Investigation Of The Effect Of Bone Marrow Mesenchymal Stem Cells On Leukemia Of Asparaginase Resistance In Vitro

Objective:1,adult acute lymphoblastic leukemia bone marrow primary cells were long-term cultured in vitro,and BM-MSCs were isolated from normal human and ALL patients cultured for proliferating to passage cells; cell morphology and growth characteristics of BM-MSCs and primary cells were observed under inverted microscope. 2,To study the influence of BM-MSCs on lymphocytic leukemia cells resistant to asparaginase.Methods:1,Adult acute lymphoblastic leukemia patients (20 cases) bone marrow primary cells were cultured for long-term, and cell morphology and growth characteristics were observed under inverted microscope. 2. BM-MSCs were isolated from normal human and ALL patients cultured for proliferating to passage cells. 3. 4 different lymphocytic leukemia cell lines were cultured (Jurkat,CEM,CA46,nalm-6), which IC50 (50% inhibition rate) were tested by MTT assay,after treated in different concentrations of asparaginase. 4. 4 cell lines were cultured with BM-MSCs of normal and ALL patients, then proliferations of cell lines after co-culture with/without treated in asparaginase were tested by MTT assay. 5. The expression of ASNS in normal cells , ALL,and leukemic celllines were detected by RT-PCR.6. The expression of ASNS in cell lines and BM-MSCs before and after co-culture and/or asparaginase treatment were detected by Real-time relative quantitive PCR.Results:1,The growth of primary bone marrow cell of adult ALL patients (20 cases):untreated patients (n = 4) bone marrow adherent growth does not appear during 1-2 weeks after mononuclear cell suspension inoculation, the cells gradually died after 3 weeks; majority relapsed patients (5 cases) bone marrow do not appear adherent cells growth, few cases can be Observed the growth of a small amount of adhered cells, which can not proliferation and would apoptosis in 4-5 weeks; partly CR patients after 1-2 times chemotherapy(3 cases) and CR patients (5 / 8) bone marrow can be Observed adherent, spindle stromal cells growth in 48-72h after they mononuclear cells Suspension inoculation, "cobblestone" areas appear in 1-2 weeks, and would get long-term survival in vitro; 3 / 8 cases performed no adherence phenomenon for too small cells; ALL bone marrow MSCs adhered the bottom of the flask and were fusiform shape, could proliferate , passage in vitro and cryopreservation.2,a.The expression of ASNS expression ALL patients with newly diagnosed low or very low; b. Compared with control group,the proliferation of different cell lines cultured with normal MCSs which tested by MTT,was significantly inhibited in 1-2 days(P<0.01),but was promoted after 3-5days.c.Jurkat has the highest IC50 concentration of asparaginase in 4 cell lines;d. Compared with control group, the inhibition rate of L-asp to lymphocytic leukemia cell lines was obviously lowed when cultured with MCSs. e.the expression of ASNS in cell lines was no difference after treated by L-aspor co-cultured with MSCs,while significantly up-regulated inMSCs.Conclusion: 1.In vitro long-term growth,bone marrow stromal cell growth of adult acute lymphoblastic leukemia in patients with newly diagnosed and relapsed was inhibited, and some chemotherapy had remission bone marrow stromal cells; 2, MSCs can help to protect lymphocytic leukemia cell lines from apoptosis induced by asparaginase resistance, which may be achieved through upregulation of MSCs asparagine synthetase expression.