融合基因mGM-CSF/hIL-2在肝癌细胞瘤苗特异性免疫治疗中的作用

背景与目的:手术切除是治疗肝癌的主要方法,但对其术后的复发转移却无更好的办法。近年来,免疫学的发展使肝癌的治疗有了更好的治疗方法。本研究旨在通过制备人白细胞介素2(hIL-2)与鼠粒-单核细胞集落刺激因子(mGM-CSF)融合基因修饰的H22肝癌瘤苗,观察其特异性抗肿瘤免疫作用。方法:用含hIL-2与mGM-CSF融合基因的真核表达载体,在体外转染H22细胞,制成疫苗,皮下接种小鼠,同时建立荷瘤小鼠模型。用51Cr释放法测定瘤苗免疫组、空载组、未转染组小鼠脾细胞对亲本H22细胞的杀伤活性。取血检测血清中IL-10、IFN-γ水平,观察小鼠存活期。结果:成功制备了含有hIL-2与mGM-CSF融合基因的H22肝癌瘤苗。免疫小鼠脾细胞体外对H22细胞的杀伤率为38.3%,显著高于对S180细胞的9.1%,以及空载组和未转染组的13.6%和7.5%(P<0.05)。转基因瘤苗免疫组血清IFN-γ为(12.83±0.75)pg/mL,较空载瘤苗组的(7.83±0.65)pg/mL明显升高(P<0.01),血清IL-10[(4.58±0.34)pg/mL]较空载瘤苗组的(8.15±0.28)pg/mL明显降低(P<0.01)。同时,转基因瘤苗免疫组小鼠存活期为(40±6)d,较对照组[空载瘤苗组(30±3)d,未转染组(19±4)d]明显延长。结论:转染hIL-2与mGM-CSF融合基因的同系肿瘤细胞瘤苗可激发特异性细胞介导的免疫反应,改善抗肿瘤免疫反应,延长荷瘤小鼠存活期。     

肿瘤生物治疗新方法的研究

本文简述“九五”攻关项目间“肿瘤生物治疗新方法”且经协作攻关主要完成的研究工作有：重组人TNF、IL－2及B7重组腺病毒表达载体的构建及其包装体系的建立：腺病毒介导的人TNF－α基因转染对人肝癌细胞凋亡和MHC－Ⅰ类分子表达的影响;瘤体内／瘤周注射TNF－α重组腺病毒和（或）IL－2T重组腺病毒对肝癌小鼠的治疗作用及其免疫机理研究。粘附LAK细胞的制备、体外扩增、冻存及复苏;转染B7、IL－2、TNF－α基因重组腺病毒的肿瘤细胞及A－LAK的生物学特性研究;人肝癌组织中MAG基因的表达及MAGE基因修饰树突状细胞体外抗肿瘤作用;转基因瘤苗临床应用安全性的初步性的初步检测,这些研究工作的完成为肿瘤基因治疗的临床应用打下基础。     

腺病毒介导IL-2基因转染的瘤苗体内抗肿瘤作用

目的 :探讨重组腺病毒介导的 IL- 2基因转染的瘤苗的体内抗肿瘤作用及其免疫学机制。方法 :应用腺病毒介导的鼠 IL- 2基因转染 CT2 6小鼠结肠癌细胞 ,灭活后用作瘤苗治疗荷瘤小鼠 ,观察皮下肿瘤生长及其存活期。采用乳酸脱氢酶释放法检测荷瘤小鼠脾细胞 CTL、L AK、NK细胞的杀伤活性。结果 :鼠 IL- 2基因转染瘤苗治疗能显著抑制荷瘤小鼠皮下肿瘤生长并明显延长其存活期 (P<0 .0 1)。体内免疫功能检测表明 ,鼠 IL- 2基因转染疫苗治疗组小鼠脾细胞 CTL 活性、L AK活性和 NK活性显著高于对照组 (P<0 .0 1)。结论 :腺病毒介导鼠 IL- 2基因转染的瘤苗体内具有较强的抗肿瘤效应 ,其机制可能是提高了荷瘤小鼠特异性和非特异性抗肿瘤免疫反应     

