK,PCR扩增可见1600bp大小的阳性条带,同时测序结果证实在pcDNA3质粒中多克隆位点前原有的CMV启动子被MDR1基因启动子代替(保留部分CMV增强子序列)。
(3)经过BamH I酶切质粒pcDNA3-CD-TK后获得CD基因,通过基因重组技术成功将其插入同样酶切后的pcDNA3-MDR1P-TK质粒TK基因的上游,形成pcDNA3-MDR1P-CD-TK;PCR结果显示1～4均为阳性克隆,初步表明重组正确,同时测序结果表明与基因bank上序列一致,表明重组正确。
(4)脂质体法将质粒pcDNA3-MDR1P-CD-TK成功稳定转染入C6和C6/ADR细胞,命名为C6/CDTK、C6/ADR/CDTK,其形态与C6、C6/ADR细胞无差异。PCR证实自杀基因在两种细胞中都有整合,RT-PCR也显示自杀基因仅在耐药细胞株C6/ADR中得到了表达。
结论:
(1)成功建立了MDR1启动子调控的CD-TK双自杀融合基因表达系统(pcDNA3-MDR1P-CD-TK)。
(2)将质粒pcDNA3-MDR1P-CD-TK成功稳定转染入C6和C6/ADR细胞,获得稳定的C6/CDTK、C6/ADR/CDTK转染细胞。
(3)证实MDR1启动子调控的CD-TK双自杀融合基因表达系统能够在耐药胶质瘤细胞(C6/ADR/CDTK)可特异性表达,而在C6/CDTK细胞中不表达。
目的:
研究多药耐药基因启动子(MDR1)靶向控制的CD-TK双自杀基因系统并加前体药物丙氧鸟苷及5-氟胞嘧啶(pcDNA3-MDR1P-CD-TK/GCV+5-FC)对MDR1高表达的耐药胶质瘤细胞(C6/ADR细胞)的体外特异性杀伤作用。
研究方法:
(1)将双自杀基因转染的C6细胞、C6/ADR细胞各分两组(一组使用前体药物,另一组不使用前体药物),分别接种于6孔培养板中,加入含或不含GCV 60μg/ml、5-FC 600μg/ml的RPMI-1640培养液。培养24h后,在倒置显微镜下观察细胞形态及生长状况。
(2)取对数生长期的C6、C6/ADR、C6/CDTK、C6/ADR/CDTK细胞,加入不同浓度的前体药物GCV和/或5-FC,对照组不加前体药物,利用MTT法,以540nm为测试波长,630nm为参考波长的酶标仪上读取用C6及C6/ADR细胞的光密度值,以细胞生存率为纵坐标,以前体药物浓度为横坐标,绘制细胞存活曲线,来观察细胞存活情况。
(3)取对数生长期的C6、C6/ADR、C6/CDTK、C6/ADR/CDTK细胞,加入含或不含GCV 60μg/ml、5-FC 600μg/ml的RPMI-1640培养液,利用流式细胞仪检测细胞周期及细胞凋亡,利用TUNNEL法用显微镜来观察细胞的死亡情况。
(4)取3×10~4/ml的C6、C6/ADR、C6/CDTK、C6/ADR/CDTK细胞,加入含或不含GCV 60μg/ml、5-FC 600μg/ml的RPMI-1640培养液,将培养板倒置并叠加一张带网络的透明胶片,在显微镜下计数大于50个细胞的克隆数,来计算克隆形成率。
(5)收集并整理实验数据,进行统计学分析,P＜0.05有统计学差异。
结果:
(1)未转染自杀基因的C6、C6/ADR细胞,应用前体药物后,生长状态良好,细胞数量和细胞增殖无明显变化;且GCV和5-FC联合用药较二者单独用药也无明显差异;而C6/ADR/CDTK细胞用药后内颗粒逐渐增多,细胞变得越来越皱缩,贴壁性能减弱,生长状态变差,细胞数量变少,且联合用药更加明显。
(2) MTT结果显示C6/CDTK细胞的存活率比C6细胞下降稍明显一些,随着药物浓度的增大细胞存活率有轻微的下降情况,C6/ADR细胞存活率与C6细胞相似,细胞存活率变化不大,而C6/ADR/CDTK细胞增殖明显受抑,细胞存活率明显下降(P＜0.05),且有明显的剂量依赖关系。
(3)流式细胞仪检测结果显示加入前体药物后与C6/CDTK细胞相比,C6/ADR/CDTK细胞的细胞凋亡率明显提高,而且联合用药比单一用药效果更显著,凋亡率达到22.64%(P＜0.05);另外,C6/ADR/CDTK细胞的细胞周期绝大部分被阻滞在G1期,且几无DNA合成。利用TUNNEL法来检测C6细胞、C6/ADR细胞、C6/CDTK细胞结果为阴性,C6/ADR/CDTK细胞结果为阳性。
(4)细胞克隆形成实验结果显示C6细胞、C6/ADR细胞、C6/CDTK细胞的克隆形成率均在90%以上,而C6/ADR/CDTK细胞的克隆形成率明显降低(P＜0.05),两种药物联合应用,克隆形成率降低更为明显,只有16.71±0.31%。
结论:
我们利用MDR1启动子调控的双自杀基因系统成功转染多药耐药和非耐药胶质瘤细胞,并加用前体药物GCV和/或5-FC进行干预实验。结果证明MDR1启动子控制的双自杀基因/前体药物系统对耐药细胞C6/ADR细胞的抗肿瘤效果比对非耐药细胞C6细胞的效果更为明显,尤其是两种前体药物联合应用比单一用药效果好,这说明了特异性启动子调控的自杀基因治疗能够进行选择性特异性杀伤,同时证实了这种方法的靶向有效性。本研究为解决胶质瘤等恶性疾病产生对化疗药物的耐药问题提供了一条新的治疗思路和途径。
Objective:
To establishment of a multidrug resistant C6/ADR cell line in vitro by stepwise selection in the presence of increasing concentration of Adriamycin and associated with high dose short explosure of ADM, and study on the morphological changes of C6 and C6/ADR cells and the detection of their characterization.
