| Human Mer tyrosine kinase(MerTK) was first cloned fromλgt-11 cDNA lymphoblastoid library by Graham,and mouse Mer was cloned successfully the next year by the same person.Comparison of the mouse and human c-mer amino acid sequences reveals an overall identity of 88%.Human Mer is so named after its original reported expression pattern—monocytes and epithelial and reproductive tissues.Mer belongs to Axl receptor tyrosine kinase(RTK) family,which is composed of Axl,Tyro3 and Mer,and they have the common ligand—Gas6.Axl RTK subfamily members each possess a combination of two immunoglobulin(Ig)-like domains and two fibroneetin typeⅢ(FNⅢ) repeats in their extracellular regions.Among these,IgG-like domains can bind their ligand-Gas6,and FNⅢrepeats play a regulation fuction in binding process.Intraeellular region of this family is tyrosine kinase domain,and also contains the special KWIAIES sequence.After binding their ligand-Gas6,each Axl RTK family member appears to signal for apoptosis,proliferation,migration and phagocytosis functional outcomes,besides these they also participate in many pathology processes,just like infammafion, autoimmunity,thrombosis and hemostasis,vascular and kidney disease and even the development of some malignant diseases.Now the focus of Axl RTK family is Axl,and many research were carried out through the Gas6/Axl axis.After cloned in 1994,there were few reports about Gas6/Mer until recnet years,more and more articles are involved in the Mer function,there includes inflammation,apoptosis,phagocytosis, thrombosis and hemostasis and abnormal expression of Mer in some malignant diseases.Preparation and function study of monoclonal antibody against Mer1.Prokaryotic expression of Mer receptor tyrosine kinase IgG-like domains The existed reports show that Mer Ig-like domains is most important during the process binding its ligand Gas6,and only binding Mer Ig-like domains,Gas6/Mer can activate its tyrosine kinase activity,so preparation of monoclonal antibody against Mer Ig-like domains has great significance to study the function of Gas6/Mer.We designed the cloning primers of Mer Ig-like domains according to the sequence in gene bank by using Primer 5 softwoare,interested fragment was amplified from K562 cDNA template,and after linked to PQE30 expression vector,we constructed Mer-Ig-PQE30 recombinated vector successfully.The recombinated vector was transformed to E.coli M15.The positive clones were chosen and recombinated Mer-Ig(rMer-Ig) prokaryotic protein was induced by IPTG.After analized by 12%SDS electrophoresis,the recombinated protein molecular weight is about 26kD,mainly existed in inclusion body and accounted for 15%of total bacterial protein.The purified rMer-Ig can be special recognized by anti-His antibody by Western blot.2.Preparation,characterization and function study of monoclonal antibody against MerBy using our established method,the recombined protein was purified by Ni-NTA agarose column and Balb/c mice were immunized.After the spleen cells of the mice and SP2/0 hybridoma cells were hybridized,positive clones was screened by ELISA assay and the selected hybridoma cells were used to prepare ascites.Western blot was used to detect whether the prepared McAb can bind prokaryotic rMer-Ig protein,eukaryotic Met extracellular region protein and natural Mer protein in cell lines.Characterization of immunize subclass and tissue specificity.And detected prepared McAb inhibition effect on platelet aggregation.The results show that:After the spleen cells and hybridoma cells SP2/0 were hybridized,we got one strain of cell that can last secreting McAb against Mer named SZ-128.the selected hybridoma cells was used to prepare ascites The titers of McAbs in ascites was 1×10-5 detected by indirect ELISA.The heavy chain of the McAbs belonged to IgG1 subclass.The concentrations of purified anti-Mer McAb were 0.8mg/ml after purifying by proteinG-Sephrose4B affinity chromatography column.The antibodies could not only identify 26kD prokaryotic rMer-Ig but also eukaryotic rMer extracellular region and natural Mer protein in Jurkat and K562 cell specially.The results of immuno-labelling show that Mer protein is high-expression in human prostatae,lung,kidney;and relative low-expression in spleen,intestine,colon,liver and tonsilla,and almost no expression in esophagus smooth muscle.In platelet aggregometry,we found that SZ-128 could inhibit platelet aggregation induced by ADP(2μmol/L) and ristomycin(1.25mg/ml),the inhibition ratio are 30.2%±8.5%and 25.6%±7.4%respectively(The final concentration of SZ-128 is 50μg/ml),but there is no significant deviation between the inhibition ratio of different dose group(20μg/ml,50μg/ml and 100μg/ml),so we couldn't observe dose-dependent effect obviously.This phenomenon may due to Gas6/Mer signal pathway doesn't participate in platelet aggregation diretly,they just plays a signal-amplification fuction role in midanaphase of platelet aggregation.The regulation effect of receptor tyrosine kinase Mer on endothelial cell1.The influence of receptor tyrosine kinase Mer on endothelial cell apoptosisApoptosis is a programmed death process in normal system,which is important to maintain the stability of internal environment.Many reports show that many receptor tyrosine kinase including Axl RTK patticipate in cell apoptosis,and most of them are growth factor receptors.To study the effect of Mer RTK on EC apoptosis process,we used the classic growth factor starvation method to induce apoptosis of HMEC-1,which is a acknowledged human microvascular endothelial cell line,realtime-PCR and cell-ELISA were used to detected Mer mRNA and protein expression alteration during apoptosis, according to the ELISA results,Mer expression increased gradually from