mGITRL 基因重组腺病毒转染DC增强Lewis肺癌移植瘤小鼠抗瘤免疫的实验研究

发布时间：2018-05-13 11:03

本文选题：GITRL + 树突状细胞　；参考：《江苏大学》2010年博士论文

【摘要】：肺癌是目前临床较为常见的恶性肿瘤,肺癌发病率约占所有癌症发病率的12%。每年全世界有超过130万人被确诊患有肺癌,超过110万人死于肺癌。尽管目前肺癌的临床治疗已取得一些进展,但总体治疗效果仍不理想,因此,有必要寻求更为有效的治疗手段。围绕着树突状细胞(dendritic cell, DC)建立的特异性肿瘤免疫治疗方法,为肺癌的生物治疗提供了新思路和新方法。糖皮质激素诱导的肿瘤坏死因子受体相关配体(glucocorticoid-induced tumor necrosis factor receptor ligand, GITRL)是最近发现的TNF超家族成员,可参与对效应性T细胞和调节性T细胞功能的调控。在本研究中,我们制备携带小鼠GITRL (mGITRL)基因的重组腺病毒(pAd-GITRL),将pAd-GITRL转染小鼠骨髓来源的树突状细胞(bone marrow-derived dendritic cell, BMDC),并研究其生物学功能变化。在建立小鼠Lewis肺癌移植瘤模型的基础上,探讨pAd-GITRL能否通过上调DC的免疫刺激功能,调控效应性T细胞和调节性T细胞的功能,从而增强荷瘤小鼠机体特异性抗瘤免疫应答。第一部分：携带小鼠GITRL基因重组腺病毒的制备与鉴定目的：构建能有效转导mGITRL基因的重组腺病毒,进行病毒包装、扩增和滴度测定,以用于mGITRL基因治疗的实验研究。方法：应用AdEasy-1腺病毒表达载体系统构建携带mGITRL基因的重组腺病毒(pAd-GITRL)和对照腺病毒(pAd-null)。首先将目的基因克隆至pAdTrack-CMV形成穿梭载体,然后与腺病毒病毒骨架质粒共转染至大肠埃希菌BJ5183中,进行细菌内同源重组,产生包含目的基因的重组腺病毒载体,由于pAdTrack-CMV携带增强型绿色荧光蛋白(eGFP)基因,因此该病毒载体转染293A细胞后,可通过荧光显微镜观察eGFP的表达来判断病毒转染效率,通过qRT-PCR和Western blot鉴定目的基因在293A细胞中的表达,病毒滴度采用TCID50法测定。结果：经酶切、PCR及测序鉴定证实,重组腺病毒载体插入片段为mGITRL基因。包装的重组腺病毒载体具有良好的感染性,细胞产生明显的细胞病变效应(cytopathic effect, CPE),可以在293A细胞中形成病毒颗粒。通过TCID50法测定pAd-GITRL病毒滴度为2.0×109 pfu/ml; pAd-null病毒滴度为2.5×109 pfu/ml。pAd-GITRL感染293A细胞48h后,荧光显微镜观察可见eGFP的表达。qRT-PCR检测结果显示,转染了pAd-GITRL的293A细胞表达GITRL的mRNA, pAd-null转染的293A细胞及未转染的293A细胞不表达GITRL的mRNA (mRNA的相对表达量用2△ct值×103表示)(2△ct值×103: 261.90±1.35 vs 0.36±0.039vs0)。Western blot结果亦显示,转染了pAd-GITRL的293A细胞表达GITRL蛋白, pAd-null转染的293A细胞及未转染的293A细胞不表达GITRL蛋白。结论：成功构建了复制缺陷型携带mGITRL基因的pAd-GITRL, pAd-GITRL能有效介导mGITRL在293A细胞内表达。第二部分：pAd-GITRL转染小鼠骨髓来源树突状细胞(BMDC)的实验研究目的：采用pAd-GITRL转染BMDC,观察转染前后BMDC表面MHCⅡ类分子和共刺激分子的表达情况。同时体外研究pAd-GITRL转染的BMDC(pAd-GITRL-BMDC)对CD4+CD25-T细胞增殖和CD4+CD25+Treg细胞免疫抑制功能的影响。方法：小鼠骨髓前体细胞经终浓度均为10ng/ml的GM-CSF和IL-4体外诱导培养7 d,并通过显微镜和流式细胞术(FCM)进行形态和纯度鉴定。7 d后分别用pAd-GITRL和pAd-null转染BMDC,通过免疫荧光染色和FCM检测pAd-GITRL转染前后BMDC表面MHCⅡ类分子、CD40、CD80和CD86的表达情况。通过体外细胞增殖试验检测pAd-GITRL-BMDC对抗CD3 mAb刺激的CD4+CD25-T细胞的增殖,以及对CD4+CD25+ Treg细胞免疫抑制功能的影响。结果：小鼠骨髓前体细胞体外诱导培养7 d后,在倒置显微镜下可观察到细胞体积增大,且呈不规则突起,形似树突状；FCM检测CDllc+比例达79.07%。通过荧光显微镜观察eGFP的表达可知pAd-GITRL能有效转染BMDC。FCM结果显示BMDC转染pAd-GITRL后,体外培养8 d仍能稳定表达GITRL。相对于BMDC组,pAd-null-BMDC和pAd-GITRL-BMDC表面CD40、CD80和CD86均有所升高,但组间无显著性差异,MHCⅡ类分子无明显变化。抗CD3 mAb刺激的CD4+CD25- T细胞增殖试验中,pAd-GITRL-BMDC组的cpm值为6888±1304,与BMDC (848±59)和pAd-null-BMDC(1231±75)相比有显著性差异(p0.05)。