生物工程学报  2018, Vol. 34 Issue (3): 396-406
http://dx.doi.org/10.13345/j.cjb.170325
中国科学院微生物研究所、中国微生物学会主办
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文章信息

范修德, 李娜, 王西强, 孙文刚, 李倩, 李汉超, 王小云, 贾皑
Fan Xiude, Li Na, Wang Xiqiang, Sun Wengang, Li Qian, Li Hanchao, Wang Xiaoyun, Jia Ai
PDGF-B免疫原的制备及其腹水抗体对HepG2细胞增殖的影响
PDGF-B immunogen preparation and the suppressive effect of anti-PDGF-B ascite antibody on the proliferation of hepG2 cells
生物工程学报, 2018, 34(3): 396-406
Chinese Journal of Biotechnology, 2018, 34(3): 396-406
10.13345/j.cjb.170325

文章历史

Received: August 17, 2017
Accepted: November 20, 2017
PDGF-B免疫原的制备及其腹水抗体对HepG2细胞增殖的影响
范修德1*, 李娜2*, 王西强3, 孙文刚4, 李倩4, 李汉超4, 王小云5, 贾皑5     
1 西安交通大学第一附属医院感染性疾病科,陕西 西安 710061;
2 西安交通大学第一附属医院康复医学科,陕西 西安 710061;
3 西安交通大学第一附属医院心血管内科,陕西 西安 710061;
4 西安交通大学第一附属医院风湿免疫科,陕西 西安 710061;
5 西安交通大学第一附属医院消化内科,陕西 西安 710061
收稿日期:2017-08-17; 接收日期:2017-11-20; 网络出版时间:2017-12-07
基金项目:陕西省社会发展攻关项目(No. S2013SF3884)资助
摘要:观察利用融合表达载体pET28-Trx合成的人血小板源性生长因子B (Human platelet-derived growth factor B,hPDGF-B)免疫原的免疫原性,及其诱导小鼠产生针对hPDGF-B的腹水抗体对人HepG2细胞增殖的抑制作用。本研究首先选择hPDGF-B的第103-118及152-167氨基酸序列作为抗原表位,构建pET28-Trx-重组原核表达载体,表达并纯化获得6×his Trx-hPDGF-BΔ103-118及6×his Trx -hPDGF-BΔ152-167重组蛋白;然后使用纯化后的重组蛋白免疫小鼠,并给予腹腔注射H22肿瘤细胞制备腹水抗体,ELISA法检测抗体滴度,Western blotting法检测纯化腹水抗体与膜结合的PDGF-B结合能力;最后通过CCK8实验检测外源性PDGF-BB和两种纯化腹水抗体对肝癌HepG2细胞增殖的作用。研究发现上述两种重组蛋白作为免疫原均可诱导小鼠产生高滴度的PDGF-B中和性抗体;两种hPDGF-B纯化腹水抗体能明显抑制PDGF-BB对肝癌HepG2细胞增殖的促进作用。结果提示,Trx-PDGF-B重组蛋白作为免疫原有望为PDGF-B疫苗的制备提供新的方法,也为临床上肝癌的治疗提供了一种新的思路。
关键词肝细胞癌     PDGF-B     多克隆抗体     细胞因子疫苗    
PDGF-B immunogen preparation and the suppressive effect of anti-PDGF-B ascite antibody on the proliferation of hepG2 cells
Xiude Fan1*, Na Li2*, Xiqiang Wang3, Wengang Sun4, Qian Li4, Hanchao Li4, Xiaoyun Wang5, Ai Jia5     
1 Department of Infectious Diseases, the First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, Shaanxi, China;
2 Department of Rehabilitation, the First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, Shaanxi, China;
3 Department of Cardiology, the First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, Shaanxi, China;
4 Department of Rheumatism, the First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, Shaanxi, China;
5 Department of Gastroenterology, the First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, Shaanxi, China
Abstract: To observe the immunogenicity of hPDGF-B immunogens that were synthesized with the fusional expression vector pET28-Trx and to test the suppressive effect of these specific antibodies induced by both of immunogens on proliferation of human HepG2 hepatoma cells. First, we chose 2 antigenic epitopes hPDGF-BΔ103-118aa and hPDGF-BΔ152-167aa from human PDGF-B and inserted these 2 coding regions into the empty vector plasmid pET28-Trx, separately. Second, mice were immunized with purified recombinant proteins to generate polyclonal antibody. Then we intraperitoneally injected mice bearing hepatoma 22 (H22) tumor cells to prepare antibody ascites. ELISA and Western blot were used to detect the titer and the utility of the antibody, respectively. Finally, HepG2 cells were exposed to PDGF-BB protein or anti-PDGF-B ascite antibody in different dilution concentrations groups and the proliferation of HepG2 cells was quantified by CCK8 assay. As the results, we identified mice that could produce high drop of neutralizing antibodies against hPDGF-B induced by both two recombinant proteins. Two anti-PDGF-B ascite antibodies could markedly inhibit the proliferation of HepG2 cells by blocking the stimulating effect of PDGF-BB protein. Our results suggest that Trx-PDGF-B recombinant protein as immunogen provides a new method for the preparation of PDGF-B vaccine, and also a new idea for the treatment of hepatocellular carcinoma in clinical practice.
Key words: hepatocellular carcinoma (HCC)     PDGF-B     polyclonal antibody     cytokine vaccine    

