葡萄胎高表达的F10基因功能初步研究
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摘要
葡萄胎是一种与妊娠有关的良性病变,发病率为1/100—1/500,部分葡萄胎可发展为侵蚀性葡萄胎,再进一步发展成绒癌。因此葡萄胎仍是一种严重危害育龄妇女生育健康及身心健康的重要疾病。葡萄胎的发生与恶性转化机制仍不明确,目前有如下几方面的学说:1、流行病学调查结果表明文化因素和葡萄胎的发生有统计学关联。提示较高的文化程度可以降低葡萄胎发病的可能性。2、葡萄胎的遗传学说:葡萄胎的发生多是异常受精所致。3、研究结果显示妊娠次数与葡萄胎的发病也是有相关关系,较多的妊娠次数可能是葡萄胎发病的一种危险因素。4、另外多次接受刮、吸宫术也是葡萄胎发生的一项重要危险因素。5、癌基因及肿瘤抑制基因和凋亡调节基因也参与葡萄胎的发生。而目前的大量研究提示葡萄胎的基因组中可能存在遗传易感基因,迄今为止国内外还未获得葡萄胎的遗传易感基因。为明确葡萄胎的基因学发病机制,前期我们从基因克隆方面入手,在早孕的绒毛组织和正常的葡萄胎组织进行抑制性消减杂交,分离和鉴定出一种功能未明的在葡萄胎中高表达的基因F10,F10是一个新基因从未在任何组织或疾病中有过研究报道。前期的研究表明F10基因与滋养细胞肿瘤及腺癌的发生有关。但在肿瘤发生发展过程中的作用未知。F10在葡萄胎组织中高表达说明该基因与此病的发生发展有着密切的关系,提供了F10研究的出发点。同时由于葡萄胎的生长与肿瘤转移浸润具有相似的过程。因此,对F10基因研究可能对肿瘤发生发展过程的研究提供重要的参考证据,为寻找绒毛膜癌分子诊断与治疗基因靶标及筛选其转移相关基因提供线索。进一步明确F10基因的功能将为我们认识葡萄胎的发生、发展机理以及寻找新的治疗靶点和预防策略提供重要的信息。
第一部分F10基因转染细胞表达谱分析
F10基因是我们在探讨葡萄胎发病机理时发现的未知功能的新基因。该基因从未在任何组织或疾病中有过功能研究报道,前期的研究表明F10基因在葡萄胎组织、侵蚀性葡萄胎、绒癌中均阳性表达且依次增强。初步预测F10可能与葡萄胎的恶性变及肿瘤的侵袭行为有关;原位杂交结果表明F10在卵巢腺癌、子宫内膜癌、乳腺癌、肝癌、胃癌等腺癌中均阳性表达。提示F10与上述肿瘤的发生有关。为进一步深入研究和明确F10基因的功能,本研究应用差异显示PCR的方法,从基因转录水平寻找并获得F10影响的差异表达下游基因,并进行其功能分析来间接预测F10基因的功能。
方法
1、F10低表达细胞株的筛选:选取Bel7402、HIC、HepG2、PC、A549、MGC、16HBE、293八种细胞,利用细胞免疫组化技术和荧光定量PCR技术筛选出F10低表达的细胞株;
2、F10基因转染其低表达的细胞株后观察其基因表达谱的变化:利用差异显示技术粗筛F10基因对细胞基因表达谱的影响,实验分四组:质粒pRc-CMV2/F10~+、pRc-CMV2、pRc-CMV2/F10~-分别瞬时转染A549细胞组及空白细胞对照组。提取各组细胞总RNA,逆转录合成cDNA为模板进行PCR扩增,扩增产物进行聚丙烯酰胺凝胶电泳和银染。回收各组之间差异显示的条带并行二次扩增后送测序。荧光定量PCR鉴定各组之间差异表达的基因。
结果
1、荧光定量PCR、细胞免疫组化结果表明:新基因F10在人的8种细胞中呈不同程度的表达。其中F10基因在MGC、PC、Bel7402中高表达,在HepG2、HIC中表达次之,在293中表达量较低,在16HBE、A549中表达量最低。
2、质粒pRc-CMV2/F10~+、pRc-CMV2、pRc-CMV2/F10~-分别瞬时转染A549细胞组及空白细胞对照组四个实验组间差异显示的条带扩增后测序获得annexinⅠ(钙依赖、磷脂结合蛋白Ⅰ)、IPLA2(Membrane associated calciumindependent phospholipase A2,膜偶联非钙依赖的磷脂酶A2)、DATF1(Deathassociate transcription factor,死亡相关的转录因子)、STAT1(信号转导子与转录激活子)、BASP1(Homosapiens brain abundant,membrane attachedsignal protein 1,人类脑中含量丰富,膜粘附信号蛋白1)5个基因。5个基因在pRc-CMV2/F10~+组的表达量与pRc-CMV2组、pRc-CMV2/F10~-组及空白对照组之问的差别有统计学意义,P=0.000。而在pRc-CMV2组、pRc-CMV2/F10~-组及空白对照组各组之间5个基因的表达量无统计学意义。
小结
1、利用细胞免疫组化技术和荧光定量PCR技术检测到F10在Bel7402、HIC、HepG2、PC、A549、MGC、16HBE、293八种细胞中呈不同水平的表达。其中F10基因在MGC、PC、Bel7402中高表达,在HepG2、HIC中表达次之,在293中表达量较低,在16HBE、A549中表达量最低。上述结果为下一步F10转基因研究提供了细胞平台。
2、利用差异显示技术粗筛F10基因转染对细胞基因表达谱的影响,获得的各组之间有统计学意义差异表达的5个基因:annexinⅠ、IPLA2、DATF1、STAT1、BASP1,根据差异表达基因的功能提示F10基因参与细胞的增殖和凋亡过程。
第二部分F10基因对转录因子活性影响
我们通过抑制性消减杂交和差异筛选法,克隆到一个新的在葡萄胎中表达上调的基因片断:F10 EST cDNA(Genbank登陆号:AB196290),利用细胞免疫组化和荧光定量PCR技术在Bel7402、HIC、HepG2、PC、A549、MGC、16HBE、293八种细胞中筛选出F10低表达的细胞株后利用差异显示技术粗筛F10基因转染对细胞基因表达谱的影响,根据获得差异表达基因的功能,提示F10基因可能与细胞的增殖和凋亡有关。基因转录是细胞生命活动的一种重要的调控方式,是当今细胞生物学研究中最为活跃的领域之一,而NF-κB、AP1是细胞增殖、凋亡、分化转录调控调节中的两个核心因子。pHSE、pCRE、pSRE、pGRE参与多种信号传导的调节。所以我们选择了上述几个转录因子来初步的观察一下F10基因在细胞转录调控方面的作用,以进一步明确F10基因的功能。
方法
1、F10基因对重要的信号传导通路的影响:质粒pRc-CMV2/F10~+(pRc-CMV2、pRc-CMV2/F10~-)和报道系统AP_1-Luc(NF-κB-Luc、pHSE-luc、pCRE-luc、pSRE-luc、pGRE-luc)共转染A549细胞,通过测定荧火虫荧光素酶活性,利用荧光素酶报道系统分析F10对细胞重要传导通路的作用。
2、EMSA实验检测AP_1和NF-κB和DNA的结合活性。
结果
1、pRc-CMV2/F10~+组、pRc-CMV2组与pRc-CMV2/F10-组细胞在F10和AP1-Luc质粒共转染24h即可检测荧光素酶的活性,并逐渐增高至转染后48h,pRc-CMV2/F10~+组较pRc-CMV2组与pRc-CMV2/F10~-组之间的荧光素酶活性高且差别有统计学意义,P=0.000。pRc-CMV2组与pRc-CMV2/F10~-组荧光素酶活性无统计学意义,转染72h后各组荧光素酶活性开始下降,各组之间无统计学意义。EMSA进一步证实了上述结果。
2、pRc-CMV2/F10~+组、pRc-CMV2组与pRc-CMV2/F10~-组细胞在F10和NF-κB-Luc质粒共转染24h即可检测荧光素酶的活性,48小时达到最高,pRc-CMV2/F10~+组较其它二组荧光素酶活性低且差别有统计学意义,P=0.000。转染72h荧光素酶活性下降,各组之间荧光素酶的活性差异无统计学意义。EMSA进一步证实了上述结果。
3、pRc-CMV2/F10~+组、pRc-CMV2组与pRc-CMV2/F10~-组细胞在F10分别和pHSE-luc、pCRE-luc、pSRE-luc、pGRE-luc质粒共转染后的荧光素酶活性差异无统计学意义。
小结
1、利用质粒pRc-CMV2/F10~+(pRc-CMV2、pRc-CMV2/F10~-)分别和报道系统AP_1-Luc共转染A549细胞,通过测定荧火虫荧光素酶活性,利用荧光素酶报道系统分析F10对细胞重要传导通路的作用。在共转染48小时,pRc-CMV2/F10~+组较其它二组之间的荧光素酶活性高。EMSA进一步证实了上述结果。F10基因上调AP1的活性。提示F10参与细胞增殖和凋亡过程。
2、利用质粒pRc-CMV2/F10~+(pRc-CMV2、pRc-CMV2/F10~-)分别和报道系统NFκB-Luc共转染A549细胞,通过测定荧火虫荧光素酶活性,利用荧光素酶报道系统分析F10对细胞重要传导通路的作用。在共转染48小时,pRc-CMV2/F10~+组较其它二组之间的荧光素酶活性低。EMSA进一步证实了上述结果。下调NF-κB的活性。提示F10参与细胞增殖和凋亡过程。