the beginning, reached the climax at 4h,and then descended gradually,and return to the normal level at 48h,the Mer mRNA alteration trend is similar to the result of ELISA.Simultaneously,human Mer full length vector and pcDNA3.1(negative control) vector were transfected into HMEC-1 by using Lipofectamine following the protocol, after identified with realtime-PCR and Western blot,high expression Mer-HMEC-1 was established.And we also remove the growth factor to induced transfected HMEC-1 apoptosis,after evacuated 48h,the apoptosis of Mer-HMEC-1 and negative control were 5.8%±3.1%and 20.8%±6.8%respectively(P＜0.01),there was significant difference between these two transfected cells.And bcl-2 mRNA expression was 178% in Mer-HMEC-1 compare to the negative control detected by realtime-PCR.We can observe from the above results that Mer expression was increased after apoptosis,and the anti-apoptosis ability of Mer-HMEC-1 is better than the negative control during growth factor starvation induced apoptosis through up-regulating the anti-apoptosis gene-bcl-2.2.Study of anti-angiogenesis effect induced by overexpressed Mer and its mechanismGas6/Axl can inhibit human umbilical vein endothelial cell angiogenesis in vitro, and there is few report about the similar function of Gas6/Mer.We used established Mer-HMEC-1 cell model,and used pcDNA3.1-HMEC-1 as a negative control.From transwell migration experiment,we can observe that the migration capacity of Mer-HMEC-1 decreased,the cell number migrated through the insert is 21±6 vs 36±11(negative control)(P＜0.05),there is statistics difference.And the angiogenesis experiment based on Matrigel show that the angiogenesis capacity of Mer-HMEC-1 decreased significantly,and the number of tube-like form was 6±4 vs 26±8(negative control),the average inhibition ratio was 76.92%,there was significant difference(P＜0.01) between these two cells.The alteration of main angiogenesis -associated growth factor VEGF-A,VEGF-B,VEGF-C,VEGF-D,VEGFR-1和VEGFR-2 were scanned by realtime-PCR,the results show that the mRNA expression of VEGF-C and VEGFR-2 decreased significantly in Mer-HMEC-1,it was 44.7%(P＜0.01)和25.6%(P＜0.01) compare to the negative control,the the expression of VEGF-A,VEGF-B,VEGF-D and VEGFR-1 were similar to negative control(P＞0.05),Study on abnormal expression of Mer in acute leukemia and its mechanism1.Abnormal expression of Mer RTK in acute leukemiaThere is no expression on normal granulocyte,T and B lymphocyte,but there is abnormal high expression on some leukemia cell lines,so we can guess is there ectope epression of Mer RTK on leukemia patient? And does Mer RTK participant in the occurrence and development of leukemia?Bone marrows sample from 57 acute leukemia patient(32 AML,and 25 ALL) and 5 adults healthy donors were collected.Flow cytometry was used to analyse Mer expression on mononuclear cell of patient and normal control bone marrow.The flow cytometry results:there was no Mer expressed on granulocyte and lymphocyte of normal bone marrow;There were 23 Mer positive patients in total 32 AML patients; Mer expression level was high in 3 patients,middle in 8 patients,low in 12 patients and very low/negative in left 9 patients.Among 25 ALL patient,there were 16 Mer positive, Mer expression level was high in 1 patients,middle in 6 patients,low in 9 patients and very low/negative in left 9 patients,among all the positive patient,5 sample were from T-ALL patient,and 11 from B-ALL patient.The results of RT-PCR was similar to flow cytometry.And we found the most of the high and middle Mer expression AML patient were M1 or M2 according to FAB group,and M3 patient were almost low expression of Mer,and some of T-ALL Mer positive patient had the immature immunize type.This implied that Mer may take part in cell differentiation and development of leukemia.2.Study on the mechanism of Mer abnormal expression in acute leukemiaWe found abnormal expression of Mer in parts of acute leukemia patient,and it was necessary to explore its molecular mechanism.We screened Mer expression on common leukemia cell lines,and selected high Mer expression cell line-Jurkat as cell model to downstream experiment.We transfected verified and effective anti-Mer siRNA into Jurkat to block Mer expression specificially,and use uncorrelate siRNA sequence as negative control.After transfected with anti-Mer siRNA,the proliferation of Jurkat was a little slowly by MTT assay,but there was no statistics difference compare to the negative control(P＞0.05).Blocking Mer expression in vitro Jurkat cell capacity of anti-apoptosis decreased significantly,after serum starvation for 48h,the apoptosis ratio of Jurkat transfected with anti-Mer siRNA was(15.3±2.3)%,while negative control was only(1.5±0.5)%,and we used realtime-PCR to analyze change of main apoptosis-associated factors-bcl-2 and Caspase-3,from the results we can observe that the bcl-2 mRNA expression of Jurkat transfected with anti-Mer siRNA was only (42.7±8.6)%compare to the negative control,and there was no obvious change of Caspase-3.All the above results suggested that Mer could increase Jurkat capacity of anti-apoptosis via bcl-2 signal pathway. From Human Mer gene was first cloned as a new member of receptor tyrosine kinase family in 1994,more and more research work were reported about its function. In the present study,we revealed that Mer participated in the apoptosis and angiogenesis process of endothelial cell;and Mer was ectopic expression in some acute leukemia patient,this indicated Mer play an important role in the occurrence and development of acute leukemia.Thus,targeting the Gas6/Mer signal patheway is hoped to become a new target of leukemia treatment in the future. |
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