Methods:
(1) The C6 cells at logarithm growth stage were cultured in RPMI1640 medium with Adriamycin by successive exposure to increasing concentration of ADM (0.001, 0.01, 0.1, 0.25, 0.5μg/ml, each concentration for 4 weeks) in a fully humidified atmosphere containing 5% CO_2/95% air at 37℃. And then, after induced respectively with high dose short explosure of ADM (0.001, 0.01, 0.1, 1, 10, 100μg/ml) for 36h, MTT assay was used to detect the inhibition ratio of C6 cells with various concentration of ADM. The final concentration was 1μg/ml for the induction of C6/ADR cells next experiment.
(2) The C6 cells at logarithm growth stage were plated in RPMI1640 medium added with 1μg/ml ADM by stepwise selection in the presence of increasing concentration of Adriamycin and associated with high dose short explosure of ADM. After 4 months, all of the cell lines were subcultured (one generation for 2 or 3 days) in RPMI1640 medium with 0.5μg/ml ADM. The C6/ADR were obtained from non-drug medium for 2 months and then used for detection by hematoxylin and eosin staining (HE staining) with light microscope and transmission electron microscope.
(3) By using the MTT assay, the optical density (OD) was measured at 540 nm on an enzyme-linked immunosorbent assay reader with reference wavelength at 630 nm. And then, the growth curve was drawn with the OD as longitudinal coordinate and the growth time as abscissa to calculate the double time of C6 and C6/ADR respectively. The viability of cells was determined by comparing the number of viable cells. To analyze potential proliferation of single cell, clone formation assay was used to detect the reproductive activity of single cell. After performed with Giemsa staining (Sigma), cloning efficiency was determined by counting the number of colonies more than 50 cells as 1 positive colony in each well.
(4) RT-PCR was performed to detect expression of MDR1 at the mRNA level in C6 or C6/ADR cells. Total RNA from C6 and C6/ADR cells was isolated using Trizol Reagent kit. One microgram of total RNA was then used in reverse transcription reactions with AMV-reverse transcriptase and Oligo dT18 non-specificity primer as described by the manufacturer. The resulting total cDNA was amplified by PCR. And then the amplified fragments were separated on 1% agarose gels for 30 min to observe on UV Transilluminator and be taken photo.
(5) All the experimental data were gathered and put in order for statistical analysis as the statistically significant level of P-value less than 0.05.
Results:
(1) The abservation of light microscope demonstrated that the C6/ADR cells were the similar in morphology as C6 cells, including that the cell growth of both cells were more fast, and both appeared fibra and transparent without intracellular grains. The results of HE staining indicated that the characterization of nucleus was round, karyomegaly with apophysis, heterogeneity, and more were monocaryon, also appeared conjugate nuclei, besides there was mitotic figure with little endochylema. However, by using the transmission electron microscope, the observation result showed that there were lots of large and malformed nucleuses with major apophysis or microvillus, abundant endocytoplasmic reticulum and mitochondria in C6/ADR cells, compared with C6 cells.
(2) By using MTT assay, the growth curve was drawn to display that the double time of C6 and C6/ADR was 59.52h and 64.58h respectively, which means that the reproductive activity of C6/ADR cells was not subjected to the multidrug resistance conspicuously. Moreover, the result of clone formation assay manifested that after induced with ADM, the influence on the reproductive activity of single cell of C6/ADR cells was not significant deviation comparing with C6 cells (P>0.05).
(3) RT-PCR was performed to detect MDR1 mRNA levels to demonstrate that MDR1 was highly expressed in C6/ADR cells, while MDR1 mRNA levels were under detectable levels in C6 cells. Furthermore, the western blot was performed to confirm that the expression of MDR1 protein was only detected in C6/ADR cells, but not in C6 cells. In addition, flow cytometry was used to carry out quantitative analysis to show that the expression of P-gp was strongly positive in C6/ADR cells (42.51±7.82%), nevertheless, it was only 4.5% in C6 cells. It was proved that the multidrug resistance gene of multidrug-resistant cell was MDR1 gene, protein product of which was P-gp.
Conclusion:
(1) Between C6 and C6/ADR cells, there were no significant differences for observation in light microscope. However, through the transmission electron microscope, the ultrastructural organization was obviously different between them, especially abundant endocytoplasmic reticulum and mitochondria in C6/ADR cells, which demonstrated that following the increasing multidrug resistance, the synthesis of enzymes was enhanced endlessly, further indicating the complexity of multidrug resistance in C6/ADR cells.
(2) After induced for MDR, there were no great differences in the cell doubing time and clone ability of single cell in C6/ADR cell compared with C6 cells, which illustrated that the reproductive activity and clone ability of single cell in C6/ADR cell were not subjected to the multidrug resistance conspicuously.
(3) The multidrug resistant protein gene was strongly expressed in C6/ADR cells, with the product called P-glycoprotein (P-gp), whereas the expression of P-gp was not detected in C6 cells.
Objective:
To clone the multiple drug resistance protein (MDR1) promoter from C6/ADR, construct the double suicide genes expressive vector controlled by MDR1 promoter, and explore its targeted expression in C6/ADR cells.
Methods:
(1) Polymerase chain reaction (PCR) was used to amplify MDR1 promoter isolated from C6/ADR cells genomic DNA, which was linked with T vector. After cut by NdeI and HindIII, the A tailing was inserted into the 3'-end of MDR1 promoter fragments using TaKaRa DNA A-Tailing Kit. And then, this fragment was inserted into pMD 18Simple-T vector resulting in the pMD 18Simple-T-MDR1-Promoter vector construction. After the vector was transformed into JM109 competent bacterium, pMD 18Simple-T-MDR1P plasmid extracted from bacterium was amplified and confirmed through electrophoresis and sequencing.
