本文選題：GITRL + 樹突狀細(xì)胞��； 參考：《江蘇大學(xué)》2010年博士論文

【摘要】：肺癌是目前臨床較為常見的惡性腫瘤,肺癌發(fā)病率約占所有癌癥發(fā)病率的12%。每年全世界有超過130萬人被確診患有肺癌,超過110萬人死于肺癌。盡管目前肺癌的臨床治療已取得一些進(jìn)展,但總體治療效果仍不理想,因此,有必要尋求更為有效的治療手段。圍繞著樹突狀細(xì)胞(dendritic cell, DC)建立的特異性腫瘤免疫治療方法,為肺癌的生物治療提供了新思路和新方法。 糖皮質(zhì)激素誘導(dǎo)的腫瘤壞死因子受體相關(guān)配體(glucocorticoid-induced tumor necrosis factor receptor ligand, GITRL)是最近發(fā)現(xiàn)的TNF超家族成員,可參與對效應(yīng)性T細(xì)胞和調(diào)節(jié)性T細(xì)胞功能的調(diào)控。在本研究中,我們制備攜帶小鼠GITRL (mGITRL)基因的重組腺病毒(pAd-GITRL),將pAd-GITRL轉(zhuǎn)染小鼠骨髓來源的樹突狀細(xì)胞(bone marrow-derived dendritic cell, BMDC),并研究其生物學(xué)功能變化。在建立小鼠Lewis肺癌移植瘤模型的基礎(chǔ)上,探討pAd-GITRL能否通過上調(diào)DC的免疫刺激功能,調(diào)控效應(yīng)性T細(xì)胞和調(diào)節(jié)性T細(xì)胞的功能,從而增強(qiáng)荷瘤小鼠機(jī)體特異性抗瘤免疫應(yīng)答。 第一部分：攜帶小鼠GITRL基因重組腺病毒的制備與鑒定 目的：構(gòu)建能有效轉(zhuǎn)導(dǎo)mGITRL基因的重組腺病毒,進(jìn)行病毒包裝、擴(kuò)增和滴度測定,以用于mGITRL基因治療的實驗研究。 方法：應(yīng)用AdEasy-1腺病毒表達(dá)載體系統(tǒng)構(gòu)建攜帶mGITRL基因的重組腺病毒(pAd-GITRL)和對照腺病毒(pAd-null)。首先將目的基因克隆至pAdTrack-CMV形成穿梭載體,然后與腺病毒病毒骨架質(zhì)粒共轉(zhuǎn)染至大腸埃希菌BJ5183中,進(jìn)行細(xì)菌內(nèi)同源重組,產(chǎn)生包含目的基因的重組腺病毒載體,由于pAdTrack-CMV攜帶增強(qiáng)型綠色熒光蛋白(eGFP)基因,因此該病毒載體轉(zhuǎn)染293A細(xì)胞后,可通過熒光顯微鏡觀察eGFP的表達(dá)來判斷病毒轉(zhuǎn)染效率,通過qRT-PCR和Western blot鑒定目的基因在293A細(xì)胞中的表達(dá),病毒滴度采用TCID50法測定。 結(jié)果：經(jīng)酶切、PCR及測序鑒定證實,重組腺病毒載體插入片段為mGITRL基因。包裝的重組腺病毒載體具有良好的感染性,細(xì)胞產(chǎn)生明顯的細(xì)胞病變效應(yīng)(cytopathic effect, CPE),可以在293A細(xì)胞中形成病毒顆粒。通過TCID50法測定pAd-GITRL病毒滴度為2.0×109 pfu/ml; pAd-null病毒滴度為2.5×109 pfu/ml。pAd-GITRL感染293A細(xì)胞48h后,熒光顯微鏡觀察可見eGFP的表達(dá)。qRT-PCR檢測結(jié)果顯示,轉(zhuǎn)染了pAd-GITRL的293A細(xì)胞表達(dá)GITRL的mRNA, pAd-null轉(zhuǎn)染的293A細(xì)胞及未轉(zhuǎn)染的293A細(xì)胞不表達(dá)GITRL的mRNA (mRNA的相對表達(dá)量用2△ct值×103表示)(2△ct值×103: 261.90±1.35 vs 0.36±0.039vs0)。Western blot結(jié)果亦顯示,轉(zhuǎn)染了pAd-GITRL的293A細(xì)胞表達(dá)GITRL蛋白, pAd-null轉(zhuǎn)染的293A細(xì)胞及未轉(zhuǎn)染的293A細(xì)胞不表達(dá)GITRL蛋白。 