| Objective: Study found that about 40%-70% follicular lymphoma(FL)patients' bone marrow were involve at first newly diagnosed,which were associated with poor prognosis.The interaction between FL tumor cells and bone marrow(BM)microenvironment further affects each other's biological behaviors.Previous studies showed that bone niche was similar in the biological activities to the FDC nets--the FL tumor microenvironment.Osteoblasts(OB),one of stromal cells in BM microenvironment,co-cultured with FL tumor cells directly in vitro,played an important role in the migration,adhesion and proliferation process of FL tumor cells,further study on the mechanism of FL patients with BM involvement has a profound significance on the targeted treatment of FL patients with BM involvement.In summary,we mainly investigate the adherion mechanism of FL tumor cells and OB niches,further to find the precise moleculars involved in the process of FL BM involvement by bloking the adhesion process,in this way,we can find a new targeted therapy of FL.Methods: In the co-culture system of FL cell lines(DOHH2)and OB,flow cytometry(FC)was used to analysis the positive rates and MFI changes of adherion molecules(ICAM-1?CXCR4?OB-cadherin and CD44)before and after the co-culture of OB and DOHH2,in this way,we got the preliminary outcome.Then,Real-time PCR(RT-q PCR)was used to analysis the gene changes of adherion molecules(CXCR4?OB-cadherin?OPN?OCN and SDF-1)before and after DOHH2 and OB co-cultured.Western blot were usd to detected protein levels of adherion molecules(CXCR4?OB-cadherin and OPN),and Enzyme linked immunosorbent assay(ELISA)were usd to detected the secretion levels of adherion molecules(OCN and SDF-1),speculating the most possible adhersion moleculars between OB and DOHH2,further targeting blocked the CXCR4,ICAM-1 on DOHH2 with monoclonal antibodies to find the effects on proliferation rates and adhesion rates of DOHH2,analysised the results and draw a conclusion.Results: 1.(1)FC detected the positive rate of CXCR4 on DOHH2 was 100%,after co-cultured with OB for 48 hours,the positive rate of CXCR4 didn't change,the MFI of CXCR4 reduced by(61.43±4.35)%,which had a significant changes(P<0.05);(2)FC found that positive rate of ICAM-1 on DOHH2 was nearly 100%,after co-cultred with OB for 48 hours,the positive rate had no changes while the MFI incresed by(36.71±1.56)%,which had a great significance(P<0.05);(3)FC detected the positive rate of ICAM-1 on OB,before and after co-cultured with DOHH2,were about 88%.the MFI of ICAM-1 increased by(13.37±13.05)%,however,it had no statistically significant(P>0.05);(4)The positive rate of OB-cadherin was(54.33±4.80)%,after OB co-cultured with DOHH2,the positive rate of OB-cadherin increased by(41.93±4.74)% and MFI increased by 170.67±20.50,which were significant both(P < 0.05).(5)FC found the positive rate of CD44 on OB was 100%,after OB co-cultured with DOHH2,neither of the positive rate and MFI changed(P>0.05);2(1)After OB and DOHH2 co-culture for 24 hours and 36 hours,RT-q PCR found that the CXCR4 m RNA of co-cultured group was(0.472±0.03)times and(0.48±0.09)times of the DOHH2 single cultured group,respectively,CXCR4 m RNA expression was significantly reduced(P < 0.05);(2)RT-q PCR detected the 24 hours' OB-cadherin m RNA expression of co-cultured group was(1.183±0.17)times of OB alone group,which had no significance(P>0.05);however,the 36 hours co-cultured group was(9.019±1.54)times of OB group,the expression of OB-cadherin was up-regulated(P < 0.05);(3)After OB and DOHH2 co-cultured for 24 hours and 36 hours,the OPN m RNA expression of co-cultured group were(1.757±0.07)times and(2.288±0.28)times of OB groups,respectively,the up-regulation of OPN were significantly(P < 0.05);(4)RT-q PCR detected that the OCN m RNA expression of 24 hours co-cultured group was the OB alone group's(1.576±0.30)times,which was no significant(P>0.05);the results showed that 36 hours experimental group was(2.578±0.31)times of control group,the expression of OCN was up-regulated(P < 0.05);(5)Co-cultured for 24 hours and 36 hours,the SDF-1 m RNA expression of co-cultured group were(1.204±0.24)times and(1.711±0.34)times of OB group,respectively,however,the up-regulation of SDF-1 m RNA had no significant(P>0.05);3(1)Collecting 12 hours,24 hours and 36 hours samples for Western blot,the CXCR4/?-actin expressiont,in co-cultured groups were(48.322±5.8)%,(36.136±2.90)% and(40.910±5.87)% of DOHH2 groups,respectively,which were significant changs(P<0.05),but had no time dependence(P>0.05);(2)After OB and DOHH2 co-cultured for 12 hours,24hours and 36 hours,the OB-cadherin/?-actin expression in co-cultured group increased by(46.312±8.13)%,(9.181±2.50)% and(28.730 ±6.76)%,respectively,the OB-cadherin changed significantly(P < 0.05);(3)Co-cultured with DOHH2 for 12 hours,24 hours and 36 hours,OB expressed OPN were incresed,OPN/?-actin expression in OB were increased by(-5.50±5.39)%,(20.630±5.77)% and(42.870±9.86)%,respectively,the OPN of 24 hours and 36 hours samples increased greatly(P<0.05);4(1)ELISA detected the OCN concentration of 12 hours,24hours and 36 hours OB groups were(291.879±17.43)ng/L,(305.605±20.06)ng/L and(440.315±22.74)ng/L,while the co-cultured group were(330.626±26.38)ng/L,(370.227±30.08)ng/L and(487.160±33.76)ng/L,respectively,the results showed the OCN concentration increased(P < 0.05);(2)ELISA showed the SDF-1 secreted by OB were(580.156±58.03)ng/L,(594.520±32.10)ng/L and(597.133±30.66)ng/L after cultured for 24 hours,48 hours and 72 hours,respectively;co-cultured with DOHH2,OB secreted SDF-1 were(601.868±54.11)ng/L,(575.012±45.33)ng/L and(566.716±37.05)ng/L at the same times,the datas had no change(P>0.05);5(1)Blocking the CXCR4 expression by anti-CXCR4 monoclone antibody for 24 hours,we found that the growth rate of the(OB+DOHH2+anti-CXCR4 antibody)group detected by CCK8 was(0.0369±0.45)times higher than the(OB+DOHH2)group,the(OB+DOHH2+anti-ICAM-1 antibody)group decreased than the(OB+DOHH2)group by(0.0156±0.11)times,and the data had no statistical significance(P > 0.05);(2)After blocking DOHH2 surface molecule CXCR4,the adhesion rate of the(OB+DOHH2)group was(43±1.00)%,and the adhesion rate of the(OB+DOHH2+anti-CXCR4 antibody)group was about(12±0.80)%,the adhesion rate was reduced by about 31%,and the(OB+DOHH2+anti-ICAM-1 antibody)group adhesion rate were(9±0.60)%,the adhesion rate decreased by about 34% and both were significant differences(P < 0.05);Conclusion: 1 DOHH2 promotes the adherent process with OB by up-regulating the expression of ICAM-1 and down-regulating the expression of CXCR4;2 OB promotes the adhesion with DOHH2 by up-regulating the expression of OPN,OB-cadherin and promting the secretion of OCN;3 CXCR4 and ICAM-1 participate in the adherent process between OB and DOHH2,but not the proliferation process... |
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