1. 甲胎蛋白在人肝癌组织、癌旁组织、正常组织中表达的差异性分析
2. 人甲胎蛋白增强子(E_AFP)、白蛋白启动子(P_ALB)的克隆
3. 人干扰素? (IFN? -2b)cDNA的克隆
4. 白介素-2(IL-2)cDNA3’端终止密码的突变和IL-2/IFN? 2b融合基因的构建

1. 含IL-2/IFN? 融合基因的重组逆转录病毒的构建
2. 重组逆转录病毒的包装及病毒滴度测定
3. 重组逆转录病毒转染人肝癌细胞
4. IL-2/IFN? 融合基因在肝癌细胞中的表达和鉴定

细胞因子在介导机体多种免疫反应，如肿瘤免疫、移植免疫、自身免疫等过程中发挥重要作用。细胞因子的作用具有多相性、网络性的特点。它们不仅可以单独发挥生物学作用，而且不同的细胞因子之间还有相互协同或制约作用。近几年来利用细胞因子这一特点，根据不同的抗感染或抗肿瘤的需要，开展细胞因子融合的研究^[4-6]。本文将人IL-2基因与人IFN? 基因融合，IL-2主要作用于T细胞，可促进T细胞增殖与分化，对NK细胞、B细胞及单核细胞也有刺激作用。IFN? 和IL-2在许多生物学特性上有协同作用，均可增强NK、CTL及LAK细胞的活性，协同杀伤肿瘤细胞，这是临床联合使用这两种细胞因子治疗肿瘤的理论依据^[7-12]。基因治疗比常规全身用药不仅副作用小，而且肿瘤局部细胞因子浓度高，抗肿瘤作用也明显增强。文献报道，这两种细胞因子基因共同修饰的肿瘤细胞的侵袭转移表型也发生了变化，如ICAM-2及CD44的表达，与ECM的亲和力等等^[13]。因此，IL-2/IFN? 融合基因的研究不仅可以探索基因融合、高效表达的经验，而且为抗肿瘤和抗肿瘤转移的基因治疗提供实验依据。

引物1: 5’-ATTCAGACTGCTGCAGCCAA-3’，

引物2: 5’-GTGCTCATGTACATGGGCCA-3’

人干扰素? (IFN? )由人淋巴细胞产生。本研究人工合成一对引物：引物1：5’-GAG GAA TTC ATG TGT GAT CTG CCT C-3’，引物2：5’-GAA GTC GAC TCA TTC CTT ACT TC-3’，用RT-PCR方法，从淋巴细胞中扩增出510bp左右片段，经限制性内切酶EcoRI和SalI酶解，插入PUC18的EcoRI和SalI位点间，重组体命名为PUC(IFN? -2b)。经DNA顺序分析，其中含人IFN? 全长cDNA，由于其23位为AGA(编码精氨酸)，故确认其为人IFN? 2b基因。

1. 为了达到IL-2cDNA与IFN? 2b框架内融合,并能表达出有各自活性的杂和蛋白，首先需去除IL-2cDNA3’端的终止密码。本实验设计一对引物，以质粒pSVK3-IL-2为模板，PCR法重新扩增IL-2cDNA，使其3’端终止密码TGA改为GAG，成为mIL-2cDNA。其两端含有EcoRI和XhoI识别位点。

引物1：5’-TCT GAA TTC ATG TAC AGG ATG CAA CTC C-3’，含有EcoR I识别顺序。

引物2：5’-AAC TCG AGA GTT AGT GTT GAG ATG ATG CTT -3’，含有Xho I识别顺序。

2. IL-2/IFN? 2b融合基因的构建：人工合成寡聚核苷酸链接头：正意链(5’-AT CTC GAG GGT GGC GGT GGA TCC GGC GGT GGA TCT GGT GGC GGC GGA TCT GAA TTC C-3’)，反意链 (5’-G GAA TTC AGA TCC GCC GCC ACC AGA TCC ACCGCC GGA TCC ACC GCC ACC CTC GAG AT-3’)，其两端含有EcoR I和Xho I识别顺序。用重组DNA技术，将mIL-2cDNA与接头连接后，再与IFN? 2b cDNA连接达到框架内融合，组装入PUC19中，得到重组体PUC19(IL-2/IFN? 2b)。用限制性内切酶鉴定其方向性。