肝细胞癌(Hepatocellular carcinoma,HCC)是最常见的原发性肝脏恶性肿瘤,有较高的发病率和死亡率,由于起病隐匿、病情发展迅速、较高的手术复发率和转移率等特点,临床治疗效果并不理想[1-2]。但是随着肿瘤发生发展过程中相关的分子和细胞机制逐渐被认识,生长因子及其受体在肿瘤发生发展中的作用已引起人们的极大关注,阻断生长因子与其受体的相互作用可能在肿瘤的治疗方面取得一定疗效[3-4]

血小板衍生生长因子(Platelet-derived growth factor PDGF)是30多年前从人的血小板中分离出来的肽类生长因子,主要包括4种不同的多肽链:PDGF-A、B、C、D[5]。四种PDGF多肽链通过二硫键的连接形成同型或异型二聚体,与血小板衍生生长因子受体(Platelet-derived growth factor receptor PDGFR)结合发挥相应的生物学效应[6]。Doolittle等发现PDGF-B链基因与猴肉瘤病毒的癌基因v-sis有92%的同源性,PDGF-B与p28V-sis结构的相似性表明,PDGF-B可能通过与膜上的受体结合,激活相应的细胞内信号转导通路,导致细胞发生癌变[7]。在5种不同的二聚体亚型PDGF-AA、PDGFAB、PDGF-BB、PDGF-CC和PDGF-DD中,PDGF-BB是肝间质细胞分泌的最主要的二聚体结构,是唯一能与3种PDGF受体PDGFR-αα、PDGFR-αβ及PDGFR-ββ均可结合的分子,也是目前与配体特异性结合位点研究最为清楚的分子[8]。正常肝脏仅表达少量的PDGF-B,但是在肝纤维化发生时PDGF-B的表达水平明显提高,研究发现PDGF-B转基因小鼠在6个月内可以自发性地发生肝纤维化,而且PDGF-B的转基因小鼠在二乙基亚硝胺(Diethylnitrosamine,DEN)诱导下,较非转基因小鼠更早发生HCC,而且肿瘤进展更快[9]。这些研究均提示PDGF-B与肝癌的发生发展密切相关,阻断PDGF-B可能在HCC的治疗方面取得一定疗效。

因此本课题组将hPDGF-B分子中与其生物学活性密切相关的关键结构域插入到融合表达载体pET28-Trx中,构建符合读框的融合基因,将表达并纯化的重组蛋白作为免疫原,主动免疫小鼠,诱导小鼠产生针对hPDGF-B的特异性抗体,观察其对人HepG2细胞增殖的抑制作用,为PDGF-B疫苗的构建及HCC治疗探索新的可行途径。

1 材料与方法 1.1 菌种、质粒、细胞及实验动物

E. coli BL21(DE3)、pGEM-T载体、pET-28a(+)质粒、pET-28a-Trx质粒、人肝癌细胞系HepG2均为本实验室保存;雄性昆明小鼠由西安交通大学医学院动物中心提供。

1.2 主要试剂

限制性内切酶(PstⅠ、BamHⅠ、XhoⅠ和NdeⅠ)、T4 DNA连接酶、PCR引物、质粒DNA试剂盒、胶回收试剂盒均购自TaKaRa宝生物工程(大连)有限公司;弗氏完全佐剂、弗氏不完全佐剂、DMSO、CCK8、胰蛋白酶购自Sigma公司;人重组细胞因子PDGF-BB购自R & D公司;pET28-PDGF-B重组蛋白由本实验室前期构建并表达;His-tag抗体购自Abcam公司;HRP辣根过氧化物酶标记山羊抗小鼠IgG购自EarthOx公司。

