【作者】 于会明；

【导师】 侯明；

【作者基本信息】 山东大学 ， 内科学， 2009， 博士

【摘要】 第一部分体内实验背景肺癌是目前发病率和病死率均很高的恶性肿瘤,严重影响着人类健康。肺癌是一种基因病,癌基因激活和抑癌基因失活是其重要的分子事件。因此,阐明肺癌发生的分子机制,从分子水平阻断其发生的相关链接具有极为重要的临床意义。K-ras是目前研究最多的癌基因之一,在许多肿瘤中均存在K-ras基因的扩增或过度表达,其中在肺腺癌中大约有50%左右的患者存在K-ras突变,说明其在肺癌发生、发展中发挥重要作用。深入研究K-ras基因在肺癌发生中的作用机制,尽早阻断其信号系统已成为近年来肺癌研究领域中一个新的热点。大约有100多种细胞内蛋白具有CAAX基序,K-ras基因编码蛋白属于其中的一种,这些蛋白可以进行多种转录后修饰,其中半胱氨酸残基（C末端CAAX基序）可直接进行异戊二烯化修饰。一部分CAAX蛋白,其末端可以在牛龙酰基转移酶Ⅰ（GGTase-I）的作用下进行牛龙基化修饰,还有一部分CAAX蛋白,例如K-Ras,在法呢酰基转移酶（FTase）的作用下进行法尼基化修饰,其中的“X”残基表明这种CAAX蛋白是被法尼基化还是牛龙基化修饰:一般情况下,如果“X”是亮氨酸,这种蛋白就会被牛龙基化修饰,否则就会被法尼基化修饰。CAAX蛋白末端进行异戊二烯化修饰后可以增强其蛋白羧基端的疏水性,进而增强膜的亲和力及与配体的结合能力,从而增强其生物学活性。FTase和GGTase-I有一个相同的α亚单位,但是分别具有各自独特的表明其底物特异性的β亚单位。例如,GGTase-I是由Fnta编码的一个α亚单位和Pggtlb编码的独特的β亚单位组成。GGTase-I是许多真核细胞的必需酶,在果蝇和酵母菌种GGTase-I酶的β亚单位的无效突变通常是致死性的。GGTase-I缺乏导致生物死亡的主要原因是Rho1p和Cdc42p蛋白无法进行牛龙基化,而这种牛龙基化在特定条件下可通过FTase进行法尼基化弥补。RAS蛋白的法尼基化已引起学者的足够重视,研究表明,Ras蛋白的法尼基化对其靶向定位于细胞膜上的结合位点和保持正常的转化能力是非常必须的。在动物模型中,法呢酰基转移酶抑制剂（FTIs）具有高效的抗肿瘤活性且毒性低,而在临床试验中,FTIs的疗效并不理想,其可能原因是FTase缺乏时RAS蛋白进行了牛龙基化。许多研究已证实GGTIs能抑制多种K-ras突变肿瘤细胞株的增殖,同时研究还表明GGTIs能改善多发性硬化症动物模型的表型以及抑制肝癌中肝炎病毒的复制等。鉴于GGTase-I和FTase在K-ras突变诱导肿瘤发生中的作用以及前期研究结果,本研究旨在应用条件性敲基因技术建立动物模型,并运用组织病理学、分子生物学、免疫学等研究手段,充分阐明条件性敲除GGTase-I的β亚单位（Pggtlb）或FTase的β亚单位（Fntb）后,阻断CAAX蛋白异戊二烯化修饰,从而抑制K-ras基因突变诱导的肺癌发生的作用,为肺癌的基因治疗提供科学依据。目的（1）利用条件性基因敲除技术建立符合人类肺癌病变特征的动物模型;（2）应用组织病理学等方法观察肺癌组织病理形态,探讨Pggtlb或Fntb基因敲除对K-ras基因突变诱导肺癌形成的抑制作用;（3）探讨Pggtlb或Fntb基因敲除抑制K-ras基因突变诱导肺癌发生的分子生物学机制。方法1.条件性敲基因动物模型的建立及其基因型验证利用Cre/lox P系统条件性敲入K-ras基因,敲除GGTase-I或FTase基因,分别建立K-ras(K^LSL)、GGTase-I(Pggtlb^fl/fl)和FTase(Fntb^fl/fl)条件性敲基因鼠模型。然后以K^LSL鼠和Pggtlb^fl/fl鼠或Fntb^fl/fl鼠杂交产生K^LSLPggtlb^fl/fl鼠和K^LSLFntb^fl/fl鼠,经基因分型后筛选出符合要求的小鼠。应用PCR技术进行基因型验证。2.实验动物分组按基因型分为三组:即K^LSL（A组）、K^LSLPggtlb^fl/fl（B组）和K^LSLFntb^fl/fl（C组）,各组根据感染病毒种类的不同又分为两个亚组即:cre腺病毒载体感染组（实验组:A1组、B1组和C1组）和β-gal腺病毒载体感染组（对照组:A2组、B2组和C2组）。3.干预方法第14天龄时应用雾化吸入法进行腺病毒载体干预,分别敲除或激活K^LSL、K^LSLPggtlb^fl/fl和K^LSLFntb^fl/fl基因。4.称量体重病毒感染前及以后每周末称量所有实验鼠体重。5.各组动物生存率测定自基因干预起,观察各组动物的生存时间。6.病理学检查不同时间点处死动物,留取肺脏、肝脏、脾脏等组织器官并称量其重量,一部分组织进行苏木素-伊红（HE）染色,观察肿瘤细胞形态;另一部分留取标本进行分子生物学检测。7.免疫组织化学染色进行CD4、CC10、CD8、SP-C、非异戊烯化Rap1A及Ki-67的免疫组织化学染色。8.Real-time PCR检测肺脏、肝脏和脾脏等组织器官中Pggtlb、Fntb和K^LSL等基因的mRNA表达水平。9.Western blot检测肺脏肿瘤组织中非异戊烯化RAP1A、总RAP1A、p-ERK1/2、p-AKT、总AKT、总ERK1/2等的蛋白表达水平。10.统计学分析连续性数据用（?）±SD表示,离散数据用例数和百分数表示。三组间生存率的比较应用Kaplan-Meier方法,其余指标应用单因素方差分析。应用SPSS统计软件进行统计学处理（version 11.0;SPSS Inc）,P＜0.05有统计学差异。结果1.实验动物基本情况腺病毒载体干预动物的共60只。最终完成基因干预的共58只,其中A1组10只,A2组9只,B1组10只,B2组9只,C1组10只,C2组10只。本文对完成实验的58只实验鼠的资料进行分析。2.各组动物的基因分型共有6个亚组,分别是A1组(K^G12D组);B1组(K^G12DPggtlb^△/△组);C1组(K^G12DFntb^△/△组)和A2组(K^LSL组);B2组(K^LSLPggtlb^fl/fl组):C2组(K^LSLFntb^fl/fl组)。3.基因敲除率测定为验证Cre介导的基因敲除效果,应用定量PCR分析了基因组DNA和cDNA。与脾脏比较,肺脏各基因的敲除率均显著增高（P均＜0.05）。4.