为了证实克隆的E_AFP－P_ALB联合调控序列具有肝癌组织相对特异性，将其插入白细胞介素-2(IL-2)cDNA上游，定向克隆入真核表达质粒pcDNA-3的多克隆位点区。将该重组体转染AFP高表达的人肝癌细胞HepG2(AFP= 845ng/ml)，AFP低表达的人肝癌细胞SMMC7721 (AFP= 2ng/ml)以及AFP不表达的人宫颈癌细胞Hela，观察IL-2单基因的表达水平差异。

用磷酸钙共沉淀法将质粒pcDNA-3、pCD(IL-2)和pFL(IL-2)分别转染HepG2、SMMC7721和Hela细胞，转染24小时后换含G418的筛选培养液，其浓度分别为800? g/ml、600? g/ml、400? g/ml，两周后细胞克隆形成，共得到九种转染细胞。

用软琼脂糖细胞克隆法观察转染基因IL-2的人肝癌细胞SMMC7721生物学特性变化：体外扩增筛选出的阳性细胞克隆，细胞数为1? 10³/ 60 mm平板，对照组为SMMC7721(细胞数同上)。37℃，5％CO₂，10天后计数细胞克隆数。实验表明，AFP表达阳性肝癌细胞转染IL-2基因后[pFL (IL-2)-SMMC7721、 pCD(IL-2) -SMMC7721]，细胞克隆形成率分别为12.6%和18.4%，而转染空载体pcDNA-3的SMMC7721和未转染DNA的SMMC7721则分别为25.1%、28.2%。

细胞因子基因治疗的发展趋势是：联合基因治疗和组织靶向性表达。这样有利于细胞因子间发挥相互协同的生物学作用，提高基因治疗的有效性和安全性。

用重组DNA技术，将IL-2/IFN? -2b融合基因置于E_AFP－P_ALB的下游，组装入缺陷型逆转录病毒LXSN(有SV40 Promoter和NeoR抗性基因)，重组体命名为L(FL-IL-2/IFN? 2b)SN，酶切鉴定。

病毒包装：用脂质体Lipofectin法转染包装细胞PA317，经G418筛选(350? g/ml)10～15天后，细胞克隆形成，挑取3～5个克隆，扩增细胞达10⁶，收集其培液上清。

病毒滴度测定：在6孔板中，接种NIH3T3细胞1? 10⁴/孔，24小时后，加入不同稀释度的培液上清(含聚季胺盐，Polybrene 8? g/ml)。36小时后，用G418 300? g/ml筛选。2周后，用Gimmsa’s染色法染色后，显微镜下计数细胞克隆形成数即为病毒滴度，留取滴度>10⁵CFU/ml上清备用。

将以上病毒滴度>10⁵CFU/ml的病毒上清液[LXSN和L(FL-IL-2/IFN ? 2b )SN]感染人肝癌细胞HepG2(AFP高表达，845ng/ml)、SMMC7721 (AFP低表达，2ng/ml)以及对照组人宫颈癌细胞Hela(AFP不表达)。感染液中含聚季胺盐(Polybrene) 8? g/ml。感染后48小时，加G418 400? g/ml筛选，2周后，细胞克隆形成。

1. PCR检测：以转染细胞的基因组DNA为模板，扩增NeoR基因、IL-2基因和IFN? 基因。NeoR基因引物1：5’-CAA GAT GGA TTG CAC GCA GG-3’，引物2：5’-CCC GCT CAG AAG AAC TCG TC-3’；IL-2基因引物1：5’-TCT GAA TTC ATG TAC AGG ATG CAA CTC G-3’，引物2：5’-AAC TCG AGA GTT AGT GTT GAG ATG ATG CTT-3’，IFN? 基因引物1：5’-GAG GAA TTC ATG TGT GAT CTG CCT C-3’，引物2：5’-GAA GTC GAC TCA TTC CTT ACT TC-3。结果表明，L(FL-IL-2/IFN? 2b)SN-HepG2和L(FL-IL-2/IFN? 2b)SN-7721细胞基因组中整合了NeoR、IL-2和IFN? 基因；而LXSN-HepG2、LXSN-7721中仅整合NeoR基因。