(2) The pcDNA3-TK (thymidine kinase) plasmid and MDR1P-T vector were digested with NdeI and HindIII enzymes, and MDR1 promoter was cloned into the upstream of pcDNA3-TK plasmid to exchange the CMV promote resulting in pcDNA3-MDR1P-TK vector construction, which was amplified by PCR and detected by direct sequencing.
(3) Both pcDNA3-MDR1P-TK and pcDNA3-CD-TK were digested by BamH I, and then, the cytosine deaminase (CD) gene in pcDNA3-CD-TK plasmid was directly cloned into pcDNA3-MDR1P-TK plasmid to construct pcDNA3-MDR1P-CD-TK vector, which was confirmed through electrophoresis and sequencing.
(4) This pcDNA3-MDR1P-CD-TK plasmid was transfected into C6 and C6/ADR cells respectively by liposome. After selection by 500μg/ml geneticin (G418), the transferred tumor cell lines were stably established. Then these cell lines were examined through PCR, and RT-PCR to respectively detect the integration and expression of TK and CD genes in C6 and C6/ADR cells.
Results:
(1) The 260bp MDR1 promoter fragment isolated by PCR from the C6/ADR cells was identified as the expected fragment from the C6/ADR cells. After cloned into T vector, the MDR1 promoter fragment amplified was confirmed by direct sequencing reactions. The result showed that the MDR1 promoter, of which the sequencing was the same as the gene bank, had been cloned successfully into pMD 18Simple-T vector.
(2) The pcDNA3-MDR1P-TK was constructed successfully by using molecular cloning technique after MDR1P was inserted into pcDNA3-TK. The result of PCR demonstrated that the 1600bp PCR product of pcDNA3-MDR1P-TK recombinant plasmid was detected by using electrophoresis. Furthermore, CMV promoter replaced with MDR1 promoter (remaining enhancer sequence partly) was confirmed by direct sequencing, meanwhile the sequence of plasmid has been proved to be correct.
(3) After enzyme digestion with BamH I and purification, CD fragment was cloned successfully into the upstream of TK gene in pcDNA3-MDR1P-TK plasmid to construct pcDNA3-MDR1P-CD-TK. The PCR indicated that the lane 1-4 was positive clone, which meaned that the recombination was proved to be correct initially. At the same time, the result of sequencing confirmed that the sequence of this fragment from this plasmid was the same as the gene bank and the recombination was right.
(4) After stably transferring double suicide genes controlled by MDR1 promoter into C6 or C6/ADR cells, two transfectants were called differently for C6/CDTK and C6/ADR/CDTK, which were similar to the previous C6 and C6/ADR cells. PCR indicated the double suicide genes were integrated into C6 and C6/ADR cells. By using RT-PCR, the result revealed that CD and TK genes expressed in C6/ADR/CDTK cells, whereas not in C6/CDTK cells.
Conclusion:
(1) The CD-TK fusion sucide genes expressive vector controled by MDR1 promoter has been constructed successfully.
(2) The pcDNA3-MDR1P-CD-TK has been transferred successfully into C6 and C6/ADR cells respectively to obtain stable C6/CDTK and C6/ADR/CDTK cells.
(3) It has been proved that the CD-TK fusion sucide genes expressive vector controled by MDR1 promoter was targeted expression in MDR1 positive cells (C6/ADR/CDTK cells), whereas not in C6/CDTK cells.
Objective:
To investigate the targeted killing effects of cytosine deaminase (CD) -thymidine kinase (TK) fusion suicide gene/prodrug therapy (GCV+5-FC) controlled by multidrug resistance gene promoter on MDR glioma cells.
Methods:
(1) Transfected C6 and C6/ADR cells were divided respectively into two groups: one group treated with prodrug and another group without prodrug. They were plated respectively in a 6-well plate and maintained in RPMI1640 medium with 60μg/ml ganciclovir (GCV) and 600μg/ml 5-fluorocytosine (5-FC). After cultured for 24h, these cells were observed by using inverted microscope.
(2) The C6, C6/ADR, C6/CDTK and C6/ADR/CDTK cells at logarithm growth stage were maintained respectively in RPMI1640 medium with various concentrations of GCV and/or 5-FC compared with control groups without prodrugs. By using the MTT assay, the OD value was measured at 540 nm on an enzyme-linked immunosorbent assay reader with reference wavelength at 630 nm to calculate the cell survival rate. And then, the cell survival curve was drawn with the cell survival rate as longitudinal coordinate and the prodrug level as abscissa to observe the cell survival state.
(3) The C6, C6/ADR, C6/CDTK and C6/ADR/CDTK cells at logarithm growth stage were maintained respectively in RPMI1640 medium with or without 60μg/ml GCV and 600μg/ml 5-FC. The cell apoptosis in different phase of the cell cycle were measured by flow cytometry in these cells and TUNNEL assay was used to detect the cell apoptosis.
(4) The C6, C6/ADR, C6/CDTK and C6/ADR/CDTK cells at 3×10~4/ml were maintained respectively in RPMI1640 medium with or without 60μg/ml GCV and 600μg/ml 5-FC. The clone formation assay was used to detect the reproductive activity of single cell. After performed with Giemsa staining (Sigma), cloning efficiency was determined by counting the number of colonies more than 50 cells as 1 positive colony in each well.
(5) All the experimental data were gathered and put in order for statistical analysis as the statistically significant level of P-value less than 0.05.