TIM2基因修饰对H22细胞的抑制作用及苦参碱的增强作用

目的 观察苦参碱对TIM2基因修饰小鼠H22肝癌细胞瘤苗的体内作用.方法 以携带小鼠TIM2基因的真核表达载体脂质体法转染H22细胞,经体外稳定筛选获得TIM2转基因H22细胞瘤苗.建立小鼠肝癌移植瘤模型,接种TIM2转基因H22细胞瘤苗(H22-TIM2组),观察成瘤情况,并设空载体转染的H22细胞稳定表达株和H22细胞作为对照(H22-EGFP组和H22组);同时,分别加用药物苦参碱治疗,观察苦参碱对不同处理组小鼠肿瘤生长情况的影响.结果成功得到TIM2转基因修饰H22细胞,鉴定有TIM2基因mRNA及EGFP的稳定表达.免疫接种小鼠后,在小鼠体内的成瘤率为41.6%,明显低于H22组和H22-EGFP组(后两者成瘤率在91.6%以上)(P＜0.01),小鼠肿瘤的生长速率也较其他各组明显缓慢,实验结束时H22-TIM2组小鼠肿瘤平均体积为(31.34±9.21)mm3,明显小于H22.EGFP组和H22组[(98.25±25.23)mm3和(114.08±36.45)mm3;P＜0.01].接种H22-TIM2细胞对小鼠肿瘤的抑制率为69.2%,加用苦参碱后,抑瘤率达90.6%,明显高于单用苦参碱组(67.5%)及H22-EGFP和苦参碱联用组(70.8%).结论 TIM2基因修饰H22细胞可显著降低H22肝癌细胞在小鼠体内的致瘤性,抑制小鼠体内H22移植瘤的发生和发展,而苦参碱可明显改善其体内抗癌活性.     

脂质体介导mIL-12基因与MHCⅠ基因联合对小鼠实验性肝癌的治疗作用

背景与目的：白细胞介素-12及MHCⅠ基因均已单独用于肿瘤基因治疗,为探索两者的抗肿瘤协同效应,本研究探讨小鼠白细胞介素-12（mIL-12)基因与同种异型的MHCⅠ（小鼠为H-2K）基因联合治疗Balb/C小鼠实验性肝癌。方法：分别应用含mIL-12基因,C57BL/6小鼠H-2K^bcDNA及绿色荧光蛋白（GFP）报告基因的真核表达质粒载体pcDNA3。体外经新型脂质体LipofectAMINE2000(LF2000)介导转染小鼠肝癌细胞株MM45T.Li,检测转染细胞外源基因的表达,将经LF2000介导pcDNA3/mIL-12与pcDNA3/H-2K^b转染的MM45T.Li细胞,注射于小鼠皮下,观察致瘤性的变化,在荷瘤Balb/C小鼠瘤内注射上述脂质体-DNA复合物,观察瘤体生长情况及小鼠生存期的改变。结果：LF2000介导pcDNA3/GFP转染MM45T.Li细胞的最佳条件为脂质体与DNA为3：1（μg：μg）,转染效率达30%,经LF2000介导pcDNA3/mIL-12与pcDNA3/H-2K^b转染的MM45T.Li细胞,RT-PCR检测有mIL-12和H-2K^bcDNA的特异扩增片段,WesternBlot检测显著有57kDaH-2K^b蛋白表达,ELISA检测mIL-12分泌量达48ng/ml/10^6细胞,经LF2000介导pcDNA3/mIL-12与pcDNA3/H-2K^b转染的MM45T.Li细胞,其致瘤性下降;荷瘤Balb/C小鼠瘤内注射该脂质体-DNA复合物,FACS检测显示小鼠脾脏淋巴细胞中CD3^ ,CD4^ 和CD8^ 的数量治疗组较对照组高,肿瘤生长相对缓慢,且pcDNA3/mILp-12与pcDNA3/H-2K^b联合治疗具有一定的正协同效应。结论：mIL-12基因与MHCⅠ基因联合治疗小鼠肝癌具有正协同效应,增强了抗肿瘤效果。     