2. SDS-PAGE(银染法)：转染细胞经无血清培养24小时，收集上清液。SDS-PAGE电泳后，银盐染色。结果显示，L(FL-IL-2/IFN? 2b)SN转染的HepG2和SMMC 7721细胞培液中有37KDa大小的蛋白条带，而在LXSN转染的HepG2和SMMC 7721细胞培液中未见此条带，这条带的大小与IL-2/IFN? 融合蛋白的大小相符。

3.生物学活性测定：用MTS/PMS法测定培液上清中的IL-2活性，用微小病变抑制法测定IFN活性。测定结果：如表2示。实验表明，E_AFP-P_ALB具有肝癌细胞特异的转录调控作用(在AFP阳性肝癌细胞中)。表达产物IL-2/IFN? 2b 杂和蛋白有IL-2和IFN? 2b 两者的生物学活性。

4. 细胞克隆形成试验：将1.2%/0.6%的琼脂糖分别与2? DMEM(含20％的小牛血清)等体积混合，铺制双层琼脂糖平板(上层0.6%，下层0.3 %)，上层掺入感染细胞L(FL-IL-2/IFN? 2b)SN -SMMC7721和LXSN-SMMC 7721。37℃，5％CO₂孵育10-15天以后，计数细胞克隆数。结果显示，含融合基因的感染细胞克隆形成数为14.6%，明显低于未含融合基因的空载体转感染细胞(27.4%)以及SMMC7721细胞(28.2 % )。

5. 裸鼠体内试验：裸鼠分三组，每组4只。将转染细胞1? 10⁷皮下接种裸鼠，1周后对照组LXSN转染细胞及空白组SMMC 7721细胞组均长出肿瘤，而L(FL-IL-2/ IFN? 2b )SN-7721组未见肿瘤块。再接种SMMC 7721 (1? 10⁷)，2周后三组均长出肿瘤，但含融合基因组的肿瘤大小(0.5? 0.1cm )明显小于另两个对照组 (1.2? 0.2 cm，p<0.05)明显小于两个对照组。这可能是IL-2和IFN? 通过激活NK、K细胞等发挥杀瘤作用。

有关肿瘤基因治疗中靶向性表达的报道很多，靶向性表达主要有以下几个面： 借助于抗肿瘤细胞抗体， 借助于肿瘤细胞表面特异性标志的配基， 利用某些肿瘤组织特异性转录调控序列构建成肿瘤靶向性基因表达载体，可以相对特异地在相应肿瘤细胞内表达目的基因^[14]。这方面的研究很有希望，在自杀基因治疗研究中应用较多，但在细胞因子基因治疗方面报道较少。

细胞因子基因治疗一直是国内外研究的热点。将细胞因子基因导入肿瘤细胞，在肿瘤局部产生的高分泌量的细胞因子，可使肿瘤细胞从无、弱免疫原性变为强免疫原性，从而激活或增强机体的特异性抗肿瘤免疫反应，使肿瘤生长受到抑制或坏死消退，达到抗肿瘤的目的。本研究的特点是将具有协同抗肿瘤作用的IL-2和IFN? 基因融合，表达的杂和蛋白具有IL-2和IFN? 两种细胞因子功能，增强了细胞因子的联合抗肿瘤效应。另外，又用肝癌细胞特异的E_AFP－P_ALB联合转录调控序列调控其在肝癌细胞中特异表达，更加强了融合细胞因子对肝癌细胞的抑制作用。文献报道，IL-2和IFN? 的联合使用对肿瘤的复发转移也有预防和治疗作用，有关方案已经进入II期临床，疗效确切，副作用小^[20]。因此，本研究有很大的临床应用前景，可为这两种细胞因子联合抗肿瘤临床应用提供可靠的实验依据。

