重組減毒沙門氏菌疫苗SL3261-pcDNA3.1VEGFR2(n1-4)解析

1、重組減毒沙門氏菌疫苗 SL3261-pcDNA3.1+/VEGFR2 （n1-4）抗小鼠大腸癌生長的實驗研究中文摘要 目的：研究血管內(nèi)皮生長因子受體 2胞外 1-4片段 （VEGFR2（n1-4） 抑制 BALB/c 小鼠皮下腫瘤生長的效應(yīng)。方法：利用重組好的重組質(zhì)粒pcDNA3.1+/VEGFR2（ni-4）轉(zhuǎn)化感受態(tài)減毒鼠沙門氏 菌株SL3261,制備成以減毒沙門氏菌株SL3261為載體的口服基因工程疫苗SL3261-pcDNA3.1+/VEGFR2（ni-4）。抽提重組疫苗菌中的重組質(zhì)粒并經(jīng)雙酶切以 及 PCR 特異性擴增重組質(zhì)粒中真核啟動子 CMV 鑒定無誤后,將該疫苗菌擴增 經(jīng)灌胃法

2、飼服 BALB/c 小鼠,對小鼠進行基因免疫。 60只 BALB/c 小鼠分為兩個 大組,每個大組內(nèi)又隨機分為疫苗組、空質(zhì)粒組和 i0% NaHCO3 對照組,兩周 內(nèi)各口服基因疫苗三次后，第一大組用CT-26細胞建立結(jié)腸癌皮下動物模型，用 于觀察免疫后各組小鼠的中位生存期； 第二大組內(nèi)小鼠分組、 免疫方法以及腫瘤 接種同第一大組， 用于觀察口服基因工程疫苗抗小鼠皮下腫瘤的實驗研究。 采用 ELISA 法檢測各組小鼠免疫前、 免疫后以及接種腫瘤以后小鼠血清中血管內(nèi)皮生 長因子（VEGF）的水平，采用流式細胞儀觀察各組小鼠淋巴細胞亞群。觀察免 疫后各組小鼠生存時間， 測量免疫后各組荷瘤小鼠皮下腫

3、瘤體積及重量， 檢測腫 瘤組織內(nèi)微血管密度，免疫組化法分析免疫后各組小鼠腫瘤組織內(nèi)的CD4+以及CD8+T 細胞水平。結(jié)果： 成功制 備以減毒 沙門氏 菌株 SL326i 為載體的口 服基因工 程疫苗 SL3261-pcDNA3.1+/VEGFR2（ni-4）。口服基因疫苗免疫后小鼠血清中血管內(nèi)皮生 長因子（VEGF）水平較對照組明顯降低，免疫后疫苗組血清 VEGF水平出現(xiàn)下 降，在接種腫瘤 2 周后，疫苗組 VEGF 水平較兩對照組明顯下降。免疫后荷瘤 小鼠CD4+T、CD8+T值維持較高水平，小鼠結(jié)腸腺癌皮下腫瘤生長明顯受抑制 且免疫組小鼠生存時間較兩對照組明顯延長， 實驗組小鼠腫瘤體積、

4、 腫瘤重量以 及皮下腫瘤組織平均微血管密度較兩對照組低。結(jié)論：血管內(nèi)皮生長因子受體2胞外1-4片段（VEGFR2（ni-4）能夠發(fā)揮抗小鼠結(jié) 腸癌皮下腫瘤生長的作用。關(guān)鍵詞： 血管內(nèi)皮細胞生長因子受體 2；胞外片段；減毒沙門氏菌；口服；基因疫苗；抗血管生成；結(jié)腸癌Recombinant attenuated salmonella typhimurium vaccineSL3261-pcDNA3.1+/VEGFR2 (n1-4) inhibits growth ofcolorectal cancer in BALB/c miceAbstractOBJECTIVE: To investigate

5、the anti-tumor effect of extracellular 1-4 region of VEGFR2. METHODS: Oral DNA vaccine SL3261-pcDNA3.1+/VEGFR2 (n1-4) was developed and BALB/c mice were immunized with gastrogavage of vaccine encoding VEGFR2 (n1-4). BALB/c mice received gastrogavage of SL3261-pcDNA3.1+ or 10%NaHCO3 were used as cont

6、rol. Serum level of VEGF was detected by ELISA. The subset of lympholeukocyte was analysised by Flow cytometer. Mouse model of CT-26 adencarcinoma of colon were treated with gastrogavage of SL3261-pcDNA3.1+/VEGFR2 (n1-4), tumor diameter and weight were measured, microvessel density (MVD) of tumor ti

7、ssue was detected by immunohistochemistry, and the survival time of mice was also investigated.RESULTS: The serum level of VEGF in vaccine group was significantly lower than that of the control groups (P0.05). After inoculation of CT-26 cells, CD4+ T and CD8+T cells were obviously higher in vaccine

8、group than that of the control groups (P0.05). Tumor diameter, weight and MVD were significantly lower in vaccine group than that of the control groups (P0.05). The survival time of vaccine group was longer than that of the control groups P( 0.05).CONCLUSIONS: The extracellular 1-4 loops of VEGFR2 can block the growth of endothelial to achieve the purpose of inhibiting the growth of tumo