CD4+CD25+ Treg细胞免疫抑制功能试验结果显示,pAd-GITRL-BMDC共培养体系中Treg细胞的抑制率为44.7±17.2%,与BMDC (63.7±17.4%)和pAd-null-BMDC (90.3±11.9%)相比明显下降(p0.05)。结论：体外成功诱导和培养BMDC, pAd-GITRL在体外能有效转染BMDC,并且持续性表达GITRL。pAd-GITRL-BMDC能显著增强CD4+CD25-T细胞的增殖,同时能部分下调CD4+CD25+T细胞的免疫抑制功能。第三部分：pAd-GITRL-BMDC增强Lewis肺癌移植瘤小鼠抗瘤免疫应答的实验研究目的：探讨pAd-GITRL-BMDC能否增强Lewis肺癌移植瘤小鼠体内抗瘤免疫应答,寻求mGITRL在调控免疫应答中的作用及其抗瘤免疫的机制。方法：通过皮下接种小鼠Lewis肺癌细胞,建立Lewis肺癌移植瘤小鼠模型。随机分为BMDC、pAd-null-BMDC和pAd-GITRL-BMDC和PBS四组。按照3：1的比例,将3.0×106 Lewis细胞的裂解液分别加入BMDC、pAd-null-BMDC和pAd-GITRL-BMDC(1.0×106/孔)培养体系中,37℃、5%CO2孵育24 h。皮下接种Lewis后第10d、第12d和第14d,每组小鼠分别瘤内多点注射1×106/100μ1肿瘤抗原负载的BMDC、pAd-null-BMDC和pAd-GITRL-BMDC, PBS组仅注射100μl PBS,观察荷瘤小鼠体内肿瘤的生长情况。部分小鼠在最后一次注射6d后,断颈处死荷瘤小鼠,FCM检测荷瘤小鼠脾脏中CD8+IFN-γ+T细胞、CD4+CD25+Foxp3+T细胞和CD4+IL-17+T细胞的数量与比例,qRT-PCR检测肿瘤组织中GITRL、IFN-γ、Foxp3、IL-17、ROR-γt和CCL20的表达情况,免疫荧光染色法检测肿瘤组织中CD8+IFN-γ+T细胞、CD4+Foxp3+T细胞和CD4+IL-17+T细胞的分布情况。结果：pAd-GITRL-BMDC组抑制肿瘤生长的作用较pAd-null-BMDC组、BMDC组和PBS组明显,皮下种植Lewis后第30 d,各组肿瘤的大小分别为210.5±20.5,327.7±39.1,419.0±91.9和646.6±98.7mm2。在70 d的观察期内,pAd-GITRL-BMDC组的生存率为75%, pAd-null-BMDC组生存率为29%,BMDC组和PBS组生存率为0%。FCM分析小鼠脾脏中CD8+IFN-γ+ T细胞的比例,pAd-GITRL-BMDC组为32.29±14.5%,明显高于pAd-null-BMDC组(24.32±6.83%,p0.05)。CD4+CD25+Foxp3+ Treg细胞的比例在各组间没有明显差异,但pAd-GITRL-BMDC组小鼠脾脏中Treg细胞的数量与pAd-null-BMDC组相比却显著降低(9.48×105±2.21×105vs2.07×106±1.21×106,p0.05)BMDC、pAd-null-BMDC、pAd-GITRL-BMDC三组小鼠脾脏中CD4+IL-17+T细胞比例均明显高于PBS组,但各治疗组间并无显著性差异。肿瘤组织免疫荧光染色结果显示,pAd-GITRL-BMDC组与其他各组相比,肿瘤组织中CD8+IFN-γ+T细胞和CD4+IL-17+T细胞明显增多,而CD4+Foxp3+T细胞明显减少。qRT-PCR检测显示(各个指标mRNA的相对表达量用2△ct值×103表示),pAd-GITRL-BMDC组与pAd-null-BMDC组的GITRL分别为55.30±26.50和16.27±5.12,IFN-γ分别为17.42±4.87和9.37±2.72,IL-17分别为13.10±4.20和8.12±0.45,ROR-γt分别为273.44±51.20和72.42±5.21,CCL20分别为273.44±51.20和72.42±5.21,表明pAd-GITRL-BMDC组与pAd-null-BMDC组肿瘤组织中GITRL. IFN-γ、IL-17、ROR-γt和CCL20的表达均有显著性增高(p0.05)；但pAd-GITRL-BMDC组与pAd-null-BMDC组肿瘤组织中的Foxp3分别为3.09±0.93和12.78±5.60(p0.05)。结论：pAd-GITRL-BMDC能显著抑制体内肿瘤的生长,延长荷瘤小鼠的生存期。其可能的机制一方面是pAd-GITRL-BMDC能降低荷瘤小鼠体内CD4+CD25+Foxp3+Treg细胞的数量,减少肿瘤组织中CD4+Foxp3+Treg细胞的浸润。另一方面是增加荷瘤小鼠体内CD8+IFN-γ+T细胞的比例,促进肿瘤组织中CD8+IFN-γ+T细胞及CD4+IL-17+T细胞的浸润,从而增强Lewis肺癌移植瘤小鼠的抗瘤免疫效应。