由于E_AFP－P_ALB联合转录调控序列的调控活性与血清AFP的水平成正相关性，而正常肝细胞中AFP表达为阴性，因此，该调控序列在正常肝细胞中不发挥作用，也即不会产生不良的副作用。另外，本研究表达的IL-2/IFN? 融合蛋白，还可用于研究IL-2和IFN? 与受体之间的作用机制。

结 论

1. 用RT-PCR方法证实了AFP是主要由肝癌细胞靶向表达。从而利用人甲胎蛋白和白蛋白的调控序列及其组织靶向性表达的特点，用PCR方法扩增E_AFP-P_ALB联合转录调控序列，与文献报道的有关肝癌细胞特异的AFP和白蛋白转录调控顺序相比，有以下优点：它不仅片段短，可以组装入包括逆转录病毒在内的所有基因治疗载体，而且保留了肝癌组织相对靶向性转录调控活性，在肝癌细胞中调控细胞因子基因的表达显示出较高的肝癌细胞靶向性，有较广泛的临床应用前景。
1. 根据国内外研究融合基因的经验，自行设计和合成引物，用PCR方法将IL-2cDNA终止密码突变，再用基因工程技术将它与IFN? 基因通过一个富含疏水氨基酸的接头连接，在结构上达到框架内融合。
1. 构建的含IL-2/IFN? 2b融合基因及E_AFP－P_ALB联合转录调控序列的重组逆转录病毒L(FL-IL-2/IFN? 2b)SN，经PA317细胞包装后得到高滴度的病毒上清。感染人肝癌细胞株后表达的融合蛋白不仅具有IL-2和IFN? 两种细胞因子的生物学活性，而且在裸鼠体内产生了一定的抗肿瘤作用。这不仅为临床联合使用IL-2和IFN? 治疗原发性肝癌提供实验依据，而且用E_AFP－P_ALB调控IL-2/IFN? 2b融合基因在肝癌细胞中表达研究，探索了肝癌基因治疗靶向性高效表达、多基因融合以及安全性等问题，为肝癌基因治疗临床应用奠定基础。

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1. Analysis of AFPmRNA in hepatoma tissuse
2. Cloning of human AFP enhancer(E_AFP) and albumin promoter(P_ALB)
3. Cloning of human IFN? -2b cDNA
4. Mutation of 3’-end stop codon of IL-2cDNA and construction of IL-2/IFN? fused cDNA

1. Construction of recombinant retrovirus containing fused IL-2/IFN? -2b cDNA
2. Packaging and titer measurement of recombinant retrovirus
3. Infection of recombinant retrovirus to hepatoma cells
4. Expression and characterization of IL-2/IFN? protein in hepatoma cells

Hepatocellular carcinoma(HCC) has long been regarded as a serious malignant tumor disease in China. The advances of experimental study about hepatoma gene therapy are very fast. Many candidate genes have been used in hepatoma gene therapy. One of the difficulties we confront to is how to get the targeted expression of therapeutic genes in hepatocellular tissues. The expression level of some genes was very low, and some gene products have serious side effect. The clinical data suggested that 60-70% of patients with hepatoma have high serum ? -fetoprotein(AFP) level,and the albumin secreted from hepatoma cells doesn’t go down. According to the expression charaters of AFP and albumin in human hepatoma tissues, we amplified the AFP enhancer (E_AFP) and albumin promoter (P_ALB) by PCR. In order to obtain targeted expression of heterogeous genes in hepatoma cells, we constructed a recombinant eucaryotic expression plasmid containing E_AFP-P_ALB transcriptional elements to drive expression of the human interleukin-2 cDNA or IL-2/IFN? 2b fused cDNA in human hepatoma cell lines. This may be a new way for gene therapy of human hepatocellular carcinoma.

