【摘要】： 目的:對小兒腎母細胞瘤新型疫苗進行初步探索、研究,為該腫瘤的治療以及替代手術后的放、化療提供新的切入點。 方法:首先對必需的實驗材料進行制備和驗證,包括抗體的制備和腫瘤細胞模型的建立。針對小兒腎母細胞瘤WT1基因,選取兩段抗原表位肽HLA-A*0201、HLA-A*2402采用構建DNA疫苗的方式,運用Balb/c小鼠免疫檢測的方法篩選最佳的抗原表位肽,為小兒腎母細胞瘤新型疫苗的研究奠定基礎。用篩選得到的表位肽HLA-A*2402構建DNA疫苗,為最終新型疫苗的研制做對照。采用基因重組技術分別克隆編碼小兒腎母細胞瘤WT1表位肽HLA-A*2402融合到乙型肝炎核蛋白基因片段(HBc)上,運用桿狀病毒表達系統(tǒng)對融合蛋白進行表達,通過電鏡觀察得到的融合蛋白呈病毒樣顆粒(VLP)。繼而采用WB技術對表達的蛋白進行鑒定。鑒定好的VLP蛋白和DNA疫苗采用細胞模型進行研究,然后分別免疫Balb/c小鼠,采用ELISA、FCM、MTS等免疫學實驗技術分別檢測,初步判斷基于WT1表位肽HLA-A*2402構建的以VLP為呈現(xiàn)載體的重組蛋白,作為該腫瘤治療以及替代手術后的放、化療的可行性。 結果: (1)制備了合格的實驗材料:對小兒腎母細胞瘤細胞進行了成功的原代培養(yǎng),建立了細胞系用于實驗研究;克隆了WT1基因部分片段的原核表達質粒,表達并純化了蛋白,免疫家兔,采集血清制備了針對WT1的多克隆抗體。 (2) MTS及FCM初步證明,短肽HLA-A*2402在刺激淋巴細胞增殖方面優(yōu)于HLA-A*0201,而前者無顯著性差異(p0.05)。因此,HLA-A*2402為我們選擇深入研究的對象。 (3)構建了含有HLA-A*2402的DNA疫苗,雙酶切和測序結果證明構建成功。 (4)構建了HBc與HLA-A*2402偶聯(lián)(couple)的以VLP為呈現(xiàn)載體的新型疫苗,雙酶切和測序結果證明構建成功。運用桿狀病毒表達系統(tǒng)對偶聯(lián)的蛋白進行表達,WB和電鏡檢測證明目的蛋白的表達。 (5)運用以VLP為呈現(xiàn)載體的新型疫苗進行Balb/c小鼠的免疫,并以構建的DNA疫苗作為對照,結果表明以VLP為呈現(xiàn)載體的新型疫苗免疫小鼠產生了有效的特異性體液免疫和細胞免疫�？贵w亞型分類表明特異性IgG1/IgG2a小于1,說明產生了Th1型為主的免疫應答。T淋巴細胞增殖試驗證實免疫小鼠脾臟T淋巴細胞對ConA誘導的增殖反應增強;ELISPOT分析表明疫苗免疫組的特異性活化的可分泌IFN-γ的CD8+T細胞數(shù)顯著多于對照組;FCM檢測表明,疫苗免疫組的CD4+/CD8+比值顯著低于對照組。這些結果提示以VLP為呈現(xiàn)載體的新型疫苗誘導了特異性CD8+T淋巴細胞應答。其激發(fā)的細胞免疫和體液免疫均明顯優(yōu)于DNA疫苗對照組。 (6)研究證實HLA-A*2402在小兒腎母細胞瘤的免疫治療過程中扮演著重要的角色,而以VLP為呈現(xiàn)載體的新型疫苗為腫瘤的免疫治療提供了新的思路。 結論:所制備的小兒腎母細胞瘤以VLP為呈現(xiàn)載體的新型疫苗在小鼠體內可同時激發(fā)體液免疫和細胞免疫,優(yōu)于相應的DNA疫苗,為腫瘤的免疫治療提供新的方法和思路。更為重要的是將病毒有關成分引入到腫瘤的免疫治療中,其良好的實驗結果有待于腫瘤動物模型的進一步驗證。
[Abstract]:Objective: To explore a new vaccine for Wilms tumor in children, and to provide a new breakthrough point for the treatment of this tumor and for postoperative radiotherapy and chemotherapy.