自体肿瘤瘤苗对荷瘤鼠Th1、Th2型细胞因子及细胞毒作用的影响

目的研究经处理的H22肝癌细胞肿瘤瘤苗作为全细胞瘤苗对H22荷瘤小鼠体内Th1/Th2细胞比例和细胞因子的影响以及CTL的杀伤活性.方法用加重组白细胞介素2、重组粒细胞单核细胞集落刺激因子及福氏不完全佐剂制成疫苗,建立荷瘤小鼠模型,用51Cr释放法测定瘤苗免疫组、荷瘤组、正常组小鼠脾细胞对亲本H22肝癌细胞的杀伤活性;流式细胞仪检测单个核细胞中的Th1和Th2细胞,并取血检测血清中IL-10、IFN-γ水平.结果效靶比为200:1时,免疫小鼠脾细胞体外杀伤亲本H22肝癌细胞的杀伤率为38.3%,显著高于荷瘤组的13.6%,正常组的7.5%,以及对S180细胞的9.1%(P均＜0.05).瘤苗免疫组Th1细胞及Th1/Th2细胞的比值显著升高(P＜0.01),血清IFN-γ较对照组明显升高(P＜0.01);血清IL-10较对照组明显降低(P＜0.01).结论肿瘤细胞加小剂量IL-2和GM-CSF及佐剂组成的肿瘤细胞瘤苗可激发特异性细胞介导的免疫反应,改善抗肿瘤免疫反应.     

Smad4过表达对人胃癌细胞增殖和NF-κB通路的影响

目的: 构建肝癌细胞特异性小鼠IL1β反义RNA表达载体,观察其对H22肝癌细胞小鼠移植瘤生长的影响及其相关机制。方法:构建由AFP最小启动子和CMV增强子嵌合序列调控的携IL1β反义RNA表达载体pafpIRES2antiIL1β1和pafpIRES2antiIL1β2,经质粒PCR、限制性酶谱分析、序列测定进行鉴定。反义RNA表达载体转染小鼠H22肝癌细胞,分H22/mock组、H22/antiIL1β1组、H22/antiIL1β2三组,RTPCR检测IL1β的表达水平。以转染后的H22细胞皮下接种建立荷肝癌小鼠模型,观察移植瘤体积和重量,MTT法检测荷瘤小鼠脾脏中分离的NK细胞对H22细胞的杀伤活性。〖HT5W〗结果: 〖HT5"SS〗 经质粒PCR、限制性酶谱分析、序列测定证实成功构建能够在肝癌细胞中特异性表达IL1β反义RNA的表达载体pafpIRES2antiIL1β1和pafpIRES2antiIL1β2,转染H22细胞后细胞中IL1β表达水平明显下降,以pafpIRES2antiIL1β2更为显著。成功建立荷肝癌小鼠模型,与H22/mock组小鼠相比, H22/antiIL1β2组小鼠移植瘤体积较小,生长显著减慢（P<0.05)。 H22/antiIL1β1、H22/antiIL1β2组荷瘤小鼠的NK细胞对H22细胞的杀伤活性明显增强(P<0.05或P<0.01)。结论: 成功构建的肝癌细胞特异性IL1β反义RNA表达载体可有效抑制小鼠移植肝癌的生长,其机制与靶向阻断IL1β表达、上调NK细胞的杀伤活性有关     

巨噬细胞过继免疫治疗的实验研究

目的 研究脂质体瘤苗激活小鼠巨噬细胞的过继免疫治疗作用.方法 制备脂质体H22肝癌瘤苗,脂质体S180瘤苗,Freund's完全佐剂H22肝癌瘤苗以及对照脂质体瘤苗并免疫小鼠.用Hanks液清洗免疫鼠的腹腔收集糖原诱导的腹腔巨噬细胞.实验鼠腹腔注射1×105个H22肝癌细胞24小时后再腹腔给予各供体巨噬细胞5×106个/只.结果 脂质体H22瘤苗和Freund's佐剂H22瘤苗与对照组相比较,其免疫鼠提供的巨噬细胞具有较强的过继免疫治疗作用.结论 脂质体瘤苗激活的小鼠巨噬细胞具有特异性的可过继转移的抗瘤作用.     