Results:
(1) After the addition of prodrugs, the light microscope demonstrated that the growth of untransfected C6 and C6/ADR cells was in good condition, and there were no obvious changes of cell population and cell proliferation, furthermore, the change with the combination of GCV and 5-FC was no greater than with single prodrugs; however, the cell endoparticle of C6/ADR/CDTK increased gradually, cell morphous were shrinking, the ability of adherence became weak, the growth condition went bad and the cell number decreased, especially treated with both GCV and 5-FC.
(2) The MTT assay demonstrated that the cell survival rate in C6/CDTK was slightly decreased compared with C6 cell following increasing drug concentration; the cell survival rate in C6 and C6/ADR cells was similar with no marked change; nevertheless, compared with others, the inhibition of cell proliferation in C6/ADR/CDTK was more predominance and the cell survival rate was reduced significantly (P<0.05), which meaned that both GCV and 5-FC had an obvious killing effect on C6/ADR/CDTK cells in a concentration-dependent manner.
(3) The flow cytometry was used to manifest that compared with C6/CDTK cells, the rate of cell apoptosis in C6/ADR/CDTK cells was increased conspicuously after the addition of prodrugs; moreover, the effect of the combination of GCV and 5-FC was more significant than single prodrug, especially the rate of cell apoptosis reaching up to 22.64% (P<0.05); in addition, the cell cycle of C6/ADR/CDTK stayed in G1-stage without DNA synthesis. At the same time, the TUNNEL assays replayed that the result of C6/ADR/CDTK was positive and others were negative.
(4) The clone formation assay expressed that cloning efficiency of C6, C6/ADR and C6/CDTK cells went up to 90% above, but the cloning efficiency degraded significantly in C6/ADR/CDTK cell with increasing prodrug concentration. And also, the cloning efficiency was more conspicuously decreased with combination of both prodrugs than single prodrug, only was 16.71±0.31%.
Conclusion:
The double suicide genes system controlled by the MDR1 promoter was transfected successfully into multidrug-resistance (MDR) and non-multidrug-resistance glioma cells which were treated with GCV and/or 5-FC prodrug. The results showed that the MDR1 promoter driven CD-TK fusion double suicide gene/prodrug system had a significant antitumor effect in the multidrug-resistance cells (C6/ADR cells) compared with non-multidrug-resistance glioma cells (C6 cells), especially that prodrugs in combination could have a more powerful killing effect than single prodrug. In conclusion, this study indicated that the suicide gene therapy controlled by the specific promoter had a selective and specific killing effect on MDR glioma cells, which was proved to be targeted effectiveness of this method. We can believe that this study will provide a sound basis for targeted gene therapy for multidrug resistance glioma.
采用逐渐增加培养基中药物浓度的方法联合短期暴露于高浓度的抗癌药物的方法,在体外建立脑胶质瘤C6耐阿霉素细胞株(C6/ADR)MDR细胞系,观察C6及C6/ADR细胞的形态学改变,并对其多药耐药特性进行鉴定,为下一步的实验打下基础。
研究方法:
(1)取对数生长期C6细胞在37℃,5%CO_2全湿度培养箱内培养于低浓度的阿霉素(0.001,0.01,0.1,0.25,0.5μg/ml),每种药物浓度维持四周后,然后暴露在高药物浓度中进行培养36小时,利用MTT法,计算不同药物(终浓度0.001,0.01,0.1,1,10,100μg/ml)对C6细胞的抑制率,来确定阿霉素药物终浓度为1μg/ml。
(2)取对数生长期的C6胶质瘤细胞培养基中加入阿霉素,使其终浓度达1μg/ml,将逐渐增加培养液中药物浓度的方法和反复短期暴露法在细胞传代过程中互相结合,4个月后C6细胞可生长于含ADM 0.5μg/ml RPMI-1640完全培养液中,每2～3天传代一次,耐药细胞系(命名C6/ADR)在无药培养液中培养2月。用光学显微镜及电子显微镜观察活细胞的形态结构及生长特点或经HE染色后观察。