第三部分F10基因对细胞增殖和细胞周期的影响
综合前二部分的研究,提示F10基因参与促进细胞增殖、抑制细胞凋亡的过程,导致细胞生长发育异常。而恶性肿瘤的发生与细胞异常增殖密切相关,因此本部分通过检测PCNA、cyclinD1二种在细胞周期中起关键作用的调节蛋白,通过观察细胞体外增殖并利用流式细胞技术来进一步证明F10基因对细胞的增殖和细胞周期的影响。
方法
1、F10对细胞增殖的影响:转染pRc-CMV2/F10和pRc-CMV2质粒于肺癌细胞系A549,通过MTT实验检测二组细胞生长增殖情况;
2、F10对细胞周期的影响:转染pRc-CMV2/F10和pRc-CMV2质粒于肺癌细胞系A549,通过流式细胞技术检测二组细胞各个细胞周期的变化情况;
3、利用免疫组化方法检测上述二组细胞中增殖核抗原(PCNA)和细胞周期素D1(cyclin D1)的表达变化。
结果
1、根据酶标仪连续6天定的570nm波长吸光值绘制出肺癌细胞A549的生长曲线,在保证培养条件、接种细胞量、观察时间一致的情况下,二组细胞之间总体生长趋势差异有统计学意义(P=0.000)。pRc-CMV2/F10组其促进细胞增殖作用比对照组明显。
2、流式细胞技术检测结果:pRc/CMV2/F10基因转染细胞48小时较对照组细胞中G2/M期细胞比例升高1.2倍、S期升高2.1倍。二组相比差异有显著性意义,P＜0.05。
3、免疫组化显示:PCNA在A549/F10组细胞中强阳性表达,在A549/空载体组细胞中呈中度阳性表达。各组细胞中cyclinD1表达水平的变化和PCNA的改变一致。表明F10基因的表达使A549细胞中PCNA和cyclinD1表达上调。
小结
1、转染pRc-CMV2-F10和pRc-CMV2质粒于肺癌细胞系A549,通过MTT实验得出二组细胞连续6天的总体生长趋势差异有显著性意义(P=0.000)。pRc-CMV2/F10~+组其促进细胞增殖作用较对照组明显。提示F10基因有促进细胞增殖的作用。
2、转染pRc-CMV2-F10和pRc-CMV2质粒于肺癌细胞系A549,采用流式细胞技术检测F10对细胞生长周期的影响,pRc/CMV2/F10基因转染细胞48小时较对照组细胞中G2/M期细胞比例升高1.2倍,S期升高2.1倍。提示F10基因通过影响细胞周期来促进细胞增殖。
3、利用免疫组化方法检测细胞中增殖核抗原(PCNA)和细胞周期素D1(cyclin D1)的表达变化。免疫组化显示,PCNA在A549/F10组细胞中强阳性表达,在A549/空载体组细胞中呈中度阳性表达。各组细胞中cyclinD1表达水平的变化和PCNA的改变一致。表明F10基因的表达使A549细胞中PCNA和cyclinD1表达上调。提示F10基因通过上调PCNA和cyclinD1的表达,参与促进细胞的生长增殖。
第四部分稳定转染F10基因A549细胞株的构建
研究探讨基因的功能利用基因转导技术将外源基因转入某一细胞,通过观察细胞生物学行为的变化来认识基因的功能,这是目前使用最广泛、技术最成熟的基因功能研究方法,而建立稳定表达外源基因的细胞模型是这一研究不可或缺的关键一步。因此,建立稳定表达外源基因F10细胞株进行其功能的研究具有重要的意义。
方法
F10基因真核细胞稳定表达系统构建与鉴定:
1.质粒pRc-CMV2/F10和pRc/CMV2提取、鉴定后转染A549细胞系。用G418筛选并扩大培养,建立阳性单克隆细胞株。
2.荧光定量PCR技术鉴定获得的阳性克隆细胞株。
结果
1.质粒pRc-CMV2/F10、pRc-CMV2转染A549细胞系后细胞经过大约三个月的筛选,得到2株稳定表达pRc/CMV2-F10基因的A549细胞和1株稳定表达pRc/CMV2基因的A549细胞。
2.荧光定量PCR结果表明:pRc-CMV2/F10~+细胞株F10表达水平较pRc-CMV2高,结果有统计学意义。P＜0.05。
小结
1.质粒pRc-CMV2/F10和pRc/CMV2提取和鉴定后转染A549细胞系。经过G418大约三个月的筛选,得到2株稳定表达pRc/CM72-F10~+基因的A549细胞和1株稳定表达pRc/CMV2基因A549细胞。
2.阳性单克隆细胞株经过荧光定量PCR鉴定结果表明:pRc-CMV2/F10细胞株F10表达水平较pRc-CMV2细胞株高。通过阳性和对照质粒的转染、阳性克隆细胞株的筛选及鉴定我们成功构建了稳定表达外源新基因F10的肺癌细胞系及对照细胞系,为进一步研究F10基因的生物学功能提供了实验材料。
全文小结
1.利用细胞免疫组化技术和荧光定量PCR技术在八种细胞中筛选出F10低表达的细胞株后利用差异显示技术粗筛F10基因转染对其低表达细胞基因表达谱的影响,各组之间获得了五个差异表达的基因。提示:F10基因与细胞的增殖和凋亡有关。
2.质粒pRc-CMV2/F10~+(pRc-CMV2、pRc-CMV2/F10~-)分别和报道系统AP1-Luc(NFκB-Luc)共转染A549细胞,利用荧光素酶报道系统分析F10对细胞重要传导通路的作用。提示:F10参与细胞增殖和凋亡过程。
3.转染pRc-CMV2-F10和pRc-CMV2转染肺癌细胞系A549,通过MTT实验、流式细胞技术及利用免疫组化方法检测细胞中增殖核抗原(PCNA)和细胞周期素D1(cyclin D1)的表达。结果提示:F10基因通过影响细胞周期和上调PCNA和cyclin D1的表达,参与促进细胞的生长和增殖。
4.质粒pRc-CMV2/F10和pRc/CMV2提取、鉴定后转染A549细胞系。经过G418大约三个月的筛选。成功构建了稳定表达外源新基因F10的肺癌细胞系及对照细胞系,为进一步研究F10基因的生物学功能提供了实验材料。
Hydatidiform mole(HM) is a type of gestational trophoblastic disease with unpredictable malignant potential and disease incidence is 1/100—1/500.The mechanism of generation and malignant transformation of Hydatidiform mole is not definite.There are several theories about HM at present as listed below:
1.Epidemic investigation reflects that the Hydatidiform mole is related to the community culture.The higher level the culture is,the lower possibility Hydatidiform mole occurs.
2.The theory of heredity concludes that Hydatidiform mole is most likely caused by abnormal fertilizations.
3.The number of individual's pregnancies is a critical factor of Hydatidiform mole generation.
4.The excessive number of uterine aspiration,dilatation and curettage is an important factor of HM.
5.The oncogene,tumor-suppressing genes and apoptosis regulator genes are involved in HM generation.However a considerable number of researches point out that inherit predisposing genes which were not reported at present possibly exist in the genome of hydatidiform mole.Suppression subtractive hybridization (SSH) was used to assess molecular pathogenesis of HM.According to comparing the gene expression pattern of the similar gestational ages of HM with normal first-trimester trophonema,we cloned F10 gene form the subtracted cDNA libraries which up-regulated in HM and its function is unknown at present.In the previous researches,F10 gene was considered to be associated with trophoblastic tumor and adenocarcinoma,but the relationship is unclear.Therefore,it is important to do further research on biological functions of F10 gene in order to reveal the generation mechanism of HM and noval treatment and precaution for HM.
