Inhibitory Effect Of IL-12 Gene Modified Human Umbilical Cord Mesenchymal Stem Cells On Ovarian Carcinoma In Vitro And Vivo

Ovarian carcinoma seriously threatens female health and the mortality caused by it is mostly in all kinds of woman malignant tumors. Its main features are malignant growth, metastasis and recurrence. Presently the treatment, including of surgery and chemotherapy, can't reach the expectant goal of prolonging patient'life farthest because the early diagnoses is difficult, the curative effect isn't good in an advanced stage, and ovarian carcinoma may be recrudesce after operation. Long-term survival rate can't be improved with routine treatment, new treatment must be found to improve prognosis. Now biotherapy is research focus for anti-ovarian cancer, include Immunotherapy and gene therapy. IL-12 is considered of the most effective therapeutic means to anti-cancer. Foreign gene easily is expressed and introduced in UC-MSCs. With transgenic approach, IL-12 is introduced in UC-MSCs to treat ovarian cancer. In this study, UC-MSCs as targeting vehicles loaded AdIL-12-MSCs. To observe the effect on growth of morphology, proliferation, apoptosis of SKOV3 xenograft with AdIL-12-MSCs in vitro. To observe the effect on angiogenesis of human ovarian carcinoma SKOV3 transplanted subcutaneously in nude mice. To observe exogenous IL-12 effect on prevention, cancer growth, lymphangiogenesis and angiogenesis of human ovarian carcinoma OVHM transplanted subcutaneously. To investigate the effect of anti-ovarian cancer of chemotherapy combined gene-therapy, to evaluate the feasibility of UC-MSCs loaded anti-cancer gene and to investigate the anti-cancer mechanism of AdIL-12-MSCs. PartⅠInhibitory Effect of IL-12 Gene Modified Human Umbilical Cord Mesenchymal Stem Cells on Ovarian Carcinoma SKOV3 in VitroObjective: To observe effect on growth morphology, proliferation, apoptosis of ovarian cancer SKOV3 cell by UC-MSCs and AdIL-12-MSCs in vitro. To investigate the effect and mechanism of AdIL-12-MSCs in Anti-ovarian Cancer and to evaluate the feasibility of UC-MSCs loaded anti-cancer gene.Methods: Ovarian cancer SKOV3 cell and UC-MSCs were cultured in vitro. UC-MSCs was targeting carrier and loaded AdIL-12-MSCs. Express of IL-12 protein was detected with Western Blotting in AdIL-12-MSCs. Express of IL-12 mRNA was detected with RT-PCR in AdIL-12-MSCs. Express of IL-12 was detected with ELISA in AdIL-12-MSCs culture fluid. Growth and morphological of SKOV3 cell were observed with light microscope in UC-MSCs and AdIL-12-MSCs culture fluid. It was detected with MTT that UC-MSCs and AdIL-12- MSCs culture fluid influenced on multiplication of SKOV3 cell. It was detected with flow cytometry that UC-MSCs and AdIL-12- MSCs culture fluid influenced on apoptosis of SKOV3 cell.Result: Protein and mRNA of IL-12 were found in AdIL-12-MSCs group. After culture 48h, IL-12 content was 79.27±18.36 ng/ml in AdIL-12-MSCs fluid. After culture 24-72h, growth of SKOV3 cell was inhibited in UC-MSCs group and AdIL-12-MSCs group. Furthermore, compared with UC-MSCs group, growth of SKOV3 cell was conspicuously inhibited in AdIL-12-MSCs group, and depressant effect potentialized as time. Capability of inhibiting multiplication of SKOV3 potentialized as time in UC-MSCs group and AdIL-12-MSCs group. Compared with UC-MSCs group, capability of inhibiting multiplication of SKOV3 was more conspicuous in AdIL-12-MSCs group (P<0.05). It was found for promoting apoptosis of SKOV3 cell in UC-MSCs group(P>0.05). It was significantly that apoptosis rate(9.72±1.38%) in AdIL-12-MSCs group was higher than (2.69±0.45)% in UC-MSCs group(P<0.05).Conclusions: AdIL-12-MSCs cell line was succeed construction. Secretory IL-12 of AdIL-12-MSCs conspicuously restraint multiplication of SKOV3 and facilitate apoptosis of SKOV3 cell. Compared with UC-MSCs PartⅠInhibitory Effect of IL-12 Gene Modified Human Umbilical Cord Mesenchymal Stem Cells on Ovarian Carcinoma SKOV3 in Vitro group, growth of SKOV3 cell was conspicuously inhibited in AdIL-12-MSCs group, and depressant effect potentialized as time. It is feasible that UC-MSCs is used to load anti-cancer gene.PartⅡInhibitory Effect of IL-12 Gene Modified