白细胞介素18对人舌鳞状细胞癌治疗作用的探讨
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摘要
舌鳞状细胞癌（tongue squamous cell carcinoma，TSCC)是口腔颌面部最常见的恶性肿瘤。它的发病率较高，且逐年上升，患病年龄趋向年轻化；因舌的血供及淋巴丰富，尤其舌肌频繁挤压使舌癌更易早期转移；舌癌预后较差，术后生活质量明显下降。因此，舌癌是严重威胁人类健康的疾病之一。
白细胞介素18(Interleukin-18，IL-18)是一种多效性炎性细胞因子，于1995年被Okamura等人发现，因能显著刺激Thl细胞产生干扰素Υ(γ-interferon, IFN-γ)，最初被命名为干扰素诱导因子，1996年正式命名为白细胞介素18。因与IL-1有相同特性，IL-18被列为IL-1家族。它可以由多种类型的细胞产生，包括巨噬细胞，树突状细胞，小神经胶质细胞，和角化细胞。IL-18最初是以前体IL-18的形式产生，分子量为24kDa，基因编码有193个氨基酸的无活性的前体蛋白，需经caspase-1/IL-1转化酶（ICE）的裂解，才能成为成熟形式的IL-18，分子量为18kDa。IL-18可以通过以下方面影响免疫系统：1、诱导由T，NK或者B细胞分泌IFN-Υ；2、促进抗CD3单克隆抗体，IL-2或者刀豆球蛋白A增殖，激活T细胞；3、提高Fas配基介导的NK细胞的细胞毒活性；4、抑制破骨细胞在体外形成；5、激活NK细胞,调节CD8~+T、CD4~+T细胞逐渐活化，使CTL发挥杀伤作用。而且，IL-18与IL-12有协同作用，诱导IFN-γ的产生，通过内源性血管生成抑制剂阻碍血管的发生。以上的生物学活性表明，IL-18可能正在成为一种特殊的有利于癌症免疫治疗的细胞因子，无论是在动物模型还是在晚期癌症病人身上，IL-18都发挥了显著的抗瘤活性。然而，IL-18对舌鳞的抗肿瘤作用还没有被阐述清楚。
目前，许多的研究都集中在对舌癌的预防和治疗上，包括使用整合素靶定肿瘤细胞使得口腔鳞癌细胞更易于进行光学拍照；使用姜黄素，通过内质网应激和线粒体依赖性途径诱导细胞凋亡。另外，舌癌耐药相关蛋白1（TCRP1）可以通过Akt信号通路而涉及口腔鳞癌细胞的放射敏感性。然而，很少有关于对舌鳞癌使用免疫治疗的报道。
本课题开展的假设:将细胞因子引入肿瘤细胞内已经成为癌症基因治疗的一种主要手段。我们用质粒载体pCDNA3.1携带IL-18基因，转染人舌鳞状细胞癌细胞系。由于IL-18具有免疫调节作用，激活机体的免疫系统，杀伤肿瘤细胞，因此推测IL-18可能会对舌鳞癌细胞系发挥抑制作用。发现并探讨新的抑瘤途径将是本课题的关键。
本课题分为两个部分：第一部分为构建及鉴定人IL-18基因真核表达载体；第二部分为将pCDNA3.1/IL-18转染入CRL-1623中，分析细胞生长状况、细胞凋亡、测定caspase3/7活性，并对相关细胞因子进行实时定量检测。
第一部分人IL-18基因真核表达载体的构建和鉴定
目的：
通过克隆人的Interleukin-18基因，构建Interleukin-18基因的哺乳动物细胞真核表达载体，为转染做好准备。
方法：
采用密度梯度法从健康志愿者的新鲜血液中分离白细胞；用Trizol法进行IL-18总RNA提取及鉴定。设计合成PCR引物，把人基因组cDNA通过PCR获得Interleukin-18，克隆至载体pMD18-T，然后转化到大肠杆菌里，最后进行重组真核表达载体pCDNA3.1/Interleukin-18（pIL-18）的构建，并对重组质粒pIL-18进行双酶切鉴定与测序。结果：
以人cDNA为模板，扩增出580bp左右的特异性条带，测序结果与GeneBank测序结果相比，除两个碱基外，其余的完全一致，翻译成的氨基酸序列相同。对重组质粒pIL-18进行双酶切鉴定并测序，结果也完全一致。