Part one The expression spectrum analysis of F10 gene transfected cell
F10 gene is a noval gene and its function is unknown.According to the results which we have obtained,we concluded that F10 gene may participate in the malignant change of HM and tumor's invasion behavior.Our research also shows that F10 gene is related to the cancer generation.In order to identify the function of F10 gene,we obtain the different downstream express genes from the genetic transcription level by DDPCR method and indirectly predict the function of F10 gene.
Methods
DDPCR technology was used when we analysised F10 gene expression spectrum and screened the F10 gene function-related genes.The cell line with lower expression of F10 gene was screened by immunohistochemistry and fluorescent quantitated PCR among the Bel7402,HIC,HepG2,PC,A549,MGC,16HBE and 293 cell lines.The coarsely screening experiment was performed after the pRc-CMV2/F10~+,pRc-CMV2, pRc-CMV2/F10~- plasmids transient transfecting A549 cells respectively.The mRNA of cell was extracted after transfecting for 24 hours.The differently expressed genes were screened by DDPCR among 4 groups.The different expression strips were amplicated and the products were connected to T-Vector and then analyzed by sequenceing.The result was confirmed by fluorescent quantitated PCR.
Results
1.The expression levels of F10 were different among the cells.The first one was Bel7402,HIC and HepG2 cells.The following cells were PC,293 and MGC.The lowest cells were 16HBE and A549.
2.The differently expressed strips were amplicated and then analysised by sequenceing.The annexinI,BASP1,STAT1 gene were Higher level expression while IPLA2,DATF1 gene were lower level expression in F10 tranfected group. The result was justified by the fluorescent quantitation PCR technology.
Summary
1.The cell which lower expression of F10 gene was screened by immunohistochemistry and fluorescent quantitated PCR among the Bel7402,HIC, HepG2,PC,A549,MGC,16HBE,293 cell lines.The first were Bel7402,HIC and HepG2 cells.The following cells were PC,293 and MGC.The lowest cells were 16HBE and A549.
2.According to the results of F10 gene expression spectrum analysis and screening the F10 gene function-related genes,we found that annexinI,STAT1,BASP1 gene were higher level expression while IPLA2,DATF1 gene were lower level expression in F10 tranfected group.We initially conclude that F10 is related to cell proliferation and apoptosis.
Part two-Effect of F10 gene on the activities of transcription factor
We cloned F10 gene which up-regulated in HM by suppression subtractive hybridization(SSH),F10 EST cDNA(Genbank code:AB196290).The cell which lower expression of F10 gene was screened by immunohistochemistry and fluorescent quantitated PCR among the Bel7402,HIC,HepG2,PC,A549,MGC,16HBE and 293 cell lines.According to F10 gene expression spectrum analysis and screening the F10 gene function-related genes by DDPCR we initially conclude that F10 is related to cell proliferation and apoptosis.Genetic transcription is a significant regulation of life and a hot spot research at present.NF-κB and AP1 are core factors about cell proliferation,apoptosis,differentiation and transcriptional control.The transcription factor pHSE,pCRE,pSRE and pRCE participate in many signal transmission regulation.Therefore we observe the effect of F10 gene on the activities of transcription factor to further study the F10 gene biological fuction.