(3)利用MTT法,以540nm为测试波长,630nm为参考波长的酶标仪上读取C6及C6/ADR细胞的光密度值,以OD值代表细胞数为纵坐标,以时间为横坐标,绘制生长曲线,来观察细胞增殖情况,并计算细胞倍增时间。并利用克隆形成试验测定C6及C6/ADR单个细胞增殖能力,将培养板倒置并叠加一张带网络的透明胶片,在显微镜下计数大于50个细胞的克隆数,来计算克隆形成率。
(4) RT-PCR法来检测C6及C6/ADR细胞MDR1基因mRNA水平变化:用UNIQ-10柱离心式Trizol总RNA抽提试剂盒提取细胞总RNA,利用非特异性引物进行逆转录反应,然后用MDR1检测引物进行PCR扩增,取PCR扩增产物在1%琼脂糖凝胶中电泳30 min,紫外透射分析仪下观察结果并拍照。同时,用Western blot法分析C6及C6/ADR细胞中MDR1蛋白的表达情况。利用流式细胞免疫学方法对多药耐药基因MDR1编码蛋白P-gp的进行定量研究。
(5)收集并整理实验数据,进行统计学分析,P＜0.05有统计学差异。
结果:
(1)形态学观察:在光学显微镜下,C6/ADR细胞与C6细胞形态相似,细胞生长较快,二者形态相同,均为成纤维样增殖,透明性强,折光性弱,细胞生长状态良好,细胞内无颗粒。HE染色显示:有突起,核大,圆形,不均质,多为单核,可见双核,可见有丝分裂像,胞浆少;在电子显微镜下,C6/ADR细胞较C6细胞相比,细胞外形不规则,有较多的突起或微绒毛,核大形态不规则,畸形核可见,内质网及线粒体丰富。
(2)利用MTT法测定细胞增殖情况,绘制生长曲线,结果显示:C6细胞倍增时间59.52h,C6/ADR细胞倍增时间64.58h。这说明C6/ADR细胞在产生耐药性后,其增殖能力未受到明显的影响。利用克隆形成试验测定C6及C6/ADR单个细胞增殖能力,结果显示:通过与C6细胞的细胞克隆形成率相比,C6/ADR细胞产生耐药后,其单个细胞增殖能力没有显著差异(P＞0.05),说明其增殖能力并未受到影响。
(3) RT-PCR检测MDR1表达结果:电泳观察C6/ADR细胞出现约170bp的阳性条带,证实其高表达MDR1 mRNA;而C6细胞则不表达。Western blot检测C6及C6/ADR细胞MDR1蛋白的表达结果显示MDR1蛋白在C6/ADR细胞中表达,而在C6细胞则不表达。采用流式细胞术进行定量分析,其阳性细胞P-gp蛋白表达率42.51±7.82%,证明该药诱导的耐药细胞其耐药基因为MDR1基因,蛋白表达P-gp。
结论:
(1) C6及C6/ADR细胞在光镜下两者差别不大,但透射电镜发现C6/ADR细胞和C6细胞的超微结构明显不同,特别是内质网及线粒体丰富,这提示随着耐药性的增加,酶类的合成也在增加,进一步提示了耐药的复杂性。
(2) C6/ADR细胞产生耐药后,其细胞倍增时间以及单个细胞的克隆能力与C6细胞没有太大差异,这说明C6/ADR细胞的增殖能力以及单个细胞的克隆能力并没有受到明显的影响。
(3)多药耐药蛋白基因能够在C6/ADR细胞中能够高表达P-gp,而在C6细胞中基本不表达。
目的:
从耐药胶质瘤C6/ADR细胞中克隆多药耐药基因启动子(MDR1P),构建MDR1P控制的双自杀基因表达载体,并初步研究其在C6/ADR中的表达情况。
研究方法:
(1)提取C6/ADR基因组DNA,PCR扩增MDR1P,克隆入T载体,NdeⅠ和HindⅢ双酶切MDR1P,获得相应粘性末端的MDR1P片段,纯化并进行加A反应,将其连接入通用载体pMD 18Simple-T中,形成MDR1P-T载体,转化JM109感受态菌后小量提取质粒,PCR及测序鉴定。
(2) NdeⅠ/HindⅢ双酶切pcDNA3-TK质粒和MDR1P-T载体,利用T4 DNA连接酶用MDR1P置换pcDNA3-TK中的CMV启动子,获得pcDNA3-MDR1P-TK,进行PCR及测序鉴定。
(3)利用BamHⅠ酶切pcDNA3-MDR1P-TK及pcDNA3-CD-TK,将pcDNA3-CD-TK中的CD基因插入到pcDNA3-MDR1P-TK中TK基因的上游,从而获得pcDNA3-MDR1P-CD-TK,进行PCR及测序鉴定。
(4)在脂质体介导下将pcDNA3-MDR1P-CD-TK转染C6和C6/ADR细胞,G418筛选获得稳定转染细胞株,用PCR鉴定自杀基因在两种细胞中的整合情况,RT-PCR鉴定CD、TK在在两种转染细胞中的表达情况。
结果:
(1)从C6/ADR基因组DNA中提取的MDR1P片段,PCR成功扩增出260bp的MDR1P并克隆入T载体后,PCR产物测序结果,与基因bank的序列一致,说明MDR1P已成功克隆。
(2)经分子克隆技术将MDR1P成功插入pcDNA3-TK质粒中,形成pcDNA3-MDR1P-TK,PCR扩增可见1600bp大小的阳性条带,同时测序结果证实在pcDNA3质粒中多克隆位点前原有的CMV启动子被MDR1基因启动子代替(保留部分CMV增强子序列)。
(3)经过BamH I酶切质粒pcDNA3-CD-TK后获得CD基因,通过基因重组技术成功将其插入同样酶切后的pcDNA3-MDR1P-TK质粒TK基因的上游,形成pcDNA3-MDR1P-CD-TK;PCR结果显示1～4均为阳性克隆,初步表明重组正确,同时测序结果表明与基因bank上序列一致,表明重组正确。
(4)脂质体法将质粒pcDNA3-MDR1P-CD-TK成功稳定转染入C6和C6/ADR细胞,命名为C6/CDTK、C6/ADR/CDTK,其形态与C6、C6/ADR细胞无差异。PCR证实自杀基因在两种细胞中都有整合,RT-PCR也显示自杀基因仅在耐药细胞株C6/ADR中得到了表达。
结论:
(1)成功建立了MDR1启动子调控的CD-TK双自杀融合基因表达系统(pcDNA3-MDR1P-CD-TK)。
(2)将质粒pcDNA3-MDR1P-CD-TK成功稳定转染入C6和C6/ADR细胞,获得稳定的C6/CDTK、C6/ADR/CDTK转染细胞。
(3)证实MDR1启动子调控的CD-TK双自杀融合基因表达系统能够在耐药胶质瘤细胞(C6/ADR/CDTK)可特异性表达,而在C6/CDTK细胞中不表达。
目的:
研究多药耐药基因启动子(MDR1)靶向控制的CD-TK双自杀基因系统并加前体药物丙氧鸟苷及5-氟胞嘧啶(pcDNA3-MDR1P-CD-TK/GCV+5-FC)对MDR1高表达的耐药胶质瘤细胞(C6/ADR细胞)的体外特异性杀伤作用。
研究方法:
(1)将双自杀基因转染的C6细胞、C6/ADR细胞各分两组(一组使用前体药物,另一组不使用前体药物),分别接种于6孔培养板中,加入含或不含GCV 60μg/ml、5-FC 600μg/ml的RPMI-1640培养液。培养24h后,在倒置显微镜下观察细胞形态及生长状况。
(2)取对数生长期的C6、C6/ADR、C6/CDTK、C6/ADR/CDTK细胞,加入不同浓度的前体药物GCV和/或5-FC,对照组不加前体药物,利用MTT法,以540nm为测试波长,630nm为参考波长的酶标仪上读取用C6及C6/ADR细胞的光密度值,以细胞生存率为纵坐标,以前体药物浓度为横坐标,绘制细胞存活曲线,来观察细胞存活情况。
(3)取对数生长期的C6、C6/ADR、C6/CDTK、C6/ADR/CDTK细胞,加入含或不含GCV 60μg/ml、5-FC 600μg/ml的RPMI-1640培养液,利用流式细胞仪检测细胞周期及细胞凋亡,利用TUNNEL法用显微镜来观察细胞的死亡情况。
(4)取3×10~4/ml的C6、C6/ADR、C6/CDTK、C6/ADR/CDTK细胞,加入含或不含GCV 60μg/ml、5-FC 600μg/ml的RPMI-1640培养液,将培养板倒置并叠加一张带网络的透明胶片,在显微镜下计数大于50个细胞的克隆数,来计算克隆形成率。
(5)收集并整理实验数据,进行统计学分析,P＜0.05有统计学差异。
结果:
(1)未转染自杀基因的C6、C6/ADR细胞,应用前体药物后,生长状态良好,细胞数量和细胞增殖无明显变化;且GCV和5-FC联合用药较二者单独用药也无明显差异;而C6/ADR/CDTK细胞用药后内颗粒逐渐增多,细胞变得越来越皱缩,贴壁性能减弱,生长状态变差,细胞数量变少,且联合用药更加明显。