各组实验动物体重的变化各组实验鼠的基础体重无显著性差异。A1组、B1组和C1组治疗后体重增加,但增加程度均显著低于对照组（P＜0.01）。在A1组体重增加缓慢,由基础状态的（9.16±0.42）增至3周末的（10.10±0.36）,B1组和C1组体重增加趋势相似,组间未见显著性差异（P＞0.05）。5.各组动物肺脏与体重比值（LW/BW）变化与A1组比较,B1组、C1组及对照组均显著降低（P均＜0.01）,在Cre感染后3周时,A1组肺体重显著增加,这可能与其肿瘤形成有关。6.各组实验动物脾脏与体重（SW/BW）比值变化3周末时,与对照组比较,A1组、B1组和C1组显著增高（P均＜0.01）;而A1组、B1组和C1组间无统计学意义。5周末时,与A1组比较,B1组、C1组及对照组均显著降低（P均＜0.01）。7.各组实验动物生存率分析对照组生存率显著高于实验组（P均＜0.01）;与A1组比较,B1和C1组生存率显著延长（P＜0.01）,而B1组和C1组间无统计学意义。A1组在Cre感染后10天内未见有肺癌相关的死亡,但其后生存状态迅速恶化,绝大部分在3周内死于呼吸困难。8.病理学检查3周时均可见非典型腺瘤性增生（AAH）、支气管上皮细胞增生（EH）和腺瘤三种病理改变,但A1组病变更为明显且出现弥漫性腺癌阻塞肺泡腔。5周时,可见弥漫性腺癌细胞堵塞了大部分肺泡腔。肿瘤细胞具有深染的、不规则的细胞核,核浆比例明显失调,癌细胞散在或融合成片,肿瘤细胞团直径显著增大,B1组和C1组病变程度较A1组明显减轻。正常的肺脏未见肿瘤组织,说明相关基因未被激活。9.免疫组织化学染色免疫组化染色显示在肺肿瘤组织中单核细胞/巨噬细胞标志CD11b、CD4/CD8染色均为阴性,细支气管细胞标志CC10染色部分为阴性,部分阳性;肺泡Ⅱ型细胞标记SP-C染色为阳性。与A1组比较,B1组和C1组SP-C和Ki-67染色明显减弱。10.Western blot检测B1组和C1组中可见非异戊烯化RAP1蛋白表达明显增高,对照组和A1组无表达;在B1组和C1组均可见P-AKT蛋白表达水平显著升高,A1组、B2组和C2组无明显变化;在实验组P-ERK1/2蛋白表达均降低,其中B1组降低最为显著。各组间总AKT和总ERK1/2蛋白表达水平无显著差异。结论（1）利用条件性敲基因技术定时、定点的敲入癌基因K-ras能建立符合人类肺癌病变特征的动物模型;（2）条件性敲除Pggtlb基因或Fntb基因能抑制K-ras突变诱导肺癌的发生和发展;（3）Pggtlb基因或Fntb基因敲除后抑制K-ras突变诱导肺癌发生和发展的分子机制是抑制Ras蛋白的转录后修饰,从而下调Ras蛋白依赖性激活的ERK的蛋白表达、抑制肿瘤细胞的增殖能力。第二部分体外部分背景基因突变是肿瘤发生、发展的重要原因,在多种肿瘤中均存在癌基因的突变。非小细胞肺癌（Non-small cell lung cancer,NSCLC）约占肺癌的70%,它是目前世界上发病率和死亡率最高的恶性肿瘤之一,严重影响着人类健康。在NSCLC中,K-ras是最常见的癌基因之一,它可促进肺癌的发生、发展。因此,K-ras基因在诱发肺癌发生、发展中的作用日益引起学者的重视。研究表明,RAS蛋白属于具有CAAX基序的细胞内蛋白的一种,这类蛋白末端的半胱氨酸羧基可在牛龙酰基转移酶Ⅰ（GGTase-I）催化下进行牛龙基化修饰,亦可在法呢酰基转移酶（FTase）催化下进行法尼基化修饰,这两种转录后修饰被称之为异戊二烯化。GGTase-I和FTase具有共同的α亚单位,而其β亚单位的差异决定了其底物的特异性。在动物实验中,利用条件性基因敲除技术敲除GGTase-I或FTase基因后可抑制K-ras诱导肺癌的发生、发展,但敲除GGTase-I或FTase基因对鼠胚胎成纤维细胞（mouse embryo fibroblast,MEF）活力影响的文献尚未见报道。因此,为进一步探讨GGTase-I或FTase基因MEF的影响,本研究旨在应用条件性敲基因技术建立K-ras、GGTase-I和FTase基因的条件性敲除细胞模型,并运用分子生物学、免疫学等研究手段,阐明GGTase-I或FTase基因对K-ras突变细胞株的影响及其分子机制,为诊治K-ras基因诱导的肺癌提供科学依据。目的（1）利用条件性敲基因技术建立建立K-ras和GGTase-I或FTase基因的条件性敲基因MEF模型;（2）观察K-ras基因突变对各组细胞株生物特性的影响;（3）研究条件性敲除GGTase-I或FTase基因后对K-ras突变生物特性的影响;（4）探讨条件性敲除GGTase-I或FTase基因后抑制K-ras突变MEF生物特性的分子机制。方法1.MEF细胞株的获取以胎龄为12.5-17.5天的K^LSL、K^LSLPggtlb^fl/fl和K^LSLFntb^fl/fl鼠为对象,取其胚胎,获取K^LSLMEF、K^LSLPggtlb^fl/fl和K^LSLFntb^fl/flMEF。2.实验细胞分组:按基因型分为三组:即K^LSL（A组）、K^LSLPggtlb^fl/fl（B组）和K^LSLFntb^fl/fl（C组）,各组根据感染病毒种类的不同又分为两个亚组即:Cre腺病毒载体感染组（实验组,A1组、B1组和C1组）和β-gal腺病毒载体感染组（对照组,A2组、B2组和C2组）。3.实验细胞腺病毒感染:应用cre和β-gal腺病毒载体分别感染各组细胞株。4.基因分型:利用PCR技术对各组细胞株进行基因分型。5.Real-time PCR:检测各组细胞株GGTase-I、FTase和K^LSL的mRNA的表达水平。6.各组MEF生物特性检测:分别检测各组细胞株的生长情况、克隆形成能力、迁移能力和凋亡情况。7.统计学分析:连续性数据用（?）±SD表示,离散数据用例数和百分数表示。组间生长曲线比较应用单因素方差分析,其余指标应用T检验。应用SPSS统计软件进行统计学处理（version 11.0;SPSS Inc）,P＜0.05有统计学差异。结果1.MEF基因敲除率检测:实验组中均可见K^LSL的活化及GGTase-I和FTase的敲除且其敲除率显著高于体内实验,而对照组中未见K^LSL的活化及GGTase-I或FTase的敲除。2.各组MEF基因干预后基因型检测各组基因分型与体内部分相同。3.各组MEF生长能力测定在实验组中,与B1组、C1组比较,A1组生长显著增快,而B1组和C1组间无显著性差异。