1.3 引物设计

利用Primer5软件设计hPDGF-BΔ103-118和hPDGF-BΔ152-167编码序列引物,引物序列见表 1。引物由TaKaRa公司合成。

表 1 hPDGF-BΔ103-118和hPDGF-BΔ152-167编码序列的引物核苷酸序列表 Table 1 Primers used for expanding hPDGF-BΔ103-118 and hPDGF-BΔ152-167
Primer name Primer sequence (5′-3′) Size (bp)
hPDGF-BΔ103-118 F CCCTCGAGGTGTTCGAGATCTCCCGGC 65
hPDGF-BΔ103-118 R CCCGGATCCGAAGTTGGCGTTGGTGCGG 65
hPDGF-BΔ152-167 F CCCTCGAGCAGGTGAGGTGAGAAAGATT 65
hPDGF-BΔ152-167 R CCCGGATCCCTTCTTAAAGATTGGCTTCTTC 65
1.4 统计学分析

应用统计软件SPSS 19.0进行统计分析,P < 0.05表示有统计学意义。所有计量资料数据均采用均数±标准差(x±s)表示;组间两两比较采用LSD-t检验法;采用单因素方差分析(One Way ANOVA)对多组样本均数进行比较。

1.5 hPDGF-B免疫原的制备 1.5.1 hPDGF-B抗原表位的预测

通过阅读文献和利用B细胞表位预测软件(http://ailab.ist.psu.edu/bcpred/predict.htmL在线预测),针对hPDGF-B与PDGFR作用的位点,设计了两段16个氨基酸的短肽,即VFEISRRLIDRTNANF (16AA,103位到118位)和QVRKIEIVRKKPIFKK (16AA,152位到167位),标记为hPDGF-BΔ103-118和hPDGF-BΔ152-167。

1.5.2 hPDGF-BΔ103-118和hPDGF-BΔ152-167编码序列的克隆

采用PCR技术以正常人的基因组cDNA为模板分别扩增hPDGF-BΔ103-118和hPDGF-BΔ152-167编码序列,DNA引物序列见表 1。其中hPDGF-BΔ103-118 1F和hPDGF-BΔ152-167 1F的5′端分别引入PstⅠ酶切位点,hPDGF-BΔ103-118 1R和hPDGF-BΔ152-167 1R的3′端分别引入BamHⅠ酶切位点。PCR扩增产物长度均为65 bp。

1.5.3 hPDGF-BΔ103-118和hPDGF-BΔ152-167原核表达载体的构建

上述扩增产物即特异hPDGF-BΔ103-118和hPDGF-BΔ152-167基因片段经PCR产物回收试剂盒纯化,与pGEM-T载体连接,经克隆筛选、PCR扩增及基因测序鉴定(生工生物工程(上海)股份有限公司)。挑选阳性克隆,pGEM-T/hPDGF-BΔ103-118和pGEM-T/hPDGF-BΔ152-167以PstⅠ和BamHⅠ双酶切,切胶纯化回收后,与同样处理的pET28-Trx表达载体在T4 DNA连接酶的作用下,转化E. coli DH5α扩增,NdeⅠ+XhoⅠ双酶切消化4 h,酶切产物进行琼脂糖凝胶电泳和基因测序,检测两种重组质粒的正确性。

1.5.4 重组蛋白的原核表达及纯化

挑取pET28-Trx-hPDGF-BΔ103-118和pET28-Trx-hPDGF-BΔ152-167阳性E. coli BL21(DE3)感受态细菌克隆,IPTG诱导表达目的蛋白,SDS-PAGE鉴定,观察有无目的条带;大量诱导表达的pET28-Trx-hPDGF-BΔ103-118重组蛋白和pET28-Trx-hPDGF-BΔ152-167重组蛋白分别通过Ni-NTA纯化,纯化后蛋白分别简写为6×his Trx-hPDGF-BΔ103-118重组蛋白和6×his Trx-hPDGF-BΔ152-167重组蛋白。