MHC半相合脾加骨髓细胞诱发H22荷瘤鼠的抗肿瘤效应

目的：观察MHC半相合脾加骨髓细胞移植抗小鼠H22实体瘤的效果。方法：以皮下接种H22肝癌细胞的BALB/c×C57BL/6杂交F1代雌性小鼠为受鼠,以健康雌性F1、雄性C57BL/6、雄性C3H小鼠为MHC全相合、半相合、不相合供鼠,观察移植后的抑瘤情况；观察供鼠细胞经~(60)Co照射的MHC半相合移植对WBC、生化和嵌合体的影响；比较供鼠细胞经与不经~(60)Co照射的MHC半相合移植的GVHD情况。结果：供鼠细胞经/不经~(60)Co照射的MHC半相合移植小鼠的肿瘤明显较小,与单纯化疗未进行移植者比较,差异具有统计学意义(P＜0．05)；但受鼠未经化疗预处理的MHC半相合细胞输注没有出现抗肿瘤效应；供鼠细胞经7．5 Gy ~(60)Co照射的MHC半相合移植能明显降低GVHD反应,且对外周血白细胞、生化无不良影响。结论：经7．5 Gy~(60)Co照射的MHC半相合脾加骨髓细胞移植能对H22肝癌细胞产生移植物抗肿瘤效应并降低GVHD反应。     

H22-pEGFP-C1-FL肝癌细胞瘤苗构建及其生物活性的研究

目的:构建H22-pEGFP-C1-FL肝癌细胞瘤苗,研究其活体内应用的生物学活性.方法:通过G418筛选能高表达hFL蛋白H22-pEGFP-C1-FL的肝癌细胞株;小鼠尾静脉注射经放射线灭活的H22-pEGFP-C1-FL的肝癌细胞,检测注射后hFL蛋白表达情况及对骨髓抑制小鼠的升白作用.结果:获得高表达hFL蛋白的H22肝癌细胞.该细胞经辐射灭活后,输注给辐射后骨髓抑制的小鼠,与对照组相比,可快速升高白细胞(P<0.01),显著提高辐射小鼠的存活率(P<0.01).结论:成功构建了H22-pEGFP-C1-FL肿瘤细胞瘤苗,为其临床应用奠定基础.     

白细胞介素2基因治疗耐三苯氧胺乳腺癌的实验研究

为探讨耐三苯氧胺（Ｔａｍｏｘｉｆｅｎ，ＴＡＭ）乳腺癌细胞因子基因治疗的可行性及效果，在体外将逆转录病毒载体介导的白细胞介素２（ＩＬ－２）基因导入耐ＴＡＭ大鼠乳腺癌细胞株Ｙ２，并进行了动物体内抗肿瘤实验研究。结果显示：同亲代Ｙ２细胞比、基因修饰的ＹＩＬ－２细胞形态和细胞生长特性发生改变。ＰＣＲ显示ＩＬ－２基因成功地整合到ＹＩＬ－２细胞内。ＹＩＬ－２细胞（２．０×１０６个／只）在大鼠右腋皮下失去致瘤性，并阻断等量混合的Ｙ２细胞的致瘤性，但阻断作用随亲代细胞数量的增多而减弱。同样数量无ＩＬ－２基因转移的Ｙ２细胞致瘤率为１００％。结果表明ＹＩＬ－２细胞具有抗肿瘤活性。     

Complete tumor prevention by engineered tumor cell vaccines employing nonviral vectors

We report that 100% mice survival after tumor challenge is achieved with cytokine-engineered cells employing nonviral lipoplexes and without using viral vectors. We describe this effect with cytokine-secreting tumor cell vaccines, based on cell clones or fresh transfected cells. Tumor cells were transfected with murine granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-4 plasmids employing the cationic lipid DOTAP, were irradiated (150 Gy) and kept frozen until use. The transfection efficacy was analyzed by qRT-PCR and flow cytometry. Vaccination induced potent antitumor rejection, resulting in 100% mice survival. Furthermore, the antitumor immunity was long lasting, since a two-fold survival delay was observed in mice after tumor rechallenge (6 months later). While cell clones secreting GM-CSF were the most effective in wild-type tumor cell rejection, little or no effect was observed with clones secreting IL-4. We found similar antitumor efficacy employing fresh transfected cells by nonviral procedures, demonstrating that cells genetically modified by nonviral vectors (both clones and fresh transfected cells) are a safe and efficient tool for antitumor vaccines. These vaccines allow us to achieve the highest antitumor efficacy based on nonviral gene therapy techniques. In addition, the vaccination success with fresh transfected cells simplifies the procedure and provides new insights into the clinical application of nonviral gene therapy procedures.     

