MDA-7/IL-24蛋白的功能研究进展

 mda-7/IL-24基因最初被命名为黑色素瘤分化相关基因-7（melanoma differentiation associated gene-7），是1995年Jiang等[1]利用减数杂交技术从β干扰素和蛋白激酶C活化剂mezerin诱导的人黑色素瘤细胞HO-1中鉴定并克隆出来的。基于结构、序列同源性、基因定位等方面的考虑，MDA-7蛋白被归为IL-10家族，并被命名为MDA-7/IL-24蛋白。mda-7/IL-24基因在进化过程中比较保守，在酵母、猴子、牛、狗和猫的基因组DNA中都发现了mda-7/IL-24基因的同源基因[2]。人mda-7/IL-24基因是单拷贝基因，定位于1号染色体（1q32.2-q41），由7个外显子和6个内含子组成，其mRNA长约2 kb，编码由206个氨基酸组成、相对分子质量约为23 800的蛋白质。序列分析表明，MDA-7/ IL-24蛋白的N端有一个由49个氨基酸组成的信号肽，与蛋白的切割和分泌有关；此外，MDA-7/IL-24蛋白序列上有3个N-糖基化位点、3个蛋白激酶C位点、6个磷酸化位点[3-4]。MDA-7/IL-24蛋白作为IL-10家族中的一员，具有重要的免疫调节作用，主要在与免疫系统相关的组织中表达，如胸腺、脾、外周血白细胞等。MDA-7/IL-24蛋白的受体属于Ⅱ型细胞因子受体，由2个异源二聚体（IL-20R1/IL-20R2和IL-22R1/IL20R2）组成。MDA-7/IL-24蛋白与受体结合后，通过激活JAK/STAT信号通路而发挥其生理功能[5]。另有研究发现，利用质粒或腺病毒表达载体将mda-7/IL-24基因转入肿瘤细胞后，mda-7/IL-24基因的异位表达能特异性抑制多种肿瘤细胞的生长，诱导肿瘤细胞凋亡，而对正常细胞没有影响[8, 10-12, 14]。本文就MDA-7/IL-24蛋白的抗肿瘤和免疫调节作用研究进展及其临床应用作一综述。     

新型细胞因子IL-24真核表达载体的构建、表达及其对肿瘤的抑制作用

目的：构建IL-24真核表达质粒，并研究其体内外表达对肿瘤细胞的生长抑制作用。方法：采用重组DNA技术构建IL-24真核表达质粒pEGFP-C1-IL-24。用脂质体法将重组质粒及空载体外转染HIC细胞，再经激光扫描共聚焦显微镜（LSCM）观察其表达，用MTT法检测HIC细胞的体外增殖能力，用流式细胞仪检测细胞周期与凋亡。小鼠皮下接种转染IL-24真核表达质粒或空载的B16细胞观察其体内致瘤性的变化。小鼠实体瘤模型研究基因转染对肿瘤的生长抑制作用。结果：pEGF-C1-IL-24转染HIC细胞后，LSCM可观察到其表达。IL-24基因转染的HIC细胞体外增殖能力明显受到抑制．G2-M期细胞比例增高，细胞被阻滞在G2-M期。转染IL-24的B16细胞体外致瘤率降低。与对照组相比．IL-24基因治疗组肿瘤生长明显受到抑制（P〈0.05）。结论：IL-24基因转染的肿瘤细胞体外生长受抑。瘤内注射pEGFP-C1-IL-24可抑制肿瘤生长，具有明显的抗肿瘤作用。     

mda-7／IL-24基因与肿瘤

在人类黑色素瘤细胞株(HO-1)中克隆了一个位于1q34，2-q41的新的黑色素瘤分化相关基因mda-7。该基因有7025个碱基组成，令7个外显子6个内含子，编码大小为23．8kDa蛋白，由206个氨基酸序列组成。对多种肿瘤细胞具有抑制生长，诱导凋亡的作用，是一个新的肿瘤抑制基因。     