(2) MTT结果显示C6/CDTK细胞的存活率比C6细胞下降稍明显一些,随着药物浓度的增大细胞存活率有轻微的下降情况,C6/ADR细胞存活率与C6细胞相似,细胞存活率变化不大,而C6/ADR/CDTK细胞增殖明显受抑,细胞存活率明显下降(P＜0.05),且有明显的剂量依赖关系。
(3)流式细胞仪检测结果显示加入前体药物后与C6/CDTK细胞相比,C6/ADR/CDTK细胞的细胞凋亡率明显提高,而且联合用药比单一用药效果更显著,凋亡率达到22.64%(P＜0.05);另外,C6/ADR/CDTK细胞的细胞周期绝大部分被阻滞在G1期,且几无DNA合成。利用TUNNEL法来检测C6细胞、C6/ADR细胞、C6/CDTK细胞结果为阴性,C6/ADR/CDTK细胞结果为阳性。
(4)细胞克隆形成实验结果显示C6细胞、C6/ADR细胞、C6/CDTK细胞的克隆形成率均在90%以上,而C6/ADR/CDTK细胞的克隆形成率明显降低(P＜0.05),两种药物联合应用,克隆形成率降低更为明显,只有16.71±0.31%。
结论:
我们利用MDR1启动子调控的双自杀基因系统成功转染多药耐药和非耐药胶质瘤细胞,并加用前体药物GCV和/或5-FC进行干预实验。结果证明MDR1启动子控制的双自杀基因/前体药物系统对耐药细胞C6/ADR细胞的抗肿瘤效果比对非耐药细胞C6细胞的效果更为明显,尤其是两种前体药物联合应用比单一用药效果好,这说明了特异性启动子调控的自杀基因治疗能够进行选择性特异性杀伤,同时证实了这种方法的靶向有效性。本研究为解决胶质瘤等恶性疾病产生对化疗药物的耐药问题提供了一条新的治疗思路和途径。
Objective:
To establishment of a multidrug resistant C6/ADR cell line in vitro by stepwise selection in the presence of increasing concentration of Adriamycin and associated with high dose short explosure of ADM, and study on the morphological changes of C6 and C6/ADR cells and the detection of their characterization.
Methods:
(1) The C6 cells at logarithm growth stage were cultured in RPMI1640 medium with Adriamycin by successive exposure to increasing concentration of ADM (0.001, 0.01, 0.1, 0.25, 0.5μg/ml, each concentration for 4 weeks) in a fully humidified atmosphere containing 5% CO_2/95% air at 37℃. And then, after induced respectively with high dose short explosure of ADM (0.001, 0.01, 0.1, 1, 10, 100μg/ml) for 36h, MTT assay was used to detect the inhibition ratio of C6 cells with various concentration of ADM. The final concentration was 1μg/ml for the induction of C6/ADR cells next experiment.
(2) The C6 cells at logarithm growth stage were plated in RPMI1640 medium added with 1μg/ml ADM by stepwise selection in the presence of increasing concentration of Adriamycin and associated with high dose short explosure of ADM. After 4 months, all of the cell lines were subcultured (one generation for 2 or 3 days) in RPMI1640 medium with 0.5μg/ml ADM. The C6/ADR were obtained from non-drug medium for 2 months and then used for detection by hematoxylin and eosin staining (HE staining) with light microscope and transmission electron microscope.
(3) By using the MTT assay, the optical density (OD) was measured at 540 nm on an enzyme-linked immunosorbent assay reader with reference wavelength at 630 nm. And then, the growth curve was drawn with the OD as longitudinal coordinate and the growth time as abscissa to calculate the double time of C6 and C6/ADR respectively. The viability of cells was determined by comparing the number of viable cells. To analyze potential proliferation of single cell, clone formation assay was used to detect the reproductive activity of single cell. After performed with Giemsa staining (Sigma), cloning efficiency was determined by counting the number of colonies more than 50 cells as 1 positive colony in each well.
(4) RT-PCR was performed to detect expression of MDR1 at the mRNA level in C6 or C6/ADR cells. Total RNA from C6 and C6/ADR cells was isolated using Trizol Reagent kit. One microgram of total RNA was then used in reverse transcription reactions with AMV-reverse transcriptase and Oligo dT18 non-specificity primer as described by the manufacturer. The resulting total cDNA was amplified by PCR. And then the amplified fragments were separated on 1% agarose gels for 30 min to observe on UV Transilluminator and be taken photo.