Based on these considerations, we constructed recombinant retrovirus vector which contains IL-2/IFN? 2b fused cDNA. Interleukin-2(IL-2) is an important cytokine in the generation of anti-tumor immunity. It stimulates the proliferation of T cells, NK cells, B cells and mononuclear cells. Interferon ? has many common biological actions with interleukin-2, such as killing tumor cells, enhancing the activity of NK, CTL and LAK cells. All these are bases for using the two cytokines together in clinical trials. Comparising with general cytokine biotherapy, gene therapy has not only less side effects, but more strong antitumor response because of tumor local high-concentration of cytokines. As reference, IL-2 and/or IFN? can also modulated the invasive and metastatic potentials of the human renal carcimoma cells including the expression of ICAM-2 and CD44 and the affinity to ECM. Therefore, this study about the specific expression of the IL-2/IFN ? fused cDNA in human hepatoma cells can explore the optimal conditions of gene fusion and high expression and can provide the experimental basis of tumor gene therapy.

It has been certified that serum ? -fetoprotein was an important tumor marker for hepatocellular carcinoma since 1971,and that the specificity of AFP survey was just less than pathological examination. Furthermore, serum AFP level is most immportant in the early detection of hepatocellular carcinoma. In our country, 60-70% HCC patients also suffered from chronic liver diseases, especially cirrhosis. Their serum AFP levels which may be secreted by hepatoma cells or by proliferating hepatocytes were often higher than normal.

AFP Primer 1: 5’-ATT CAG ACT GCT GCA GCC AA-3’

Primer 2: 5’-GTG CTC ATG TAC ATG GGC CA-3’

Studies about the transcriptional regulation and biological significance of human ? -fetoprotein and albumin in hepatocellular carcinoma have become a hot point. These studies suggested that the 5.0 Kb of AFP enhancer on upstream of AFP gene was hepatoma cell-specific transcriptional regulation sequence. It is difficult to insert the 5.0Kb AFP enhancer into most vectors of gene therapy, so we just isolated the central sequence of the AFP enhancer(-3.3Kb? -3.7Kb) and combined it to the albumin promoter in order to drive the specific expression of foreign genes in hepatoma cells.

Human interferon ? (IFN? ) cDNA was amplified from lymphocyte mRNA by RT-PCR. The 510bp fragment cut by restriction endonuclease EcoRI and SalI was inserted into PUC18 [PUC18(IFN? 2b)]. It was identified as IFN? 2b by DNA sequencing because the sequences coding 23rd amino acid is AGA (arginine).

To obtain a fused protein which has bifunction of IL-2 and IFN? , the stop code (TGA) of IL-2 cDNA was mutated into GAG. The mutated IL-2 cDNA (mIL-2cDNA) was amplified from pSVK3-IL-2 plasmid by PCR method. The mIL-2cDNA was linked to IFN? cDNA by inserting a 57bp linker and then ligated with PUC19.The recombinant plasmid was named as PUC19(IL-2/IFN? 2b).

Primer 1::5’-TCT GAA TTC ATG TAC AGG ATG CAA CTC C-3’

Primer 2: 5’-AAC TCG AGA GTT AGT GTT GAG ATG ATG CTT -3’

Sense strain 5’-AT CTC GAG GGT GGC GGT GGA TCC GGC GGT GGA

Antisense strain 5’-G GAA TTC AGA TCC GCC GCC ACC AGA TCC

The sequences of E_AFP-P_ALB, IFN? 2b cDNA and IL-2cDNA were cloned and confirmed. The stop codon (TGA) of IL-2 cDNA was mutated into GAG . The IFN? 2bcDNA was fused in frame into IL-2 cDNA with linker.

In order to certify the specific transcriptional activity of E_AFP-P_ALB in hepatoma cells, E_AFP-P_ALB was inserted to the upstream of IL-2 cDNA which cloned into the eukarytic expression plasmid pcDNA-3. This recombinant plasmid named as pFL(IL-2) was transfected to two human hepatoma cell lines (HepG2 and SMMC7721) and control cell line Hela. Different expression levels of IL-2 were observed among these three transfected cells.