[Abstract]:Lung cancer is a common malignant tumor at present. The incidence of lung cancer accounts for about 12%. of all cancer incidence. More than 1 million 300 thousand people in the world have been diagnosed with lung cancer every year and more than 1 million 100 thousand people have died of lung cancer. Although some progress has been made in the clinical treatment of lung cancer, the overall treatment effect is still not ideal. Therefore, it is necessary to seek more. Effective therapy. The specific tumor immunotherapy based on dendritic cell (DC) provides new ideas and new methods for the biological treatment of lung cancer.
The glucocorticoid induced tumor necrosis factor receptor related ligand (glucocorticoid-induced tumor necrosis factor receptor ligand, GITRL) is a recently discovered member of the TNF superfamily, which can participate in the regulation of the functional T cell and regulatory T cell function. In this study, we have prepared the recombinant of the GITRL (mGITRL) gene in mice. Adenovirus (pAd-GITRL), transfect pAd-GITRL into mouse bone marrow derived dendritic cells (bone marrow-derived dendritic cell, BMDC), and study their biological function changes. On the basis of establishing a mouse Lewis lung cancer transplant tumor model, it is explored whether pAd-GITRL can regulate the effect of T cells and regulatory T by up regulation of the immune stimulation function of DC. The function of cells enhances the specific anti-tumor immune response of tumor bearing mice.
Part one: preparation and identification of recombinant adenovirus carrying mouse GITRL gene
Objective: to construct a recombinant adenovirus which can effectively transduce mGITRL gene and to carry out viral packaging, amplification and titer determination for mGITRL gene therapy.
Methods: recombinant adenovirus (pAd-GITRL) and control adenovirus (pAd-null) carrying mGITRL gene were constructed by AdEasy-1 adenovirus expression vector system. First, the target gene was cloned to pAdTrack-CMV to form a shuttle vector and then co transfected with the adenovirus vector to the BJ5183 of Escherichia coli to carry out homologous recombination in bacteria. The recombinant adenovirus vector containing the target gene, because pAdTrack-CMV carries the enhanced green fluorescent protein (eGFP) gene, and then transfected to 293A cells by the vector, the expression of eGFP can be observed by fluorescence microscopy to determine the transfection efficiency of the virus. The expression of the target gene in 293A cells is identified by qRT-PCR and Western blot. The toxic titer was determined by TCID50.
