L428-MVC/EVC细胞及其NF-κB表型和功能初步研究
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摘要
研究背景
因特网上生物信息资源的快速查找与充分利用,为生物医学研究者提供了有价值的信息和研究手段。GeneSifter软件是一个以网络为基础集统计分析和生物学功能分析为一体的基因芯片数据分析系统,对预测肿瘤易感基因及其相关的信号通路,具有重要的价值,已有不少学者通过GeneSifter软件寻找感兴趣的致癌基因或抑癌基因,为进一步的科学研究和相关实验提供了科学的依据。
近年关于霍奇金淋巴瘤(Hodgkin lymphoma,HL)肿瘤细胞-H/RS细胞的形成过程及其相关的调节机制研究有了很大的进展,初步研究表明H/RS细胞起源于“残疾B淋巴细胞”,其中潜伏膜蛋白-1(LMP-1)的表达、CD99基因缺失和核因子kB(nuclear factor-kappa B,NF-kB)的持续活化是H/RS细胞形成的三大要素,特别是mic2/CD99基因缺失与H/RS细胞的发生有直接关系。
用SiRNA技术将人B淋巴瘤细胞株BJAB和IM9的CD99基因沉默,结果可出现像HL一样的H/RS细胞的典型特征,出现H/RS样“镜影细胞”,高表达CD30和CD15;当再将CD99基因转入上述H/RS样细胞中,又可使出现的H/RS样细胞的形态特征消失,该细胞又恢复到B细胞淋巴瘤原有的表型特征,提示H/RS细胞的发生及免疫表型的变化与CD99基因表达缺失有关。
由于经典型HL(classical Hodgkin lymphoma,cHL)来源于前凋亡生发中心B(germinal center B-cell,GCB)细胞,调节B细胞分化和存活的重要转录因子核因子kB基因(NF-kB)也成为研究H/RS细胞的重要因素。NF-kB属于一个高度保守的转录因子家族,作为重要的转录调控因子,NF-kB调节很多的基因表达,这些基因与细胞增殖、分化、免疫反应、凋亡、转化有着密切关系。在培养的HL细胞株和原代H/RS细胞核中检测到NF-kB与DNA结合的活性持续升高。在HL病人的活检标本免疫组化染色也证实存在大量活化的NF-kB/RelA。NF-kB的持续活化是H/RS细胞生存的必要条件。
本课题组前期研究将高表达mCD99L2基因的小鼠B淋巴瘤细胞株A20细胞的mCD99L2基因沉默,下调CD99基因的表达,可诱导出H/RS样细胞的产生,并从免疫表型、生物学特性等方面验证其具有H/RS细胞部分特性。
如果本研究将低表达CD99基因的人HL细胞株L428细胞上调CD99基因表达,能否在基因修饰后使其H/RS细胞特性消失或改变?与NF-kB的关系又如何呢?L428细胞株主要由大细胞(直径≥25um)、小细胞(直径≤10um)和介于两者之间的细胞混杂组成,一种细胞系中为什么会出现大小不同的细胞混合共生?是否与NF-kB活化有关?如果抑制NF-kB活化,又会怎样的改变呢?
鉴于此,本研究首先利用生物信息学探究CD99基因与NF-kB的关系,然后追踪观测比较L428大小细胞的生长周期及转化规律,上调低表达CD99基因的L428细胞株CD99基因,在该细胞获得稳定表达CD99基因克隆株的基础上,连续观测该株细胞中H/RS细胞的形态及表型改变,并探究bortezomib抑制NF-kB后CD99基因与NF-kB在HL中调节关系,为进一步研究HL中H/RS细胞形成的分子机制奠定实验基础。
目的
1.借助生物信息学软件对CD99基因与HL相关性的基因进行检索和分析。利用当前常用的基因数据库分析CD99基因和HL瘤组织中各种基因的表达及差异,探寻CD99基因与HL之间的相互关系。
2.以探究CD99基因对HLH/RS细胞的影响,对比转染CD99基因前后、bortezomib干预前后CD99基因与NF-kB的相关关系,探讨CD99基因是否通过NF-κB起作用,并为本研究后续工作奠定基础。
3.将L428-MVC、L428-EVC细胞亚系进行细胞周期、凋亡、生长曲线分析,及体内成瘤情况观察,探究CD99基因在HL中所起的作用。
方法
1.从GEO DataSets下载CD99基因与HL细胞株及组织中的基因芯片数据,提交至基因芯片在线分析工具GeneSifter后,对CD99基因与HL所表达的NF-κB进行基因表达差异分析、KEGG通路分析。
2.用计数板确定细胞数量,并根据细胞数量用含胎牛血清的1640将培养的L428细胞株持续稀释成单个大细胞(直径>25um)、单个小细胞(直径≤10um)于96孔板培养细胞,并在光镜、扫描电镜下进行连续观察其生长周期,并检测NF-κB的活化情况。