所以，重组质粒pIL-18由真核载体pCDNA3.1和Interleukin-18片段组成。且插入的Interleukin-18片段与已知序列完全相同。
结论：
成功构建了pIL-18重组质粒。
第二部分IL-18基因转染人舌鳞癌及体外抗肿瘤作用的实验研究
目的：
研究IL-18在舌鳞状细胞癌系CRL623生长调控中的作用，探讨抑瘤机制。
方法：
1、构建pCDNA3.1/IL-18重组质粒载体；
2、培养癌细胞系CRL1623；
3、将人IL-18基因克隆至pcDNA3.1(+)并转染CRL1623细胞，进行总RNA提取并进行实时定量检测；
4、随后用MTS法分析癌细胞的生长状况、使用锚定蛋白-异硫氰酸荧光素（FITC）凋亡试剂盒检测细胞凋亡；
5、用Apo-ONE Homogeneous试剂盒检测Caspase3/7的活性；
6、实时定量检测CCND1(cyclin D1), CCNA1(cyclin A1)和IFNG(interferon-γ)；
7、用SPSS16.0统计软件进行组别间t检验，p<0.05认为具有统计学意义。
结果：
1、成功构建了pCDNA3.1/IL-18载体。
2、我们使用pRFP转染细胞，分别在24、48和72小时检测发光率，在CRL-1623转染pRFP48h时，达到了一个较高的转染效率。
3、我们分别对转染后24、48和72小时的形态进行观察，细胞凋亡的数目从24h到72h逐渐上升。
4、与未转染细胞相比，转染pIL-18后细胞的活力显著下降（48h，p=0.031；72h，p=0.007)；凋亡细胞数目从0到72h之间显著上升(24h，p=0.046；48h，p=0.037；72h，p=0.016)；caspase-3/7的活性显著增加（p<0.05,72h）。
5、实时定量检测结果显示，与未转染细胞相比，pIL-18转染后，IL-18和IFN-γ mRNA的水平上调(p=0.009和p=0.014)；在pIL-18转染细胞中，cyclin D1mRNA水平下降，而cyclin A1mRNA水平并未发生明显改变。
结论：
1、过表达外源IL-18基因，可以直接在体外干涉舌鳞癌细胞系的生长，引起细胞凋亡；
2、肿瘤细胞凋亡的诱导可能是通过一种caspase依赖性通路发生的；
3、IL-18诱导干扰素γ产生，在宿主对细胞内微生物的防御中扮演着重要角色；
4、IL-18基因可能干涉了染色体在S期过程中的复制，减缓细胞增殖的速度。
因此，IL-18在调控舌鳞状细胞癌中扮演着重要角色，并可能在该癌症的治疗中起着重要的作用。
Tongue squamous cell carcinoma(TSCC) is the most common malignanttumor in oral and maxillofacial region. A higher incidence of tongue cancer,and increased year by year, the prevalence of age is getting younger. Due torich blood supply and lymphatic of the tongue, particularly genioglossusfrequent extrusion tongue cancer more likely to early transfer. Tongue cancerwith poor prognosis, postoperative lifequality has declined markedly. Therefore,tongue cancer is a disease of a serious threating to human health.