1.6 hPDGF-B抗体的制备及纯化 1.6.1 动物免疫

将4周龄的昆明种雄性小鼠15只,随机分成6×his Trx-hPDGF-BΔ103-118重组蛋白免疫组、6×his Trx-hPDGF-BΔ152-167重组蛋白免疫组及对照组各5只;将透析好的两种hPDGF-B重组蛋白分别与弗氏完全佐剂按1︰1比例混合,充分混匀至完全乳化,腹腔注射相应蛋白混合物0.2 mL,对照组注射PBS溶液0.2 mL;2周后,将两种hPDGF-B重组蛋白分别与弗氏不完全佐剂按1︰1比例混合,完全乳化后每只小鼠注射0.2 mL,对照组注射PBS溶液0.2 mL,每2周免疫一次;以后每隔1周通过断尾静脉采血的方法,以ELISA法测定hPDGF-B抗体滴度。

1.6.2 ELISA方法检测小鼠血清抗体效价

原核表达的PDGF-B重组蛋白定量后按20 ng/孔包被96孔酶标板,封闭后将上述鼠尾血清用10%的牛血清/PBST稀释液按一定的比例稀释后,第一孔稀释比为1︰200,依次倍比稀释,最后一孔为阴性对照,以HRP辣根过氧化物酶标记的山羊抗小鼠IgG二抗(1︰5 000)进行检测,孵育后各孔依次加入底物A液和显色B液各1滴,5 min向各反应孔中加入终止C液1滴,观察各孔颜色并记录抗体滴度。

1.6.3 hPDGF-B多克隆抗体腹水的制备和纯化

注射小鼠H22肝癌细胞前,6×his Trx-hPDGF-BΔ103-118重组蛋白免疫组及6×his Trx-hPDGF-BΔ152-167重组蛋白免疫组的抗体滴度需达1︰8 000以上;用生理盐水稀释H22细胞浓度为1×107个/mL;将H22细胞悬液按0.5 mL/只的剂量注入免疫过的小鼠腹腔;注入H22细胞后7 d左右,小鼠体重逐渐停止增加,腹腔明显膨隆,行动迟缓,皮毛无光泽,对照组小鼠精神及一般状态均正常。取小鼠眼血后将小鼠处死抽取腹水,获得PDGF-B多抗血清及多抗腹水,间接ELISA检测小鼠抗体滴度,而后用双层滤纸过滤腹水;4 ℃、12 000 r/min离心15 min,收集上清,精确定量腹水体积;采用辛酸硫酸铵法纯化腹水抗体,取少量纯化后的抗体适当稀释后,ELISA方法检测纯化腹水抗体效价(方法同前)。

1.6.4 Western blotting法验证纯化腹水抗体与PDGF-B的结合能力

收集的纯化腹水经过ELISA检测含有高滴度的抗体,还需验证其与PDGF-B的结合能力,上样蛋白选择pET28-PDGF-B重组蛋白,通过制样,电泳,转膜,用10 %的脱脂奶粉封闭2 h,分别加入一抗Anti-hPDGF-BΔ103-118纯化腹水抗体(1︰500),Anti-hPDGF-BΔ152-167纯化腹水抗体(1︰500)及His-tag抗体(1︰1 000),4 ℃孵育过夜,分别加入二抗山羊抗小鼠IgG (1︰10 000)于37 ℃孵育1 h,ECL化学发光试剂盒显色,采用超灵敏多功能成像仪(美国GE Amersham Imager 600;型号AI600)采集蛋白条带图像。

1.7 hPDGF-B抗体对肝癌HepG2细胞增殖的抑制作用 1.7.1 PDGF-BB对人肝癌HepG2细胞增殖的影响

取对数生长期HepG2细胞,以每孔1×103个细胞接种于96孔板,每孔加150 μL,周围孔则加入PBS,置于37 ℃、含5% CO2、饱和湿度的细胞培养箱中培养;实验分组:①无血清DMEM组;②1 ng/mL PDGF-BB组;③2 ng/mL PDGF-BB组;④4 ng/mL PDGF-BB组;⑤8 ng/mL PDGF-BB组;⑥16 ng/mL PDGF-BB组,每组设5个复孔;细胞接种24 h后,按照分组分别加入不同培养基,继续培养48 h;48 h后每孔加入CCK-8溶液10 μL,继续培养1-2 h;在酶标仪上测量各孔吸光度值,测定波长450 nm,重复CCK-8实验3次,记录结果并绘制细胞增殖曲线。

