段斐斐，殷月蘭，康美琴，談衛(wèi)軍，陶成武，潘志明，黃金林，焦新安

1 LM感染后誘導(dǎo)的免疫應(yīng)答

1.1天然免疫應(yīng)答 LM感染宿主細(xì)胞以后可以導(dǎo)致多種前炎性因子以及炎癥趨化因子的產(chǎn)生，其中最重要的早期細(xì)胞因子有IL-12p70、γ-干擾素(IFN-γ)及白細(xì)胞介素18(IL-18)[4]。這些細(xì)胞因子及級聯(lián)反應(yīng)產(chǎn)生的趨化因子會吸引嗜中性粒細(xì)胞浸潤至感染部位，以此來活化細(xì)胞介導(dǎo)的殺傷作用[5]。白細(xì)胞介素12(IL-12)可刺激NK細(xì)胞和T細(xì)胞產(chǎn)生IFN-γ，而產(chǎn)生的IFN-γ反過來又可以活化巨噬細(xì)胞，同時刺激攜帶細(xì)菌抗原的樹突狀細(xì)胞(DC)聚集至感染部位，從而進一步的誘發(fā)調(diào)節(jié)抗原特異性的細(xì)胞毒性T淋巴細(xì)胞效應(yīng)(CTL)[6]。LM感染宿主后誘導(dǎo)產(chǎn)生的IFN-γ可觸發(fā)攜帶細(xì)菌抗原的DC細(xì)胞正調(diào)節(jié)共刺激分子，并產(chǎn)生功能性的IL-12，而IL-12作為重要的信號分子可以誘導(dǎo)未致敏的輔助性T細(xì)胞分化成輔助性T細(xì)胞，以此來推進細(xì)胞介導(dǎo)的免疫應(yīng)答[7]。

2 減毒LM作為外源抗原表達(dá)載體的優(yōu)點

研究者將弱毒化的LM改造成為可以容納外源性抗原的載體，從而成為可誘導(dǎo)較強細(xì)胞免疫應(yīng)答的預(yù)防性或治療性活載體疫苗。減毒李斯特菌作為疫苗載體具有以下優(yōu)勢：直接感染抗原遞呈細(xì)胞，具有兩類抗原加工和提呈途徑；利用染色體系統(tǒng)可以穩(wěn)定表達(dá)多個基因產(chǎn)物，操作容易；作為革蘭陽性菌，不含有內(nèi)毒素，使用方便[10]。鑒于LM作為外源抗原表達(dá)載體的這些優(yōu)點，近幾年來，LM已成為分子生物學(xué)和免疫學(xué)的研究熱點。LM已被廣泛作為攜帶病毒和腫瘤抗原的載體來激發(fā)細(xì)胞介導(dǎo)的免疫應(yīng)答[11]。以減毒LM為運送載體，在小鼠、兔、貓、雞和猴子等實驗動物模型，已對許多外源抗原進行了研究，并取得較好的結(jié)果。

3 LM作為腫瘤疫苗載體的應(yīng)用

1992年，首次報道應(yīng)用LM運送外源抗原能夠誘導(dǎo)抗原特異性CTL應(yīng)答[12]。Pan 等最早報道應(yīng)用LM運送模式腫瘤抗原，可以保護小鼠抵抗致死性腫瘤細(xì)胞的攻擊，并且能引起肉眼可見的腫瘤消退，證明LM作為載體運送腫瘤抗原方面具有很強的優(yōu)勢[13]。目前，運用LM作為多種病毒或腫瘤相關(guān)抗原的疫苗載體，已如火如荼進行，這些相關(guān)抗原主要包括人乳頭瘤病毒16型E7蛋白(HPV16 E7)、酪氨酸酶相關(guān)蛋白(Trp2)、高分子量黑色素瘤相關(guān)抗原(HMW-MAA)、前列腺特異性抗原(PSA)以及酪氨酸激酶表皮生長因子(HER-2/neu)等[14-16]。

