Effects Of Umbilical Cord Mesenchymal Stem Cells On The Proliferation And Apoptosis Of Ovarian Cancer

OBJECTIVE:Human umbilical cord mesenchymal stem cells(UC-MSCs)are a kind of stem cells with multiple differentiation potential.They have many advantages,such as wide source,simple collection,rapid amplification,strong migration ability,ability to synthesize a variety of cytokines and regulate immunity,so they have attracted more and more attention.UC-MSCs have strong homing ability and broad application prospects in cancer targeted therapy.In this part,primary UC-MSCs were isolated from human umbilical cord tissue by tissue explants adherent method,and identified from cell morphology,cell immune phenotype and proliferation ability in vitro.This part laid a foundation for further research.METHODS:Umbilical cord was taken from healthy full-term caesarean section fetus of Shanghai General Hospital of Nanjing Medical University.Fresh umbilical cord tissue was put into sterile PBS solution containing penicillin and streptomycin.UC-MSCs were separated by tissue explants adherence method.The growth and morphological characteristics of UC-MSCs were observed under microscope.The immunophenotypic expression of UC-MSCs was detected by flow cytometry.UC-MSCs of P3 generation were inoculated into 24-well plate.Three holes were digested randomly every 24h.Cells were collected and counted.Mean values of three experimental results were taken to draw cell growth curve and observe the proliferation of UC-MSCs in vitro.RESULTS:Under inverted microscope,adherent cells formed communities on the 7th to 8th days of tissue culture.After 14 days,cell fusion could reach 80%-90%.Under the microscope,the cells were mononuclear,and formed special whirlpool-like arrays and developed a defined spindle-shaped fibroblastic morphology.Flow cytometry was used to detect the immunophenotype of UC-MSCs.The cells overexpressed CD106,CD29,CD105,CD44 and CD73,but did not express CD34,CD45,CD14,CD31 and HLA-DR,which belonged to the typical immunophenotype expression of UC-MSCs.The growth curve of UC-MSCs cells in the P3 generation experienced latentperiod,logarithmic and plateau period.The growth curve of UC-MSCs cells was "S" type,which accorded with the general growth law of cells.CONCLUSION:Mesenchymal stem cells isolated from fresh human umbilical cord tissue by tissue explants adherent method conform to the characteristics of UC-MSCs and can be used in future research.OBJECTIVE:Ovarian cancer is the most lethal gynecological malignancy.Because of the asymptomatic early stage,most patients are in the advanced stage when diagnosed,leading to poor prognosis.The morbidity of ovarian cancer is increasing year by year.The mortality rate is the highest among gynecological malignant tumors,and it is still the main malignant disease threatening female health.Proliferation and metastasis of ovarian cancer are the main causes of malignant clinical outcomes.UC-MSCs have the ability to target tumor microenvironment,which makes them become a new hotspot in cancer targeted therapy research.At present,the anti-tumor activities of UC-MSCs in the proliferation and metastasis of human ovarian cancer cells were rarely reporte.In this part,we observed the homing characteristics of UC-MSCs targeting ovarian cancer in vitro,and explored the possible molecular mechanism of UC-MSCs inhibiting the proliferation of ovarian cancer cells.METHODS:Transwell co-cultured UC-MSCs and SKOV3 cells for 24 and 48 hours,respectively,to observe the targeted homing characteristics of UC-MSCs to ovarian cancer cells in vitro.Transwell migration experiment verified the effect of UC-MSCs conditioned medium on inhibiting the migration of SKOV3 cells.MTT assay was used to detect the inhibitory effect of UC-MSCs conditioned medium on the proliferation of SKOV3 cells.RT-PCR was used to detect the expression of proliferation-related gene Ki-67 at RNA level.RESULTS:Transwell co-culture results showed that after co-culture for 24 hours,the migrated UC-MSCs in experimental group and control group were 14.67±7.04 and 96.17±23.42,respectively.After co-culture for 48 hours,they were 19.5±7.46 and 205.17±52.44,respectively.With the increase of co-culture time,a large number of UC-MSCs migrated to the lower chamber,indicating UC-MSCs had tumor homing characteristic,which was significantly different from the control group(P<0.01).Transwell migration experiment indicated that after 48 hours of treatment with 75%UC-MSCs conditioned medium,the migrating SKOV3 cells in the experimental group and the control group were 56.50±8.54 and 108.67±20.73,respectively,suggesting that UC-MSCs could dramatically inhibit the migration ability of SKOV3 cells,with a significant difference compared with the control group(P<0.01).MTT results showed that the proliferation activity of SKOV3 cells treated with 25%,50%and 75%UC-MSCs conditioned medium was 89.80±7.68%,59.57±11.52%and 42.05±9.27%,respectively.The proliferation of SKOV3 