3.利用本课题前期将CD99/mic2基因成功转染至L428细胞中的细胞株命名为L428-MVC(L428-mic2 vector construct,L428-MVC)的基础上,通过免疫组化对L428与L428-MVC克隆株细胞的形态及表型进行对比分析,免疫组化检测CD99、CD30、CD15、CD2、CD3、CD20、CD79在L428-MVC和L428细胞中的表达;流式细胞术检测L428-MVC、L428细胞CD30和CD15的表达,光镜、扫描电镜、透射电镜下观察L428与L428-MVC细胞形态特征,以转染空质粒的L428细胞株命名为L428-EVC(L428-empty vector construct,L428-EVC)为对照。应用流式细胞分析技术、MTT法以及体内成瘤试验,观察CD99基因对L428细胞细胞周期、凋亡、增殖的影响;免疫荧光细胞化学染色方法、Westernblot分析L428转染CD99基因前后NF-κB的变化,观察bortezomib对L428细胞、NF-κB抑制的影响、与CD99基因的调节关系,以及接种BALB/c小鼠和BALB/c-nu/nu裸鼠体内成瘤试验的影响。
结果
1.差异基因分析中发现,与CD99基因相比,从GeneSifter中挖掘出与HL有关的显著高丰度基因1568种,除发现NF-κB基因外,还发现了脂肪酸合成相关基因。从差异基因KEGG通路分析中发现NF-kB参与MAPK及TOLL样受体等信号通路中。
2.经持续稀释成单个大细胞、单个小细胞的L428细胞,小细胞可分裂转化成大细胞,大细胞亦可生成小细胞;常见单个大细胞周围出现多个小细胞围绕的现象,在此过程中NF-kB一直持续活化。
3.细胞计数结果显示,L428组中的大细胞或H/RS样细胞(直径≥25μm)比例为(11.6±1.5)%,L428-MVC组细胞中H/RS样细胞的比例为(4.6±0.7)%,L428-EVC组为(13.1±1.3)%。L428、L428-EVC细胞CD30和CD15表达阳性,CD99表达则为阴性;而L428-MVC表达CD99则为阳性,同时发现L428-MVC所表达CD2、CD3、CD20、CD79仍为阴性。透射电镜发现转染CD99基因后的L428细胞胞质内脂质体增多,扫描电镜发现L428-MVC细胞表面突起减少。L428-MVC细胞株生长增殖高于L428细胞株和L428-EVC细胞株(P<0.01),其细胞凋亡亦显著增加,细胞周期阻滞在G2/M期。与L428-MVC细胞相比,免疫组化及Westernblot均显示L428、L428-EVC表达的NF-kB显著升高。120nmol/L的bortezomib对L428细胞株抑制后发现NF-kB阻断明显,但并未引起CD99重新表达。L428、L428-EVC及L428-MVC细胞株均未在BALB/c小鼠和BALB/c-nu/nu裸鼠体内成瘤。
结论
1.NF-kB基因持续活化与H/RS细胞的增殖与转化密切相关。H/RS细胞则显示明显的NF-kB活性,在HL细胞中灭活NF-kB将引起细胞凋亡。
2.CD99基因可导致L428大细胞形态变小,其特征性的细胞表面标记CD15、CD30消失,CD99基因对L428细胞增殖起正调节作用,但同时引起细胞凋亡亦增加,CD99基因对NF-kB起调控作用,而NF-kB对CD99基因无反调节作用,同时CD99基因不增加致瘤性。
3.L428大小两种肿瘤细胞,小细胞经历从小至大,大细胞可分裂成众多小细胞的过程,在单个大细胞周围常出现多个小细胞围集的现象。
4.上调HL细胞的CD99基因可导致胞质内脂质体含量增多,可能与脂肪酸合成相关基因高表达有关。
创新之处
从生物信息学分析出发,提出并证实人HL细胞株L428中CD99基因与NF-kB的关系,CD99基因是通过下调NF-kB活性并使HL细胞株L428细胞的细胞形态、免疫表型和生物学特性发生改变的重要分子。
Backgroud
It has provided the valuable information and the research tool for the biomedicine researcher by looking for and by fully using biology information resource's on the Internet.The GeneSifter software is one of the software to take the network as the foundation collection statistical analysis and the biology functional analysis,in which a body's gene chip data were analysed in the system.Many scholars have been looking for the signifivcant carcinogenic gene or damps the cancer gene by GeneSifter software.It is an efficient tool that provided the science basis for the further research experiment.