3.2HER-2/neu抗原 Her-2/neu是酪氨酸激酶表皮生長因子家族中的一員，在20%～40%的乳腺癌中過度表達(dá)，并且還可引發(fā)卵巢癌、肺癌、胰腺癌及胃腸道癌癥[24]。Paterson等構(gòu)建了5種表達(dá)不同片段的鼠Her-2/neu和LLO融合蛋白的LM載體疫苗，研究表明這5種重組疫苗株能夠誘導(dǎo)小鼠體內(nèi)抗Her-2/neu的CTL應(yīng)答，并且對NT-2腫瘤顯示出有效的預(yù)防性和治療性效果[16]。研究還報道這些疫苗同時對表達(dá)Her-2/neu的轉(zhuǎn)基因小鼠腫瘤模型有同樣的作用，該轉(zhuǎn)基因小鼠對Her-2/neu有顯著耐受。免疫LM重組疫苗后可延遲Her-2/neu轉(zhuǎn)基因小鼠中乳頭瘤的出現(xiàn)。為了使這些研究成果更好的應(yīng)用于臨床，Seavey等進一步完善疫苗，研發(fā)出表達(dá)Her-2/neu的HLA表位和LLO融合蛋白的嵌合疫苗[25-26]。

3.5PSA抗原 PSA抗原又被稱為前列腺特異抗原，是由前列腺腺泡和導(dǎo)管的上皮細(xì)胞分泌的一種單鏈糖蛋白，由于PSA在惡性的前列腺細(xì)胞中過量表達(dá)，而在正常的前列腺上皮細(xì)胞和其他器官中低表達(dá)，因此已成為前列腺癌的免疫靶抗原[31]。Shahabi等構(gòu)建表達(dá)融合蛋白的重組李斯特菌，并在pCa小鼠模型上鑒定LM-LLO-PSA疫苗株，結(jié)果顯示該疫苗株能誘導(dǎo)腫瘤浸潤性T調(diào)節(jié)型細(xì)胞數(shù)量的下降，導(dǎo)致80%小鼠的腫瘤完全消退。ELISPOT和胞內(nèi)染色試驗表明LM-LLO-PSA能夠誘導(dǎo)小鼠產(chǎn)生分泌IFN-γ細(xì)胞的數(shù)量增加，另外，CTL實驗表明PSA特異性的T細(xì)胞能夠識別和裂解PSA靶細(xì)胞[32]。對于應(yīng)用于臨床的疫苗而言，安全性是一個重要問題。Wallecha等對減毒重組疫苗的安全性進行了有意義的探索，他們應(yīng)用減毒李斯特菌(LM△dal/dat/actA)運送PSA抗原，該菌必須在添加丙氨酸的條件下才能生存。研究表明雖然減毒重組菌在體內(nèi)的存活率降低，但是仍然能有效誘導(dǎo)清除腫瘤所必須的細(xì)胞免疫應(yīng)答。實驗結(jié)果進一步表明，用該減毒疫苗免疫小鼠后，可誘導(dǎo)產(chǎn)生大量的PSA特異性浸潤T細(xì)胞，同時研究顯示該疫苗還可引起表達(dá)PSA抗原的腫瘤消退，表明該減毒重組疫苗是非常有效和安全的前列腺癌免疫治療候選疫苗，并且LM△dal/dat/actA是非常有潛力的疫苗載體，可以運送其它單抗原或融合抗原用于癌癥免疫治療[33]。

4 展 望

LM獨特的胞內(nèi)生活史及其誘導(dǎo)強烈的天然免疫應(yīng)答和特異性免疫應(yīng)答，使得它具有活載體疫苗的眾多優(yōu)勢，運用LM作為多種腫瘤疫苗載體的研究也在進行，并且取得了一定的預(yù)防性和治療性作用。但是也存在需進一步克服的問題，例如疫苗的抗性、外源抗原的表達(dá)水平等，此外在應(yīng)用LM做載體時如何將其毒力降至最低，這也是研究工作者主要考慮的問題。我們相信隨著研究的不斷深入，LM作為疫苗載體將具有更廣闊的應(yīng)用前景。

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