Human Umbilical Cord Mesenchymal Stem Cells on Ovarian Carcinoma SKOV3 xenograft in nude miceObjective: To establish the model of human ovarian carcinoma SKOV3 transplanted subcutaneously in nude mice. To observe exogenous IL-12 effect on cancer growth, vascularization of xenograft. To investigate the effect of anti-ovarian cancer of chemotherapy combined gene-therapy, to evaluate the feasibility of UC-MSCs loaded anti-cancer gene and to investigate the anti-cancer mechanism of AdIL-12-MSCs.Methods: SKOV3 cells(Human ovarian carcinoma cell lines) were cultured in vitro, to establish the model of human ovarian carcinoma SKOV3 transplanted subcutaneously in nude mice. Divided randomly 56 nude mice into 7 groups. After bearing cancer 7d, vena caudalis transplantation was done(1time/5d). (1) control group: 0.2ml PBS;(2) UC-MSCs group:1×10~6 ~UC-MSCs in 0.2ml PBS;(3) Ad-MSCs group: 1×10~6 Ad-MSCs in 0.2ml PBS;(4) AdIL-12 group: 109PFU AdIL-12 in 0.2ml PBS; (5) AdIL-12-MSCs group: 1×10~6 AdIL-12-MSCs in 0.2ml PBS; (6) cyclophosphamide group: 0.2ml PBS vena caudalis transplanta -tion; after inoculation 8d, 100mg/kg cyclophosphamide intraperitoneal injection 2 time/2 week; (7) combination group (AdIL-12-MSCs+ cyclophosphamide): 1×10~6 AdIL-12-MSCs in 0.2ml PBS plus 100mg/kg cyclophosphamide (2 time/2 week).The tumor volumes are measured every 3 days and draw the tumor growth curve by the ending of the experiment on 30 days. The formation rate, tumor weight and inhibitory rate of each group were observed. Apoptosis rate and cell cycle of the SKOV3 transplanted tumor were assessed in each group. Immunohist -ochemsitry was used to investigate the influence on the expression of IFN-γ, VEGF and CD34 in tumor tissue.Result: 1 The growth curve of human ovarian carcinoma SKOV3 transpl -anted subcutaneously in nude mice: each therapy group all showed sup -pressive effect on tumor growth and the most obviously is combination group. 2 Tumor weight and inhibitory rate: significant deviation was found between control group and treatment group, AdIL-12-MSCs group and AdIL-12 group, combination group and cyclophosphamide group(P<0.05). Inhibitory rate of UC-MSCs group, Ad-MSCs group, AdIL-12 group, AdIL-12-MSCs group, cyclophosphamide group and combination group respectively was 14.42%, 12.10%, 43.81%, 59.08%, 69.74% and 81.84%. 3 Apoptosis rate of UC-MSCs group, Ad-MSCs group, AdIL-12 group, AdIL-12-MSCs group, cyclophosphamide group and combination group respectively was (10.02±0.17)%, (9.76±0.25)%, (14.32±1.08)%, (19.25±1.22)%, (22.56±0.32)% and (30.21±0.56)%. Significant deviation of apoptosis rate was found between control group and treatment group, AdIL-12-MSCs group and AdIL-12 group, combination group and cyclophosphamide group(P<0.05). 4 The influence of AdIL-12-MSCs on the expression of IFN-γ: Significant deviation of IHS was found between AdIL-12-MSCs group with other groups(P < 0.05). Significant deviation of IHS was found between combination group and cyclophosphamide group (P<0.05). AdIL-12-MSCs had promotion to expression of IFN-γ. 5 AdIL-12-MSCs influence on vascularization of transplanted tumor: Significant deviation of IHS and MVD rate was found between treatment group and control group, AdIL-12-MSCs group and AdIL-12 group, combination group and cyclophosphamide group(P<0.05). AdIL-12-MSCs had obviously depressant effect to expression of VEGF proteinum, Vascularization of transplantation tumor decreased.Conclusions: AdIL-12-MSCs can inhibit growth and induce apoptosis for SKOV3 xenograft in nude mouse. Mechanism of anti-vascularization maybe that AdIL-12-MSCs induce production of IFN-γand down regulation expression of VEGF protein. AdIL-12-MSCs combined with chemotherapeutics could enhance inhibiting tumor. UC-MSCs could be used to load antioncogene.PartⅢInhibitory Effect of IL-12 Gene Modified Human Umbilical Cord Mesenchymal Stem Cells on Ovarian Carcinoma OVHM xenograft in vivoObjective: To establish the model of human ovarian carcinoma SKOV3 transplanted subcutaneously in mouse. To observe exogenous IL-12 effect on prevention, cancer growth, lymphangion genesis and angiogenesis of human ovarian carcinoma OVHM transplanted subcutaneously. To