Recent years had witnessed the very big progress about the H/RS cell's forming process and the relating research,in which the mechanism were adjusted.The preliminary study indicated that the H/RS cell's origin major part comes from“the disabled B lymphocyte”,latent membrane protein 1,LMP-1 expression,CD99 gene flaw and nuclear factorκB(NF-κB)the continually activation.Specially,the mic2/CD99 gene flaw and the H/RS cell's occurrence have the direct relation.After the person B cell lymphoma BJAB and IM9 cell's CD99 gene were silenced by SiRNA technology,these cells presented typical characteristic of H/RS cell's, presented the“mirror shade cell”of H/RS type,expressed CD30 and CD15. Restored cell morphology by exogenous expression of CD99 in H/RS type cell's cells,in which characteristics of typical characteristic of H/RS cell's were vanished. This cell restores to the B cell lymphoma original phenotype characteristic otherwise prompted the H/RS cell's occurrence.It suggested that the immunity phenotype change were related to the deficited expression for CD99 gene.
Because cHL originates pre-perishes weakly the germinal center B-cell,GCB cell, adjusting the B cell differentiation and the survival important transcription factorκgene unifies the nuclear factor-κB,NF-κB become important factor to study the H/RS cell's.The NF-κB belongs to a highly conservative transcription factor family.Being the important duplication regulation factor,NF-kB adjusts many gene expressions, the cell multiplication,split up,the immune response,perish and the transformation, in which have the close relationship.Examining NF-κB and DNA in HL cell line and in the original generation of H/RS cell's cell nucleus union,activeness of NF-κB continues to elevate.It is also confirmed the existence massive activation of NF-κB/RelA by immunohistochemistry in the HL patient's biopsy specimen.NF-κB continually activation is the H/RS cell survival essential condition.
The mouse B lymphoma cell line mCD99L2 gene had been silenced in our research group recently,which result in declined expression for CD99.These changes may induce the characteristics with H/RS.It has been identified its partial characteristics with H/RS cell from aspects of immunity phenotype to some biology function.
If CD99 gene was upregulted into the person HL cell line to express,whether or nor is to cause its H/RS cell characteristic vanishing or the change? How does with NF-kB relations? L428 cells is consisted of large cells(diameter≥25m)and small cells(diameter≤10m).Why does the phenomenon of mix large cells and small cells in L428 cells appear? Does it relate to inactivation of NF-κB? If NF-κB was inhabited,how does changes in L428 cells?
In view of this,CD99 gene was firstly analyzed using the biological information sciences related to NF-κB.Secondly,The life of large cell and small cells of L428 cells were observed.Thirdly,CD99 gene was successful expressed in L428-MVC cells clone.We continue to observe this cell change of the H/RS cell's shape and the phenotype,to discuss the relation between CD99 and NF-kB after inactivation of NF-κB was inhibited by bortezomib,to study the H/RS cell's origin and to form the molecular mechanism.
Objective
1.Analyzed relation between CD99 gene and NF-κB in HL using the biological information sciences to understand that role CD99 gene plays in HL.
2.To explore L428 living signal large,small cells and muti-cells life cycle under light microscope and it's morphological property by scanning electron microscope as well as activation of NF-κB.
3.To observe the L428-mic2/CD99 vector construct,named as L428-MVC clone cell shape,the structure and the function phenotype under the light microscope,the scanning electron microscope,transmission electron microscope and to confirm relation between CD99 gene and NF-κB.The cell cycle,apoptosis,and rumour formation were to explore.It is to understand the relation between CD99 gene and NF-κB and to study whether does the tumor develop when L428-MVC cells were inoculated into BALB/c mice and BALB/c-nu/nu bare mice in vivo.
Method
1.Downloads CD99 gene and cell lines and tissue gene of HL chip data from GEO DataSets.The chip data was submissed to gene chip online analysis tool GeneSifter.The relationship between the CD99 gene and NF-kB were analysed using gene chip analysis tool of GeneSifter Gene expression variance analysis and KEGG signal passway analysis.
2.With the counting board definite cell quantity,the L428 live cell lines was diluted into single large cells(diameter≥25μm)and small cells(diameter≤10um),muti-cells with 1640 including the fetal calf serum according to the cell quantity.These cells life was continuously observes under the light microscope and scanning electronic microscope.The activation of NF-κB in L428 cells was analyzed.