Methods
1.Plasmids pRc-CMV2/F10~+,pRc-CMV2,pRc-CMV2/F10~- and reporter gene AP1-Luc,NFκB-Luc,pHSE-luc,pCRE-luc,pSRE-luc,pGRE-luc were cotransfected A549 cell respectively.After transfecting for 24h,48h and 72h measuring and compareing the lucifrease activity of different groups were used to evaluate the effect of the F10 gene on the important signal transduction pathways.
2.The activity of DNA bingding AP1 and NF-κB were detected by EMSA.
Results
1.The lucifrease activity can be detected after F10 and AP1-Luc plasmids are transfected to four groups for 24 hours and increased gradually.After transfecting for 48 hours the lucifrease of pRc-CMV2/F10~+ group has been obviously higher than the other groups.After transfecting for 72 hours the lucifrease of three groups has no difference and the results were confirmed by the EMSA.
2.The lucifrease activity can be detected after F10 and NFκB-Luc plasmids transfecting for 24 hours and increased gradually among three groups.After transfecting for 48 hours the lucifrease of pRc-CMV2/F10~+ group has obviously lower than the other groups.After transfecting for 72 hours the lucifrease of three groups has no distinction and the results were confirmed by the EMSA.
3.The lucifrease activity has no visible difference among the three groups transfected with the lucifrease report plasmid of pHSE-luc,pCRE-luc,pSRE-luc, pRC-luc.
1.Plasmids pRc-CMV2/F10~+,pRc-CMV2,pRc-CMV2/F10~- and reporter gene AP1-Luc were cotransfected A549 cell respectively.After being transfected for 24h,48h,72h we measured and compared the lucifrease activity of different groups to evaluate the effect of the F10 gene on the important signal transduction pathways.We found that after transfecting for 48 hours the lucifrease of pRc-CMV2/F10~+ group has been obviously lower than the other groups.We can conclude that F10 gene can be up-regulated the transcription activity of AP1.
2.Plasmids pRc-CMV2/F10~+,pRc-CMV2,pRc-CMV2/F10~- and reporter gene NFκB-Luc were cotransfected A549 cell respectively.After being transfected for 24h,48h and 72h we measured and compared the lucifrease activity of different groups to evaluate the effect of F10 gene on the important signal transduction pathways.We found that after transfecting for 48 hours the lucifrease of pRc-CMV2/F10~+ group has been obviously higher than the other groups.We can conclude that F10 gene can down-regulated the transcription activity of NFκB.
3.Plasmids pRc-CMV2/F10~+,pRc-CMV2,pRc-CMV2/F10~- and reporter gene pHSE-luc,pCRE-luc,pSRE-luc,pGRE-luc cotransfected A549 cell respectively. Lucifrease activity of different groups has no obvious difference.
Part three Effect of F10 Gene on the activities of cell growth
According to the first two parts results F10 gene possibly can promote cell promation and inhibit cell apoptosis and lead to cell development abnormally.The malignant tumors are related to the cell growth and development abnormally.So we detected the PCNA and cyclinD1 two types of protein which play important role in the cell cycle.The cell proliferation in vitro and flow cytometry technology were used to observe F10 gene's function.
Methods
1.The influence of F10 gene on the cell multiplication and cell life cycle,plasmids pRc-CMV2/F10~+ and pRc-CMV2 transfected A549 cell respectively.The influence of F10 on cell multiplication and cell life cycle were detected by MTT and flow cytometry.
2.The expression of PCNA and cyclin D1 were detected by immunohisochemistry.
Results
1.According to the A549 cell's 6 days growth curve the two groups growth tendency has obvious difference.The pRc-CMV2/F10 group's cell proliferation was higher than the control.
2.The pRc-CMV2/F10 group's the ratio of G2/M cell was 1.2 times higher and the ratio S cell was 2.1 times higher than the control after transfecting 48 hours.The result of two groups was obviously different.
3.The result of immunohisochemistry showed that the expression of PCNA in the A549/F10 group was higher than the control.The expression of cyclin D1 was the same as the PCNA's among the groups.
Summary
1.The plasmids pRc-CMV2/F10~+,pRc-CMV2 transfected A549 cell respectively.
According to the A549 cell's 6 days growth curve the two groups growth tendency was obvious difference.The pRc-CMV2/F10 group's cell proliferation was higher than the control.The results reflected that F10 gene can promote cell multiplication.
2.The plasmids pRc-CMV2/F10~+,pRc-CMV2 transfected A549 cell respectively.
We detected the influence of F10 on the cell cycle by flow cytometry technology. The pRc-CMV2/F10 group's the ratio of G2/M cell is 1.2 times higher and the ratio S cell is 2.1 times higher than the control after transfecting 48 hours.The results reflect cue that F10 gene can promote cell multiplication by influence the cell cycle.
3.The expression of PCNA and cyclin D1 were detected by immunohisochemistry.
The expression of PCNA in the A549/F10 group is higher than the control.The same as the expression of cyclin D1.The results cue that F10 gene can promote cell multiplication by up-regulating the expression of PCNA and cyclin D1.
Part four Construction and identification the stable expression system of F10 gene
The gene transduction is a technology widely used when studying the gene function.Constructing the stable expression system of gene is necessary step.So constructing the cell line which stable express F10 gene has significant role on study the F10 gene function.
Methods
1.The A549 cell was transfected with plasmids pRc-CMV2/F10~+,pRc-CMV2 respectively in order to Construct and identify the stable expression system of F10 gene in eukaryocytes.The positive cell clones were screened by G418.
2.The insertion and expression of F10 gene in the A549 cells were analyzed by fluorescent quantitation PCR technology.
Results
1.The plasmids pRc-CMV2/F10~+,pRc-CMV2 transfected A549 cell respectively.
The positive cell clones were screened by G418 for three months.We obtain two cell lines of stable express pRc-CMV2/F10~+ and one cell line of stable express pRc-CMV2.
2.The result of fluorescent quantitation PCR shows that the level of express F10 gene in pRc-CMV2/F10~+ group is higher than the pRc-CMV2 group.The result is obvious difference.
Summary
1.The plasmids pRc-CMV2/F10~+,pRc-CMV2 transfected A549 cell respectively.
The positive cell clones were screened by G418 for three months.We obtain two cell lines of stable express pRc-CMV2/F10~+ and one cell line of stable express pRc-CMV2.
2.The result of fluorescent quantitation PCR reflects that the level of express F10 gene in pRc-CMV2/F10~+ group is higher than the pRc-CMV2 group's.
3.The pulmonary carcinoma cell line A549 with stable expression of F10 gene was established,which will be a good model for further study on biological functions of F10 gene.