The eukarytic expression plasmid pcDNA-3 contains CMV (Cytomegalovirus) immediate/early promoter and NeoR selective gene. A 812bp HindIII/EcoRI fragment containing E_AFP-P_ALB sequences from pUH-3 and 469bp EcoRI/XhoI fragment containing human IL-2 cDNA from pSVK-3-IL-2 were isolated and directly ligated with pcDNA-3 vector and then constructed an expression plasmid pFL(IL-2). Another expression plasmid pCD(IL-2) was constructed just by ligating 479bp IL-2 cDNA fragment to pcDNA-3 vector. These two plasmids were characterized by digesting of restriction endonucleases.

Plasmids pcDNA-3, pCD(IL-2) and pFL(IL-2) were transfected into HepG2, SMMC7721 and Hela cells by a modified version of DNA- CaPO₃ co-precipitation protocol. At 24hr post-transfection, medium was replaced with selective medium containing G418(800? g/ml, 600? g/ml and 400? g/ml, respectively). Two weeks later, nine cell clones were obtained.

Each of these nine cell clones was proliferated to 10⁶ cells and samples of supernatant (1ml) were harvested at 24hr, 48hr, 5days, 15days, 30days and 60days post-transfections. The IL-2 activity of them was measured by MTS/PMS method(Table 1).

The biologic character of SMMC7721 cells transfected with pFL(IL-2) and pCD(IL-2) was studied by soft-agarose cell colony formation test. The transfected and untransfected SMMC7721 cells were proliferated to 1×10³ cells/60mm plated and cultured at 5% CO₂ and 37℃ for 10 days. The results showed that the cell colony formation rates of pFL(IL-2)-SMMC7721 and pCD(IL-2)-SMMC7721 were 12.6% and 18.4% respectively, while the rates of pcDNA-3-SMMC7721 and SMMC7721 were 25.1% and 28.2% respectively.

The trends of cytokine gene therapy are combined gene therapy and targeted expression. This aids to enhance the antitumor effects of cytokines.

The fragment of IL-2/IFN? 2b fused cDNA was ligated to the dowmstream of E_AFP-P_ALB sequences and then inserted to retroviral vector LXSN SV40 by recombinant DNA technology. This recombinant retrovirus named as L(FL- IL-2/IFN? 2b)SN was certified by digesting of restriction endonuclease.

Packaging of viral particle: the recombinat retrovirus L(FL-IL-2/IFN? 2b)SN and LXSN were transfected into PA317 cell line by lipofectinAmine method and then selected by G418(350? g/ml) for 10 to 15 days. Three to five cell clones were choosed and proliferated to 10⁶cells and the supernatants of these cells were harvested for measurement.

Measurement of retrovirus titer: 1×10⁴ NIH3T3 cells were innoculated into 60mm tissue culture plates and various diluted retrovirus supernatants which contains 8? g/ml polybrene were overlaid onto the cells. At 36hr post-infection, culture medium was replaced with 3ml of medium supplemented with G418 to a final concentration of 300? g/ml. After two weeks of G418 selection, colony numbers were determined by ethanol fixation and Gimmsa’s staining. Titer (colony-formation units/ml) was calculated as:(number of colonies)×(dilution of infection retrovirus)/total volume of diluted vector overlaid onto cells). The virus supernatants which have higher virus titer than 10⁵ CFU/ml were collected and stored in -80℃.

The human hepatoma cell lines HepG2 (AFP=845ng/ml), SMMC7721 (AFP=2ng/ml) and control cell line Hela(no AFP) were infected by the high titer retrovirus supernatants(>10⁵CFU/ml). At 48hr post-infection culture medium was replaced with 5ml medium supplemented with G418 to a final concentration of 400? g/ml. After two weeks of G418 selection, the cell colonies were formed.

1. PCR

Primers for amplifing NeoR cDNA, IL-2 cDNA and IFN? cDNA were synthesized(see below). The fragments of NeoR, IL-2 and IFN? cDNA can all be amplified from the genomic DNA of HepG2 and SMMC7721 infected with L(FL-IL-2/IFN? 2b)SN, and only NeoR cDNA fragment was amplified from the genomic DNA of HepG2 and SMMC7721 infected with LXSN. The results suggested that the fused IL-2/IFN? cDNA was intergrated into the genomic DNA of the infected cells.