3.Clones cells shape and the phenotype were examed through immunohistochemistry to L428 and L428-MVC cells.The characteristic of L428-MVC cellular form was analyzed under the light microscope,scanning electronic microscope,transmission electron microscopy.The CD99,CD30,CD15, CD2,CD3,CD20,CD79 expression in L428-MVC and in L428 cells were observed. Flows cytometry technique examined CD30 and the CD15 expression in L428-MVC and L428 cell also.The experiment was taken the L428-empty vector construct (named as L428-EVC)as control.Using the flows cytometry technique,the MTT, the cell cycle,apoptosis as well as in vivo experiment were examed.The NF-κB of L428 and L428-MCV cells were analyzed by the immunity fluorescence cytochemistry staining method and Westernblot.The relation between NF-κB and the CD99 gene were observed with bortezomib.And the inoculated experiment were observed in BALB/c and BALB/c-nu/nu mice in L428 and L428-MVC cells.
Result
1.Compared with the CD99 gene group,a number of remarkable high abundance gene including NF-κB and it participated in MAPK and in the TOLL like acceptor signal circuit in HL cells by bioinformatics softerware of GeneSifter of Gene expression variance analysis and KEGG circuit analysis.However,the lipoic acid synthetase also were significantly increased in CD99 group contrast with HL group.
2.The living cell of L428 cell has experienced from monoclonal to the multi-cell forming process,and presents a big cell around many small clone cell encystation phenomenon.finally,gigantic cells is dead in the life time.
3.The cell counting result showed that the proportion of big cell or the H/RS type cell(diameter≥25um)is for(11.6±1.5)%in the L428 group,for(4.6±0.7)%in L428-MVC group and for(13.1±1.3)%in L428-EVC group,respectively.The CD30 and CD15 expressed in L428 and L428-EVC cells.However,expression for CD99 were negative in L428 and L428-EVC cells.Interestingly,the CD30 and CD15 were negative and CD99 was positive in L428-MVC cells by immunohistochemistry and by flow cytometry,respectively.The expressing for CD2, CD3,CD20,CD79 in L428-MCV cells were negative.The lipin body increases obviously were discovered in L428-MCV cells by the transmission electron microscope after the CD99 gene was transfected into the L428 cells.There were granulate-like particles on the membranes in L428 cell and more smooth on the membranes in L428-MVC by scanning electronic microscope.The L428-MVC cell lines growth is higher than that in L428,L428-EVC cells(P＜0.01).But the L428-MVC cell lines apoptosis obviously increases also,blocked in the G2/M stage. Compared with the L428-MVC cell,the immunohistochemistry and westernblot demonstrated that L428,L428-EVC expression NF-κB obviously elevated.The effect of suppresses is most great with 120nmol/L bortezomib and NF-κB was found to block.However,blocking NF-κB did not to cause CD99 to express.These cell lines did not develop the lump in the BALB/c mice and BALB/c-nu/nu bare mice in vivo.
Conclusion
1.A lots of remarkable high abundance genes including NF-κB were found and it participated in MAPK and in the TOLL like acceptor signal passway in HL cells by bioinformatics softerware of GeneSifter.Meanwhile,the lipoic acid synthetase also were observed in CD99 gene group.
2.The living cell of L428 cell has experienced from monoclonal to the multi-cell forming process,and presented characteristic phenomenon whice a big cell(diameter≥5μm)was embranced by many small clone cells small cells(diameter≤10μm).The NF-κB was continue actived.
3.The size of L428-MVC cells become smaller by transfectent of CD99 gene and the immunophenotypic criteria for diagnosis of HD such as CD15,CD30 were vanished.However,it had not characteristic mark of T cell orientation.The L428-MVC cells multiplication capacity were increased by transferctent of CD99 gene,but it causes the cell to apopatosis accelerated also.The CD99 gene plays the roles to regulated NF-κB and oncogenicity of CD99 gene does not increase in vivo.
New point
Based on the biological information sciences analysis,we propose and confirm the relations between the CD99 gene and NF-κB in cell line L428 of person HL.CD99 gene is influential role molecular to change HL cellular form,the immunity phenotype and the biology characteristic through inactivation of NF-κB.