(5) All the experimental data were gathered and put in order for statistical analysis as the statistically significant level of P-value less than 0.05.
Results:
(1) The abservation of light microscope demonstrated that the C6/ADR cells were the similar in morphology as C6 cells, including that the cell growth of both cells were more fast, and both appeared fibra and transparent without intracellular grains. The results of HE staining indicated that the characterization of nucleus was round, karyomegaly with apophysis, heterogeneity, and more were monocaryon, also appeared conjugate nuclei, besides there was mitotic figure with little endochylema. However, by using the transmission electron microscope, the observation result showed that there were lots of large and malformed nucleuses with major apophysis or microvillus, abundant endocytoplasmic reticulum and mitochondria in C6/ADR cells, compared with C6 cells.
(2) By using MTT assay, the growth curve was drawn to display that the double time of C6 and C6/ADR was 59.52h and 64.58h respectively, which means that the reproductive activity of C6/ADR cells was not subjected to the multidrug resistance conspicuously. Moreover, the result of clone formation assay manifested that after induced with ADM, the influence on the reproductive activity of single cell of C6/ADR cells was not significant deviation comparing with C6 cells (P>0.05).
(3) RT-PCR was performed to detect MDR1 mRNA levels to demonstrate that MDR1 was highly expressed in C6/ADR cells, while MDR1 mRNA levels were under detectable levels in C6 cells. Furthermore, the western blot was performed to confirm that the expression of MDR1 protein was only detected in C6/ADR cells, but not in C6 cells. In addition, flow cytometry was used to carry out quantitative analysis to show that the expression of P-gp was strongly positive in C6/ADR cells (42.51±7.82%), nevertheless, it was only 4.5% in C6 cells. It was proved that the multidrug resistance gene of multidrug-resistant cell was MDR1 gene, protein product of which was P-gp.
Conclusion:
(1) Between C6 and C6/ADR cells, there were no significant differences for observation in light microscope. However, through the transmission electron microscope, the ultrastructural organization was obviously different between them, especially abundant endocytoplasmic reticulum and mitochondria in C6/ADR cells, which demonstrated that following the increasing multidrug resistance, the synthesis of enzymes was enhanced endlessly, further indicating the complexity of multidrug resistance in C6/ADR cells.
(2) After induced for MDR, there were no great differences in the cell doubing time and clone ability of single cell in C6/ADR cell compared with C6 cells, which illustrated that the reproductive activity and clone ability of single cell in C6/ADR cell were not subjected to the multidrug resistance conspicuously.
(3) The multidrug resistant protein gene was strongly expressed in C6/ADR cells, with the product called P-glycoprotein (P-gp), whereas the expression of P-gp was not detected in C6 cells.
Objective:
To clone the multiple drug resistance protein (MDR1) promoter from C6/ADR, construct the double suicide genes expressive vector controlled by MDR1 promoter, and explore its targeted expression in C6/ADR cells.
Methods:
(1) Polymerase chain reaction (PCR) was used to amplify MDR1 promoter isolated from C6/ADR cells genomic DNA, which was linked with T vector. After cut by NdeI and HindIII, the A tailing was inserted into the 3'-end of MDR1 promoter fragments using TaKaRa DNA A-Tailing Kit. And then, this fragment was inserted into pMD 18Simple-T vector resulting in the pMD 18Simple-T-MDR1-Promoter vector construction. After the vector was transformed into JM109 competent bacterium, pMD 18Simple-T-MDR1P plasmid extracted from bacterium was amplified and confirmed through electrophoresis and sequencing.
(2) The pcDNA3-TK (thymidine kinase) plasmid and MDR1P-T vector were digested with NdeI and HindIII enzymes, and MDR1 promoter was cloned into the upstream of pcDNA3-TK plasmid to exchange the CMV promote resulting in pcDNA3-MDR1P-TK vector construction, which was amplified by PCR and detected by direct sequencing.
(3) Both pcDNA3-MDR1P-TK and pcDNA3-CD-TK were digested by BamH I, and then, the cytosine deaminase (CD) gene in pcDNA3-CD-TK plasmid was directly cloned into pcDNA3-MDR1P-TK plasmid to construct pcDNA3-MDR1P-CD-TK vector, which was confirmed through electrophoresis and sequencing.
(4) This pcDNA3-MDR1P-CD-TK plasmid was transfected into C6 and C6/ADR cells respectively by liposome. After selection by 500μg/ml geneticin (G418), the transferred tumor cell lines were stably established. Then these cell lines were examined through PCR, and RT-PCR to respectively detect the integration and expression of TK and CD genes in C6 and C6/ADR cells.
Results:
(1) The 260bp MDR1 promoter fragment isolated by PCR from the C6/ADR cells was identified as the expected fragment from the C6/ADR cells. After cloned into T vector, the MDR1 promoter fragment amplified was confirmed by direct sequencing reactions. The result showed that the MDR1 promoter, of which the sequencing was the same as the gene bank, had been cloned successfully into pMD 18Simple-T vector.
(2) The pcDNA3-MDR1P-TK was constructed successfully by using molecular cloning technique after MDR1P was inserted into pcDNA3-TK. The result of PCR demonstrated that the 1600bp PCR product of pcDNA3-MDR1P-TK recombinant plasmid was detected by using electrophoresis. Furthermore, CMV promoter replaced with MDR1 promoter (remaining enhancer sequence partly) was confirmed by direct sequencing, meanwhile the sequence of plasmid has been proved to be correct.
(3) After enzyme digestion with BamH I and purification, CD fragment was cloned successfully into the upstream of TK gene in pcDNA3-MDR1P-TK plasmid to construct pcDNA3-MDR1P-CD-TK. The PCR indicated that the lane 1-4 was positive clone, which meaned that the recombination was proved to be correct initially. At the same time, the result of sequencing confirmed that the sequence of this fragment from this plasmid was the same as the gene bank and the recombination was right.