第一部分F10基因转染细胞表达谱分析
F10基因是我们在探讨葡萄胎发病机理时发现的未知功能的新基因。该基因从未在任何组织或疾病中有过功能研究报道,前期的研究表明F10基因在葡萄胎组织、侵蚀性葡萄胎、绒癌中均阳性表达且依次增强。初步预测F10可能与葡萄胎的恶性变及肿瘤的侵袭行为有关;原位杂交结果表明F10在卵巢腺癌、子宫内膜癌、乳腺癌、肝癌、胃癌等腺癌中均阳性表达。提示F10与上述肿瘤的发生有关。为进一步深入研究和明确F10基因的功能,本研究应用差异显示PCR的方法,从基因转录水平寻找并获得F10影响的差异表达下游基因,并进行其功能分析来间接预测F10基因的功能。
方法
1、F10低表达细胞株的筛选:选取Bel7402、HIC、HepG2、PC、A549、MGC、16HBE、293八种细胞,利用细胞免疫组化技术和荧光定量PCR技术筛选出F10低表达的细胞株;
2、F10基因转染其低表达的细胞株后观察其基因表达谱的变化:利用差异显示技术粗筛F10基因对细胞基因表达谱的影响,实验分四组:质粒pRc-CMV2/F10~+、pRc-CMV2、pRc-CMV2/F10~-分别瞬时转染A549细胞组及空白细胞对照组。提取各组细胞总RNA,逆转录合成cDNA为模板进行PCR扩增,扩增产物进行聚丙烯酰胺凝胶电泳和银染。回收各组之间差异显示的条带并行二次扩增后送测序。荧光定量PCR鉴定各组之间差异表达的基因。
结果
1、荧光定量PCR、细胞免疫组化结果表明:新基因F10在人的8种细胞中呈不同程度的表达。其中F10基因在MGC、PC、Bel7402中高表达,在HepG2、HIC中表达次之,在293中表达量较低,在16HBE、A549中表达量最低。
2、质粒pRc-CMV2/F10~+、pRc-CMV2、pRc-CMV2/F10~-分别瞬时转染A549细胞组及空白细胞对照组四个实验组间差异显示的条带扩增后测序获得annexinⅠ(钙依赖、磷脂结合蛋白Ⅰ)、IPLA2(Membrane associated calciumindependent phospholipase A2,膜偶联非钙依赖的磷脂酶A2)、DATF1(Deathassociate transcription factor,死亡相关的转录因子)、STAT1(信号转导子与转录激活子)、BASP1(Homosapiens brain abundant,membrane attachedsignal protein 1,人类脑中含量丰富,膜粘附信号蛋白1)5个基因。5个基因在pRc-CMV2/F10~+组的表达量与pRc-CMV2组、pRc-CMV2/F10~-组及空白对照组之问的差别有统计学意义,P=0.000。而在pRc-CMV2组、pRc-CMV2/F10~-组及空白对照组各组之间5个基因的表达量无统计学意义。
小结
1、利用细胞免疫组化技术和荧光定量PCR技术检测到F10在Bel7402、HIC、HepG2、PC、A549、MGC、16HBE、293八种细胞中呈不同水平的表达。其中F10基因在MGC、PC、Bel7402中高表达,在HepG2、HIC中表达次之,在293中表达量较低,在16HBE、A549中表达量最低。上述结果为下一步F10转基因研究提供了细胞平台。
2、利用差异显示技术粗筛F10基因转染对细胞基因表达谱的影响,获得的各组之间有统计学意义差异表达的5个基因:annexinⅠ、IPLA2、DATF1、STAT1、BASP1,根据差异表达基因的功能提示F10基因参与细胞的增殖和凋亡过程。
第二部分F10基因对转录因子活性影响
我们通过抑制性消减杂交和差异筛选法,克隆到一个新的在葡萄胎中表达上调的基因片断:F10 EST cDNA(Genbank登陆号:AB196290),利用细胞免疫组化和荧光定量PCR技术在Bel7402、HIC、HepG2、PC、A549、MGC、16HBE、293八种细胞中筛选出F10低表达的细胞株后利用差异显示技术粗筛F10基因转染对细胞基因表达谱的影响,根据获得差异表达基因的功能,提示F10基因可能与细胞的增殖和凋亡有关。基因转录是细胞生命活动的一种重要的调控方式,是当今细胞生物学研究中最为活跃的领域之一,而NF-κB、AP1是细胞增殖、凋亡、分化转录调控调节中的两个核心因子。pHSE、pCRE、pSRE、pGRE参与多种信号传导的调节。所以我们选择了上述几个转录因子来初步的观察一下F10基因在细胞转录调控方面的作用,以进一步明确F10基因的功能。
方法
1、F10基因对重要的信号传导通路的影响:质粒pRc-CMV2/F10~+(pRc-CMV2、pRc-CMV2/F10~-)和报道系统AP_1-Luc(NF-κB-Luc、pHSE-luc、pCRE-luc、pSRE-luc、pGRE-luc)共转染A549细胞,通过测定荧火虫荧光素酶活性,利用荧光素酶报道系统分析F10对细胞重要传导通路的作用。
2、EMSA实验检测AP_1和NF-κB和DNA的结合活性。
结果
1、pRc-CMV2/F10~+组、pRc-CMV2组与pRc-CMV2/F10-组细胞在F10和AP1-Luc质粒共转染24h即可检测荧光素酶的活性,并逐渐增高至转染后48h,pRc-CMV2/F10~+组较pRc-CMV2组与pRc-CMV2/F10~-组之间的荧光素酶活性高且差别有统计学意义,P=0.000。pRc-CMV2组与pRc-CMV2/F10~-组荧光素酶活性无统计学意义,转染72h后各组荧光素酶活性开始下降,各组之间无统计学意义。EMSA进一步证实了上述结果。
2、pRc-CMV2/F10~+组、pRc-CMV2组与pRc-CMV2/F10~-组细胞在F10和NF-κB-Luc质粒共转染24h即可检测荧光素酶的活性,48小时达到最高,pRc-CMV2/F10~+组较其它二组荧光素酶活性低且差别有统计学意义,P=0.000。转染72h荧光素酶活性下降,各组之间荧光素酶的活性差异无统计学意义。EMSA进一步证实了上述结果。
3、pRc-CMV2/F10~+组、pRc-CMV2组与pRc-CMV2/F10~-组细胞在F10分别和pHSE-luc、pCRE-luc、pSRE-luc、pGRE-luc质粒共转染后的荧光素酶活性差异无统计学意义。
小结
1、利用质粒pRc-CMV2/F10~+(pRc-CMV2、pRc-CMV2/F10~-)分别和报道系统AP_1-Luc共转染A549细胞,通过测定荧火虫荧光素酶活性,利用荧光素酶报道系统分析F10对细胞重要传导通路的作用。在共转染48小时,pRc-CMV2/F10~+组较其它二组之间的荧光素酶活性高。EMSA进一步证实了上述结果。F10基因上调AP1的活性。提示F10参与细胞增殖和凋亡过程。
2、利用质粒pRc-CMV2/F10~+(pRc-CMV2、pRc-CMV2/F10~-)分别和报道系统NFκB-Luc共转染A549细胞,通过测定荧火虫荧光素酶活性,利用荧光素酶报道系统分析F10对细胞重要传导通路的作用。在共转染48小时,pRc-CMV2/F10~+组较其它二组之间的荧光素酶活性低。EMSA进一步证实了上述结果。下调NF-κB的活性。提示F10参与细胞增殖和凋亡过程。