因特网上生物信息资源的快速查找与充分利用,为生物医学研究者提供了有价值的信息和研究手段。GeneSifter软件是一个以网络为基础集统计分析和生物学功能分析为一体的基因芯片数据分析系统,对预测肿瘤易感基因及其相关的信号通路,具有重要的价值,已有不少学者通过GeneSifter软件寻找感兴趣的致癌基因或抑癌基因,为进一步的科学研究和相关实验提供了科学的依据。
近年关于霍奇金淋巴瘤(Hodgkin lymphoma,HL)肿瘤细胞-H/RS细胞的形成过程及其相关的调节机制研究有了很大的进展,初步研究表明H/RS细胞起源于“残疾B淋巴细胞”,其中潜伏膜蛋白-1(LMP-1)的表达、CD99基因缺失和核因子kB(nuclear factor-kappa B,NF-kB)的持续活化是H/RS细胞形成的三大要素,特别是mic2/CD99基因缺失与H/RS细胞的发生有直接关系。
用SiRNA技术将人B淋巴瘤细胞株BJAB和IM9的CD99基因沉默,结果可出现像HL一样的H/RS细胞的典型特征,出现H/RS样“镜影细胞”,高表达CD30和CD15;当再将CD99基因转入上述H/RS样细胞中,又可使出现的H/RS样细胞的形态特征消失,该细胞又恢复到B细胞淋巴瘤原有的表型特征,提示H/RS细胞的发生及免疫表型的变化与CD99基因表达缺失有关。
由于经典型HL(classical Hodgkin lymphoma,cHL)来源于前凋亡生发中心B(germinal center B-cell,GCB)细胞,调节B细胞分化和存活的重要转录因子核因子kB基因(NF-kB)也成为研究H/RS细胞的重要因素。NF-kB属于一个高度保守的转录因子家族,作为重要的转录调控因子,NF-kB调节很多的基因表达,这些基因与细胞增殖、分化、免疫反应、凋亡、转化有着密切关系。在培养的HL细胞株和原代H/RS细胞核中检测到NF-kB与DNA结合的活性持续升高。在HL病人的活检标本免疫组化染色也证实存在大量活化的NF-kB/RelA。NF-kB的持续活化是H/RS细胞生存的必要条件。
本课题组前期研究将高表达mCD99L2基因的小鼠B淋巴瘤细胞株A20细胞的mCD99L2基因沉默,下调CD99基因的表达,可诱导出H/RS样细胞的产生,并从免疫表型、生物学特性等方面验证其具有H/RS细胞部分特性。
如果本研究将低表达CD99基因的人HL细胞株L428细胞上调CD99基因表达,能否在基因修饰后使其H/RS细胞特性消失或改变?与NF-kB的关系又如何呢?L428细胞株主要由大细胞(直径≥25um)、小细胞(直径≤10um)和介于两者之间的细胞混杂组成,一种细胞系中为什么会出现大小不同的细胞混合共生?是否与NF-kB活化有关?如果抑制NF-kB活化,又会怎样的改变呢?
鉴于此,本研究首先利用生物信息学探究CD99基因与NF-kB的关系,然后追踪观测比较L428大小细胞的生长周期及转化规律,上调低表达CD99基因的L428细胞株CD99基因,在该细胞获得稳定表达CD99基因克隆株的基础上,连续观测该株细胞中H/RS细胞的形态及表型改变,并探究bortezomib抑制NF-kB后CD99基因与NF-kB在HL中调节关系,为进一步研究HL中H/RS细胞形成的分子机制奠定实验基础。
目的
1.借助生物信息学软件对CD99基因与HL相关性的基因进行检索和分析。利用当前常用的基因数据库分析CD99基因和HL瘤组织中各种基因的表达及差异,探寻CD99基因与HL之间的相互关系。
2.以探究CD99基因对HLH/RS细胞的影响,对比转染CD99基因前后、bortezomib干预前后CD99基因与NF-kB的相关关系,探讨CD99基因是否通过NF-κB起作用,并为本研究后续工作奠定基础。
3.将L428-MVC、L428-EVC细胞亚系进行细胞周期、凋亡、生长曲线分析,及体内成瘤情况观察,探究CD99基因在HL中所起的作用。
方法
1.从GEO DataSets下载CD99基因与HL细胞株及组织中的基因芯片数据,提交至基因芯片在线分析工具GeneSifter后,对CD99基因与HL所表达的NF-κB进行基因表达差异分析、KEGG通路分析。
2.用计数板确定细胞数量,并根据细胞数量用含胎牛血清的1640将培养的L428细胞株持续稀释成单个大细胞(直径>25um)、单个小细胞(直径≤10um)于96孔板培养细胞,并在光镜、扫描电镜下进行连续观察其生长周期,并检测NF-κB的活化情况。