Methods: first, the necessary experimental materials were prepared and verified, including the preparation of the antibody and the establishment of the tumor cell model. According to the WT1 gene of the nephroblastoma in children, the two segment epitope peptide HLA-A*0201 was selected, and HLA-A*2402 was used to construct the DNA vaccine, and the best epitope peptide was screened by the method of immune detection in Balb/c mice. For the study of a new type of vaccine for nephroblastoma, the DNA vaccine was constructed with the selected epitope peptide HLA-A*2402, which was used as the control for the development of the final new vaccine. The gene recombination technique was used to clone the WT1 epitope peptide HLA-A*2402 of the children's nephroblastoma to the hepatitis B nucleoprotein gene fragment (HBc) and use the pole shape. The fusion protein was expressed by the virus expression system. The fusion protein obtained by electron microscopy was virus like particles (VLP). Then WB technology was used to identify the expressed protein. The identified VLP protein and DNA vaccine were studied by cell model, and then immune Balb/ c mice were immunized with ELISA, FCM, MTS and other immunoassay techniques. The preliminary determination of the recombinant protein based on the WT1 epitope peptide HLA-A*2402, based on the VLP as the carrier, was used as the tumor therapy and the possibility of chemotherapy after the replacement of the tumor.
Result:
(1) the qualified experimental materials were prepared: the primary culture of the children's nephroblastoma cells was successfully cultured, the cell line was established for experimental study, the prokaryotic expression plasmid of the partial fragment of WT1 gene was cloned, the protein was expressed and purified, the rabbit was immunized, and the polyclonal antibody against WT1 was prepared by the collection of serum.
(2) MTS and FCM preliminarily proved that the short peptide HLA-A*2402 is superior to HLA-A*0201 in stimulating lymphocyte proliferation, but the former has no significant difference (P0.05). Therefore, HLA-A*2402 is the object of our study.
(3) a DNA vaccine containing HLA-A*2402 was constructed. Double enzyme digestion and sequencing showed that the recombinant DNA vaccine was successfully constructed.
(4) a new vaccine was constructed with HBc and HLA-A*2402 coupling (couple) with VLP as the carrier. The results of double enzyme digestion and sequencing proved that the construction was successful. The protein expression of the coupling was expressed by the baculovirus expression system. The expression of the target protein was proved by WB and electron microscopy.
(5) the immunization of Balb/c mice was carried out with the new vaccine of VLP as the carrier, and the constructed DNA vaccine was used as the control. The results showed that the new vaccine immunized with VLP as the carrier produced effective specific humoral immunity and cell immunity. The specific IgG1/IgG2a of the antibody subtype was less than 1, indicating that the Th1 type was produced. The immune response.T lymphocyte proliferation test proved that the proliferation response of spleen T lymphocyte in immunized mice was enhanced by ConA, and ELISPOT analysis showed that the number of CD8+T cells that secreted IFN- gamma in the vaccine immunization group was significantly more than that of the control group; FCM detection showed that the CD4+/CD8+ ratio of the immunization group was significantly lower than that of the control group. These results suggest that a new type of vaccine with VLP as the carrier induces specific CD8+T lymphocyte response. Both the stimulated cell immunity and the humoral immunity are obviously superior to that of the DNA vaccine control group.
(6) the study confirms that HLA-A*2402 plays an important role in the immunotherapy of children with nephroblastoma, and a new vaccine with VLP as the carrier provides a new way of thinking for the immunotherapy of tumor.
Conclusion: the new vaccine with VLP as the carrier can stimulate both humoral and cellular immunity in mice. It is superior to the corresponding DNA vaccine and provides new methods and ideas for the immunotherapy of tumor. The results need to be further validated by animal models of tumor.
【學位授予單位】：內蒙古農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R392

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