The Effects Of Human Embryonic Germ Cells On The Growth Inhibition Of Ovarian Cancer

【Background】Ovarian cancer is one of the three most common malignant gynecologic tumors, whose morbidity is the third in the women's genital cancers.Because of deficiency of special symptoms and effective earlier diagnosis,it is an advanced tumor when diagnosed.Ovarian cancer is generally characterized by poor prognosis,a high rate of recurrence and early metastases.The 5-year overall survival rate is approximately 30%.The cause of ovarian cancer is unknown.In recent years,the hypothesis of cancer stem cells has been put forward because of the similarities between the stem cells and cancer cells.It is supposed that there are cancer stem cells in the tumor which contribute to the development of cancer and cancer stem cells may come from the mutation of normal stem cells.Now some kinds of side population cells which have some characteristics resemble to stem cells have been found in several kinds of cancers including ovarian cancer.The hypothesis of cancer stem cells brings new perspectives for the treatment and plasticity of tumors.Pluripotent stem cells are the most popular one in the stem cell treatment.They demonstrate self-renewal and display the ability to differentiate into a variety of normal cell types of all three primary cell lineages based on different microenvironments.They operate normally under the control of many factors.This comparison evokes our question:do the human pluripotent stem cells,that follow this normal developmental process,have the potential to keep the growth of tumor cells under control? There is some evidence that embryonic stem(ES)cells may inhibit somatic cell growth.Lightfoot et al.reported that mouse ES cells could inhibit the growth of human pancreatic carcinoma cells in vitro.A recent study by Hendrix et al. demonstrated that metastatic melanoma cells could be reverted to a normal,skin cell-like type with the ability to form colonies similar to human embryonic stem cells (hESCs)under the microenvironment of two hESCs lines.The human embryonic germ cell(hEGC),that has similar characteristics to embryonic stem cells coming from the inner cells,belongs to the pluripotent stem cell and comes from primordial germ cells.Our current study investigated the potential effect of hEGCs on a human ovarian cancer cell line(SKOV3)both in vitro and in vivo.PART 1THE ISOLATION,CULTURE AND IDENTIFICATION OF HUMAN EMBRYONIC GERM CELLSObjective:To isolate human primary germ cells from the gonads of abortion fetuses and turn them into human embryonic germ cells through conditioned culture.Methods:1,The mouse embryonic fibroblast coming from day 13 post-coitus fetuses of KM mouse was used as feeder layer cells.2,Human primary germ cells were isolated from the gonads of abortion embryos at 5-7 weeks postconception.Human embryonic germ cells were cultured and passaged under conditioned medium.During the passage,we compared the effect of collagenase IV + mechanical digestion and trypsin.Whether LIF has effect on the culture of human embryonic germ cells was simply investigated.3,Some hEGCs clones were taken out for characterization.Alkaline phosphatase (AKP)activity and Antibodies for stage-specific embryonic antigens(SSEA-1,3, 4),OCT-4,TRA-1-60,TRA-1-81 were detected.Spontaneous differentiation was also tested.Results: 1,The mouse embryonic fibroblasts were cultured and passaged.Three to five passage were used as feeder layer cells.2,After 2-3 days' primary culture,groups of tightly packed cells with distinct cell borders were recognized.HEGCs could be cultured and passaged to passage 11 under conditioned medium.Collagenase IV + mechanical digestion was fit for the passage of hEGCs.Little difference was observed after LIF was taken out of the conditioned medium.3,Analysis was performed on some hEG clones at different passages and high level of AP activity was shown in EG clones.In addition to AKP activity,EG clones were characterized by a range of cell surface markers including:SSEA-1,SSEA-3, SSEA-4,OCT-4,TRA-1-60,TRA-1-81.When we replaced the conditioned culture medium with DMEM/F-12 and 10%heat-inactivated fetal calf serum, hEGCs could spontaneous differentiate into nerve-sphere liked cells or cells which could contract automatically.Conclusion:We successfully isolated and cultured hEGCs which conform to the characterization