对照组中各亚组间无显著性差异。A1组和A2组、B1组和B2组、C1组和C2组间比较均有显著性差异。4.各组MEF迁移能力测定12h和24h时A1组与A2组、B1组与B2组和C1组与C2组之间均有显著性差异;A1组显著高于B1组和C1组,而对照组中各亚组间无显著性差异。5.各组MEF克隆形成检测只有A1组形成克隆,而B1组和C1组细胞未见克隆形成。对照组中亦未见克隆形成。6.各组MEF凋亡检测A1组MEF的凋亡率为3.31±1.34%,B1组为13.91%±1.88%,C1组为9.57%±2.45%。与A1组比较,B1组和C1组的细胞凋亡率显著增高,且B1组显著高于C1组,而对照组中各亚组间无显著性差异。7.Western blot与A2组比较,A1组Caspase-3的蛋白表达水平显著降低,在B组和C组中,与各自亚组比较,B1组和C1组Caspase-3的蛋白表达水平显著增高。磷酸化-P70蛋白表达水平在各组间无显著性差异。在B组和C组中,与各自亚组比较,B1组和C1组PARP的蛋白表达水平显著增高,而A1组与A2组间无显著性差异。结论（1）应用条件性敲基因技术可建立同时伴有K-ras活化和GGTase-I或FTase基因敲除的细胞株;（2）K-ras基因突变显著增强细胞的增殖能力、迁移能力和克隆形成能力并抑制其凋亡能力;（3）条件性敲除GGTase-I或FTase基因后可显著抑制细胞的增殖能力、迁移能力和克隆形成能力并增强其凋亡能力;（4）GGTase-I或FTase可作为治疗K-ras基因活化诱发的肺癌的靶基因;（5）敲除GGTase-I或FTase基因作用于K-ras突变MEF的主要机制是:抑制K-ras蛋白转录后修饰,增强凋亡基因表达,促进细胞凋亡。更多还原

【Abstract】 PartⅠstudy in vivoBackgroundRecent studies have demonstrated that lung cancer is the leading morbility and fatality rate among malignance tumor in the worldwide.Lung cancer is a gene disease; its genesis is a multistep process involving the cooperation of oncogenic mutations and antioncogenic inactivation.Therefore,elucidating the molecular mechanism and blockage the correlated link of lung cancer is very significance in clinical study.K-ras and P53 gene are the most common oncogenes and anti-oncogene.For example,K-ras mutations occur with high frequency in human colon,pancreas and lung cancer,and the presence of this strongly correlates with disease progression to malignancy. Moreover,there is recent evidence that activation of Ras to promote cancer progression in mouse models for lung.Recently,the study of the role of K-ras gene in the genesis of lung cancer has been the hot study.More than 100 intracellular proteins contain a CAAX motif that directs isoprenylation at a carboxyterminal cysteine（the "C" of the CAAX motif）.Some CAAX proteins,such as RHOA,cell division cycle 42（CDC42）,and RAP1,are geranylgeranylated by protein geranylgeranyltransferase typeⅠ（GGTase-I）.Others, such as K-RAS and N-RAS,are farnesylated by protein farnesyltransferase（FTase）. If the "X" of the CAAX motif is a leucine,the protein is generally geranylgeranylated; otherwise,it is farnesylated.Isoprenylation renders the carboxyl terminus of the CAAX proteins more hydrophobic,enhancing their ability to bind to membranes within cells,and also regulates protein-protein interactions.GGTase-I and FTase share a commonαsubunit but have uniqueβsubunits that dictate their substrate specificities.Theirβsubunits are called Pggtlb or Fntbs,respectively.In some eukaryotic cells,GGTase-I is an essential enzyme.Null mutations in theβsubunit of GGTase-I are lethal in both Drosophila melanogaster and Saccharomyces cerevisiae.The