抑癌基因mda-7／IL-24与肿瘤治疗

mda 7/IL 2 4是在黑色素瘤细胞中发现的一个与黑色素瘤分化相关的基因 ,位于染色体 1q3 2 .2～ 1q41区域。编码蛋白MDA 7/IL 2 4通过与受体IL 2 0R1,IL 2 2R1与IL 2 0R2形成的 2种异二聚体复合物结合进行信号转导 ,对多种组织起源的肿瘤细胞均有抑制作用 ,还能在IFN β和分化诱导剂MEZ的作用下间接诱导黑素瘤细胞向终末期分化 ,但对正常细胞并无明显的毒副作用 ,在肿瘤的基因治疗上具有应用前景     

人泡沫病毒介导IL-24对肿瘤细胞的抑制作用

目的:利用重新构建的人逆转录病毒四载体系统共转染人胚肾细胞获得携带IL-24的复制缺陷型重组人泡沫病毒(HFV-IL24)颗粒,探讨复制缺陷型人泡沫病毒介导IL-24对肿瘤细胞生长分化的抑制作用。方法:重组构建含IL-24的人泡沫病毒载体质粒(pΔΦ-IL24);与辅助质粒共转染人胚肾HEK293T细胞后获取重组病毒颗粒(HFV-IL24);RT-PCR检测目的基因的表达;MTT法检测人宫颈癌HeLa细胞的体外增殖水平,台盼蓝染色计数检测细胞存活率,流式细胞术检测细胞活性和周期变化。结果:成功构建含IL-24的人泡沫病毒载体(pΔΦ-IL24);收获得到复制缺陷型重组人泡沫病毒颗粒(HFV-IL24);感染后的HeLa细胞显示出明显的生长抑制现象(P0.01)并呈现周期变化和较高的凋亡(死亡)率。结论:所构建的重组病毒载体pΔΦ-IL24及由此而产生的重组人泡沫病毒颗粒(HFV-IL24)的功能性检测加深对HFV和抗癌基因IL-24的了解,为泡沫病毒载体的应用和IL-24抗肿瘤的基因治疗提供了研究方向和理论依据。     

重组腺相关病毒介导黑色素瘤分化相关基因7对人肝癌细胞体内外抑制效应的研究

目的观察PEG启动子调控腺相关病毒介导的黑色素瘤分化相关基因7（MDA-7）在体内外抑制肝癌细胞的生物学活性，探讨重组腺相关病毒介导MDA-7基因用于肝癌基因治疗的应用前景。方法构建重组腺相关病毒rAAV-PEG-MDA-7表达系统，体外感染人肝癌细胞株HepG2细胞。Western印迹检测转染细胞内MDA-7蛋白；MTT法检测细胞增殖抑制率；流式细胞术分析细胞周期和细胞凋亡变化。构建肝癌细胞HepG2裸鼠皮下移植瘤模型，尾静脉注射rAAV-PEG-MDA-7，观察其对肝癌生长的抑制作用；ELISA方法检测血浆MDA-7蛋白浓度；TUNEL法分析MDA-7对肿瘤细胞的凋亡诱导情况；免疫组织化学分析MDA-7在肿瘤组织中的表达。结果重组腺相关病毒rAAV-PEG-MDA-7可特异性转染HepG2细胞，MDA-7蛋白在HepG2细胞中高效表达，并呈时间依赖性。重组腺相关病毒rAAV-PEG-MDA-7可抑制HepG2细胞增殖并诱导其凋亡，G0／G1期细胞百分比明显增多，G2／M期的细胞显著减少（P〈0、05）。全身系统性给予rAAV-PEG-MDA-7后，血清中可持续检测到MDA-7蛋白，且注射后2周浓度达高峰（200ng／ml）；肿瘤生长受抑制，抑瘤率为62％（P〈0、05）；免疫组化结果显示MDA-7在肿瘤组织中表达；TUNEL结果显示rAAV-PEG-MDA-7可诱导肿瘤细胞凋亡。结论构建出的重组腺相关病毒rAAV-PEG-MDA-7表达系统具有良好的肿瘤靶向性，通过抑制肝癌细胞增殖和诱导其凋亡发挥抗肝癌作用。     