表达白细胞介素-12质粒DNA抗小鼠肝癌皮下移植瘤的作用

目的 探讨瘤内注射mIL-12质粒DNA抗小鼠肝癌皮下移植瘤的作用。方法：构建真核表达质粒载体pDC511mIL-12，ELISA方法检测质粒载体在真核细胞中的表达，淋巴母细胞增殖法检测mIL-12的生物学活性；分别于小鼠肝癌H22皮下移植瘤内直接注射质粒DNA，观察各组小鼠存活时间、肿瘤体积变化及各组小鼠脾脏细胞毒T淋巴细胞(CTL)的活性：注射质粒DNA后1月进行瘤体组织病理学观察：结果：mIL-12基因治疗组与空载体对照组相比，肿瘤生长显著受抑制(F=4．10，P=0．03)，小鼠存活期显著延长(X^2=4．48，P=0．03)．并且小鼠脾细胞CTL杀伤活性增强。质粒DNA瘤内注射1月后，pDC511mIL-12组肿瘤病灶炎性细胞浸润明显，病灶内肿瘤细胞广泛坏死。结论：瘤内注射mIL-12表达质粒DNA可抑制小鼠肝癌皮下移植瘤生长，能提高机体抗肿瘤免疫应答。     

阳离子脂质体介导HSV-TK/ACV基因系统抑制人肺癌生长的实验研究

目的:探讨阳离子脂质体介导的HSV-TK/ACV基因系统治疗人肺癌的实验研究的意义.方法:利用已构建的真核表达质粒pCR3-TK,用阳离子脂质体LipofectAMINE为载体,将pCR3-TK转染Anip973细胞株,然后给予不同浓度的Aciclovir(ACV).结果:经MTT检测证实转染了TK基因的肺癌细胞对ACV的杀伤敏感性明显提高,FCM检测可以看到凋亡峰的出现,且相应的S期细胞比率增高.结论:HSV-TK/ACV系统对人类肺癌细胞株Anip973具有良好的抑制作用;阳离子脂质体LipofectAMINE对人类肺癌细胞株Anip973安全、低毒,有可能成为基因治疗应用到临床试验的有价值的载体.     

Vaccination with allogeneic GM-CSF gene-modified lung cancer cells: antitumor activity comparing with that induced by autologous vaccine

OBJECTIVE: The aim of this study was to investigate the whole allogeneic (differing tissue-type) tumor cells as vaccine in the mouse lung cancer model. The immunogenic and antitumor activity of allogeneic vaccine was compared with that of autologous cancer cell vaccine. METHODS: C57/BL mice inoculated with Lewis lung cancer (LLC) cells were used as the animal model to test the effects of allogeneic vaccination. LA795 and LLC lung cancer cell lines, which were transfected with the mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) gene, were administered as allogeneic and autologous tumor vaccine, respectively. The irradiated tumor cells were administered as subcutaneous vaccines before the tumor challenge. The immunity of cancer vaccine was tested by mouse interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) lactate dehydrogenase (LDH) assays. The serum level of IFN-gamma and interleukin (IL)-4 was tested using the enzyme-linked immunosorbent assay method. RESULTS: Prophylactic vaccination with allogeneic LA795 cells protected against the LLC tumor challenge in C57/BL. The tumor growth was inhibited and the survival was accordingly prolonged. The cytotoxicity of the spleen cells or the purified CD(8)(+) T-cells against LLC cells in the mice immunized with either the autologous or allogeneic cancer cell vaccine was significantly increased, relative to that of the control, untreated group (p<0.05). ELISPOT IFN-gamma assays showed that spleen cells from mice immunized with LA795 cells could be activated after coculture with irradiated LLC cells. In addition, the serum level of Th1-king cytokine IFN-gamma significantly increased after vaccination; however, no statistically difference was found in Th2-kind cytokine IL-4. CONCLUSIONS: The allogeneic cancer vaccine could induce immune responses and protection against lung cancer, which had no significant difference with that of autologous vaccine.     

Induction of antitumor immunity with combination of HER2/neu DNA vaccine and interleukin 2 gene-modified tumor vaccine.

