Application of lrsam1 as a molecular marker for hepatocellular carcinoma
A hepatocellular carcinoma and drug technology, applied in the field of biomedicine, can solve the problems of unclear expression and function of hepatocellular carcinoma
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Embodiment 1
[0041] Example 1: Under normal in vitro culture conditions, the effect of LRSAM1 on the growth of human hepatocellular carcinoma cells
[0042] 1. Using siRNA to down-regulate the expression level of LRSAM1 in hepatocellular carcinoma cells
[0043] 1) Cell seeding and transfection
[0044] The cells of the logarithmic growth phase hepatocellular carcinoma cell lines Huh7, HepG2, SK-Hep1, and BEL-7404 were respectively taken, digested with 0.25% trypsin, and after centrifugation, the cell mass at the bottom of the glass test tube was bounced by hand; Prepared DMEM medium containing 10% common fetal bovine serum, resuspended the cells, slowly and repeatedly pipetting the cells to make a single cell suspension, and then 2×10 per well 5 Cells were seeded into a six-well plate, so that the final volume of each well of the six-well plate was 1.5 mL; the six-well plate was then placed in an incubator for overnight culture; the cells were observed under an inverted microscope the ne...
Embodiment 2
[0080] Example 2: Effect of LRSAM1 on the growth of human hepatocellular carcinoma cells under soft agar colony formation experimental conditions
[0081] 1. Construction of HepG2 cell line with stable low expression of LRSAM1
[0082] Digest the HepG2 cells in the logarithmic growth phase with 0.25% trypsin, collect them in a glass centrifuge tube, cover with a sterile rubber stopper, centrifuge at 1200rpm for 4min, and resuspend with new DMEM medium supplemented with 10% FBS after centrifugation Cells (pay attention to gently pipetting the cell suspension, try to make a single cell suspension). Dilute the cells to an appropriate concentration according to the cell concentration, draw 10 μL of the diluted cell suspension, count the cells under a microscope, plant them into a six-well plate after calculation, and the number of cells per well is 5×10 4 After 24 hours, the state of the cells and the density of the cells were observed under a microscope.
[0083] When the cells...
Embodiment 3
[0115] Example 3: The effect of LRSAM1 on the growth of human hepatocellular carcinoma cells in vivo
[0116] Take cloned cells in logarithmic growth phase, such as stable cell lines of GFP-HepG2 and GFP-LRSAM1-HepG2; or HepG2 cell lines with stable expression of NC shRNA and stable low expression of LRSAM1 (LRSAM1 shRNA 549# or LRSAM1 shRNA 1636#) ;
[0117] First wash the cells with sterile normal saline, digest the cells with 0.25% trypsin, make a single cell suspension, put it into a glass test tube, centrifuge at 1200rpm / min for 4min, discard the old medium, and then use serum-free Resuspend the cells in DMEM medium and perform a cell count.
[0118] According to the number of nude mice used in the experiment and the number of cells per nude mouse, obtain the number of cells you need, centrifuge in a horizontal centrifuge, discard the old medium, and then use serum-free DMEM medium Resuspend the cells, then insert the Matrigel matrigel that was placed in a 4°C refrigera...
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