Preliminary Study On Morphology And Functional Characteristics And Expression Of NF-κB In L428-MVC/EVC Cells

BackgroudIt 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.Objective1.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.Method1.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.Result1.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.Conclusion1.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 pointBased 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.