Interleukin-18is a pleiotropic inflammatory cytokine. In the year of1995,Okamura et al purified a new protein. Because it could significantly stimulateThl cell to produce IFN-γ，it was named IGIF (IFN-γ-inducing factor). Due toIGIF possessing many kinds of biological function, it was formally namedIL-18in1996. IL-18, a member of the IL-1superfamily of cytokines and likeIL-1β, is produced by several cell types, including macrophages, dendritic cells,microglial cells, and keratinocytes. IL-18is initially produced as pro-IL-18(24kDa), which encodes a193–amino acid biologically inactive precursorprotein. It is then processed by caspase-1, which is an IL-1β-converting enzyme(ICE), to a mature form with a molecular weight of18kDa. IL-18affects theimmune system through：1.inducing IFN-γ secretion by T, NK, or B cells;2.enhancing proliferation of anti-CD3monoclonal antibody, IL-2, orconcanavalin A-stimulated T cells;3.augmenting Fas ligand-mediated NK cellcytotoxic activity;4. inhibiting osteoclast formation in vitro;5. reinforceing theactivity of NK cell, regulating the activation of CD8~+T and CD4~+T cell, andCTL giving free rein of cytolytic function. Moreover, IL-18has a synergisticeffect with IL-12for IFN-γ production. Markedly retarded blood vessel growth are likely the result of more effective inhibition of angiogenesis by thecombination of the two cell types. Because of the above biologic activity，IL-18is probably becoming an antineoplastic cytokine. It has exhibited significantantitumor activities in multiple animal models and patients with advancedcancer. However, an antitumor property of IL-18on tongues quamous cellcarcinoma has not been directly shown.
At present, a large volume of research has been performed regarding theprevention and treatment of TSCC, including targeting tumour cell-basedintegrins to enable optical imaging of oral SCC, and the use of curcumin toinduce apoptosis via endoplasmic reticulum stress and mitochondria-dependentpathways. In addition, tongue cancer resistance-associated protein1(TCRP1)has been implicated in the radiation sensitivity of oral squamous cell carcinomacells through the Akt signaling pathway. There is, however, limited dataregarding immunotherapy in TSCC.Hypothesis of the research:
The introduction of cytokine genes into tumor cells has become one of themain techniques in cancer gene therapy. We transfected into human tonguesquamous cell carcinoma cell line CRL-1623with pcDNA3.1(+) vectorcontaining the human interleukin18gene. Because of the immunoregulationand antitumor property of IL-18, we presume that IL-18probably inhibitgrowth of CRL1623. It may be a critical problem of approaching a newantineoplastic pathway in our study.
Our research is divided into two parts. In the first part we construct andidentify the eukaryotic expression vector of human Interleukin-18. In thesecond part, we use pcDNA3.1(+) vector containing the human interleukin18gene and transfected into human tongues quamous cell carcinoma cell lineCRL-1623. Analysis of cell growth, detection of apoptosis, assessment ofcaspase3/7activity, and real-time reverse transcription–polymerase chain reaction were performed to assess the related genes expression of correlatedcytokine.
Part one Construction and Identification of Eukaryotic
Expression Vector of Human Interleukin-18
Object
The eukaryotic expression vector of Interleukin-18was constructedaccording to the Interleukin-18gene of human．
Method
We isolated leukocyte from fresh blood of one healthy donor withdensity gradient centrifugation,then extracted and assessed IL-18total RNAusing TRIzol reagent according to manufacturer’s instruction. Aftersynthetized primer for PCR, interleukin-18gene was obtained from humangenome DNA by PCR. Vector pMD18-T/Interleukin-18was cloned andtransformed into Escherichia coli，and the recombinant pCDNA3.1/Interleukin-18was constructed and identified．
Result
The result of agarose gel electrophoresis showed that an expected580bpband was amplified. The result of enzyme-disgetion identification indicated aband was in accordance with the expected length. Sequencing result ofpCDNA3.1/Interleukin-18showed that the reading frame was not changedexcept two base compared with the GeneBank, and amino acid sequence wasthe same.
Conclusion
The eukaryotic expression vector pCDNA3.1/IL-18was constructedsuccessfully.
Part two Construction of human tongue squamous cell
carcinoma cells transducted by IL-18and study of its
antitumor effect in vitro
Objective
This study investigated the role of interleukin-18(IL-18) in regulating thegrowth of the human tongue squamous cell carcinoma cell line CRL-1623.
Methods:
1Constructing the eukaryotic expression vector pCDNA3.1/IL-18;
2Culturing of tongue carcinoma cell line CRL1623;
3The human IL18gene was cloned and transfected into CRL-1623cellsusing the transfection vector pcDNA3.1(+), then real-time reversetranscription–polymerase chain reaction was performed;
4Analysis of cell growth using MTS method, detection of apoptosis usingannexin V–fluorescein isothiocyanate,
5Assessment of caspase3/7activity using Apo-ONE Homogeneous Kit
6Real-time reverse transcription–polymerase chain reaction to assessexpression of the IL18, CCND1(cyclin D1), CCNA1(cyclin A1) and IFNG(interferon-γ) genes.