mda-7/IL-24基因与肿瘤

在人类黑色素瘤细胞株 (HO- 1)中克隆了一个位于 1q34.2 - q4 1的新的黑色素瘤分化相关基因 m da- 7。该基因有70 2 5个碱基组成 ,含 7个外显子 6个内含子 ,编码大小为 2 3.8k Da蛋白 ,由 2 0 6个氨基酸序列组成。对多种肿瘤细胞具有抑制生长 ,诱导凋亡的作用 ,是一个新的肿瘤抑制基因。     

IL-24基因重组腺病毒载体转染的树突状细胞促进宫颈癌CaSki细胞凋亡

 目的：探讨白细胞介素24(interleukin-24,IL-24) 基因重组腺病毒载体转染的树突状细胞(dendri-tic cells,DCs)是否在体外诱导细胞毒性T淋巴细胞(cytotoxic T-lymphocytes,CTLs)对宫颈癌CaSki细胞的杀伤效应。方法：分离培养小鼠未成熟DCs,将已成功构建的带有IL-24基因的重组腺病毒感染体外培养的小鼠未成熟DCs,提取CaSki细胞裂解物负载DCs,制备DC疫苗,Western blotting检测IL-24蛋白的表达;PE-Annexin V和Western blotting检测细胞凋亡及cleaved caspase-3表达;克隆形成实验检测克隆形成能力;裸鼠荷瘤模型体内研究带有IL-24基因的重组腺病毒载体转染DCs在体外诱导的CTLs对宫颈癌CaSki细胞的杀伤效应。结果：Western blotting检测IL-24蛋白高表达;DCs疫苗诱导产生的CTLs后与CaSki细胞共培养,对CaSki细胞生长具有明显的抑制作用, DCs疫苗诱导产生的CTLs促进CaSki细胞凋亡,增加cleaved caspased-3蛋白表达,克隆形成实验检测该疫苗具有抑制CaSki细胞形成克隆的能力。裸鼠的成瘤实验表明,带有IL-24基因的重组腺病毒载体转染DCs可以抑制体内肿瘤的形成,促进肿瘤细胞凋亡。结论：IL-24基因重组腺病毒感染DCs制备的DCs疫苗可诱导CTLs,从而抑制宫颈癌CaSki细胞增殖,并促进其凋亡。     

白介素24抗肿瘤作用机制的研究进展

黑色素瘤分化相关基因-7(MDA-7)/白介素24(IL24),是IL-10家族的成员之一,主要表达于人体脾脏、胸腺、外周血白细胞等免疫组织器官和正常黑色素细胞。IL24是一种独特的抑制肿瘤细胞因子,通过自分泌旁分泌方式调控,抑制肿瘤生长、侵袭转移及血管生成,诱导凋亡,可以选择性的杀伤多种肿瘤细胞,产生放化疗增敏效应,而不伤害正常细胞。但是目前对于其作用的具体机制尚不明确。     

Enhancing the apoptotic effect of IL-24/mda-7 on the human hepatic stellate cell through RGD peptide modification