1.7.2 hPDGF-B抗体对肝癌HepG2细胞增殖的抑制作用

结合上一实验结果,PDGF-BB浓度在4 ng/mL对HepG2细胞促增殖能力最强,故PDGF-BB浓度定为4 ng/mL;取对数生长期HepG2细胞,每孔1×103个细胞接种于96孔板中,每孔加150 μL,周围孔则加入PBS,置于37 ℃、含5% CO2、饱和湿度的细胞培养箱中培养;实验分组:①无血清DMEM组;②4 ng/mL PDGF-BB组:培养液中加入hPDGF-BB 4 ng/mL;③Anti-hPDGF-BΔ103-118纯化腹水抗体组(1︰10始,4倍系列稀释);④PDGF-BB+Anti-hPDGF-BΔ103-118纯化腹水抗体组(培养液中同时加入PDGF-BB 4 ng/mL和3种不同稀释度(1︰10始,4倍系列稀释)的小鼠PDGF-B纯化腹水抗体);⑤Anti-hPDGF-BΔ152-167纯化腹水抗体组(1︰10始,4倍系列稀释);⑥PDGF-BB+Anti-hPDGF-BΔ152-167纯化腹水抗体组(培养液中同时加入PDGF-BB 4 ng/mL和3种不同稀释度(1︰10始,4倍系列稀释)的小鼠PDGF-B纯化腹水抗体);⑦3种不同稀释度(1︰10始,4倍系列稀释) Trx抗体组,每组设5个复孔;细胞接种24 h后,按照分组分别加入不同培养基,继续培养48 h;48 h后每孔加入CCK-8溶液10 μL,继续培养1-2 h;在酶标仪上测量各孔吸光度值,测定波长450 nm,重复CCK-8实验3次,记录结果并绘制细胞增殖曲线。

2 结果与分析 2.1 pET28-Trx-hPDGF-BΔ103-118和pET28-Trx-hPDGF-BΔ152-167原核表达载体的构建

采用PCR技术以正常人的基因组cDNA为模板分别克隆出65 bp的hPDGF-BΔ103-118和hPDGF-BΔ152-167编码序列(图 1A),重组质粒pET28-Trx-hPDGF-BΔ103-118和pET28-Trx-hPDGF-BΔ152-167经NheⅠ和XhoⅠ双酶切后,消化产物经琼脂糖电泳可见394 bp酶切片段(图 1B);基因测序结果正确。

图 1 目的基因的扩增及重组质粒的PCR和酶切鉴定 Figure 1 Amplification products of target genes and identification of the recombinant plasmids by PCR and restriction enzyme analysis. (A) Amplification products of hPDGF-BΔ103-118 and hPDGF-BΔ152-167 gene with PCR. M: 50 bp Ladder DNA marker; 1, 2: PDGF-BΔ103-118 and hPDGF-BΔ152-167 amplified fragments. (B) Identification of the recombinant plasmids pET28-Trx-hPDGF-BΔ103-118 and pET28-Trx-hPDGF-BΔ152-167 by PCR and restriction enzyme analysis. M: DNA ladder mix; 1-3: pET28-Trx, pET28-Trx-hPDGF-BΔ103-118, pET28-Trx-hPDGF-BΔ152-167.
2.2 6×his Trx-hPDGF-BΔ103-118及6×his Trx-hPDGF-BΔ152-167重组蛋白的原核表达和纯化

重组质粒pET28-Trx-hPDGF-BΔ103-118和pET28-Trx-hPDGF-BΔ152-167以及对照空质粒pET28转化入BL21(DE3)感受态细菌,次日LB半固体平板生长菌落数较多,挑取单克隆菌落在卡那霉素抗性LB液体培养基中大量扩增后加入IPTG诱导表达,12% SDS-PAGE可见约16.3 kDa的6×his Trx-hPDGF-BΔ103-118重组蛋白和16.4 kDa的6×his Trx-hPDGF-BΔ152-167重组蛋白表达条带;BL21(DE3)-pET28-Trx-hPDGF-BΔ103-118和BL21(DE3)-pET28-Trx-hPDGF-BΔ152-167表达蛋白经Ni-NTA纯化后可见相同大小的重组蛋白纯化条带(图 2)。