Results: by enzyme digestion, PCR and sequencing confirmed that the recombinant adenovirus vector was inserted into the mGITRL gene. The recombinant adenovirus carrying the recombinant adenovirus vector had good infectivity, the cells produced obvious cytopathic effect (cytopathic effect, CPE), and the virus particles could be formed in the 293A cells. The TCID50 assay was used to determine the titer of pAd-GITRL virus to 2 .0 x 109 pfu/ml, pAd-null virus titer was 2.5 x 109 pfu/ml.pAd-GITRL infected 293A cells 48h, the fluorescence microscope observed that the eGFP expression.QRT-PCR detection results showed that the 293A cells transfected with pAd-GITRL expressed GITRL mRNA, and the transfected cells and untransfected cells did not express the relative expression. The 2 Delta CT value * 103 (2 Delta CT value * 103: 261.90 + 1.35 vs 0.36 + 0.039vs0).Western blot results also showed that the 293A cells transfected with pAd-GITRL expressed GITRL protein, pAd-null transfected 293A cells and untransfected cells did not express the protein.
Conclusion: the replication defective pAd-GITRL carrying mGITRL gene has been successfully constructed, and pAd-GITRL can effectively mediate the expression of mGITRL in 293A cells.
The second part: pAd-GITRL transfected mouse bone marrow derived dendritic cells (BMDC).
Objective: to transfect BMDC with pAd-GITRL and observe the expression of MHC class II and co stimulator on BMDC surface before and after transfection. The effect of pAd-GITRL transfected BMDC (pAd-GITRL-BMDC) on the proliferation of CD4+CD25-T cells and the immunosuppressive function of CD4+CD25+Treg cells was studied in vitro.
Methods: the mouse bone marrow precursor cells were induced and cultured in vitro by GM-CSF and IL-4, which were all 10ng/ml, and were cultured in vitro for 7 d. The morphology and purity of.7 D were identified by microscope and flow cytometry (FCM),.7 D was transfected with pAd-GITRL and pAd-null respectively, and the molecules were detected by immunofluorescence staining and FCM before and after pAd-GITRL. The expression of CD80 and CD86. The proliferation of pAd-GITRL-BMDC against CD3 mAb stimulated CD4+CD25-T cells and the effect on the immunosuppressive function of CD4+CD25+ Treg cells were detected by cell proliferation test in vitro.
Results: after the mouse bone marrow precursor cells were induced and cultured in vitro for 7 d in vitro, the volume of the cells could be observed under the inverted microscope, and the cells were irregular protruding and resembling the dendritic shape. The ratio of FCM to CDllc+ was 79.07%. through the fluorescence microscope to observe the expression of eGFP. It can be found that pAd-GITRL can effectively transfer the BMDC.FCM results to show that BMDC transfected pAd-GITRL after the transfection. 8 D can still express GITRL. relative to group BMDC, CD40, CD80 and CD86 on the surface of pAd-null-BMDC and pAd-GITRL-BMDC, but there is no significant difference between the groups, and there is no obvious change in the MHC class II. In the CD4+CD25- cell proliferation test of anti CD3 mAb stimulation, the value of the group is 6888 + 1304, and that is (848 + 59). L-BMDC (1231 + 75) showed significant difference (P0.05).CD4+CD25+ Treg cell immunosuppressive test results showed that the inhibitory rate of Treg cells in the pAd-GITRL-BMDC co culture system was 44.7 + 17.2%, compared with BMDC (63.7 + 17.4%) and pAd-null-BMDC (90.3 + 11.9%) significantly decreased (P0.05).
Conclusion: the successful induction and culture of BMDC in vitro, pAd-GITRL can effectively transfect BMDC in vitro, and the continuous expression of GITRL.pAd-GITRL-BMDC can significantly enhance the proliferation of CD4+CD25-T cells, and can partly reduce the immunosuppressive function of CD4+CD25+T cells.
The third part: pAd-GITRL-BMDC enhances the anti-tumor immune response of Lewis lung cancer xenograft mice.
Objective: To investigate whether pAd-GITRL-BMDC can enhance the anti tumor immune response in Lewis lung cancer transplanted mice, and seek the role of mGITRL in the regulation of immune response and the mechanism of anti-tumor immunity.