3.利用本课题前期将CD99/mic2基因成功转染至L428细胞中的细胞株命名为L428-MVC(L428-mic2 vector construct,L428-MVC)的基础上,通过免疫组化对L428与L428-MVC克隆株细胞的形态及表型进行对比分析,免疫组化检测CD99、CD30、CD15、CD2、CD3、CD20、CD79在L428-MVC和L428细胞中的表达;流式细胞术检测L428-MVC、L428细胞CD30和CD15的表达,光镜、扫描电镜、透射电镜下观察L428与L428-MVC细胞形态特征,以转染空质粒的L428细胞株命名为L428-EVC(L428-empty vector construct,L428-EVC)为对照。应用流式细胞分析技术、MTT法以及体内成瘤试验,观察CD99基因对L428细胞细胞周期、凋亡、增殖的影响;免疫荧光细胞化学染色方法、Westernblot分析L428转染CD99基因前后NF-κB的变化,观察bortezomib对L428细胞、NF-κB抑制的影响、与CD99基因的调节关系,以及接种BALB/c小鼠和BALB/c-nu/nu裸鼠体内成瘤试验的影响。
结果
1.差异基因分析中发现,与CD99基因相比,从GeneSifter中挖掘出与HL有关的显著高丰度基因1568种,除发现NF-κB基因外,还发现了脂肪酸合成相关基因。从差异基因KEGG通路分析中发现NF-kB参与MAPK及TOLL样受体等信号通路中。
2.经持续稀释成单个大细胞、单个小细胞的L428细胞,小细胞可分裂转化成大细胞,大细胞亦可生成小细胞;常见单个大细胞周围出现多个小细胞围绕的现象,在此过程中NF-kB一直持续活化。
3.细胞计数结果显示,L428组中的大细胞或H/RS样细胞(直径≥25μm)比例为(11.6±1.5)%,L428-MVC组细胞中H/RS样细胞的比例为(4.6±0.7)%,L428-EVC组为(13.1±1.3)%。L428、L428-EVC细胞CD30和CD15表达阳性,CD99表达则为阴性;而L428-MVC表达CD99则为阳性,同时发现L428-MVC所表达CD2、CD3、CD20、CD79仍为阴性。透射电镜发现转染CD99基因后的L428细胞胞质内脂质体增多,扫描电镜发现L428-MVC细胞表面突起减少。L428-MVC细胞株生长增殖高于L428细胞株和L428-EVC细胞株(P<0.01),其细胞凋亡亦显著增加,细胞周期阻滞在G2/M期。与L428-MVC细胞相比,免疫组化及Westernblot均显示L428、L428-EVC表达的NF-kB显著升高。120nmol/L的bortezomib对L428细胞株抑制后发现NF-kB阻断明显,但并未引起CD99重新表达。L428、L428-EVC及L428-MVC细胞株均未在BALB/c小鼠和BALB/c-nu/nu裸鼠体内成瘤。
结论
1.NF-kB基因持续活化与H/RS细胞的增殖与转化密切相关。H/RS细胞则显示明显的NF-kB活性,在HL细胞中灭活NF-kB将引起细胞凋亡。
2.CD99基因可导致L428大细胞形态变小,其特征性的细胞表面标记CD15、CD30消失,CD99基因对L428细胞增殖起正调节作用,但同时引起细胞凋亡亦增加,CD99基因对NF-kB起调控作用,而NF-kB对CD99基因无反调节作用,同时CD99基因不增加致瘤性。
3.L428大小两种肿瘤细胞,小细胞经历从小至大,大细胞可分裂成众多小细胞的过程,在单个大细胞周围常出现多个小细胞围集的现象。
4.上调HL细胞的CD99基因可导致胞质内脂质体含量增多,可能与脂肪酸合成相关基因高表达有关。
创新之处
从生物信息学分析出发,提出并证实人HL细胞株L428中CD99基因与NF-kB的关系,CD99基因是通过下调NF-kB活性并使HL细胞株L428细胞的细胞形态、免疫表型和生物学特性发生改变的重要分子。
Backgroud
It has provided the valuable information and the research tool for the biomedicine researcher by looking for and by fully using biology information resource's on the Internet.The GeneSifter software is one of the software to take the network as the foundation collection statistical analysis and the biology functional analysis,in which a body's gene chip data were analysed in the system.Many scholars have been looking for the signifivcant carcinogenic gene or damps the cancer gene by GeneSifter software.It is an efficient tool that provided the science basis for the further research experiment.