7Statistical analyses were carried out using the SPSS statistical package,version16.0for Windows. The Student’s t-test was used to compare thedifferences between groups. A P-value <0.05was considered to be statisticallysignificant.
Result
1Constructing the eukaryotic expression vector successfully.
2Transfection of CRL-1623cells with pRFP appeared qualitatively toresult in high transfection efficiency at48h after transfection, as determined by fluorescence microscopy.
3The number of apoptosis and dead cells increased from24to72h,following transfection with pIL18.
4.Transfection with pIL18reduced cell viability compared withuntransfected cells and this difference was statistically significant at48h (P=0.031) and72h (P=0.007); Cell viability was also reduced slightly bytransfection with empty vector pcDNA3.1(+) from24h to72h compared withuntransfected cells, although this difference was not statistically significant.The number of apoptotic cells in the pIL18transfected group also increasedsignificantly between0and72h compared with untransfected cells (P=0.046at24h; P=0.037at48h; P=0.016at72h). Transfection with pIL18but notpcDNA3.1(+) resulted in increased caspase-3/7activity between0and72h.although this difference was only statistically significant at72h.
5.The levels of both IL18and IFNγ mRNA were increased in cellstransfected with pIL18compared with untransfected cells (P=0.009and P=0.014, respectively). Cyclin D1mRNA was reduced in pIL18-transfected cellscompared with untransfected cells (P=0.024). Transfection with pIL18did notinduce any change in the level of cyclin A1mRNA.
Conclusion
1Over-expression of an exogenous IL18gene directly inhibited thegrowth of, and induced apoptosis in the human tongue squamous cell carcin-oma cell line CRL1623；
2The induction of apoptosis might occurr via a caspase-dependentpathway；
3IL-18stimulates Th1-mediated immune responses, which play a criticalrole in host defence against intracellular microbes through the induction ofIFN-γ；
4IL-18signal transduction may result in interference with the regulation of the cyclin proteins and/or the replication of chromosome during S phase，leading to a slow proliferative rate。
These findings suggest that IL-18plays a role in the regulation of tonguesquamous cell carcinoma and may represent a potential therapeutic target inthis cancer.
白细胞介素18(Interleukin-18，IL-18)是一种多效性炎性细胞因子，于1995年被Okamura等人发现，因能显著刺激Thl细胞产生干扰素Υ(γ-interferon, IFN-γ)，最初被命名为干扰素诱导因子，1996年正式命名为白细胞介素18。因与IL-1有相同特性，IL-18被列为IL-1家族。它可以由多种类型的细胞产生，包括巨噬细胞，树突状细胞，小神经胶质细胞，和角化细胞。IL-18最初是以前体IL-18的形式产生，分子量为24kDa，基因编码有193个氨基酸的无活性的前体蛋白，需经caspase-1/IL-1转化酶（ICE）的裂解，才能成为成熟形式的IL-18，分子量为18kDa。IL-18可以通过以下方面影响免疫系统：1、诱导由T，NK或者B细胞分泌IFN-Υ；2、促进抗CD3单克隆抗体，IL-2或者刀豆球蛋白A增殖，激活T细胞；3、提高Fas配基介导的NK细胞的细胞毒活性；4、抑制破骨细胞在体外形成；5、激活NK细胞,调节CD8~+T、CD4~+T细胞逐渐活化，使CTL发挥杀伤作用。而且，IL-18与IL-12有协同作用，诱导IFN-γ的产生，通过内源性血管生成抑制剂阻碍血管的发生。以上的生物学活性表明，IL-18可能正在成为一种特殊的有利于癌症免疫治疗的细胞因子，无论是在动物模型还是在晚期癌症病人身上，IL-18都发挥了显著的抗瘤活性。然而，IL-18对舌鳞的抗肿瘤作用还没有被阐述清楚。
