郭路生　張麗娜　楊寧江

樹突狀細(xì)胞疫苗的免疫學(xué)效應(yīng)實(shí)驗(yàn)研究

郭路生1張麗娜2楊寧江1

【摘要】目的 探討樹突狀細(xì)胞疫苗的免疫學(xué)效應(yīng)和抑瘤機(jī)制。方法 制備負(fù)載MB 49細(xì)胞粗提全抗原的DC疫苗；建立小鼠MB 49膀胱癌皮下移植瘤模型。造模后第7 d、14 d，疫苗組小鼠于右前腋皮下注射疫苗，PBS對(duì)照組相應(yīng)注射PBS，正常組正常飼養(yǎng)。造模第21 d，流式細(xì)胞術(shù)檢測(cè)小鼠脾CD 4+、CD 4+CD 69+T細(xì)胞數(shù)；取分離后的T細(xì)胞培養(yǎng)48 h，取上清，ELISA法檢測(cè)IFN-γ水平。應(yīng)用免疫組化方法檢測(cè)瘤組織內(nèi)CD 4+、CD 8+細(xì)胞數(shù)。結(jié)果 小鼠脾CD 4+、CD 4+CD 69+細(xì)胞數(shù)，DC疫苗組高于PBS對(duì)照組和正常組，P＜0.01，差異具有統(tǒng)計(jì)學(xué)意義。（其中1組P＜0.05）。T細(xì)胞培養(yǎng)上清IFN-γ水平，DC疫苗組高于PBS對(duì)照組和正常組，P＜0.01，差異具有統(tǒng)計(jì)學(xué)意義。瘤組織內(nèi)CD 4+、CD 8+T細(xì)胞數(shù)，DC疫苗組高于PBS對(duì)照組，P＜0.01，差異具有統(tǒng)計(jì)學(xué)意義。結(jié)論 DC 2.4疫苗刺激T細(xì)胞活化和增殖，使脾內(nèi)CD 4+、CD 4+CD 69+細(xì)胞數(shù)增加，IFN-γ分泌增多，同時(shí)趨化遷移至瘤組織的CD 4+、CD 8+T細(xì)胞數(shù)增多，使模型小鼠自身殺傷清除腫瘤細(xì)胞能力增強(qiáng)，從而抑制了模型小鼠腫瘤的生長(zhǎng)。

【關(guān)鍵詞】樹突狀細(xì)胞疫苗；流式細(xì)胞術(shù)；ELISA；

作者單位：1 132013吉林，吉林醫(yī)藥學(xué)院附屬醫(yī)院；2 132013吉林，吉林醫(yī)藥學(xué)院護(hù)理學(xué)院

基金資助：吉林市科技計(jì)劃項(xiàng)目（201133103）

Experimental Study of Immunology Domino Effect on Dendritic Cell Vaccine

GUO Lusheng1ZHANG Li'na2YANG Ningjiang11 the Affiliated Hospital of Jilin Medical College, Jilin 132013, China. 2 Jilin Medicine College of Nursing, Jilin 132013, China

[Abstract]Objective To explore the immunological effects and mechanisms of dendritic cell vaccine. Methods DC vaccine against MB 49 cells were prepared by loading MB 49 cells, and the model was established in mice. After making the model, the 7 days, 14 days, the vaccine were injected in right anterior axillary of vaccine group mice, and the mice of PBS control group were injected with PBS, normal feeding to normal group.21 days after modeling, flow cytometry was used to detect the number of CD 4+and CD 4+CD 69+T cells in spleen of mice. The T cells were cultured for 48 hours, and IFN-γ was detected by ELISA. The number of CD 4+and CD 8+cells in tumor tissues was detected by immunohistochemical method. Results Mouse spleen, CD 4+andCD 4+CD 69+cells count, DC vaccine group was higher than the PBS control group and normal group, P<0.01, had difference statistically significance.(one of them P<0.05). The level of IFN-γof T cell culture supernatant , DC vaccine group was higher than that of PBS control group and the normal group, P<0.01, had difference statistically significance. The number of CD 4+, CD 8+T cells in tumor tissue, DC vaccine group was higher than that of PBS control group, P<0.01, had difference statistically significance. Conclusion DC vaccine stimulated T cell activation and proliferation, increased the number of CD 4+, CD 4+CD 69+cells in the spleen,increased IFN-γsecretion, and increased the number of CD 4+, CD 8+T cells in the tumor tissues. DC 2.4 vaccine can inhibit the growth of tumor cells in mice by killing the tumor cells.

[Key words]Dendritic cell vaccine, Flow cytometry, ELISA, Immunohistochemical staining

樹突狀細(xì)胞（DC）具有超強(qiáng)的抗原提呈能力，且能夠誘導(dǎo)初始T細(xì)胞活化，在免疫應(yīng)答的誘導(dǎo)中具有重要作用[1]。近年來，DC疫苗在實(shí)驗(yàn)室中廣泛應(yīng)用于治療腫瘤的研究[2-3]。探討DC 2.4細(xì)胞疫苗在MB 49小鼠膀胱癌皮下移植瘤模型中的免疫學(xué)效應(yīng)。

1　材料

（1）DC 2.4、MB 49細(xì)胞，受贈(zèng)于遼寧醫(yī)學(xué)院張佩教授。（2）C 57 BL/6近交系雌性小鼠36只，8～12周齡，體重18～20 g，雌性，由上海斯萊克實(shí)驗(yàn)動(dòng)物有限公司提供，許可證編號(hào)：SCXK（滬）2012-0005。（3）FITC anti-Mouse CD 4、PE anti-Mouse CD 69購(gòu)自美國(guó)BD公司。Anti-CD 4、Anti-CD 8、Goat Anti-Rabbit IgG/HRP購(gòu)自北京博奧森生物技術(shù)有限公司。