lethality of GGTase-I deficiency in eukaryotic cells was likely due to the failure to geranylgeranylate Rho1p and Cdc42p,as the lethality could be overcome by expressing mutant Rho1p and Cdc42p proteins engineered to undergo farnesylation by FTase.The realization that the RAS proteins are farnesylated has fueled interest in protein isoprenylation.Farnesylation is important for the proper membrane targeting of RAS proteins and for their transforming ability. In mouse models,farnesyltransferase inhibitors（FTIs） have significant antitumor activity and minimal toxicity.In human clinical trials,however,FTIs have been disappointing,at least for the treatment of solid tumors,likely because K-ras and N-ras -the Ras isoforms most often implicated in human cancer-can be geranylgeranylated in the presence of an FTI.At the same time,inhibition of GGTase-I ameliorated disease phenotypes in a mouse model of multiple sclerosis, inhibited hepatitis C viral replication in hepatoma cell.According to the inhibitory role of GGTase-I or FTase gene in K-ras mutation induced lung cancer and the results of fundament study.The present study was designed to test the hypothesis that the role of inactivation of Pggtlb or Fntb gene in K-ras induced lung cancer due to its anti-proliferation in a mouse model of lung cancer created mice with a conditional knockout allele.The result of this study will provide science evidence for gene therapy.Objective（1） To establish an animal model of lung cancer that are mimic to human pathological changes and convenient for study;（2） To investigate the inhibitory role of knockout GGTase-I or FTase gene on K-ras induced lung cancer transformation;（3） To investigate the molecular mechanisms of GGTase-I or FTase gene on the genesis of K-ras induced lung cancer in vivo.Methods1.Animal model:we created mice with a conditional knockout or knockin allele for GGTase-I(Pggtlb^fl/fl)、FTase(Fntb^fl/fl) and K-ras(K^LSL).And then K^LSL mice were bred with Pggtlb^fl/fl mice or Fntb^fl/flmice,generating K^LSLPggtlb^fl/fl mice and K^LSL Fntb^fl/flmice.2.According the genotype of mice,they were divided into three groups:K^LSL（A group）、K^LSLPggtlb^fl/fl（B group） and K^LSLFntb^fl/fl（C group）,then they were divided into two subgroups according to different adenovirus:cre-adenovirus groups（Study groups:A1 group、B1 group and C1 group） and gal-adenovirus groups（Control groups:A2 group、B2 group and C2 group）.3.Adenovirus infection of animals:On the day of 14,all animals were infected with Cre-adenovirus orβ-gal-adenovirus by aerosol rebreathing method.4.Genotype:The K^LSL,Fntb^fl and Pggtlb^fl allele was genotyped by PCR amplification of genomic DNA from tail biopsies.5.Body