The therapeutic effects of both cytokine-secreting tumor vaccine and DNA vaccine were studied using mouse MBT-2 bladder cancer cells as a model. Cytokine-secreting MBT-2 cells were obtained by infecting cells with retroviral particles containing interleukin (IL) 2-, IL-4-, or granulocyte-macrophage colony-stimulating factor (GM-CSF)-expression vector. The MBT-2-IL-2 cells were not tumorigenic in syngenic C3H mice at all. Tumor formation decreased significantly for the MBT-2-GM-CSF cells. MBT-2-IL-2, -IL-4, and -GM-CSF cells were killed by irradiation and tested as tumor vaccines. The irradiated MBT2-IL-2 cells could complete protect mice from the growth of the preexisting tumor cells, and the immune memory lasted for 8 months. On the other hand, irradiated MBT-2-IL-4 and MBT-2-GM-CSF cells were less effective. When the loading tumor mass increased, all tumor vaccines lost protective effects. DNA vaccine encoding the tumor antigen neu was additionally tested to improve the therapeutic efficacy. Coinjection of 60 microg pSV-neu DNA was effective in enhancing the antitumor effects of MBT2-IL-2; however, DNA vaccine alone cannot prevent the progression of the preexisting tumor. Immunohistochemical analysis of tumor infiltrate revealed massive increase of CD4+ lymphoid cells in the group of mice treated with both DNA vaccine and IL-2-secreted tumor vaccine. Western blotting demonstrated the presence of anti-neu antibody in the serum from immunized mice. In contrast, combination of DNA vaccine and MBT-2-GM-CSF has no additive effect. The results indicate the combination of DNA vaccine and IL-2-secreting tumor vaccine can additionally improve therapeutic efficacy, and the efficacy is correlated with the increase of CD4+ T lymphocytes and anti-neu antibody.     

A convenient cancer vaccine therapy with in vivo transfer of interleukin 12 expression plasmid using gene gun technology after priming with irradiated carcinoma cells

We studied interleukin (IL)-12 gene therapy using a gene gun as a new autologous vaccination strategy for cancer. In the first experiment, BALB/c mice were inoculated with syngeneic murine renal cancer cells (Renca) intradermally in the abdomen. This was followed by an injection of IL-12 expression plasmid using the gene gun. About 40% of the mice exhibited rejection of the tumor after the treatment and these mice also acquired immunological resistance against a secondary challenge with Renca cells. Based on these results, we examined whether antitumor activity can be potentiated when mice undergo combination treatment with intradermal inoculation of irradiated Renca cells and transfection with IL-12 gene. Inoculation of irradiated Renca cells alone was partially effective in inducing antitumor immunity, whereas the combined treatment remarkably intensified this effect. Moreover, this combined treatment inhibited tumor establishment and enhanced survival of the mice with tumor infiltration by CD4(+) and CD8(+) T cells, even when the treatment was started after tumor-implantation at a distant site. This antitumor effect was antigen specific and we confirmed the induction of antitumor cytotoxic T cells by this treatment. These results show that local cutaneous transfer of IL-12 expression plasmid using gene gun technology enhances systemic and specific antitumor immunity primed by irradiated tumor cells.     

p14~(ARF)对照射后肺癌细胞加速再群体化抑制效应

目的　探讨抑癌蛋白p14ARF功能恢复对照射后肺癌细胞加速再群体化的影响。方法　以ARF基因纯合缺失但表达野生型p5 3的人肺腺癌A5 49和H46 0细胞系为靶细胞 ,应用Fugene 6对照射后的细胞进行pCI neo p14ARF 表达载体基因转染 ,检测照射前后和转染前后细胞潜在倍增时间(Tpot)、细胞周期分布及克隆存活率变化。结果　 6GyX射线照射后 96h处 ,A5 49和H46 0细胞Tpot分别缩短 2 5 .4%和 2 9.2 % ,与照射前比较差异有显著性意义 (P <0 .0 1)。此时进行p14ARF表达载体基因转染 ,恢复p14ARF功能 ,可使A5 49和H46 0细胞Tpot延长并接近照射前水平 ,同时伴随G0 G1 期和G2 M期细胞显著增加 ,S期细胞显著下降 ,克隆存活率下降。结论　A5 49和H46 0细胞照射后存在加速再群体化。p14ARF功能恢复对其照射后加速再群体化有抑制作用 ,该作用与p14ARF的G1 、G2 期阻滞功能密切相关