目前，许多的研究都集中在对舌癌的预防和治疗上，包括使用整合素靶定肿瘤细胞使得口腔鳞癌细胞更易于进行光学拍照；使用姜黄素，通过内质网应激和线粒体依赖性途径诱导细胞凋亡。另外，舌癌耐药相关蛋白1（TCRP1）可以通过Akt信号通路而涉及口腔鳞癌细胞的放射敏感性。然而，很少有关于对舌鳞癌使用免疫治疗的报道。
本课题开展的假设:将细胞因子引入肿瘤细胞内已经成为癌症基因治疗的一种主要手段。我们用质粒载体pCDNA3.1携带IL-18基因，转染人舌鳞状细胞癌细胞系。由于IL-18具有免疫调节作用，激活机体的免疫系统，杀伤肿瘤细胞，因此推测IL-18可能会对舌鳞癌细胞系发挥抑制作用。发现并探讨新的抑瘤途径将是本课题的关键。
本课题分为两个部分：第一部分为构建及鉴定人IL-18基因真核表达载体；第二部分为将pCDNA3.1/IL-18转染入CRL-1623中，分析细胞生长状况、细胞凋亡、测定caspase3/7活性，并对相关细胞因子进行实时定量检测。
第一部分人IL-18基因真核表达载体的构建和鉴定
目的：
通过克隆人的Interleukin-18基因，构建Interleukin-18基因的哺乳动物细胞真核表达载体，为转染做好准备。
方法：
采用密度梯度法从健康志愿者的新鲜血液中分离白细胞；用Trizol法进行IL-18总RNA提取及鉴定。设计合成PCR引物，把人基因组cDNA通过PCR获得Interleukin-18，克隆至载体pMD18-T，然后转化到大肠杆菌里，最后进行重组真核表达载体pCDNA3.1/Interleukin-18（pIL-18）的构建，并对重组质粒pIL-18进行双酶切鉴定与测序。结果：
以人cDNA为模板，扩增出580bp左右的特异性条带，测序结果与GeneBank测序结果相比，除两个碱基外，其余的完全一致，翻译成的氨基酸序列相同。对重组质粒pIL-18进行双酶切鉴定并测序，结果也完全一致。
所以，重组质粒pIL-18由真核载体pCDNA3.1和Interleukin-18片段组成。且插入的Interleukin-18片段与已知序列完全相同。
结论：
成功构建了pIL-18重组质粒。
第二部分IL-18基因转染人舌鳞癌及体外抗肿瘤作用的实验研究
目的：
研究IL-18在舌鳞状细胞癌系CRL623生长调控中的作用，探讨抑瘤机制。
方法：
1、构建pCDNA3.1/IL-18重组质粒载体；
2、培养癌细胞系CRL1623；
3、将人IL-18基因克隆至pcDNA3.1(+)并转染CRL1623细胞，进行总RNA提取并进行实时定量检测；
4、随后用MTS法分析癌细胞的生长状况、使用锚定蛋白-异硫氰酸荧光素（FITC）凋亡试剂盒检测细胞凋亡；
5、用Apo-ONE Homogeneous试剂盒检测Caspase3/7的活性；
6、实时定量检测CCND1(cyclin D1), CCNA1(cyclin A1)和IFNG(interferon-γ)；
7、用SPSS16.0统计软件进行组别间t检验，p<0.05认为具有统计学意义。
结果：
1、成功构建了pCDNA3.1/IL-18载体。
2、我们使用pRFP转染细胞，分别在24、48和72小时检测发光率，在CRL-1623转染pRFP48h时，达到了一个较高的转染效率。
3、我们分别对转染后24、48和72小时的形态进行观察，细胞凋亡的数目从24h到72h逐渐上升。
4、与未转染细胞相比，转染pIL-18后细胞的活力显著下降（48h，p=0.031；72h，p=0.007)；凋亡细胞数目从0到72h之间显著上升(24h，p=0.046；48h，p=0.037；72h，p=0.016)；caspase-3/7的活性显著增加（p<0.05,72h）。
5、实时定量检测结果显示，与未转染细胞相比，pIL-18转染后，IL-18和IFN-γ mRNA的水平上调(p=0.009和p=0.014)；在pIL-18转染细胞中，cyclin D1mRNA水平下降，而cyclin A1mRNA水平并未发生明显改变。
结论：
1、过表达外源IL-18基因，可以直接在体外干涉舌鳞癌细胞系的生长，引起细胞凋亡；
2、肿瘤细胞凋亡的诱导可能是通过一种caspase依赖性通路发生的；
3、IL-18诱导干扰素γ产生，在宿主对细胞内微生物的防御中扮演着重要角色；
4、IL-18基因可能干涉了染色体在S期过程中的复制，减缓细胞增殖的速度。
因此，IL-18在调控舌鳞状细胞癌中扮演着重要角色，并可能在该癌症的治疗中起着重要的作用。
Tongue squamous cell carcinoma(TSCC) is the most common malignanttumor in oral and maxillofacial region. A higher incidence of tongue cancer,and increased year by year, the prevalence of age is getting younger. Due torich blood supply and lymphatic of the tongue, particularly genioglossusfrequent extrusion tongue cancer more likely to early transfer. Tongue cancerwith poor prognosis, postoperative lifequality has declined markedly. Therefore,tongue cancer is a disease of a serious threating to human health.