結(jié)論：成功構(gòu)建了復(fù)制缺陷型攜帶mGITRL基因的pAd-GITRL, pAd-GITRL能有效介導(dǎo)mGITRL在293A細(xì)胞內(nèi)表達(dá)。 第二部分：pAd-GITRL轉(zhuǎn)染小鼠骨髓來源樹突狀細(xì)胞(BMDC)的實驗研究 目的：采用pAd-GITRL轉(zhuǎn)染BMDC,觀察轉(zhuǎn)染前后BMDC表面MHCⅡ類分子和共刺激分子的表達(dá)情況。同時體外研究pAd-GITRL轉(zhuǎn)染的BMDC(pAd-GITRL-BMDC)對CD4+CD25-T細(xì)胞增殖和CD4+CD25+Treg細(xì)胞免疫抑制功能的影響。 方法：小鼠骨髓前體細(xì)胞經(jīng)終濃度均為10ng/ml的GM-CSF和IL-4體外誘導(dǎo)培養(yǎng)7 d,并通過顯微鏡和流式細(xì)胞術(shù)(FCM)進(jìn)行形態(tài)和純度鑒定。7 d后分別用pAd-GITRL和pAd-null轉(zhuǎn)染BMDC,通過免疫熒光染色和FCM檢測pAd-GITRL轉(zhuǎn)染前后BMDC表面MHCⅡ類分子、CD40、CD80和CD86的表達(dá)情況。通過體外細(xì)胞增殖試驗檢測pAd-GITRL-BMDC對抗CD3 mAb刺激的CD4+CD25-T細(xì)胞的增殖,以及對CD4+CD25+ Treg細(xì)胞免疫抑制功能的影響。 結(jié)果：小鼠骨髓前體細(xì)胞體外誘導(dǎo)培養(yǎng)7 d后,在倒置顯微鏡下可觀察到細(xì)胞體積增大,且呈不規(guī)則突起,形似樹突狀；FCM檢測CDllc+比例達(dá)79.07%。通過熒光顯微鏡觀察eGFP的表達(dá)可知pAd-GITRL能有效轉(zhuǎn)染BMDC。FCM結(jié)果顯示BMDC轉(zhuǎn)染pAd-GITRL后,體外培養(yǎng)8 d仍能穩(wěn)定表達(dá)GITRL。相對于BMDC組,pAd-null-BMDC和pAd-GITRL-BMDC表面CD40、CD80和CD86均有所升高,但組間無顯著性差異,MHCⅡ類分子無明顯變化�？笴D3 mAb刺激的CD4+CD25- T細(xì)胞增殖試驗中,pAd-GITRL-BMDC組的cpm值為6888±1304,與BMDC (848±59)和pAd-null-BMDC(1231±75)相比有顯著性差異(p0.05)。CD4+CD25+ Treg細(xì)胞免疫抑制功能試驗結(jié)果顯示,pAd-GITRL-BMDC共培養(yǎng)體系中Treg細(xì)胞的抑制率為44.7±17.2%,與BMDC (63.7±17.4%)和pAd-null-BMDC (90.3±11.9%)相比明顯下降(p0.05)。 結(jié)論：體外成功誘導(dǎo)和培養(yǎng)BMDC, pAd-GITRL在體外能有效轉(zhuǎn)染BMDC,并且持續(xù)性表達(dá)GITRL。pAd-GITRL-BMDC能顯著增強(qiáng)CD4+CD25-T細(xì)胞的增殖,同時能部分下調(diào)CD4+CD25+T細(xì)胞的免疫抑制功能。 第三部分：pAd-GITRL-BMDC增強(qiáng)Lewis肺癌移植瘤小鼠抗瘤免疫應(yīng)答的實驗研究 目的：探討pAd-GITRL-BMDC能否增強(qiáng)Lewis肺癌移植瘤小鼠體內(nèi)抗瘤免疫應(yīng)答,尋求mGITRL在調(diào)控免疫應(yīng)答中的作用及其抗瘤免疫的機(jī)制。 方法：通過皮下接種小鼠Lewis肺癌細(xì)胞,建立Lewis肺癌移植瘤小鼠模型。隨機(jī)分為BMDC、pAd-null-BMDC和pAd-GITRL-BMDC和PBS四組。按照3：1的比例,將3.0×106 Lewis細(xì)胞的裂解液分別加入BMDC、pAd-null-BMDC和pAd-GITRL-BMDC(1.0×106/孔)培養(yǎng)體系中,37℃、5%CO2孵育24 h。