Experimental Investigation On The Effects Of K562 On The Growth Characteristics And The Secretory Of Negative Regulation Factor Of BMSCs

Objective: To investigate the effects of K562 on BMSCs by observing the changes of the proliferation and the secretory of negative regulation factor of BMSCs in separated culture or contact culture, and to clarify the effects of K562 on the biology chapter of BMSCs and the effects of BMSCs on the proliferation of K562.Methods: 1. Investigation object: Normal bone marrow (totol 9 samples) and CML bone marrow (totol 15 samples) were from volunteer of Southwest hospital and Xinqiao hospital. 2.Experiment material: K562 was from the department of molecular genetics. Insert-well were self-restraint. 3. Experiment method: (1) Cell culture: K562 was cultured on BMSCs after cell confluence at 10d in normal BMSCs and at 15d in CML BMSCs. (2) Cell group: The experimental cell were divided into normal BMSCs group (3 samples) and CML BMSCs group (6 samples). Each group was divided into: Control; separated culture group,SCG; contact culture group,CCG. (3) Detect method: On 2d,4d,7d and 12d after culture, the growth of BMSCs was observed under optical microscope; the K562 cells were counted with optical microscope; the level of TGF-β1 and MIP-1α in supernatant of the culture medium was measured by an enzyme-Linked immunosorbent assays(ELISA); the expression of TGF-β1 and MIP-1α mRNA on BMSCs was determined by hybridization in situ; the apoptosis of BMSCs and K562 was detected by TUNEL. Cell cycle of BMSCs was determined by flow cytometry at 2d,3d,5 dand 7d after coculture.Results: 1.The effects of K562 on normal BMSCs in separated culture. (1) Changes of the growth of BMSCs: There was no significant change in the quantity of control. The quantity of normal BMSCs in separated culture was significantly lower than that of control on 12d. (2) Changes of the secretory of TGF-β1 and MIP-1α: There was no significant change of TGF-β1 and MIP-1αin control. The level of TGF-β1 on normal BMSCs in separated culture was significantly higher than that of TGF-β1 on control and K562 after 4d(P<0.01). The level of MIP-1αon normal BMSCs in separated culture was significantly higher than that of control after 4d(P<0.01). (3) Changes of TGF-β1 mRNA and MIP-1αmRNA: The positive rate of TGF-β1 mRNA and MIP-1αmRNA on normal BMSCs in separated culture was significantly higher than that of control after 4d(P<0.01).(4) Changes of cell cycle: the proportion of cells in G2 phase rised on 3d and 7d(p<0.05), and reached the highest level on 5d (p<0.01). (5) Changes of apoptosis: There was no significant change of apoptosis in separated culture. 2. The effects of K562 on CML BMSCs in separated culture. (1) Changes of the growth of BMSCs: There was significant change in the quantity of control on 12d. The quantity of BMSCs in separated culture was significantly lower than that of control on 12d. (2) Changes of the secretory of TGF-β1 and MIP-1α: There was no significant change of TGF-β1 and MIP-1αin control. The level of TGF-β1 on CML BMSCs in separated culture was significantly higher than that of TGF-β1 on control and K562 after 4d(P<0.01). MIP-1αon CML BMSCs was significantly higher than that of control after 4d(P<0.01). (3) Changes of TGF-β1 mRNA and MIP-1αmRNA: The positive rate of TGF-β1 mRNA and MIP-1αmRNA on CML BMSCs in separated culture was significantly higher than that of control after 4d(P<0.01).(4) Changes of cell cycle: the proportion of cells in G2 phase was significantly higher than that of control after 2d(P<0.01). (5) Changes of apoptosis: the apoptosis of CML BMSCs was significantly higher than that of control after 4d(P<0.01). 3. The effects of K562 on normal BMSCs in contact culture. (1) Changes of the growth of BMSCs: The quantity of BMSCs in contact culture was significantly lower than that of control and that in separated culture on 12d. (2) Changes of the secretory of TGF-β1 and MIP-1α: The level of TGF-β1 on BMSCs in contact culture was significantly higher than that of TGF-β1 on control and K562 after 4d(P<0.01). MIP-1αon BMSCs was significantly higher than that of c...