weight:after infection with adenovirus,the weight of all mousse was measured every week.6.Survival for all animals:the life span of all animals was detected from the treatment.7.Histopathological analysis:The lung,spleen and liver were processed and examined by hematoxylin and eosin staining.8.Immunohistochemical staining was performed and the expressions of CD11b、SP-C、CC10 and Ki-67 were detected.9.Real-time PCR:The mRNA expressions of Pggtlb^fl/fl、Fntb^fl/fl and K^LSL in the lung,liver,cell lines and spleen tissue were analyzed using real-time PCR technique.10.Western blot:The protein expressions of nonprenylated RAP1、total RAP1、RHOA、phosphorylated-ERK1/2、phosphorylated-AKT、total AKT and total ERK1/2 in lung,liver,cell lines and spleen tissue were analyzed using western blot technique.11.Statistical analysis:Data are expressed as means for continuous variables and by frequency count and percentage for qualitative variables.Survival rates of animal were compared with Kaplan-Meier curve test and other indexes were compared with One-Way ANOVA comparison test.P＜0.05 was considered statistically significant.Results1.General state of the experimental animals:60 mice were infected with adenovirus in all groups.19 mice in group A,19 mouse in group B and 20 mouse in group C.2.Cre-adenovirus infection rate:After infected with Cre-adenovirus,K-ras gene was activation and GGTase-I or FTased was silencing in the tumor tissue.3.Body weight:body weights in the control groups were essentially equal.Since two weeks after infection,body weight in group B1 and C1 were significantly higher than that in group A1（P all＜0.01）.Compared with the control group,body weight in the group A1,group B1,and group C1 were significantly decreased（P all＜0.01）.4.The rate of lung or spleen weight on body weightCompared A1 group,the lung weight relative to total body weight（LW/BW） of group B1 and group C1 were significantly decreased（P all＜0.01）,while the spleen weight relative to total body（SW/BW） of group B1 and group C1 were significantly decreased（P all＜0.01）.Compared the group B1,the LW/BW of group C1 is significantly increased（P＜0.01）,and the SW/BW of group C1 is significantly increased（P＜0.01）.5.Pathologic staining:Most mouse in the trial groups developed tumor cells with large and trachychromatic nuclear,diffuse hyperplasia and leukocyte infiltration were seen in the lung tumor tissue.Compared with group A1,there are little tumor cells and leukocyte infiltration in the group B1 and C1.In the group A1,the diffuse adenocarcinoma has obliterated the majority of alveolar spaces in lung.6.Immunohistochemical staining:The lung tumors of K^LSL、K^LSLPggtlb^fl/fl and K^LSLFntb^fl/fl mice