Study On The Method Of Serum-free Suspending Culture To Enrich Human Gastric Cancer Cell Line SGC-7901Stem Cells And Stemness Identification

Objective：Preliminary enrich human gastric cancer SGC-7901stem cells with serum-freesuspending culture method and investigate its possibility, in which try to find out themost proper condition. Identify the stemness of acquired cells.Methods：1．Enrich the human gastric cancer SGC-7901stem cells by serum-free suspendingculture method involving supplements under ultralow attachment condition. Makesure the best group of supplements through combining different couples of thevarieties and concentrations.2．Detect the ability of self-renewal by sphere forming essay in acquired cells. Testthe ability of cloning by colony forming essay in acquired cells. Inspect the ability ofmulti-differentiation by immunofluorescence essay in acquired cells. Check out theability of chemoresistance by MTT. Examine the ability of high tumorigenicity bytumor forming essay in Balb/c nude mice which has been considered the goldstandard of cancer stem cells. Detect the expression of CD44/Oct4in acquired cellsby western blot and flow cytometry.Results：1．It is possible for human gastric cancer SGC-7901cells to form cancer stem cellspheres in the circumstance with supplements (EGF：20ng/ml、bFGF：10ng/ml、B27：2%、N-2：1%) and ultralow attachment condition. The rate and volume of formingspheres are related to the cell generations. The descendants of sphere-forming cellsseem more stereo under microscope.2．Sphere forming essay indicated that the efficiency of sphere forming insphere-forming cells were more outstanding than that in SGC-7901cells（28.6%vs 4.1%）.Colony forming essay indicated that the cloning ability of acquired cells weremore outstanding than SGC-7901cells（78.3%vs20.4%）.Chemo resistant essayindicated that acquired cells were resistant to fluorouracil compare to SGC-7901cells(P＜0.05,Resistance Index=5.68). Tumor forming essay in vivo indicated thattumors from acquired cells grew faster, they had larger volume（3127mm3vs424mm3）and heavier weight. Immunofluorescence staining indicated that acquiredcells can differentiate to SGC-7901cells when serum was supplied to cancer stem cellculture. Flow cytometry essay indicated that compared to SGC-7901cell, the part ofOct4and CD44positive in acquired cells were more remarkable (Oct4:96.03%vs4.69%;CD44:30.17%vs2.58%). Western blot indicated that Oct4and CD44expression is more outstanding in acquired cells.（P＜0.05）Conclusions：1.Gastric cancer suspending spheres can be enriched by serum-free suspendingculture with supplements(EGF：20ng/ml、bFGF：10ng/ml、B27：2%、N-2：1%) underultralow attachment condition which had been considered the best.2.The sphere-forming cells possess stemness characters of self-renewal,differentiation, chemoresistance and tumorigenicity which belong to gastric cancerstem cells. That is, serum-free suspending culture is the effective way to enrich gastriccancer stem cells.3.CD44was higher expression on the surface of sphere-forming cells compared toSGC-7901cells. Oct4was higher expression in the nuclear of sphere-forming cellscompared to SGC-7901cells.CD44/Oct4may be the potential mark group of gastriccancer stem cells.