Recent years had witnessed the very big progress about the H/RS cell's forming process and the relating research,in which the mechanism were adjusted.The preliminary study indicated that the H/RS cell's origin major part comes from“the disabled B lymphocyte”,latent membrane protein 1,LMP-1 expression,CD99 gene flaw and nuclear factorκB(NF-κB)the continually activation.Specially,the mic2/CD99 gene flaw and the H/RS cell's occurrence have the direct relation.After the person B cell lymphoma BJAB and IM9 cell's CD99 gene were silenced by SiRNA technology,these cells presented typical characteristic of H/RS cell's, presented the“mirror shade cell”of H/RS type,expressed CD30 and CD15. Restored cell morphology by exogenous expression of CD99 in H/RS type cell's cells,in which characteristics of typical characteristic of H/RS cell's were vanished. This cell restores to the B cell lymphoma original phenotype characteristic otherwise prompted the H/RS cell's occurrence.It suggested that the immunity phenotype change were related to the deficited expression for CD99 gene.
Because cHL originates pre-perishes weakly the germinal center B-cell,GCB cell, adjusting the B cell differentiation and the survival important transcription factorκgene unifies the nuclear factor-κB,NF-κB become important factor to study the H/RS cell's.The NF-κB belongs to a highly conservative transcription factor family.Being the important duplication regulation factor,NF-kB adjusts many gene expressions, the cell multiplication,split up,the immune response,perish and the transformation, in which have the close relationship.Examining NF-κB and DNA in HL cell line and in the original generation of H/RS cell's cell nucleus union,activeness of NF-κB continues to elevate.It is also confirmed the existence massive activation of NF-κB/RelA by immunohistochemistry in the HL patient's biopsy specimen.NF-κB continually activation is the H/RS cell survival essential condition.
The mouse B lymphoma cell line mCD99L2 gene had been silenced in our research group recently,which result in declined expression for CD99.These changes may induce the characteristics with H/RS.It has been identified its partial characteristics with H/RS cell from aspects of immunity phenotype to some biology function.
If CD99 gene was upregulted into the person HL cell line to express,whether or nor is to cause its H/RS cell characteristic vanishing or the change? How does with NF-kB relations? L428 cells is consisted of large cells(diameter≥25m)and small cells(diameter≤10m).Why does the phenomenon of mix large cells and small cells in L428 cells appear? Does it relate to inactivation of NF-κB? If NF-κB was inhabited,how does changes in L428 cells?
In view of this,CD99 gene was firstly analyzed using the biological information sciences related to NF-κB.Secondly,The life of large cell and small cells of L428 cells were observed.Thirdly,CD99 gene was successful expressed in L428-MVC cells clone.We continue to observe this cell change of the H/RS cell's shape and the phenotype,to discuss the relation between CD99 and NF-kB after inactivation of NF-κB was inhibited by bortezomib,to study the H/RS cell's origin and to form the molecular mechanism.
Objective
1.Analyzed relation between CD99 gene and NF-κB in HL using the biological information sciences to understand that role CD99 gene plays in HL.
2.To explore L428 living signal large,small cells and muti-cells life cycle under light microscope and it's morphological property by scanning electron microscope as well as activation of NF-κB.
3.To observe the L428-mic2/CD99 vector construct,named as L428-MVC clone cell shape,the structure and the function phenotype under the light microscope,the scanning electron microscope,transmission electron microscope and to confirm relation between CD99 gene and NF-κB.The cell cycle,apoptosis,and rumour formation were to explore.It is to understand the relation between CD99 gene and NF-κB and to study whether does the tumor develop when L428-MVC cells were inoculated into BALB/c mice and BALB/c-nu/nu bare mice in vivo.
Method
1.Downloads CD99 gene and cell lines and tissue gene of HL chip data from GEO DataSets.The chip data was submissed to gene chip online analysis tool GeneSifter.The relationship between the CD99 gene and NF-kB were analysed using gene chip analysis tool of GeneSifter Gene expression variance analysis and KEGG signal passway analysis.
2.With the counting board definite cell quantity,the L428 live cell lines was diluted into single large cells(diameter≥25μm)and small cells(diameter≤10um),muti-cells with 1640 including the fetal calf serum according to the cell quantity.These cells life was continuously observes under the light microscope and scanning electronic microscope.The activation of NF-κB in L428 cells was analyzed.