were negative for the monocyte/macrophage marker,the Clara cell marker,and the T cell markers but were positive for the typeⅡpneumocyte marker SP-C as judged by immunohistochemistry.Compared with group A1,the expression of SP-C in lung tumor tissue was significantly decreased in group B1 and C1,while Ki-67 was significantly enhanced.7.Real-time PCR:The mRNA expressions of Pggtlb^fl/fl,Fntb^fl/fl and K^LSLmRNA in the tumor tissue of the lung,liver and spleen in group B1 and group C1 were significantly lower than those in group B2 and C2（P＜0.01 or 0.05）.Comparison the group A2,the group A1 had higher K^LSL mRNA expressions（P＜0.01）.8.Western blot:The protein expression of P-AKT in the tissues of trial group were increased,and P-ERK1/2 were decreased.The nonprenylated RAP1 was only detected in the group B1 and C1.No difference was found about the total AKT and total ERK1/2.9.Survival rate:Kaplan-Meier curve showing the survival of mouse in the group B1 and group C1 was significantly improved than that in group A1,while there is no significant difference between the group B1 and group C1.Compared with the trial groups,the survival the control group was significantly improved（P＜0.001）.Conclusions （1） The method of conditional knockout allele technique is an efficient and time-saving way to establish lung cancer model induced by K-ras;（2） The special gene can be expressed in lung by inhale adenovirus.（3） Inactivation of Pggtlb or Fntb by conditional knockout technique can reduce the tumor formation and improves survival in mice with K-ras-induced lung cancer;（4） GGTase-I or FTase can be as the target to treated the lung cancer induced by K-ras;（5） The molecular mechanisms of Pggtlb or Fntb inactivation to treat the lung cancer induced by K-ras is to inhibit the post-transcriptional modification of K-ras protein. PartⅡin vitro studyBackgroundGene mutation is the main cause of the genesis of cancer and there is gene mutation in many solid tumor.Non-small cell lung cancer（NSCLC） may be occupy more than 70%of all the pulmonary carcinoma.It is the leading morbility and fatality rate among malignance tumor in the worldwide and shorten the life span.K-ras gene is the most common oncogene in NSCLC.Therefore,the study of the role of K-ras in the genesis of lung cancer has been the hot study.Recent studies have demonstrated that RAS protein belong to the intracellular proteins contain a CAAX motif.Parts of these proteins can be geranylgeranylated by protein geranylgeranyltransferase typeⅠ（GGTase-I） and the others can farnesylated by protein farnesyltransferase（FTase） alternatively.This is the prenylation process by post-transcriptional modification.GGTase-I and FTase