(4) After stably transferring double suicide genes controlled by MDR1 promoter into C6 or C6/ADR cells, two transfectants were called differently for C6/CDTK and C6/ADR/CDTK, which were similar to the previous C6 and C6/ADR cells. PCR indicated the double suicide genes were integrated into C6 and C6/ADR cells. By using RT-PCR, the result revealed that CD and TK genes expressed in C6/ADR/CDTK cells, whereas not in C6/CDTK cells.
Conclusion:
(1) The CD-TK fusion sucide genes expressive vector controled by MDR1 promoter has been constructed successfully.
(2) The pcDNA3-MDR1P-CD-TK has been transferred successfully into C6 and C6/ADR cells respectively to obtain stable C6/CDTK and C6/ADR/CDTK cells.
(3) It has been proved that the CD-TK fusion sucide genes expressive vector controled by MDR1 promoter was targeted expression in MDR1 positive cells (C6/ADR/CDTK cells), whereas not in C6/CDTK cells.
Objective:
To investigate the targeted killing effects of cytosine deaminase (CD) -thymidine kinase (TK) fusion suicide gene/prodrug therapy (GCV+5-FC) controlled by multidrug resistance gene promoter on MDR glioma cells.
Methods:
(1) Transfected C6 and C6/ADR cells were divided respectively into two groups: one group treated with prodrug and another group without prodrug. They were plated respectively in a 6-well plate and maintained in RPMI1640 medium with 60μg/ml ganciclovir (GCV) and 600μg/ml 5-fluorocytosine (5-FC). After cultured for 24h, these cells were observed by using inverted microscope.
(2) The C6, C6/ADR, C6/CDTK and C6/ADR/CDTK cells at logarithm growth stage were maintained respectively in RPMI1640 medium with various concentrations of GCV and/or 5-FC compared with control groups without prodrugs. By using the MTT assay, the OD value was measured at 540 nm on an enzyme-linked immunosorbent assay reader with reference wavelength at 630 nm to calculate the cell survival rate. And then, the cell survival curve was drawn with the cell survival rate as longitudinal coordinate and the prodrug level as abscissa to observe the cell survival state.
(3) The C6, C6/ADR, C6/CDTK and C6/ADR/CDTK cells at logarithm growth stage were maintained respectively in RPMI1640 medium with or without 60μg/ml GCV and 600μg/ml 5-FC. The cell apoptosis in different phase of the cell cycle were measured by flow cytometry in these cells and TUNNEL assay was used to detect the cell apoptosis.
(4) The C6, C6/ADR, C6/CDTK and C6/ADR/CDTK cells at 3×10~4/ml were maintained respectively in RPMI1640 medium with or without 60μg/ml GCV and 600μg/ml 5-FC. The clone formation assay was used to detect the reproductive activity of single cell. After performed with Giemsa staining (Sigma), cloning efficiency was determined by counting the number of colonies more than 50 cells as 1 positive colony in each well.
(5) All the experimental data were gathered and put in order for statistical analysis as the statistically significant level of P-value less than 0.05.
Results:
(1) After the addition of prodrugs, the light microscope demonstrated that the growth of untransfected C6 and C6/ADR cells was in good condition, and there were no obvious changes of cell population and cell proliferation, furthermore, the change with the combination of GCV and 5-FC was no greater than with single prodrugs; however, the cell endoparticle of C6/ADR/CDTK increased gradually, cell morphous were shrinking, the ability of adherence became weak, the growth condition went bad and the cell number decreased, especially treated with both GCV and 5-FC.
(2) The MTT assay demonstrated that the cell survival rate in C6/CDTK was slightly decreased compared with C6 cell following increasing drug concentration; the cell survival rate in C6 and C6/ADR cells was similar with no marked change; nevertheless, compared with others, the inhibition of cell proliferation in C6/ADR/CDTK was more predominance and the cell survival rate was reduced significantly (P<0.05), which meaned that both GCV and 5-FC had an obvious killing effect on C6/ADR/CDTK cells in a concentration-dependent manner.
(3) The flow cytometry was used to manifest that compared with C6/CDTK cells, the rate of cell apoptosis in C6/ADR/CDTK cells was increased conspicuously after the addition of prodrugs; moreover, the effect of the combination of GCV and 5-FC was more significant than single prodrug, especially the rate of cell apoptosis reaching up to 22.64% (P<0.05); in addition, the cell cycle of C6/ADR/CDTK stayed in G1-stage without DNA synthesis. At the same time, the TUNNEL assays replayed that the result of C6/ADR/CDTK was positive and others were negative.
(4) The clone formation assay expressed that cloning efficiency of C6, C6/ADR and C6/CDTK cells went up to 90% above, but the cloning efficiency degraded significantly in C6/ADR/CDTK cell with increasing prodrug concentration. And also, the cloning efficiency was more conspicuously decreased with combination of both prodrugs than single prodrug, only was 16.71±0.31%.
Conclusion:
The double suicide genes system controlled by the MDR1 promoter was transfected successfully into multidrug-resistance (MDR) and non-multidrug-resistance glioma cells which were treated with GCV and/or 5-FC prodrug. The results showed that the MDR1 promoter driven CD-TK fusion double suicide gene/prodrug system had a significant antitumor effect in the multidrug-resistance cells (C6/ADR cells) compared with non-multidrug-resistance glioma cells (C6 cells), especially that prodrugs in combination could have a more powerful killing effect than single prodrug. In conclusion, this study indicated that the suicide gene therapy controlled by the specific promoter had a selective and specific killing effect on MDR glioma cells, which was proved to be targeted effectiveness of this method. We can believe that this study will provide a sound basis for targeted gene therapy for multidrug resistance glioma.
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