Induction of apoptosis or quiescent hepatic stellate cells (HSCs) can be an attractive molecular strategy due to the importance of activation of HSCs during hepatic fibrogenesis. Interleukin-24/melanoma differentiation-associated gene-7 (IL-24/mda-7) is a cytokine that has attracted a great deal of attention in the tumor killing as well as pathophysiology of the diseases. In this study, the Pro-apoptotic and senescence inductive properties of IL-24/mda-7 were assessed in human-derived HSCs. Three plasmids expressing natural mda-7, peptide modified version, mda-7-RGD genes beside a recombinant IL-24 protein, were added or transfected into activated LX-2 cells. Cell viability and the amount of apoptosis were analyzed using MTT and Annexin V staining method, respectively. Hence, the expression levels of apoptotic genes and PPARγ in different groups were also compared by real-time PCR analysis. Furthermore, the senescence effect of IL-24/mda-7 by a β-galactosidase (SA-β-gal) senescence assay, was evaluated. The viability assessment showed that pmda-7-RGD had the most significant growth inhibitory effect when compared to the control group, pcDNA3.1 (P = 0.0002). The apoptosis analysis also revealed a significant impact of different mda-7 forms in apoptosis induction. The measuring of cell senescence also indicated that IL-24/mda-7 in plasmid and protein forms exhibited a senescence inductive activity as determined by an increase in PPARγ gene expression and beta-galctosidase activity. In conclusion, our findings demonstrated that both endogenous and soluble forms of IL-24/mda-7 induced apoptosis and senescence in activated LX-2 cells and more importantly, fusion of RGD peptide to this cytokine enhanced these activities. So, RGD-modified IL-24/mda-7 could be a suitable candidate for further molecular therapy of fibrosis.     

IL-24/MDA-7 Suppressed the Transplantation Tumor in BALB/c Mice

It is well-documented that interleukin-24 （IL-24） can induce apoptosis in a large spectrum of human cancer derived cell lines, but the effect of MDA-7/IL-24 gene transfer on mouse melanoma cells remains unknown. The eukaryotic expressing plasmid of IL-24 （pEGFP-IL-24） was constructed by DNA recombination technique. The recombination plasmid and empty vector were transfected into B16F0 cells and the expressions of IL-24 were determined by LSM, the proliferation of B16F0 cells was measured by MTT assay, and apoptosis rate and cell-cycle distribution of B16F0 cells were measured by FCM. The inhibitory effect of IL-24 gene transfection in mouse solid tumor was observed and measured. Compared with the control, the proliferation of B16F0 cells was inhibited by transfection with pEGFP-IL-24 and the G2/M phase of the transfected cells was also increased. Moreover, the percentage of mice with detectable tumor was decreased after inoculated with B16F0 cells transfected with pEGFP-IL-24. Growth rate of tumor in mouse model was significantly inhibited in IL-24 gene therapy group compared with the control. Proliferation of B16F0 cells was inhibited by pEGFP-IL-24 transfection. The intratumor injection of pEGFP-IL-24 could inhibit the growth of solid tumor in mice remarkably. Cellular ＆ Molecular Immunology.     

Interleukin-7 (IL-7)-induced proliferation of CD8+ T-chronic lymphocytic leukemia cells

Interleukin-7 (IL-7) is a growth factor for pro-B cells, pre-B cells, and thymocytes and is known to induce the proliferation of normal human peripheral T cells. Moreover, human B and T acute leukemia cells with immature surface markers proliferate in response to IL-7. Here we describe a case of T-chronic lymphocytic leukemia, in which the leukemic cells showed a proliferative response to human recombinant IL-7in vitro. The patient was a 74-year-old woman with anemia and thrombocytopenia, whose bone marrow was fibrosed and infiltrated with pathologic cells. Surface markers of the leukemic cells were CD2(+), CD3(+), CD5(+), CD7(+), CD8(+), and CD4(–). Both T-cell receptor -chain and -chain genes were found to be rearranged by immunogenotypic analysis. The leukemic cells proliferated in response to IL-7 dose dependently. The DNA synthesis of CLL cells was stimulated by not only IL-7 but also IL-2 and IL-4. The IL-7-induced proliferation was not inhibited by antibodies to IL-2 receptors or the anti-IL-4 antibody. These findings indicate that IL-7 may induce the proliferation of peripheral CD8⁺ T cells, even on its pathological counterpart.     