3.Clones cells shape and the phenotype were examed through immunohistochemistry to L428 and L428-MVC cells.The characteristic of L428-MVC cellular form was analyzed under the light microscope,scanning electronic microscope,transmission electron microscopy.The CD99,CD30,CD15, CD2,CD3,CD20,CD79 expression in L428-MVC and in L428 cells were observed. Flows cytometry technique examined CD30 and the CD15 expression in L428-MVC and L428 cell also.The experiment was taken the L428-empty vector construct (named as L428-EVC)as control.Using the flows cytometry technique,the MTT, the cell cycle,apoptosis as well as in vivo experiment were examed.The NF-κB of L428 and L428-MCV cells were analyzed by the immunity fluorescence cytochemistry staining method and Westernblot.The relation between NF-κB and the CD99 gene were observed with bortezomib.And the inoculated experiment were observed in BALB/c and BALB/c-nu/nu mice in L428 and L428-MVC cells.
Result
1.Compared with the CD99 gene group,a number of remarkable high abundance gene including NF-κB and it participated in MAPK and in the TOLL like acceptor signal circuit in HL cells by bioinformatics softerware of GeneSifter of Gene expression variance analysis and KEGG circuit analysis.However,the lipoic acid synthetase also were significantly increased in CD99 group contrast with HL group.
2.The living cell of L428 cell has experienced from monoclonal to the multi-cell forming process,and presents a big cell around many small clone cell encystation phenomenon.finally,gigantic cells is dead in the life time.
3.The cell counting result showed that the proportion of big cell or the H/RS type cell(diameter≥25um)is for(11.6±1.5)%in the L428 group,for(4.6±0.7)%in L428-MVC group and for(13.1±1.3)%in L428-EVC group,respectively.The CD30 and CD15 expressed in L428 and L428-EVC cells.However,expression for CD99 were negative in L428 and L428-EVC cells.Interestingly,the CD30 and CD15 were negative and CD99 was positive in L428-MVC cells by immunohistochemistry and by flow cytometry,respectively.The expressing for CD2, CD3,CD20,CD79 in L428-MCV cells were negative.The lipin body increases obviously were discovered in L428-MCV cells by the transmission electron microscope after the CD99 gene was transfected into the L428 cells.There were granulate-like particles on the membranes in L428 cell and more smooth on the membranes in L428-MVC by scanning electronic microscope.The L428-MVC cell lines growth is higher than that in L428,L428-EVC cells(P＜0.01).But the L428-MVC cell lines apoptosis obviously increases also,blocked in the G2/M stage. Compared with the L428-MVC cell,the immunohistochemistry and westernblot demonstrated that L428,L428-EVC expression NF-κB obviously elevated.The effect of suppresses is most great with 120nmol/L bortezomib and NF-κB was found to block.However,blocking NF-κB did not to cause CD99 to express.These cell lines did not develop the lump in the BALB/c mice and BALB/c-nu/nu bare mice in vivo.
Conclusion
1.A lots of remarkable high abundance genes including NF-κB were found and it participated in MAPK and in the TOLL like acceptor signal passway in HL cells by bioinformatics softerware of GeneSifter.Meanwhile,the lipoic acid synthetase also were observed in CD99 gene group.
2.The living cell of L428 cell has experienced from monoclonal to the multi-cell forming process,and presented characteristic phenomenon whice a big cell(diameter≥5μm)was embranced by many small clone cells small cells(diameter≤10μm).The NF-κB was continue actived.
3.The size of L428-MVC cells become smaller by transfectent of CD99 gene and the immunophenotypic criteria for diagnosis of HD such as CD15,CD30 were vanished.However,it had not characteristic mark of T cell orientation.The L428-MVC cells multiplication capacity were increased by transferctent of CD99 gene,but it causes the cell to apopatosis accelerated also.The CD99 gene plays the roles to regulated NF-κB and oncogenicity of CD99 gene does not increase in vivo.
New point
Based on the biological information sciences analysis,we propose and confirm the relations between the CD99 gene and NF-κB in cell line L428 of person HL.CD99 gene is influential role molecular to change HL cellular form,the immunity phenotype and the biology characteristic through inactivation of NF-κB.
引文
2.以探究CD99基因对HLH/RS细胞的影响,对比转染CD99基因前后、bortezomib干预前后CD99基因与NF-kB的相关关系,探讨CD99基因是否通过NF-kB起作用,并为本研究后续工作奠定基础。
3.将L428-MVC、L428-EVC细胞亚系进行细胞周期、凋亡、生长曲线分析,及体内成瘤情况观察,探究CD99基因在HL中所起的作用。
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3.将L428-MVC、L428-EVC细胞亚系进行细胞周期、凋亡、生长曲线分析,及体内成瘤情况观察,探究CD99基因在HL中所起的作用。
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