| Part1: The isolation, culture and biological characteristics identification ofhuman umbilical cord-derived mesenchymal stem cellsObjective: To isolate human umbilical cord-derived mesenchymal stem cells fromnewborn human umbilical cord and indentify the surface marker and multipledifferentiation potential.Methods: The fresh human umbilical cord were washed. Atery and vein wereremoved remaining Wharton’s jelly. MSCs were isolated and amplified viatissue-cultivation. Morphology and adherent condition of hUC-MSCs were observedunder the optical microscope．Cell growth curve was obtained by WST-1assay. Cellsurface markers were analyzed by flow cytometry．The hUC-MSCs were induceddifferentiate into osteoblast and adipocyte to test the differetiation potential.Results: Morphology observation showed that hUC-MSCs were fusiform,whirlpool-like, and grown at plastic wall, gradually purified with passage. Cell growthcurve shape was similar to "S". Surface marker analysis showed that hUC-MSCsstrongly expressed CD90, CD29, CD73, CD105, weakly expressed CD106,negatively expressed CD45, CD34and HLA-DR. Experiments of differentiationindicated that the hUC-MSCs were capable of differentiating into osteoblast andadipocyte.Conclusion: Pure hUC-MSCs can be isolated from human umbilical cord viatissue-cultivation and after subculture its surface marker and differentiation potentialmeet the mesenchymal stem cell biological characteristics. It can be the stable sourceof cells for the follow-up experiments. Part2: hUC-MSCs effect on liver cancer cell growth in vitroObjective: To explore the effect of hUC-MSCs on the proliferation and migration ofHep3B via Transwell co-culture system in vitro.Methods: Hep3B and hUC-MSCs in logarithmic growth phase were cultured. Cellproliferation assay: Established the0.4μm Transwell non-contacted co-culture system,the upper well contained hUC-MSCs, lower well contained Hep3B. The experimentwas divided into three groups: low-dose hUC-MSCs group (1hUC-MSCs:1Hep3B),high-dose hUC-MSCs group (2hUC-MSCs:1Hep3B), blank control group. After24hours,48hours and72hours co-culture Hep3B proliferation was tested by WST-1.Cell migration assay: The experiment was divided into three groups: low-dosehUC-MSCs group (1hUC-MSCs:1Hep3B), high-dose hUC-MSCs group (2hUC-MSCs:1Hep3B), blank control group. After24hours of culture, Hep3Bmigrated to lower wells were determined by crystal violet staining assay. Cells werecounted randomly selected five field at high magnification.Results: The proliferation assay: After24hours co-culture cell proliferation was theame in each group (P>0.05). After48hours and72hours co-culture, compared withthe control group, the cell proliferation of two experiment groups decreased (P <0.05).Cell migration assay: the number of migrated cell showed: low-dose group:92.50±11.36, the high dose group:134.76±16.43, the control group:67.33±8.72.There were significant differences between the groups (P <0.05).Conclusion: After co-culture of hUC-MSCs and Hep3B in vitro, hUC-MSCs caninhibit the proliferation of Hep3B. The hUC-MSCs can promote Hep3B cellmigration in vitro. Part3: Effect of hUC-MSCs on the growth of Hep3B in vivoObjective: To explore the effect of hUC-MSCs injected by the tail vein on the growthof Hep3B by establishing human hepatocellular carcinoma xenograft model in nudemice.Methods: The model: logarithmic growth phase of Hep3B cells.2×107cells wereinjected into the femoral subcutaneous of the nude mice. After the tumor growed to a certain size, the mices were randomly divided into two group: experiment group andcontrol group. The mice in experiment group were injected0.5ml1×106cells/ml ofhUC-MSCs cell, the control group was injected with an equal volume of PBS,injected once weekly for three weeks. The tumor volume was measured every4days.After3weeks the mice were sacrificed, stripped out the tumors, the lungs and thelivers were tested by biopsy. The tumor tissues were carried on Ki-67immunohistochemistry.Results: The mean tumor volume (2257.34±279.42mm3) of experiment group wassmaller than the control group (3215.20±408.25m3)(p <0.05). Ki-67in theexperimental group were lower than the control group (p <0.05). Lung and liver tissuesections were not found the evidence of metastases.Conclusion: The hUC-MSCs injected by the tail vein can inhibit the growth of thetumor in nude mice, but have no significant effect on the metastases of the tumor. |
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