第三部分F10基因对细胞增殖和细胞周期的影响
综合前二部分的研究,提示F10基因参与促进细胞增殖、抑制细胞凋亡的过程,导致细胞生长发育异常。而恶性肿瘤的发生与细胞异常增殖密切相关,因此本部分通过检测PCNA、cyclinD1二种在细胞周期中起关键作用的调节蛋白,通过观察细胞体外增殖并利用流式细胞技术来进一步证明F10基因对细胞的增殖和细胞周期的影响。
方法
1、F10对细胞增殖的影响:转染pRc-CMV2/F10和pRc-CMV2质粒于肺癌细胞系A549,通过MTT实验检测二组细胞生长增殖情况;
2、F10对细胞周期的影响:转染pRc-CMV2/F10和pRc-CMV2质粒于肺癌细胞系A549,通过流式细胞技术检测二组细胞各个细胞周期的变化情况;
3、利用免疫组化方法检测上述二组细胞中增殖核抗原(PCNA)和细胞周期素D1(cyclin D1)的表达变化。
结果
1、根据酶标仪连续6天定的570nm波长吸光值绘制出肺癌细胞A549的生长曲线,在保证培养条件、接种细胞量、观察时间一致的情况下,二组细胞之间总体生长趋势差异有统计学意义(P=0.000)。pRc-CMV2/F10组其促进细胞增殖作用比对照组明显。
2、流式细胞技术检测结果:pRc/CMV2/F10基因转染细胞48小时较对照组细胞中G2/M期细胞比例升高1.2倍、S期升高2.1倍。二组相比差异有显著性意义,P＜0.05。
3、免疫组化显示:PCNA在A549/F10组细胞中强阳性表达,在A549/空载体组细胞中呈中度阳性表达。各组细胞中cyclinD1表达水平的变化和PCNA的改变一致。表明F10基因的表达使A549细胞中PCNA和cyclinD1表达上调。
小结
1、转染pRc-CMV2-F10和pRc-CMV2质粒于肺癌细胞系A549,通过MTT实验得出二组细胞连续6天的总体生长趋势差异有显著性意义(P=0.000)。pRc-CMV2/F10~+组其促进细胞增殖作用较对照组明显。提示F10基因有促进细胞增殖的作用。
2、转染pRc-CMV2-F10和pRc-CMV2质粒于肺癌细胞系A549,采用流式细胞技术检测F10对细胞生长周期的影响,pRc/CMV2/F10基因转染细胞48小时较对照组细胞中G2/M期细胞比例升高1.2倍,S期升高2.1倍。提示F10基因通过影响细胞周期来促进细胞增殖。
3、利用免疫组化方法检测细胞中增殖核抗原(PCNA)和细胞周期素D1(cyclin D1)的表达变化。免疫组化显示,PCNA在A549/F10组细胞中强阳性表达,在A549/空载体组细胞中呈中度阳性表达。各组细胞中cyclinD1表达水平的变化和PCNA的改变一致。表明F10基因的表达使A549细胞中PCNA和cyclinD1表达上调。提示F10基因通过上调PCNA和cyclinD1的表达,参与促进细胞的生长增殖。
第四部分稳定转染F10基因A549细胞株的构建
研究探讨基因的功能利用基因转导技术将外源基因转入某一细胞,通过观察细胞生物学行为的变化来认识基因的功能,这是目前使用最广泛、技术最成熟的基因功能研究方法,而建立稳定表达外源基因的细胞模型是这一研究不可或缺的关键一步。因此,建立稳定表达外源基因F10细胞株进行其功能的研究具有重要的意义。
方法
F10基因真核细胞稳定表达系统构建与鉴定:
1.质粒pRc-CMV2/F10和pRc/CMV2提取、鉴定后转染A549细胞系。用G418筛选并扩大培养,建立阳性单克隆细胞株。
2.荧光定量PCR技术鉴定获得的阳性克隆细胞株。
结果
1.质粒pRc-CMV2/F10、pRc-CMV2转染A549细胞系后细胞经过大约三个月的筛选,得到2株稳定表达pRc/CMV2-F10基因的A549细胞和1株稳定表达pRc/CMV2基因的A549细胞。
2.荧光定量PCR结果表明:pRc-CMV2/F10~+细胞株F10表达水平较pRc-CMV2高,结果有统计学意义。P＜0.05。
小结
1.质粒pRc-CMV2/F10和pRc/CMV2提取和鉴定后转染A549细胞系。经过G418大约三个月的筛选,得到2株稳定表达pRc/CM72-F10~+基因的A549细胞和1株稳定表达pRc/CMV2基因A549细胞。
2.阳性单克隆细胞株经过荧光定量PCR鉴定结果表明:pRc-CMV2/F10细胞株F10表达水平较pRc-CMV2细胞株高。通过阳性和对照质粒的转染、阳性克隆细胞株的筛选及鉴定我们成功构建了稳定表达外源新基因F10的肺癌细胞系及对照细胞系,为进一步研究F10基因的生物学功能提供了实验材料。
全文小结
1.利用细胞免疫组化技术和荧光定量PCR技术在八种细胞中筛选出F10低表达的细胞株后利用差异显示技术粗筛F10基因转染对其低表达细胞基因表达谱的影响,各组之间获得了五个差异表达的基因。提示:F10基因与细胞的增殖和凋亡有关。
2.质粒pRc-CMV2/F10~+(pRc-CMV2、pRc-CMV2/F10~-)分别和报道系统AP1-Luc(NFκB-Luc)共转染A549细胞,利用荧光素酶报道系统分析F10对细胞重要传导通路的作用。提示:F10参与细胞增殖和凋亡过程。
3.转染pRc-CMV2-F10和pRc-CMV2转染肺癌细胞系A549,通过MTT实验、流式细胞技术及利用免疫组化方法检测细胞中增殖核抗原(PCNA)和细胞周期素D1(cyclin D1)的表达。结果提示:F10基因通过影响细胞周期和上调PCNA和cyclin D1的表达,参与促进细胞的生长和增殖。
4.质粒pRc-CMV2/F10和pRc/CMV2提取、鉴定后转染A549细胞系。经过G418大约三个月的筛选。成功构建了稳定表达外源新基因F10的肺癌细胞系及对照细胞系,为进一步研究F10基因的生物学功能提供了实验材料。
Hydatidiform mole(HM) is a type of gestational trophoblastic disease with unpredictable malignant potential and disease incidence is 1/100—1/500.The mechanism of generation and malignant transformation of Hydatidiform mole is not definite.There are several theories about HM at present as listed below:
1.Epidemic investigation reflects that the Hydatidiform mole is related to the community culture.The higher level the culture is,the lower possibility Hydatidiform mole occurs.