MDA-7/IL-24对乳腺癌MDA-MB-231细胞生长抑制和凋亡的影响

 摘要：目的 探讨IL-24基因转染乳腺癌MDA-MB-231细胞的增殖抑制和促凋亡作用。方法 利用脂质体将穿梭质粒pDC316-hIL-24-EGFP瞬时转染至乳腺癌MDA-MB-231细胞,通过RT-PCR检测转染后hIL-24基因mRNA的转录,Western blot检测转染后IL-24和Caspase-3蛋白的表达,MTT比色法测定细胞增殖的抑制, 流式细胞仪检测细胞的凋亡和细胞周期的变化,Hoechst 33258染色检测细胞的凋亡情况。结果IL-24基因可在MDA-MB-231细胞中成功转录及表达。IL-24可上调MDA-MB-231细胞中Caspase-3蛋白表达。IL-24基因的表达使得乳腺癌MDA-MB-231细胞出现增殖抑制。流式细胞仪检测显示MDA-MB-231细胞DNA 合成受到抑制,细胞周期主要抑制在G2/M期。Hoechst 33258染色显示IL-24基因转染后MDA-MB-231细胞出现凋亡。结论IL-24基因转染乳腺癌MDA-MB-231细胞后能够抑制细胞增殖并促进其凋亡,其机制之一可能是上调Caspase-3的表达。     

白细胞介素24(IL-24)的生物学功能及信号通路

白细胞介素24(IL-24)是细胞因子白细胞介素10(IL-10)家族的成员,具有多种生物学功能:抗肿瘤特性,参与机体免疫调节,影响微血管形成.IL-24的信号通路可能与内质网应激途径,神经酰胺介导途径,丝氨酸苏氨酸激酶依赖途径及半胱天冬酶介导途径有关.文章综述了IL-24的生物学功能及信号通路,为临床工作者在肿瘤、炎症等疾病的治疗中提供有效的基因治疗途径.     

Interleukin-7 (IL-7) Knockout Mice. Implications for Lymphopoiesis and Organ-Specific Immunity

Interleukin-7 (IL-7) is produced by both immune and non-immune cells including stromal cell lines, B-cells, monocytes/macrophages, follicular dendritic cells, keratinocytes, and gut epithelial cells. The development of IL-7 knockout mice aided to elucidate the role of this multifaceted cytokine in lymphopoiesis. Additionally, IL-7 genedeleted mice may represent an excellent model in order to define the functional role of locally secreted IL-7 in organ-specific immunity and in anti-microbial responses as well. For instance, analysis of IL-7 gene-deleted mice revealed reduced numbers of total T-lymphocytes with preservation of the CD4/CD8 ratio and increased ratio of αβ⁺ T-cells compared to γδ⁺ T-cells. Transition of pro-T-cells to pre-T-cells was impaired. Cell marker analysis of thymocytes in IL-7-/-mice suggested that IL-7 may induce expression of as yet unidentified cytokine receptors, and that IL-7 may also be critically involved in T-cell differentiation. However, there are clear differences in the requirements of αβ or γβ T-cells for IL-7. In general, IL-7 appears to serve as the major growth and differentiation factor for γβ T-cells. IL-7-/-mice are characterized by a block of maturation of Vγ3^low, CD24⁺ T-cells to Vγ3^high, CD24^low T-cells. Thus, IL-7 does not only represent a ‘maintenance factor’, but rather a cytokine required for sucessful thymic and extrathymic development and maturation of γδ T-cells. γδ⁺ intestinal intraepithelial lymphocytes (iIEL) are absent in IL-7-/-animals. In contrast, αβ⁺ iIEL can be detected in IL-7 gene-deleted animals, but not in γc, or in JAK-3 deficient mice suggesting that alternative cytokines may be involved in development of iIEL αβ⁺ T-cells, but not necessarily for γδ T-cells. To this end, IL-7 has predominantly been studied in the context of B- and T-cell development. With the availability of IL-7 gene-deleted mice, the paracrine effects of IL-7, which may be secreted in vivo by non-immune cells including keratinocytes or gut epithelial cells, can now be critically examined.