Methods: the mice model of Lewis lung cancer transplanted by subcutaneous inoculation of Lewis lung cancer cells was randomly divided into four groups: BMDC, pAd-null-BMDC, pAd-GITRL-BMDC and PBS. According to the proportion of 3:1, the lysate of 3 x 106 Lewis cells was added to BMDC, pAd-null-BMDC and pAd-GITRL-BMDC (1 * 106/ hole) culture system, 37 degrees centigrade. 24 h. subcutaneous inoculation of Lewis, 10d, 12D and 14d, each group of mice were injected with 1 x 106/100 mu 1 tumor antigen loaded BMDC, pAd-null-BMDC and pAd-GITRL-BMDC, and PBS group only injected 100 mu L PBS, to observe the tumor growth in the tumor bearing mice. The number and proportion of CD8+IFN- gamma +T cells, CD4+CD25+Foxp3+T cells and CD4+IL-17+T cells in the spleen of the tumor bearing mice were detected. QRT-PCR was used to detect the expression of GITRL, IFN- gamma, Foxp3, IL-17, ROR- gamma T and CCL20, and the distribution of tumor cells and cells in tumor tissues by immunofluorescence staining. Situation.
Results: the effect of pAd-GITRL-BMDC group on tumor growth was compared with that in group pAd-null-BMDC, in group BMDC and in group PBS, thirtieth d after subcutaneous implantation, and the size of the tumor in each group was 210.5 + 20.5327.7 + 39.1419.0 + 91.9 and 646.6 + 98.7mm2. in the observation period of 70 D respectively. The survival rate of pAd-GITRL-BMDC group was 75%, and the survival rate of pAd-null-BMDC group was 29%. The rate of survival of group BMDC and group PBS was 0%.FCM analysis of CD8+IFN- gamma + T cells in mice, and that in group pAd-GITRL-BMDC was 32.29 + 14.5%, obviously higher than that of pAd-null-BMDC group (24.32 + 6.83%, P0.05).CD4+CD25+Foxp3+ Treg cells, but the number of Treg cells in the spleen of the pAd-GITRL-BMDC group Compared with group MDC (9.48 * 105 + 2.21 * 105vs2.07 * 106 + 1.21 * 106, P0.05) BMDC, pAd-null-BMDC, pAd-GITRL-BMDC three, the proportion of CD4+IL-17+T cells in the spleen of mice was significantly higher than that in PBS group, but there was no significant difference between the treatment groups. The results of immunofluorescence staining in tumor tissue showed that the pAd-GITRL-BMDC group was compared with the other groups. In tumor tissue, CD8+IFN- gamma +T cells and CD4+IL-17+T cells were significantly increased, while CD4+Foxp3+T cells significantly decreased.QRT-PCR detection (the relative expression of mRNA was expressed as 2 Delta CT value by 103), pAd-GITRL-BMDC group and pAd-null-BMDC group GITRL were 55.30 + 26.50 and 16.27 + 5.12 respectively, IFN- gamma was 17.42 + 4.87 and 9.37 + 2.72, respectively. L-17 was 13.10 + 4.20 and 8.12 + 0.45 respectively, and ROR- gamma t was 273.44 + 51.20 and 72.42 + 5.21 respectively. CCL20 was 273.44 + 51.20 and 72.42 + 5.21, respectively, indicating that GITRL. IFN- gamma, IL-17, ROR- gamma T and CCL20 were significantly higher in pAd-GITRL-BMDC and pAd-null-BMDC group tumor tissues. The Foxp3 in tumor tissues was 3.09 + 0.93 and 12.78 + 5.60 (P0.05) respectively.
Conclusion: pAd-GITRL-BMDC can significantly inhibit the growth of tumor in vivo and prolong the survival time of tumor bearing mice. The possible mechanism is that pAd-GITRL-BMDC can reduce the number of CD4+CD25+Foxp3+Treg cells in the tumor bearing mice and reduce the infiltration of CD4+Foxp3+Treg cells in the tumor tissue. On the other hand, it is the increase of CD8+IFN- gamma +T in the tumor bearing mice. The proportion of cells promotes the infiltration of CD8+IFN- gamma +T cells and CD4+IL-17+T cells in tumor tissues, thereby enhancing the anti-tumor immune effect of Lewis lung cancer xenograft mice.

【学位授予单位】：江苏大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

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