cells was significantly inhibited by 50%and 75%UC-MSCs conditioned medium.With the increased UC-MSCs conditioned medium concentration,the inhibition rate increased and the proliferation activity of SKOV3 cells was remarkablely decreased(P<0.01).RT-PCR results showed that the expression of Ki-67 in control group was 1.01±0.11,and in SKOV3 cells treated with 25%,50%and 75%conditioned medium were 1.00±0.25,0.83±0.28 and 0.69±0.31,respectively.75%conditioned medium could significantly inhibit the expression of Ki-67,which was significantly different from the control group(P<0.01).CONCLUSION:UC-MSCs can target SKOV3 cells in vitro,inhibit the migration of SKOV3 and significantly reduce the expression of proliferation-related factor Ki-67 in ovarian cancer cell SKOV3.UC-MSCs may be an ideal mesenchymal stem cell for targeted treatment of ovarian cancer.OBJECTIVE:Inactivation of apoptosis is the center of tumorigenesis and development.Escaping apoptosis may be an important reason for insensitivity to treatment and poor prognosis of ovarian cancer.The main participants of apoptotic pathway include Bax gene which promotes apoptosis and Bcl-2 gene which inhibits apoptosis.In this part,the potential molecular mechanism of UC-MSCs promoting apoptosis of ovarian cancer SKOV3 cells was explored through in vitro experiments.METHODS:After treated with UC-MSCs conditioned medium for 48h,the morphological changes of SKOV3 cells were observed by PI staining.Annexin V-FITC/PI double staining was used to detect apoptosis of SKOV3 cells.RT-PCR was used to detect the expression of Bcl-2 and Bax genes.RESULTS:PI staining showed that after treated with 50%and 75%UC-MSCs conditioned medium for 48h,the proliferation of SKOV3 cells was significantly inhibited,cell density was decreased,and apoptotic positive cells were increased.Flow cytometry showed that UC-MSCs conditioned medium promoted SKOV3 cells apoptosis.Annexin V-FITC/PI double staining showed that the apoptotic rates of SKOV3 cells treated with 75%UC-MSCs conditioned medium for 24 and 48 hours were 7.47±1.17%and 31.57±2.73%respectively,which were significantly higher than those of control group 4.87±0.75%and 14.10±0.82%,the differences were statistically significant(P<0.05).RT-PCR results showed that UC-MSCs conditioned medium could significantly induce SKOV3 cell apoptosis via inhibiting the expression of Bcl-2 gene and up-regulating the expression of Bax gene.With the increased concentration of conditioned medium,the effect of promoting apoptosis was increased,with a significant difference compared with the control group(P<0.01).CONCLUSION:UC-MSCs can promote the apoptosis of ovarian cancer SKOV3 cells by regulating the expression of apoptosis-related genes Bcl-2 and Bax in vitro.OBJECTIVE: Ovarian cancer is prone to recurrence and metastasis,seriously affecting the quality of patients' life.Rapid cell proliferation and significant reduction of apoptosis result in unsatisfactory treatment and prognosis.In this part,clinical data were used to explore the relationship between the expression of cell proliferation-related gene Ki-67 and apoptosis-related gene Bcl-2 and the clinical stage,pathological grade and lymph node metastasis of ovarian cancer,so as to find a reasonable therapeutic target for ovarian cancer.METHODS: Immunohistochemistry was carried out to stain histopathological sections.Ki-67 positive expression was localized in the nucleus of the tumor cells,showing brown-yellow granules.The positive expression of Bcl-2 protein was located in the cytoplasm,which was brown or dark yellow.Both of them were determined by the percentage of positive cells.The percentage of positive cells was divided into 2grades: weak positive(<30%)and strong positive(>30%).Compare the differences of expression levels between different clinical stage,pathological grade and lymph node metastasis.RESULTS: The expression of Ki-67 was correlated with the clinical stage of ovarian cancer and lymph node metastasis.The strong positive expression in stage III/IV was significantly higher than that in stage I/II(P<0.05).The expression of Ki-67 in patients with positive lymph node metastasis was significantly higher than that in non-metastasis patients(P<0.05).There was no significant correlation between Ki-67 expression and pathological grade(P>0.05).The expression of Bcl-2 is closely related to the stage and grade of ovarian cancer.The strong positive expression in stage III/IV was significantly higher than that in stage I/II(P<0.05).The strong positive expression of G2/G3 was significantly higher than that of G1(P<0.05).There was no significant correlation between the expression of Bcl-2 and lymph node metastasis(P>0.05).CONCLUSION: The expression of Ki-67 is correlated with FIGO stage and lymph node metastasis of ovarian cancer.The expression of Bcl-2 is correlated with FIGO stage and pathological grade of ovarian cancer.Ki-67 and Bcl-2 can be used as important targets for experimental study of ovarian cancer.