2.The theory of heredity concludes that Hydatidiform mole is most likely caused by abnormal fertilizations.
3.The number of individual's pregnancies is a critical factor of Hydatidiform mole generation.
4.The excessive number of uterine aspiration,dilatation and curettage is an important factor of HM.
5.The oncogene,tumor-suppressing genes and apoptosis regulator genes are involved in HM generation.However a considerable number of researches point out that inherit predisposing genes which were not reported at present possibly exist in the genome of hydatidiform mole.Suppression subtractive hybridization (SSH) was used to assess molecular pathogenesis of HM.According to comparing the gene expression pattern of the similar gestational ages of HM with normal first-trimester trophonema,we cloned F10 gene form the subtracted cDNA libraries which up-regulated in HM and its function is unknown at present.In the previous researches,F10 gene was considered to be associated with trophoblastic tumor and adenocarcinoma,but the relationship is unclear.Therefore,it is important to do further research on biological functions of F10 gene in order to reveal the generation mechanism of HM and noval treatment and precaution for HM.
Part one The expression spectrum analysis of F10 gene transfected cell
F10 gene is a noval gene and its function is unknown.According to the results which we have obtained,we concluded that F10 gene may participate in the malignant change of HM and tumor's invasion behavior.Our research also shows that F10 gene is related to the cancer generation.In order to identify the function of F10 gene,we obtain the different downstream express genes from the genetic transcription level by DDPCR method and indirectly predict the function of F10 gene.
Methods
DDPCR technology was used when we analysised F10 gene expression spectrum and screened the F10 gene function-related genes.The cell line with lower expression of F10 gene was screened by immunohistochemistry and fluorescent quantitated PCR among the Bel7402,HIC,HepG2,PC,A549,MGC,16HBE and 293 cell lines.The coarsely screening experiment was performed after the pRc-CMV2/F10~+,pRc-CMV2, pRc-CMV2/F10~- plasmids transient transfecting A549 cells respectively.The mRNA of cell was extracted after transfecting for 24 hours.The differently expressed genes were screened by DDPCR among 4 groups.The different expression strips were amplicated and the products were connected to T-Vector and then analyzed by sequenceing.The result was confirmed by fluorescent quantitated PCR.
Results
1.The expression levels of F10 were different among the cells.The first one was Bel7402,HIC and HepG2 cells.The following cells were PC,293 and MGC.The lowest cells were 16HBE and A549.
2.The differently expressed strips were amplicated and then analysised by sequenceing.The annexinI,BASP1,STAT1 gene were Higher level expression while IPLA2,DATF1 gene were lower level expression in F10 tranfected group. The result was justified by the fluorescent quantitation PCR technology.
Summary
1.The cell which lower expression of F10 gene was screened by immunohistochemistry and fluorescent quantitated PCR among the Bel7402,HIC, HepG2,PC,A549,MGC,16HBE,293 cell lines.The first were Bel7402,HIC and HepG2 cells.The following cells were PC,293 and MGC.The lowest cells were 16HBE and A549.
2.According to the results of F10 gene expression spectrum analysis and screening the F10 gene function-related genes,we found that annexinI,STAT1,BASP1 gene were higher level expression while IPLA2,DATF1 gene were lower level expression in F10 tranfected group.We initially conclude that F10 is related to cell proliferation and apoptosis.
Part two-Effect of F10 gene on the activities of transcription factor
We cloned F10 gene which up-regulated in HM by suppression subtractive hybridization(SSH),F10 EST cDNA(Genbank code:AB196290).The cell which lower expression of F10 gene was screened by immunohistochemistry and fluorescent quantitated PCR among the Bel7402,HIC,HepG2,PC,A549,MGC,16HBE and 293 cell lines.According to F10 gene expression spectrum analysis and screening the F10 gene function-related genes by DDPCR we initially conclude that F10 is related to cell proliferation and apoptosis.Genetic transcription is a significant regulation of life and a hot spot research at present.NF-κB and AP1 are core factors about cell proliferation,apoptosis,differentiation and transcriptional control.The transcription factor pHSE,pCRE,pSRE and pRCE participate in many signal transmission regulation.Therefore we observe the effect of F10 gene on the activities of transcription factor to further study the F10 gene biological fuction.
Methods
1.Plasmids pRc-CMV2/F10~+,pRc-CMV2,pRc-CMV2/F10~- and reporter gene AP1-Luc,NFκB-Luc,pHSE-luc,pCRE-luc,pSRE-luc,pGRE-luc were cotransfected A549 cell respectively.After transfecting for 24h,48h and 72h measuring and compareing the lucifrease activity of different groups were used to evaluate the effect of the F10 gene on the important signal transduction pathways.
2.The activity of DNA bingding AP1 and NF-κB were detected by EMSA.
Results
1.The lucifrease activity can be detected after F10 and AP1-Luc plasmids are transfected to four groups for 24 hours and increased gradually.After transfecting for 48 hours the lucifrease of pRc-CMV2/F10~+ group has been obviously higher than the other groups.After transfecting for 72 hours the lucifrease of three groups has no difference and the results were confirmed by the EMSA.
2.The lucifrease activity can be detected after F10 and NFκB-Luc plasmids transfecting for 24 hours and increased gradually among three groups.After transfecting for 48 hours the lucifrease of pRc-CMV2/F10~+ group has obviously lower than the other groups.After transfecting for 72 hours the lucifrease of three groups has no distinction and the results were confirmed by the EMSA.
3.The lucifrease activity has no visible difference among the three groups transfected with the lucifrease report plasmid of pHSE-luc,pCRE-luc,pSRE-luc, pRC-luc.
1.Plasmids pRc-CMV2/F10~+,pRc-CMV2,pRc-CMV2/F10~- and reporter gene AP1-Luc were cotransfected A549 cell respectively.After being transfected for 24h,48h,72h we measured and compared the lucifrease activity of different groups to evaluate the effect of the F10 gene on the important signal transduction pathways.We found that after transfecting for 48 hours the lucifrease of pRc-CMV2/F10~+ group has been obviously lower than the other groups.We can conclude that F10 gene can be up-regulated the transcription activity of AP1.
2.Plasmids pRc-CMV2/F10~+,pRc-CMV2,pRc-CMV2/F10~- and reporter gene NFκB-Luc were cotransfected A549 cell respectively.After being transfected for 24h,48h and 72h we measured and compared the lucifrease activity of different groups to evaluate the effect of F10 gene on the important signal transduction pathways.We found that after transfecting for 48 hours the lucifrease of pRc-CMV2/F10~+ group has been obviously higher than the other groups.We can conclude that F10 gene can down-regulated the transcription activity of NFκB.
3.Plasmids pRc-CMV2/F10~+,pRc-CMV2,pRc-CMV2/F10~- and reporter gene pHSE-luc,pCRE-luc,pSRE-luc,pGRE-luc cotransfected A549 cell respectively. Lucifrease activity of different groups has no obvious difference.
