【作者】 武立华；

【导师】 艾军； 单保恩；

【作者基本信息】 河北医科大学 ， 肿瘤学， 2010， 硕士

【摘要】 目的:采用小鼠结肠癌细胞为研究对象。研究转染CD40L基因的小鼠结肠癌细胞抗肿瘤效应,以及表达CD40L基因的肿瘤细胞诱导骨髓起源的树突状细胞(dendritic cells,DC)产生免疫抗肿瘤作用的情况,分析CD40L基因及其瘤苗在结肠癌治疗中的效果及作用机制,探讨表达CD40L基因的肿瘤细胞和DC细胞相互作用的关系,为CD40L基因修饰疫苗在结肠癌的临床治疗研究提供理论依据。方法:1 CD40L真核表达载体的扩增、鉴定与大量制备将pMKITneo-CD40L质粒转化DH5ɑ感受态菌株进行扩增,采用质粒提取试剂盒提取质粒,用限制性内切核酸酶EcoR I酶切鉴定、PCR鉴定及质粒测序分析鉴定。将鉴定符合实验条件的质粒进行大量制备。2细胞培养和质粒转染及筛选稳定转染的细胞系采用脂质体转染法将全长的小鼠CD40L基因转染入结肠癌细胞株colon26中,用G418筛选抗药性克隆,并以相同的方法得到空载体转染细胞。3细胞转染效率测定用流式细胞术检测各组细胞CD40L的表达。分别用半定量RT-PCR法、激光共聚焦显微镜、Western印迹法检测各组细胞CD40L mRNA及蛋白的表达。4转染CD40L基因对细胞增殖的影响测定用MTS法测定各组细胞的增殖情况,并绘制细胞生长曲线。5转染CD40L基因对细胞侵袭能力的影响测定用半定量RT-PCR法检测各组细胞MMP-2 mRNA的表达。用Transwell法测定各组细胞的侵袭能力。6小鼠骨髓源性DC的制备取BALB/c雌性小鼠的胫骨和股骨骨髓细胞,在含GM-CSF、IL-4的10% FCS RPMI-1640中培养7天。收集非黏附的细胞并用CD11c+磁珠进行筛选,筛选后细胞用作DC。流式细胞术检测DC细胞表面CD11c的表达。7肿瘤细胞与DC共培养对DC功能的影响测定将各组细胞与DC共培养24 h后,流式细胞术检测DC细胞表面CD80、CD86、MHCⅠ、MHCⅡ的表达;用半定量RT-PCR法检测IL-12、IL-23、IL-18、IFN-γ、IFN-γ(Mig)基因mRNA的表达;用ELISA法检测共培养上清中IL-12、IL-23、IFN-γ的释放量。8荷瘤小鼠模型的建立收集呈对数生长期的colon26细胞,制成细胞悬液,调整细胞浓度为1×107/ml,于BALB/c小鼠右侧肩背部皮下注射细胞悬液0.1 ml,1×106/只,建立荷瘤小鼠模型。9 CD40L基因瘤苗的体内抑瘤作用在荷瘤小鼠模型建立后,于荷瘤小鼠腹腔分别注射DC、CD40L基因瘤苗、DC联合CD40L基因瘤苗(实验组)或PBS(对照组),每组5只。观察肿瘤大小变化及治疗效果,经3次治疗后处死小鼠,留取血清、肿瘤、肝、脾组织用于后续实验。10用ELISA法检测荷瘤小鼠外周血中IL-12、IL-23、IFN-γ、IL-10、TGF-β的水平。11经HE染色,显微镜下观察肿瘤组织有无炎细胞浸润,观察肝、脾的组织病理学变化以及有无肿瘤细胞浸润。结果:1经酶切和PCR鉴定扩增质粒及含目的基因分子大小与已知质粒相同,扩增质粒所测核苷酸序列与已知的核苷酸序列相同。2经筛选分别得到了稳定转染CD40L基因和空载体基因的细胞系,转染细胞形态较野生型细胞无明显变化,细胞呈梭形生长,形态饱满。3经流式细胞术检测,转染CD40L基因后colon26/CD40L细胞CD40L表达率较colon26和colon26/vector增加;与colon26/vector和colon26相比,colon26/CD40L组细胞中CD40L mRNA表达量分别上调了45.32%和44.81%(P<0.05),而colon26/vector和colon26相比,差异无统计学意义(P>0.05);在激光共聚焦显微镜下可观察到colon26/CD40L组细胞CD40L表达较colon26/vector和colon26高;同时,Western印迹法也证实colon26/CD40L组细胞CD40L蛋白较colon26/vector和colon26分别上调了52.34%和53.58%(P<0.05),而colon26/vector和colon26相比,差异无统计学意义(P>0.05)。4利用MTS法检测结果显示, colon26、colon26/vector和colon26/CD40L三组细胞在体外生长及细胞增殖无明显差别(P>0.05)。5经半定量RT-PCR法检测结果显示,colon26/CD40L组细胞较colon26/vector和colon26组细胞MMP-2 mRNA的表达减弱(P<0.05);经苏木素染色观察,colon26/CD40L组细胞穿过Transwell小室较colon26/vector和colon26组细胞少(P<0.05)。6小鼠骨髓源性DC在体外培养过程中,细胞逐渐呈悬浮状态,细胞边缘可见不规则树突状突起,在培养第7天时用CD11c+磁珠进行筛选,筛选后CD11c+ DC纯度可达92.88%。7与colon26/CD40L共培养后,DC细胞表面CD80、CD86、MHCⅠ、MHCⅡ分子表达显著提高,与其它组比较有显著性差异;DC与colon26/CD40L共培养组有IL-12、IL-23、IL-18、IFN-γ、IFN-γ(Mig)基因mRNA的表达,在其它组未见;DC与colon26/CD40L共培养组上清中IL-12、IL-23、IFN-γ的释放量高于DC与colon26共培养组和DC组(P< 0.05)。8成功建立colon26荷瘤小鼠模型。9经过3次治疗后,colon26/CD40L与DC联合治疗组荷瘤小鼠肿瘤比colon26/CD40L治疗组、DC治疗组、对照组减小(P<0.05),colon26/CD40L与DC联合治疗组抑瘤率为47.92%;colon26/CD40L治疗组、DC治疗组、对照组抑瘤率分别为32.95%、32.26%和0%。10 colon26/CD40L与DC联合治疗组荷瘤小鼠外周血血清中IL-12、IL-23、IFN-γ高于colon26/CD40L治疗组、DC治疗组和对照组,IL-10和TGF-β则呈较低水平(P<0.05)。11 HE染色结果显示,colon26/CD40L瘤苗治疗组、DC治疗组的肿瘤组织切片中可见到大量的肿瘤细胞,病灶周围有少量炎性细胞浸润,有坏死灶;而在联合治疗组肿瘤病灶内有大量炎性细胞浸润,并伴有大片坏死,肿瘤细胞散在分布,肿瘤局限化。以上三组均未发现肝、脾转移。对照组肿瘤组织切片中可见大量肿瘤细胞,但只见极少量炎细胞浸润,并可在肝、脾组织中发现转移灶。结论:表达CD40L的结肠癌细胞有利于DC和肿瘤细胞的相互作用,促进了DC的成熟,诱导了Th1型细胞因子的分泌,产生了T细胞依赖的抗肿瘤免疫反应。DC-CD40L-肿瘤细胞瘤苗有望成为结肠癌免疫治疗的一种新策略。更多还原