NeoR cDNA: Primer1: 5’-CAA GAT GGA TTG CAC GCA GG-3’

Primer2: 5’-CCC GCT CAG AAG AAC TCG TC-3’

IL-2 cDNA: Primer1：5’-TCT GAA TTC ATG TAC AGG ATG CAA CTC G-3’ Primer2: 5’-AAC TCG AGA GTT AGT GTT GAG ATG ATG CTT-3’

IFN? cDNA: Primer1:5’-GAG GAA TTC ATG TGT GAT CTG CCT C-3’ Primer 2: 5’-GAA GTC GAC TCA TTC CTT ACT TC-3’

The supernatants were harvested after the infected cells were incubated in free-serum medium at 37℃ for 24hr. The protein bands were visualized after SDS-PAGE by silver staining. The result showed that the 37kD protein band was found in the culture medium of HepG2 and SMMC7721 transfected with L(FL-IL-2/IFN? 2b)SN, but none protein band of the same molecular weight was found in that of HepG2 and SMMC7721 with LXSN.The molecular weight of the 37KD protein is the same as that of IL-2/IFN? fused protein.

The activity of IL-2 was measured by MTS/PMS method. The activity of IFN? was measured by CPE method. The results showed in Table2.

These data suggested that E_AFP-P_ALB has specific transcriptional regualtion effects in AFP-producing hepatoma cells and also that IL-2/IFN? 2b chimeric protein has both of IL-2 and IFN? 2b activities.

1.2% and 0.6% of agarose were mixed with the same volume of 2×DMEM (containing 20% calf serum) and prepared bilayer agarose plates. The top agarose contains 1×10³ of infected SMMC7721 cells. The plates were incubate at 37℃ and 5% CO₂. After 10-14 days the cell colony numbers were counted by nake eye. The results showed that the cell colony formation rates of the infected SMMC7721 cells which contains IL-2/IFN? 2b fused gene(14.6%) was lower than that of control group(LXSN-7721 27.4%, SMMC7721 28.2%).

5. Antitumor activity of IL-2/IFN? fused protein in HCC-bearing nude mice

As mentioned above, transfer the IL-2/IFN? fused gene into hepatoma cells using retroviral vector has been found to be a potential approach for the treatment of HCC. In this part , we investigated whether the IL-2/IFN? -infected hepatoma cell lines could induce tumor suppression of HCC solid tumor in vivo. The SMMC7721 transfected with IL-2/IFN? fused gene was found to have lower proliferation rate and can significantly suppress the tumor growth of re-inoculation of SMMC7721(0.5? 0.1cm vs. 1.2? 0.2cm) as compared with control group(p<0.05). The results suggested the IL-2 and IFN? can kill tumor cells by stimulating the activity of NK and K cells.

A recombinant retrovirus L(FL-IL-2/IFN? 2b)SN which contains hepatoma cell-specific transcriptional elements E_AFP-P_ALB and IL-2/IFN? 2b fused gene was successfully constructed. Packaged by PA317 cell line, high titer recombinant retrovirus can infect hepatoma cell lines(HepG2 and SMMC7721) and Hela cell. Only AFP-producing hepatoma cells can effectively express IL-2/IFN? 2b protein, and the chimeric protein not only has the activities of il-2 and IFN? 2b, but also has ability to suppress the growth of hepatoma cells in nude mice.

(2) IL-2/IFN? cDNA expression system has been constructed and expressed in hepatoma cells. It not only provides an experience for studies of poly-gene fusion in cancer gene therapy, but also helps to investigate the antitumor mechanism of IL-2 and IFN? such as the interaction of IL-2 and IFN-? with their receptors.

(3) Retroviral vector-mediated gene transfer of IL-2/IFN? fused cDNA is a potential approach to the gene therapy of HCC.

Key words: Hepatoma, Gene therapy, Expression regulation, Enhancer, Promoter, Interleukin-2, Interferon-? , alpha-fetoprotein, IL-2/IFN? fused cDNA, eukarytic expression plasmid, retroviral vector

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