图 2 SDS-PAGE分析纯化前后的6×his Trx-hPDGF-BΔ103-118及6×his Trx-hPDGF-BΔ152-167重组蛋白 Figure 2 SDS-PAGE analysis of expressed and purified 6×his Trx-hPDGF-BΔ103-118 and 6×his Trx-hPDGF-BΔ152-167 proteins. M: unstained protein marker; 1: pET28-E. coli BL21(DE3); 2: pET28-Trx-PDGF103-118-E. coli BL21(DE3); 3: pET28-Trx-PDGF103-118 (purified by Ni-NTA); 4: pET28-Trx-PDGF152-167-E. coli BL21(DE3); 5: pET28-Trx-PDGF152-167 (purified by Ni-NTA).
2.3 ELISA法测定血清hPDGF-B抗体滴度结果

分别用6×his Trx-hPDGF-BΔ103-118重组蛋白和6×his Trx-hPDGF-BΔ152-167重组蛋白免疫小鼠,这两种重组蛋白免疫小鼠后抗体滴度很快就达到1︰8 000,而且组间差异较小(表 2表 3)。

表 2 6×his Trx-hPDGF-BΔ103-118重组蛋白免疫小鼠血清抗体滴度(pET28-PDGF-B重组蛋白包板) Table 2 pET28-PDGF-B recombinant protein detected antibody titers in 6×his Trx-hPDGF-BΔ103-118 mice antisera
Groups Number First immunization Second immunity Third immunization
6×his Trx-hPDGF-BΔ103-118 recombinant protein group 1 1:800 1:3 200 1:12 800
2 1:800 1:3 200 1:12 800
3 1:800 1:6 400 1:25 600
4 1:400 1:3 200 1:12 800
5 1:200 1:3 200 1:6 400
表 3 6×his Trx-hPDGF-BΔ152-167重组蛋白免疫小鼠血清抗体滴度(pET28-PDGF-B重组蛋白包板) Table 3 pET28-PDGF-B recombinant protein detected antibody titers in 6×his Trx-hPDGF-BΔ152-167 mice antisera
Groups Number First immunization Second immunity Third immunization
6×his Trx-hPDGF-BΔ152-167 recombinant protein group 1 1:200 1:6 400 1:25 600
2 1:800 1:1 600 1:6 400
3 1:200 1:6 400 1:25 600
4 1:400 1:3 200 1:12 800
5 1:400 1:1 600 1:25 600
2.4 成功制备并纯化hPDGF-B腹水抗体 2.4.1 ELISA法测定hPDGF-B纯化前后腹水抗体滴度结果。

纯化后Anti-hPDGFΔ103-118组和Anti-hPDGFΔ152-167组腹水抗体滴度均达到1︰16 000及以上,具有较为理想的抗体滴度(表 4)。

表 4 纯化前后Anti-hPDGFΔ103-118组和Anti-hPDGFΔ152-167组腹水抗体滴度(pET28-PDGF-B重组蛋白包板) Table 4 pET28-PDGF-B recombinant protein detected ascite antibody titers of Anti-hPDGFΔ103-118 and Anti-hPDGFΔ152-167 groups before and after purification
Groups Anti-hPDGFΔ103-118 antibody group Anti-hPDGFΔ152-167 antibody group
Non purified group 1:8 000 1:8 000
Purified group 1:16 000 1:32 000
2.4.2 Western blotting验证hPDGF-B纯化腹水抗体结果

Western blotting法检测发现,两种hPDGF-B纯化腹水抗体(1︰500)均能和pET28-PDGF-B重组蛋白发生反应,在33 kDa处可见PDGF-B重组蛋白条带。纯化后的hPDGF-B腹水抗体可以与膜结合PDGF-B结合,可用于下游实验(图 3)。

图 3 Western blotting检测纯化后的hPDGF-B腹水抗体与pET28-PDGF-B重组蛋白的结合反应 Figure 3 Western blotting analysis of purified hPDGF-B ascites antibody responses to pET28-PDGF-B proteins. 1: anti-hPDGFΔ103-118 antibody; 2: anti-hPDGFΔ152-167 antibody; 3: His tag antibody.
2.5 PDGF-BB对肝癌HepG2细胞增殖的促进作用

CCK-8实验结果显示,PDGF-BB对HepG2细胞增殖有促进作用,PDGF-BB各浓度组与对照组相比,细胞体外生长速度明显加快(P < 0.05)。增殖曲线显示PDGF-BB促细胞增殖的作用与生长因子浓度呈峰形状相关,4 ng/mL浓度时作用最强(图 4)。