2　方法

2.1小鼠膀胱癌MB 49細(xì)胞皮下移植瘤模型的建立

將36只小鼠按隨機(jī)數(shù)字表法分為3組，疫苗組、PBS對(duì)照組和正常對(duì)照組。用PBS調(diào)整對(duì)數(shù)生長(zhǎng)期MB 49細(xì)胞濃度至5×106/ ml，注射于各組小鼠右前腋皮下，每只0.2 ml，建立MB 49細(xì)胞皮下移植瘤模型[4]。

2.2疫苗制備與接種

凍融法制備MB 49粗提全抗原，按原細(xì)胞的比例10：1負(fù)載DC 2.4，制備DC 2.4疫苗。調(diào)整疫苗細(xì)胞濃度至1×106/ml，各組小鼠分別于建模第7 d、14 d給予相應(yīng)治療：疫苗組給予疫苗0.1 ml＋PBS 0.1 ml，PBS對(duì)照組給予PBS 0.2 ml，注射于小鼠接種瘤細(xì)胞處皮下。正常組正常飼養(yǎng)。

2.3疫苗免疫學(xué)效應(yīng)檢測(cè)

尼龍毛柱法分離各組小鼠脾T細(xì)胞，RPMI-1 640調(diào)整細(xì)胞濃度至1×107/ml。以FITC-CD 4和PE-CD 69雙標(biāo)；流式細(xì)胞術(shù)檢測(cè)T細(xì)胞CD 4、CD 69表達(dá)。取分離的T淋巴細(xì)胞，PBS調(diào)整細(xì)胞濃度為5×107/ml，24孔板，37℃培養(yǎng)箱培養(yǎng)48 h，取上清，ELISA法檢測(cè)IFN-γ含量。石蠟包埋瘤組織切片，Envision兩步法免疫組化染色。免疫組化圖像分析系統(tǒng)分析，計(jì)數(shù)CD 4+和CD 8+細(xì)胞數(shù)。

2.4統(tǒng)計(jì)學(xué)方法

應(yīng)用SPSS13.0軟件進(jìn)行數(shù)據(jù)處理和統(tǒng)計(jì)學(xué)分析，計(jì)量資料采用t檢驗(yàn)，計(jì)數(shù)資料采用χ2檢驗(yàn)，P＜0.05，差異具有統(tǒng)計(jì)學(xué)意義。

3　實(shí)驗(yàn)結(jié)果

3.1流式細(xì)胞術(shù)檢測(cè)比較各組小鼠脾CD 4+、CD 4+CD 69+T細(xì)胞數(shù)

流式檢測(cè)小鼠脾CD 4+、CD 4+CD 69+T細(xì)胞數(shù)，疫苗組均高于PBS對(duì)照組和正常組，P＜0.01，差異具有統(tǒng)計(jì)學(xué)意義，（其中1 組P＜0.05），見表1。各組取1張流式圖進(jìn)行比較，見圖1。3.2ELISA法檢測(cè)比較各組小鼠脾T細(xì)胞培養(yǎng)上清IFN-γ水平

脾T細(xì)胞培養(yǎng)上清IFN-γ水平，疫苗組高于PBS對(duì)照組和正常組，P＜0.01，差異具有統(tǒng)計(jì)學(xué)意義。見表2。

3.3免疫組化檢測(cè)比較各組小鼠瘤組織CD 4+、CD 8+細(xì)胞數(shù)

免疫組化檢測(cè)小鼠瘤組織CD 4+、CD 8+細(xì)胞數(shù)，疫苗組CD 4+和CD 8+T細(xì)胞數(shù)均高于PBS對(duì)照組，P＜0.01，差異具有統(tǒng)計(jì)學(xué)意義。見表3。

4　討論

CD 69是一個(gè)早期淋巴細(xì)胞活化的標(biāo)志，近年的研究表明[5]，CD 69抗原的表達(dá)在T細(xì)胞活化的過程中起重要作用。DC 2.4疫苗刺激T細(xì)胞活化和增殖，使脾內(nèi)CD 4+、CD 4+CD 69+細(xì)胞數(shù)增加。DC 2.4能通過分泌細(xì)胞因子和趨化因子趨化T細(xì)胞，通過血管內(nèi)皮屏障而增加腫瘤部位的效應(yīng)T細(xì)胞數(shù)量[6]。CD 4+T細(xì)胞在IL-12等細(xì)胞因子促進(jìn)下可極化為Th 1細(xì)胞，Th 1細(xì)胞產(chǎn)生IL-2、IFN-γ等細(xì)胞因子，促進(jìn)Th 1細(xì)胞、CTL的增殖，從而放大免疫效應(yīng)。CD 8+CTL通過溶細(xì)胞作用直接殺傷腫瘤細(xì)胞和通過分泌IFN-γ、TNF間接殺傷腫瘤細(xì)胞。

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·實(shí)驗(yàn)研究·

doi：10.3969/j.issn.1674-9308.2015.26.012

【文章編號(hào)】1674-9308（2015）26-0018-02

【文獻(xiàn)標(biāo)識(shí)碼】B

中圖分類號(hào)】【免疫組織化學(xué)染色R392