皮下接種Lewis后第10d、第12d和第14d,每組小鼠分別瘤內(nèi)多點注射1×106/100μ1腫瘤抗原負(fù)載的BMDC、pAd-null-BMDC和pAd-GITRL-BMDC, PBS組僅注射100μl PBS,觀察荷瘤小鼠體內(nèi)腫瘤的生長情況。部分小鼠在最后一次注射6d后,斷頸處死荷瘤小鼠,FCM檢測荷瘤小鼠脾臟中CD8+IFN-γ+T細(xì)胞、CD4+CD25+Foxp3+T細(xì)胞和CD4+IL-17+T細(xì)胞的數(shù)量與比例,qRT-PCR檢測腫瘤組織中GITRL、IFN-γ、Foxp3、IL-17、ROR-γt和CCL20的表達(dá)情況,免疫熒光染色法檢測腫瘤組織中CD8+IFN-γ+T細(xì)胞、CD4+Foxp3+T細(xì)胞和CD4+IL-17+T細(xì)胞的分布情況。 結(jié)果：pAd-GITRL-BMDC組抑制腫瘤生長的作用較pAd-null-BMDC組、BMDC組和PBS組明顯,皮下種植Lewis后第30 d,各組腫瘤的大小分別為210.5±20.5,327.7±39.1,419.0±91.9和646.6±98.7mm2。在70 d的觀察期內(nèi),pAd-GITRL-BMDC組的生存率為75%, pAd-null-BMDC組生存率為29%,BMDC組和PBS組生存率為0%。FCM分析小鼠脾臟中CD8+IFN-γ+ T細(xì)胞的比例,pAd-GITRL-BMDC組為32.29±14.5%,明顯高于pAd-null-BMDC組(24.32±6.83%,p0.05)。CD4+CD25+Foxp3+ Treg細(xì)胞的比例在各組間沒有明顯差異,但pAd-GITRL-BMDC組小鼠脾臟中Treg細(xì)胞的數(shù)量與pAd-null-BMDC組相比卻顯著降低(9.48×105±2.21×105vs2.07×106±1.21×106,p0.05)BMDC、pAd-null-BMDC、pAd-GITRL-BMDC三組小鼠脾臟中CD4+IL-17+T細(xì)胞比例均明顯高于PBS組,但各治療組間并無顯著性差異。腫瘤組織免疫熒光染色結(jié)果顯示,pAd-GITRL-BMDC組與其他各組相比,腫瘤組織中CD8+IFN-γ+T細(xì)胞和CD4+IL-17+T細(xì)胞明顯增多,而CD4+Foxp3+T細(xì)胞明顯減少。qRT-PCR檢測顯示(各個指標(biāo)mRNA的相對表達(dá)量用2△ct值×103表示),pAd-GITRL-BMDC組與pAd-null-BMDC組的GITRL分別為55.30±26.50和16.27±5.12,IFN-γ分別為17.42±4.87和9.37±2.72,IL-17分別為13.10±4.20和8.12±0.45,ROR-γt分別為273.44±51.20和72.42±5.21,CCL20分別為273.44±51.20和72.42±5.21,表明pAd-GITRL-BMDC組與pAd-null-BMDC組腫瘤組織中GITRL. IFN-γ、IL-17、ROR-γt和CCL20的表達(dá)均有顯著性增高(p0.05)；但pAd-GITRL-BMDC組與pAd-null-BMDC組腫瘤組織中的Foxp3分別為3.09±0.93和12.78±5.60(p0.05)。 結(jié)論：pAd-GITRL-BMDC能顯著抑制體內(nèi)腫瘤的生長,延長荷瘤小鼠的生存期。其可能的機(jī)制一方面是pAd-GITRL-BMDC能降低荷瘤小鼠體內(nèi)CD4+CD25+Foxp3+Treg細(xì)胞的數(shù)量,減少腫瘤組織中CD4+Foxp3+Treg細(xì)胞的浸潤。另一方面是增加荷瘤小鼠體內(nèi)CD8+IFN-γ+T細(xì)胞的比例,促進(jìn)腫瘤組織中CD8+IFN-γ+T細(xì)胞及CD4+IL-17+T細(xì)胞的浸潤,從而增強(qiáng)Lewis肺癌移植瘤小鼠的抗瘤免疫效應(yīng)。
[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.

【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R392

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