【Abstract】 Objective: colonic cancer cell line colon26 was used as research agent. To study the anti-tumor activity of colon26 transfected with full-length mouse CD40L cDNA in vitro and in vivo. To analyse the anti-tumor effects of the co-culture of CD40L-expressed tumors and bone marrow-derived dendritic cells.Methods:1 The amplification of full-length mouse CD40L cDNA. The pMKITneo CD40L plasmid was transformated into the competence Escherichia coli DH5ɑ, and abundantly amplificated the Escherichia coli. The plasmid abstracted from the Escherichia coli was verified by enzyme digestion and sequencing, and the product expressed was detected by RT-PCR.2 Transfection of CD40L expression vector into tumor cells. Colon26 cells were transfected with the full-length mouse CD40L cDNA by lipofectamineTM2000 and then G418 resistant cells were selected. G418 resistant colon26 cells transfected with vector DNA pMKITneo were used as a control.3 Detection of the expression of CD40L. Transcription level of CD40LmRNA in colon26 transfected cells and the membrane binding rate of PE-labelled CD40L antibody were detected with RT-PCR and FCM respectively. The protein expressional level of CD40L in colon26 transfected cells were detected with western blot and laser scanning confocal microscope respectively.4 The proliferation of the transfected cells was determined. The cells proliferation was detected by MTT assay. To determinate proliferation response by MTS assay and draw curve of cells growth. 5 Detection of the invasion of the transfected cells. The expression level of MMP-2 mRNA in cells was detected through semi-quantitative RT-PCR assay. Using invasion experiment to detect the cells’invasive abilities.6 The preparation of dendritic cells. Bone marrow cells from tibias and femurs were depleted of erythrocytes with spallation buffer. These cells were further cultured in RPMI1640 medium supplemented with recombinant mouse GM-CSF and IL-4, and 10% fetal calf serum for 7 days. Nonadherent cells were harvested and used as DCs. CD11c+ DCs were separated by CD11c-conjugated magnetic beads. The expressions of CD11c on DCs were examined with flow cytometry.7 Test of in vitro immunity function of DC culture with tumor cells expressing CD40L. Tumor cells were cultured with DCs for 24 hours and the phenotype of DCs was examined with flow cytometry. The expression level of IL-12、IL-23、IL-18、IFN-γand IFN-(γMig)mRNA in co-cultured DCs- tumor cells were detected by RT-PCR. The level of IL-12、IL-23 and IFN-γin supernatant of co-cultured DCs- tumor cells were detected by ELISA assay.8 The BALB/C mouse was inoculated colon26 cells to establish tumor model. Logarithmic phase of colon26 cells were collected into single-cell suspension. To adjust cell concentration 1×107/ml. Colon26 cells were injected in the BALB/C mice’s right department of shoulder (0.1ml, 1×106 once) to estabilish mouse xenograft model.9 Anti-tumor effects of tumor vaccine expressing CD40L in vivo. After mice were injected tumor cells 3 days, the DCs, tumor vaccine expressing CD40L, co-cultured DCs-tumor cells expressing CD40L (experiment group) or PBS (control group) was re-infused through peritoneal cavity in tumor barring mice. Every group has 5 mice. Tumor changing and treatment effect were detected. Peripheral blood of tumor barring mice, tumor tissue, liver and spleen were collected after treatmented in four groups.10 The level of IL-12, IL-23, IFN-γ, IL-10 and TGF-βin peripheral blood of tumor barring mice was detected by ELISA assay.11 After HE staining, the histophological changed and tumor cells infiltrated of liver and spleen were observed by microscope.Results:1 Identification of inserted CD40L DNA sequence in this plasmid was performed by enzyme digestion, PCR and DNA sequencing. The results showed that it fully matched the parent