share a commonαsubunit but have uniqueβsubunits that dictate their substrate specificities.In mouse study,We have proved that inactivation of GGTase-I or FTase can inhibit the development of lung cancer induced by K-ras.But we do not know the effects of inactivation of GGTase-I or FTase on the mouse embryo fibroblast（MEF） with K-ras mutation.To investigate the effects of the inactivation of GGTase-I or FTase on K-ras mutation MEF,The present study was designed to test the role of the inactivation GGTase-I or FTase on K-ras induced MEF through a conditional knockout technique.Objective（1） To establish the cell model with the K-ras and GGTase-I or FTase knockout; （2） To observe the effects of K-ras on the bionomics in vitro,such as curve growth, clone formation in soft agar and migration;（3） To investigate the inhibitory role of the inactivation of GGTase-I or FTase on K-ras induced MEF;（4） To investigate the molecular mechanisms of the inactivation of GGTase-I or FTase on the K-ras induced MEF.Methods1.Isolation of embryonic fibroblasts:The mouse embryonic fibroblasts（MEF） were isolated from K^LSL、K^LSLPggtlb^fl/fl and K^LSLFntb^fl/fl embryos on day 15.5 post coitum.2.cell lines groups:According the genetype of cell lines,they were divided into three groups:K^LSL（A group）、K^LSLPggtlb^fl/fl（B group） and K^LSLFntb^fl/fl（C group）, then they were divided into two subgroups according to different adenovirus:creadenovirus groups（Trial groups,A1 group、B1 group and C1 group） andβ-gal-adenovirus groups（Control groups,A2 group、B2 group and C2 group）.3.Adenovirus infection of cell lines:To inactivate Pggtlb,Fntb^fl and activate K-RAS^G12D expression,all cells were seeded and infected with Cre-adenovirus orβ-gal-adenovirus for two times.4.Genotype:The K^LSL,Fntb^fl and Pggtlb^fl allele was genotyped by PCR amplification of genomic DNA from tail biopsies.5.Real-time PCR:The mRNA expressions of GGTase-I、FTase and K^LSL in the cell lines were analyzed using real-time PCR technique.6.Bionomics of cell lines:The bionomics of cell lines was detected,such as cell growth curve,clone formation in soft agar,migration and apoptosis.7.Statistical analysis:Data are expressed as means for continuous variables and by frequency count and percentage for qualitative variables.Survival curve of cell lines were compared with One-Way ANOVA comparison test and other indexes were compared with T test.P＜0.05 was considered statistically significant.Results1.Cre-adenovirus infection rate:After infected with Cre-adenovirus,K-ras gene was activation and GGTase-I and FTased was inactived.2.Real-time PCR:The mRNA expressions of GGTase-I,FTase and K^LSL mRNA in the tumor tissue of the lung,liver