图 4 PDGF-BB对HepG2细胞增殖的影响 Figure 4 Positive effect of PDGF-BB on HepG2 cells proliferation. Compared with the control group, the growth rate of the HepG2 cells was obviously accelerated in different PDGF-BB concentration groups (P < 0.05).
2.6 hPDGF-B腹水抗体对肝癌HepG2细胞增殖的抑制作用

CCK-8检测结果发现,4 ng/mL PDGF-BB组和对照组相比明显促HepG2细胞增殖,同时两种PDGF-B纯化腹水抗体均能明显抑制PDGF-BB所诱导的细胞增殖,随着抗体浓度的增加,对细胞增殖的抑制作用越明显,组间差异有显著的统计学意义(P < 0.05) (表 5)。

表 5 hPDGF-B抗体对HepG2细胞增殖的影响 Table 5 Inhibitory effect of Anti-hPDGF-B antibodies on HepG2 cell proliferation
Groups Concentration Sample number OD value (A)
Serum-free DMEM 0 5 0.318 3±0.026 0
PDGF-BB 4 ng/mL 5 0.554 9±0.028 6
Anti-hPDGF-BΔ103-118 antibody 1:10 dilution 5 0.200 3±0.014 4Δ
1:40 dilution 5 0.234 3±0.018 4Δ
1:160 dilution 5 0.302 3±0.011 0
PDGF-BB+Anti-hPDGF-BΔ103-118 antibody 1:10 dilution 5 0.300 3±0.019 0ΔΔ
1:40 dilution 5 0.335 1±0.019 8ΔΔ
1:160 dilution 5 0.497 4±0.009 0
Anti-hPDGF-BΔ152-167 antibody 1:10 dilution 5 0.202 3±0.013 7Δ
1:40 dilution 5 0.212 5±0.002 0Δ
1:160 dilution 5 0.304 3±0.006 7
PDGF-BB+Anti-hPDGF-BΔ152-167 antibody 1:10 dilution 5 0.292 3±0.010 1ΔΔ
1:40 dilution 5 0.339 1±0.012 1ΔΔ
1:160 dilution 5 0.466 2±0.014 8
Anti-Trx-antibody 1:10 dilution 5 0.313 1±0.023 7
1:40 dilution 5 0.332 1±0.016 0
1:160 dilution 5 0.326 3±0.025 4
Compared with serum-free medium group, ΔP < 0.05; compared with 4 ng/mL PDGF-BB group, ΔP < 0.05.
图 5 hPDGF-B抗体对HepG2细胞增殖的影响 Figure 5 Inhibitory effect of Anti-hPDGF-B antibodies on HepG2 cell proliferation. Compared with serum-free medium group, the growth rate of the HepG2 cells was obviously inhibited in Anti-hPDGF-BΔ103-118 antibody groups (1:10 dilution and 1:40 dilution) and Anti-hPDGF-BΔ152-167 antibody groups (1:10 dilution and 1:40 dilution; P < 0.05). Compared with 4 ng/mL PDGF-BB group, the growth rate of the HepG2 cells was obviously inhibited in PDGF-BB+Anti-hPDGF-BΔ103-118 antibody groups (1:10 dilution and 1:40 dilution) and PDGF-BB+Anti-hPDGF-BΔ152-167 antibody groups (1:10 dilution and 1:40 dilution; P < 0.05).
3 讨论

肝细胞癌(HCC)是最常见的原发性肝脏恶性肿瘤,由于起病隐匿、病情发展迅速、手术复发率和转移率高等特点,中晚期肝癌的治疗效果并不理想,故现在迫切需要探索新的有效治疗方法。