Interleukin-18is a pleiotropic inflammatory cytokine. In the year of1995,Okamura et al purified a new protein. Because it could significantly stimulateThl cell to produce IFN-γ，it was named IGIF (IFN-γ-inducing factor). Due toIGIF possessing many kinds of biological function, it was formally namedIL-18in1996. IL-18, a member of the IL-1superfamily of cytokines and likeIL-1β, is produced by several cell types, including macrophages, dendritic cells,microglial cells, and keratinocytes. IL-18is initially produced as pro-IL-18(24kDa), which encodes a193–amino acid biologically inactive precursorprotein. It is then processed by caspase-1, which is an IL-1β-converting enzyme(ICE), to a mature form with a molecular weight of18kDa. IL-18affects theimmune system through：1.inducing IFN-γ secretion by T, NK, or B cells;2.enhancing proliferation of anti-CD3monoclonal antibody, IL-2, orconcanavalin A-stimulated T cells;3.augmenting Fas ligand-mediated NK cellcytotoxic activity;4. inhibiting osteoclast formation in vitro;5. reinforceing theactivity of NK cell, regulating the activation of CD8~+T and CD4~+T cell, andCTL giving free rein of cytolytic function. Moreover, IL-18has a synergisticeffect with IL-12for IFN-γ production. Markedly retarded blood vessel growth are likely the result of more effective inhibition of angiogenesis by thecombination of the two cell types. Because of the above biologic activity，IL-18is probably becoming an antineoplastic cytokine. It has exhibited significantantitumor activities in multiple animal models and patients with advancedcancer. However, an antitumor property of IL-18on tongues quamous cellcarcinoma has not been directly shown.
At present, a large volume of research has been performed regarding theprevention and treatment of TSCC, including targeting tumour cell-basedintegrins to enable optical imaging of oral SCC, and the use of curcumin toinduce apoptosis via endoplasmic reticulum stress and mitochondria-dependentpathways. In addition, tongue cancer resistance-associated protein1(TCRP1)has been implicated in the radiation sensitivity of oral squamous cell carcinomacells through the Akt signaling pathway. There is, however, limited dataregarding immunotherapy in TSCC.Hypothesis of the research:
The introduction of cytokine genes into tumor cells has become one of themain techniques in cancer gene therapy. We transfected into human tonguesquamous cell carcinoma cell line CRL-1623with pcDNA3.1(+) vectorcontaining the human interleukin18gene. Because of the immunoregulationand antitumor property of IL-18, we presume that IL-18probably inhibitgrowth of CRL1623. It may be a critical problem of approaching a newantineoplastic pathway in our study.
Our research is divided into two parts. In the first part we construct andidentify the eukaryotic expression vector of human Interleukin-18. In thesecond part, we use pcDNA3.1(+) vector containing the human interleukin18gene and transfected into human tongues quamous cell carcinoma cell lineCRL-1623. Analysis of cell growth, detection of apoptosis, assessment ofcaspase3/7activity, and real-time reverse transcription–polymerase chain reaction were performed to assess the related genes expression of correlatedcytokine.