and spleen in group B1 and group C1 were significantly lower than those in group B2 and C2（P＜0.01 or 0.05）.Comparison of the group A1 and group A2 revealed that group A1 had higher K^LSL mRNA expressions than group A2（P＜0.01）.2.Genetype of the MEF linesThe genetype of the cell lines are the same as that in the animal.3.The growth of MEF linesIn the trial group,compared with group B1 and group C1,the group A1 grows significantly quickly.No significant difference was found between the group B1 and group C1.No significant difference was found between the trial group and the control group.There is no significant difference in the control group.4.Migration of the MEF linesCompared with the control group,the ability of migration in the trial group was increased significantly.In the trial group,the number of migration cells in the group A1 is much more than that in the group B1 and C1.There is no significant difference in the control group.5.Clone formation of MEF linesThe clone formation was only seen in the group A1,and no clone in the group B1 and C1.There is also no clone in the control group.6.Apoptosis of MEF linesThe rate of apoptosis in the group A1 is 3.31±1.34%,the group B1 is 13. 91%±1.88%,and the group C1 is 9.57%±2.45%.Compared with the group A1,the rates of apoptosis in the group B1 and C1 are increased significantly,and the group B1 is significant higher than that in the group C1.There is no significant difference in the control group.7.Western blotCompared with the group A2,the expression of Caspase-3 in the group A1 was significantly decreased.The expression of Caspase-3 in the group B1 and C1 was significantly lower than that in the group B2 and C2.The expression of phos-P70 in all the cell lines was no significant difference.The expression of PARP in the group B1 and C1 was significantly higher than that in the group B2 and C2,and no significant difference in the group A1 and A2.Conclusions（1） The method of conditional knockout allele technique is an efficient and time-saving way to establish MEF model induced by K-ras;（2） Activation of K-ras can promote the ability of cancer cell growth,migration,clone formation,and inhibit the apoptosis;（3） Inactivation of GGTase-I or FTase by conditional knockout technique can reduce the K^LSL cell viability,proliferation and enhanced the apoptosis; （4） GGTase-I or FTase can be as the target to treated the lung cancer induced by K-ras; （5） The molecular mechanisms of the inactivation of GGTase-I or FTase to affect the MEF induced by K-ras is to inhibit the post-transcriptional modification of K-ras protein,enhance the expression of apoptosis gene,promote the apoptosis,and suppressor cell division.更多还原