Part three Effect of F10 Gene on the activities of cell growth
According to the first two parts results F10 gene possibly can promote cell promation and inhibit cell apoptosis and lead to cell development abnormally.The malignant tumors are related to the cell growth and development abnormally.So we detected the PCNA and cyclinD1 two types of protein which play important role in the cell cycle.The cell proliferation in vitro and flow cytometry technology were used to observe F10 gene's function.
Methods
1.The influence of F10 gene on the cell multiplication and cell life cycle,plasmids pRc-CMV2/F10~+ and pRc-CMV2 transfected A549 cell respectively.The influence of F10 on cell multiplication and cell life cycle were detected by MTT and flow cytometry.
2.The expression of PCNA and cyclin D1 were detected by immunohisochemistry.
Results
1.According to the A549 cell's 6 days growth curve the two groups growth tendency has obvious difference.The pRc-CMV2/F10 group's cell proliferation was higher than the control.
2.The pRc-CMV2/F10 group's the ratio of G2/M cell was 1.2 times higher and the ratio S cell was 2.1 times higher than the control after transfecting 48 hours.The result of two groups was obviously different.
3.The result of immunohisochemistry showed that the expression of PCNA in the A549/F10 group was higher than the control.The expression of cyclin D1 was the same as the PCNA's among the groups.
Summary
1.The plasmids pRc-CMV2/F10~+,pRc-CMV2 transfected A549 cell respectively.
According to the A549 cell's 6 days growth curve the two groups growth tendency was obvious difference.The pRc-CMV2/F10 group's cell proliferation was higher than the control.The results reflected that F10 gene can promote cell multiplication.
2.The plasmids pRc-CMV2/F10~+,pRc-CMV2 transfected A549 cell respectively.
We detected the influence of F10 on the cell cycle by flow cytometry technology. The pRc-CMV2/F10 group's the ratio of G2/M cell is 1.2 times higher and the ratio S cell is 2.1 times higher than the control after transfecting 48 hours.The results reflect cue that F10 gene can promote cell multiplication by influence the cell cycle.
3.The expression of PCNA and cyclin D1 were detected by immunohisochemistry.
The expression of PCNA in the A549/F10 group is higher than the control.The same as the expression of cyclin D1.The results cue that F10 gene can promote cell multiplication by up-regulating the expression of PCNA and cyclin D1.
Part four Construction and identification the stable expression system of F10 gene
The gene transduction is a technology widely used when studying the gene function.Constructing the stable expression system of gene is necessary step.So constructing the cell line which stable express F10 gene has significant role on study the F10 gene function.
Methods
1.The A549 cell was transfected with plasmids pRc-CMV2/F10~+,pRc-CMV2 respectively in order to Construct and identify the stable expression system of F10 gene in eukaryocytes.The positive cell clones were screened by G418.
2.The insertion and expression of F10 gene in the A549 cells were analyzed by fluorescent quantitation PCR technology.
Results
1.The plasmids pRc-CMV2/F10~+,pRc-CMV2 transfected A549 cell respectively.
The positive cell clones were screened by G418 for three months.We obtain two cell lines of stable express pRc-CMV2/F10~+ and one cell line of stable express pRc-CMV2.
2.The result of fluorescent quantitation PCR shows that the level of express F10 gene in pRc-CMV2/F10~+ group is higher than the pRc-CMV2 group.The result is obvious difference.
Summary
1.The plasmids pRc-CMV2/F10~+,pRc-CMV2 transfected A549 cell respectively.
The positive cell clones were screened by G418 for three months.We obtain two cell lines of stable express pRc-CMV2/F10~+ and one cell line of stable express pRc-CMV2.
2.The result of fluorescent quantitation PCR reflects that the level of express F10 gene in pRc-CMV2/F10~+ group is higher than the pRc-CMV2 group's.
3.The pulmonary carcinoma cell line A549 with stable expression of F10 gene was established,which will be a good model for further study on biological functions of F10 gene.
引文
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13.付欣,周问渠,等.F10基因与绿色荧光蛋白融合载体的构建和亚细胞定位。广州医学院学报.2005,33(5):1-5
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4.李苏宜,高非,靳平,et al.侵蚀性葡萄胎危险因素的研究.实用妇科与产科杂志,1990,6(6):329
5.吴宜林,张琼英,汤虹。应用PCR-SSCP技术检测妊娠滋养细胞肿瘤P53基因突变.湖南医科大学学报,1997,22(4):338-340
6.Fulop V,Mok SC,Genest DR,et al.c-myc,c-erbB-2,c-fms and bcl-2 oncop roteinsl Expression in normal placenta,partial and complete mole,and choriocarcinomalJ Rep rodMed,1998,43(2):101-110
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13.付欣,周问渠,等.F10基因与绿色荧光蛋白融合载体的构建和亚细胞定位。广州医学院学报.2005,33(5):1-5
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15.Pencil SD,Toth M.Elevated levels of annexin 1 protein in vitro in rat and human mammary adenocarcinoma[J].Clin Exp Metastasis,1999,16(2):113-121
16.Bromberg J,Darnell JE Jr,The role of STATs intranscriptional control and their impact on cellular function[J].Oncogene,2000,19(21):2468-2473
17.Bowman T,Garcia R,Tukson J.STATS in ongenesis[J].oncongene 2000,19(21):2474-2488
18.Buettner R,Mora LB,Jove R.Activated STAT signaling in human tumors provides novel molecular targets for therapeutic intervention[J]. Clinc Cancer Res,2002,8(4):945-954
19.谷俊侠,陈燕。STAT蛋白异常活化和恶性淋巴瘤[J]。临床血液学.2003,16(5):233-236
20.Mosevitskii MI,Caponi JP,Skladchikova GIu,et al.Specific properties and primary structure of BASP1 protein,initially detected in neuronal axonal terminals[J].Biokhimiia.1996,61(7):1209-1220.
21.Fitzgibbon J,Neat M.J,Foot N,et al.Assignmentl of brain acid-soluble protein 1(BASP1) to human chromosome 5p15.1→p14,differential expression in human cancer cell lines as a result of alterations in gene dosage[J].cytogenetic and Genone Res,2004,89(3-4):147-149
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23.Mancuso DJ,Jenkins CM,Sims HF,et al.Complex transcriptional and translational regulation of iPLAgamma resulting in multiple gene products containing dual competing sites for mitochondrial or peroxisomal localization[J].Eur J Biochem.2004,271(23-24):4709-4724
24.Seleznev K,Zhao C,Zhang XH,et al.Calcium-independent phospholipase A2 localizes in and protects mitochondria during apoptotic induction by staurosporine[J].J Biol Chem.2006,281(31):22275-22288.
25.Atsumi G,Murakami M,Kojima K,et al.Distinct roles of two intracellular phospholopase A2s in fatty acid release in the cell
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