investigate the effect of anti-ovarian cancer of chemotherapy combined gene-therapy, to evaluate the feasibility of UC-MSCs loaded anti-cancer gene and to investigate the anti-cancer mechanism of AdIL-12-MSCs.Methods: OVHM cells were cultured in vitro, to establish the model of human ovarian carcinoma OVHM transplanted subcutaneously in mouse. Divided randomly 80 nude mice into 8 groups. Prevention group: intrap -eritoneal injection 1×106 AdIL-12-MSC in 0.2ml PBS. After 1 week, bearing cancer was done. After bearing cancer 7d, vena caudalis transplantation was done(1time/5d) in other groups. (1) control group: 0.2ml PBS;(2) UC-MSCs group:1×106 UC-MSCs in 0.2ml PBS;(3) Ad-MSCs group: 1×106 Ad-MSCs in 0.2ml PBS;(4) AdIL-12 group: 109PFU AdIL-12 in 0.2ml PBS; (5) AdIL-12-MSCs group: 1×106 AdIL-12-MSCs in 0.2ml PBS; (6) cyclophosphamide group: 0.2ml PBS vena caudalis transplantation; after inoculation 8d, 100mg/kg cyclophosphamide intraperitoneal injection 2 time/2 week; (7) combination group (AdIL-12-MSCs+ cyclophosphamide): 1×106 AdIL-12-MSCs in 0.2ml PBS plus 100mg/kg cyclophosphamide (2 time/2 week).The tumor volumes are measured every 3 days and draw the tumor growth curve by the ending of the experiment on 30 days. The formation rate, tumor weight and inhibitory rate of each group were observed. Apoptosis rate and cell cycle of the SKOV3 transplanted tumor were assessed in each group. Immunohistochemistry was used to investigate the influence on the expression of CD4+, CD8+, IFN-γ, LYVE-1 and CD31 in tumor tissue.Result: 1 The growth curve of human ovarian carcinoma OVHM transpl -anted subcutaneously: apart from UC-MSCs and Ad-MSCs groups, other therapy groups all showed suppressive effect on tumor growth and the most obviously is combination group.2 Tumor weight and inhibitory rate: significant deviation was found between control group and treatment group, AdIL-12-MSCs group and AdIL-12 group, combination group and cyclophosphamide group(P < 0.05). Inhibitory rate of AdIL-12 group, AdIL-12-MSCs group, cyclophosphamide group and combination group respectively was 32.34%, 58.42%, 68.32%, 87.79%. 3 infiltrating leukomonocyte of each group: major CD4+ and CD8+ infiltration was found in AdIL-12-MSCs and AdIL-12 group. CD4+ and CD8+ infiltration obviously grown downwards in other groups. 4 generating absorbent vessels: UC-MSCs could have auxoaction to expression of LYVE-l protein. AdIL-12-MSCs had conspicuous depressant effect to expression of LYVE-l protein. In positive expression of LYVE-l,It shown significantly for treatment group to compare with control group, AdIL-12-MSCs group compare with AdIL-12 group, combination group and cyclophosphamide group (P<0.05). 5 AdIL-12-MSCs influence on vascularization of transplanted tumor: Significant deviation of IFN-γexpression was found between AdIL-12-MSCs group and AdIL-12 group(P<0.05). It didn't show significantly for combination group and cyclophosphamide group (P>0.05). AdIL-12-MSCs had conspicuous depressant effect to expression of IFN-γprotein. UC-MSCs and Ad-MSCs can't inhibit vascularization of xenograft, expression of CD31 markedly degraded in other treatment groups. Significant deviation of CD31expression was found between AdIL-12-MSCs group and AdIL-12 group, combination group and cyclophosphamide group(P<0.05). AdIL-12-MSCs can inhibit vascularization of xenograft, combination chemotherapeutics can enhance inhibiting vascularization. 6 growth of xenograft, lymphization, vascularization in prevention group: 7out of 10 mice had xenograft after innoculation OVHM cell and tumor growth slow. Body weigh, food and drin, motoricity didn't change. Expression of CD4+, CD8+ and IFN-γheighten, and expression of LYVE-1 and CD31 cut down in prevention group.Conclusions: AdIL-12-MSCs can inhibit growth of OVHM xenograft, mechanism maybe promotion T cell multiplication and induction expression of IFN-γ, then, lymphization and Vascularization of xenograft are inhibitted . AdIL-12-MSCs plus chemotherapeutics can enhance anti-tumor. UC-MSCs could be used to load antioncogene. AdIL-12-MSCs can prevent tumor formation, and mechanism maybe that IL-12 can interfere lymphization and Vascularization of tumor in early period.