reported.PART 2THE EFFECT OF HUMAN EMBRYONIC GERM CELLS ON SKOV3 CELLS IN VITRO AND IN VIVOObjective:To investigate the effect of human embryonic germ cells on SKOV3 in vitro through coculture and in vivo using a xenograft model in SCID mouse.Methods:1,In vitro experiment1),The SKOV3 cells and hEGCs were seeded orderly onto the 6-well plates for coculture.Only SKOV3 cells were cultured in the control groups.After coculturing for 48h,HE and cell counting were performed to compare the proliferation of SKOV3 cells between the two groups.2),Tunel apoptosis and caspase-9 activity were detected by immtmocytochemistry.3),The expression of AKT and p-AKT were examined through western blot.At the same time,akt mRNA was measured by real-time PCR.2,In vivo experiment1),Forty mice were randomly separated into two groups.The SKOV3 cells were subcutaneously injected into the fight flank of the SCID mice to form a xenograft model.2),Seven days after SKOV3 cells injection,2×10~5 EG cells in 0.1ml PBS were injected into the tumors in the experiment groups,only 0.1ml PBS was injected into the tumors in control groups.Tumor volume was assessed by measuring two axes(R1,R2)and calculated using the formula:V=1/6πR12 R2.3),The mice were sacrificed respectively at day 14,24,35.Tumor tissues were fixed in 10%formaldehyde,and embedded in paraffin wax,then cut into 6-μm-thick sections.The sections were also taken for tunel apoptosis assay.Results:1,In vitro experiment1),After coculturing,HE staining exhibited that SKOV3 cells near the EG clones were sparser than those far off.Cell counting showed that there was a 1.5-fold growth reduction for SKOV3 cells in the coculture group.There were(7.8±2.4)×10~4 in the coculture group,while(11.5±2.3)×10~4 in the control group.2),There were more positive signals of tunel and caspase-9 in the coculture group, especially near the clones,than the control group.3),SKOV3 cells in the coculture group showed less p-AKT and akt mRNA than those in the control group.2,In vivo experiment1),The time-course of tumor volume change was detected.It is noteworthy that the rumors in the EG group grew dramatically slower than those in the control group.2),More positive signals of the tunel apoptosis were detected in the experiment group.Conclusion: The hEGCs could inhibit the growth of SKOV3 cells in vitro by inducing apoptosis by inhibiting AKT pathway and activating caspase-9.The transplantation of EG cells suppressed the growth of tumor which may be caused by inducing apoptosis of SKOV3 cells.PART 3COCULTURE HUMAN EMBRYONIC GERM CELLS WITH SKOV3 CELLS ON A MICROFLUIDIC CHIPObjective:A well-designed microfluidic device with unidirectional-perfusion has been developed to observe the effect of human embryonic germ(hEG)cells on SKOV3 cells.Methods:1,The microfluidic chip was designed according our experiment and fabricated as reported.2,The hEGCs and SKOV3 cells in the experiment group were seeded in the inlet reserviors and the outlet reserviors separately,and were cocultured for two days. In the control group,just SKOV3 cells were seeded in the outlet reservoirs.The medium was perfused unidirectionally from the inlet to the outlet reserviors.3,The growth of SKOV3 cells were observed online.4,Tunel apoptosis was detected after coculturing.5,Coculture on 6-well plate was used as a parallel group to compare the differences between the two cell culture methods.Results:1,A microfluidic chip suitable for cell culturing was designed according to the experiment.The barrier makes it possible that medium can flow slowly and come to the balance between the inlet reserviors and the outlet reservoirs after 12h. 2,Using our device,the growth of SKOV3 cells could be observed online.The growth inhibition of SKOV3 cells by hEG cells was monitored intuitionally.3,More apoptosis signals in SKOV3 cells culture area were detected in the coculture group,which decreased along flowing of the medium.4,Compared with the traditional coculture,microfluidic chip showed many advantages in the experiment.Conclusion:In conclusion,the results demonstrated that microfluidic chip might be a potential tool to invest the effect of stem cells on cancer cells with intuitionistic cell-based screens.The perfused microfluidic system could control the change of culture microenvironment better and assure uniform perfusion of the cell culture media throughout the cell culture chamber.