plasmid’s nucleic acid database with right direction.2 Colon26 cells were transfected with the mouse CD40L gene and a clone expressing a large amount of CD40L on cell surface was selected. The shape of transfected cells has no change with wild cells. The cells were rhombus, full shape.3 Expression of CD40L on colon26/CD40L cells was much higher than that of colon26 cells, were detected with RT-PCR, FCM, western blot and laser scanning confocal microscope respectively. It showed the recombinant vector could play a normal role in translation of CD40L, transcription and protein synthesis.4 In vitro the proliferation rates of parent and CD40L expressed colon26 cells were not different were detected with MTS assay.5 Expression of MMP-2 mRNA on colon26/CD40L cells was gradually decreased than that of colon26 cells, were detected with RT-PCR. The results of invasion experiment shows that the invasive ability of colon26/CD40L cells decrease remarkable, compared with colon26 cells.6 The bone marrow dendritic cells were cultured by different cytokines in vitro. The cells grew nonadherently, and showed branching-like and pseudopod-like under invert microscope. DCs were separated by CD11c-conjugated magnetic beads at 7 days, and the purity of CD11c may reach above 92.88%.7 The Expression of the CD80, CD86, MHCⅠand MHCⅡof DCs that were cultured with colon26/CD40L was gradually increased than that of in control group. The Expression of the IL-23, IL-12, IL-18, IFN-γand Mig (monokine induced by IFN-γ) genes was induced in the DCs that were cultured with colon26/CD40L but not with colon26 cells. The expression level of the IL-23, IL-12 and IFN-γin co-cultured DCs-colon26/CD40L cells was gradually increased than that of in control group.8 After subcutaneous injected colon26 cells 3 days, the mouse xenograft rate was 100%. The mouse xenograft model was successful established.9 The tumor size in the injecting co-cultured DCs-tumor cells expressing CD40L group was smaller than that in the injecting DCs group, the injecting tumor vaccine expressing CD40L group and PBS. The inhibitory rates were 47.92%, 32.26%, 32.95% and 0%, respectiveily.10 The level of IL-12、IL-23 and IFN-γin the injecting co-cultured DCs-tumor cells expressing CD40L group was higher than that in the injecting DCs group, the injecting tumor vaccine expressing CD40L group and PBS. But the level of IL-10 and TGF-βwas decresed.11 The results of HE showed that the sections of tumor tissues in the injecting DCs group and tumor vaccine expressing CD40L group, and there were a small quantity inflammatory cells and necrosis. However in the injecting co-cultured DCs-tumor cells expressing CD40L group has many inflammatory cells infliltrated and mass necrosis, and tumor cells scattered and circumscribed. Cancer nodules were not found in spleen and sliver in that three group. Many tumor cells and less inflammatory cells were detected in the section of tumor tissues in the PBS group, and cancer nodules were found in spleen and sliver in this group.Conclusions: These data directly showed that the expression of CD40L in colon cancer cells facilitated the interaction between DCs and tumors, enhanced the maturation of DCs, induced secretion of cytokines, and consequently produced T-cell-dependent systemic immunity. DC-CD40L -tumor cells may be a useful strategy for cancer immunotherapy.更多还原