Effect Of Astragalus Polysaccharides On The Proliferation And Expression Of TAFs Related Molecules Of BMSCs In The Co-culture System With Lung Cancer Cells

OBJECTIVE: To study the proliferation, cell cycle and expression of the tumor-associated fibroblasts protein of bone marrow mesenchymal stem cells(BMSCs) under Lewis lung cancer(LLC) microenvironment through establishing a co-cultured system, and analyse the protective effect and molecular mechanism of Astragalus polysaccharides(APS) on BMSCs in tumor microenvironment in order to provide experimental evidence for safely clinical application of BMSCs. METHODS: 1. The effect of different concentration(0~200 μg/m L) of APS at different time points(12~96h) on the proliferation of the BMSCs and LLC cells was investigate by CCK-8, and the effective concentration of APS was screened out. Then we screened out the most appropriate time to administer APS with phenol-sulfuric acid method at different time points. 2. After established a co-cultured system of BMSCs and LLC cells, the cultured system was divided into 4 groups: BMSCs group, LLC group, Co-BMSCs group and APS-Co-BMSCs group. Meanwhile, 3 treatments, exchanging DMEM/F12 at 48 h, exchanging DMEM/ F12 at 72 h and no-treatment, were set up to each group. 3. CCK-8 was used to mesure BMSCs cells in lung-cancer cells co-culture system changes in growth and the cell cycle was tested by flow cytometric analysis.4. The expression of TAFs related molecules α-SMA and FAP protein of BMSCs in lung-cancer cells co-culture system was detected by Western bolt. RESULTS:1. Compared with control group, 50 μg/m L APS could promote the proliferation of BMSCscells and inhibit LLC cells after incubating 24 h(P<0.01), with statistical significance.However, high concentration of APS(100 μg/m L and 200 μg/m L) could inhibit the proliferation of LLC cells after incubating 24 h(P<0.01), with statistical significance, but promot BMSCs cells after incubating 60 h(P<0.01), with statistical significance. 2. There was a negative correlation between the contents of DMEM/F12 with 50 μg/m L APS and the incubation time in vitro. As time went on, the glucose content of DMEM/F12 with 50 μg/m L APS reduced gradually(P<0.05), with statistical significance. It reached a half between 36~48 h. 3. Under phase-contrast microscopy, BMSCs cells showed fibroblast-like, spindle, orderly, regular and swirling growth; Co-BMSCs cells growed irregular, polygonous, agglomerate, slender and cytoplasmic contraction; APS-Co-BMSCs cells were mostly spindle and agglomerate occasionally. 4. Compared with BMSCs groups, Co-BMSCs groups growed faster from the 3rd day to 7th day(P<0.05), with statistical significance. Compared with Co-BMSCs groups, APS-Co- BMSCs groups growed slower from the 5th day to 7th day(P<0.05), with statisti- cal significance. 5. The results of flow cytometric analysis showed the proportion of cells in G1 phase of Co-BMSCs groups was significant decreased,while the proportion of cells in S phase was increased(P<0.05), with statistical significance; Compared with Co-BMSCs groups, the proportion of cells in G1 phase of APS-Co-BMSCs groups was increased, and the proportion of cells in S phase was significant decreased(P<0.05), with statistical significance.6. The expression of α-SMA and FAP protein in Co-BMSCs groups raised(P<0.05); Meanwhile in APS-Co-BMSCs groups the expression of α-SMA and FAP protein declined in comparision with Co-BMSCs groups(P<0.05), with statistical significance. CONCLUSION: BMSCs under lung cancer microenvironment could have genetic stability changes, and the mechanism may be related to the differentiation of BMSCs under tumor microenvironment into TAFs in vitro; APS could inhibit these changes of BMSCs in lung-cancer cells co-culture system, and exchanging DMEM/F12 every 48 hours could improve the effect of APS, the mechanism may be related to improving the tumor environment and restraining the differentiation of BMSCs under tumor microenvironment into TAFs in vitro.