Part one Construction and Identification of Eukaryotic
Expression Vector of Human Interleukin-18
Object
The eukaryotic expression vector of Interleukin-18was constructedaccording to the Interleukin-18gene of human．
Method
We isolated leukocyte from fresh blood of one healthy donor withdensity gradient centrifugation,then extracted and assessed IL-18total RNAusing TRIzol reagent according to manufacturer’s instruction. Aftersynthetized primer for PCR, interleukin-18gene was obtained from humangenome DNA by PCR. Vector pMD18-T/Interleukin-18was cloned andtransformed into Escherichia coli，and the recombinant pCDNA3.1/Interleukin-18was constructed and identified．
Result
The result of agarose gel electrophoresis showed that an expected580bpband was amplified. The result of enzyme-disgetion identification indicated aband was in accordance with the expected length. Sequencing result ofpCDNA3.1/Interleukin-18showed that the reading frame was not changedexcept two base compared with the GeneBank, and amino acid sequence wasthe same.
Conclusion
The eukaryotic expression vector pCDNA3.1/IL-18was constructedsuccessfully.
Part two Construction of human tongue squamous cell
carcinoma cells transducted by IL-18and study of its
antitumor effect in vitro
Objective
This study investigated the role of interleukin-18(IL-18) in regulating thegrowth of the human tongue squamous cell carcinoma cell line CRL-1623.
Methods:
1Constructing the eukaryotic expression vector pCDNA3.1/IL-18;
2Culturing of tongue carcinoma cell line CRL1623;
3The human IL18gene was cloned and transfected into CRL-1623cellsusing the transfection vector pcDNA3.1(+), then real-time reversetranscription–polymerase chain reaction was performed;
4Analysis of cell growth using MTS method, detection of apoptosis usingannexin V–fluorescein isothiocyanate,
5Assessment of caspase3/7activity using Apo-ONE Homogeneous Kit
6Real-time reverse transcription–polymerase chain reaction to assessexpression of the IL18, CCND1(cyclin D1), CCNA1(cyclin A1) and IFNG(interferon-γ) genes.
7Statistical analyses were carried out using the SPSS statistical package,version16.0for Windows. The Student’s t-test was used to compare thedifferences between groups. A P-value <0.05was considered to be statisticallysignificant.
Result
1Constructing the eukaryotic expression vector successfully.
2Transfection of CRL-1623cells with pRFP appeared qualitatively toresult in high transfection efficiency at48h after transfection, as determined by fluorescence microscopy.
3The number of apoptosis and dead cells increased from24to72h,following transfection with pIL18.
4.Transfection with pIL18reduced cell viability compared withuntransfected cells and this difference was statistically significant at48h (P=0.031) and72h (P=0.007); Cell viability was also reduced slightly bytransfection with empty vector pcDNA3.1(+) from24h to72h compared withuntransfected cells, although this difference was not statistically significant.The number of apoptotic cells in the pIL18transfected group also increasedsignificantly between0and72h compared with untransfected cells (P=0.046at24h; P=0.037at48h; P=0.016at72h). Transfection with pIL18but notpcDNA3.1(+) resulted in increased caspase-3/7activity between0and72h.although this difference was only statistically significant at72h.
5.The levels of both IL18and IFNγ mRNA were increased in cellstransfected with pIL18compared with untransfected cells (P=0.009and P=0.014, respectively). Cyclin D1mRNA was reduced in pIL18-transfected cellscompared with untransfected cells (P=0.024). Transfection with pIL18did notinduce any change in the level of cyclin A1mRNA.
Conclusion
1Over-expression of an exogenous IL18gene directly inhibited thegrowth of, and induced apoptosis in the human tongue squamous cell carcin-oma cell line CRL1623；
2The induction of apoptosis might occurr via a caspase-dependentpathway；
3IL-18stimulates Th1-mediated immune responses, which play a criticalrole in host defence against intracellular microbes through the induction ofIFN-γ；
4IL-18signal transduction may result in interference with the regulation of the cyclin proteins and/or the replication of chromosome during S phase，leading to a slow proliferative rate。
These findings suggest that IL-18plays a role in the regulation of tonguesquamous cell carcinoma and may represent a potential therapeutic target inthis cancer.
引文
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