PDGF作为已有30余年历史的肽类生长因子,最初发现其为一种重要的促有丝分裂因子,主要作用于成纤维细胞、平滑肌细胞、神经胶质细胞等。随着研究的深入,发现PDGF既参与重要的生理活动,如胚胎发育、免疫调节及组织修复等,同时又与多种疾病的发生密切相关,如肝纤维化、心肌过度纤维化、结肠癌、恶性胶质瘤、前列腺癌等[10-11]。PDGF及其受体的过度表达是肿瘤常见的特征之一,在皮肤癌、胃癌、结肠癌、宫颈癌、胰腺癌、前列腺癌、肺癌等多种肿瘤细胞中均可检测到高表达水平的PDGF及其受体[8]。研究发现PDGF与其受体结合后可使受体酪氨酸激酶激活,从而激活细胞内多条信号转导通路,主要通过自分泌的形式促进肿瘤细胞的生长,也可通过旁分泌的形式促进血管和淋巴管的生成,调控细胞外基质的生成和降解[12-14]。目前认为PDGF可能主要通过3个方面促进肿瘤的发生和发展:肿瘤细胞自分泌刺激;刺激血管生成;肿瘤微环境的调控。近年来国内外研究发现PDGF在肝癌的发生、发展及转移过程可能发挥重要作用[15-17],这提示我们PDGF及其受体可能作为肿瘤治疗的靶点。

肝癌的发生和发展与多种细胞因子有关,而且涉及多条信号通路,如何在疗效和毒副作用之间选择最佳的治疗方法是当前研究的热点。目前对于中晚期肝癌临床上尚缺乏安全、有效的治疗方法,虽然多靶点、多激酶抑制剂索拉非尼通过对酪氨酸激酶和丝氨酸/苏氨酸激酶的抑制作用明显延长了晚期肝癌患者的生存期[4],但是由于价格较高,而且在用药过程中存在较多的毒副作用,限制了其在临床上的应用。鉴于PDGF在肿瘤细胞自分泌刺激、促进血管生成及对调控肿瘤微环境的作用,我们认为有必要研究和开发高效、方便、安全、价廉的阻断PDGF的药物或手段。考虑到完全阻断PDGF/PDGFR信号通路技术难度较大,而且可能引起较多的毒副作用,仔细查阅文献发现PDGF-B相较于其他几种亚型结构研究最为清楚,而且在肝癌的发生和发展过程中发挥重要的作用,我们认为选择性地阻断PDGF-B有望达到理想效果。目前阻断PDGF信号转导的方法主要有基因沉默、DNA适配子、PDGF可溶性受体及受体后信号转导通路阻断剂等[18-21],但是由于技术难度大,价格高昂、副作用大等缺点难以在临床推广使用。相较于上述几种阻断PDGF信号转导的方法,细胞因子疫苗通过基因工程改造过的大肠杆菌表达,刺激机体产生特异性PDGF抗体,不会整合到免疫动物基因组,也不会产生强烈的过敏反应;而且国内外动物实验发现细胞因子疫苗不会造成其他正常器官的损伤,因此PDGF-B疫苗有治疗高效、制作方法简单、使用方便、作用持久等无可比拟的优点,在肿瘤治疗中有良好的临床应用前景。

本实验通过PDGF-B重组蛋白主动免疫的方式,刺激机体产生hPDGF-B抗体,观察hPDGF-B抗体对肝癌HepG2细胞增殖的抑制作用,旨在探讨PDGF-B疫苗构建及阻断PDGF/PDGFR信号转导在HCC治疗中的可行性。实验中选用硫氧还蛋白融合蛋白表达体系作为原核表达载体,其在大肠杆菌中可高产量地表达可溶性目的蛋白质,即通过pET28-Trx空载质粒作为载体,向其中分别连入2段来自PDGF-B的关键编码序列,经原核表达纯化出两种重组蛋白6×his Trx-hPDGF-BΔ103-118和6×his Trx-hPDGF-BΔ152-167,通过主动免疫小鼠成功制备并纯化出2种PDGF-B多克隆抗体,纯化后的腹水抗体滴度可达1︰16 000以上,而且抗体能与膜结合的PDGF-B反应,证实两种重组蛋白具有良好的免疫原性;通过CCK8实验发现2种PDGF-B纯化腹水抗体(≥1︰40稀释度)均可有效抑制PDGF-BB对HepG2细胞增殖的促进作用。上述实验结果表明6×his Trx-hPDGF-BΔ103-118及6×his Trx-hPDGF-BΔ152-167重组蛋白具有较高的免疫原性,其作为免疫原均可以诱导小鼠产生高滴度的PDGF-B中和性抗体,而且PDGF-B纯化腹水抗体具有良好的中和活性,可有效抑制PDGF-BB对肝癌细胞增殖的促进作用,这为利用不同表位组合构建PDGF-B疫苗提供了新的方法,也为临床上HCC的治疗提供了一种新的思路。

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