| Objective: In this paper,the anti-tumor effect and its mechanism of C-phycocyanin of Spirulina platensis were studied in vitro and in vivo.In vitro,this paper mainly studied whether C-phycocyanin has direct inhibitory activity on tumor cells,and to study the anti-tumor mechanism of C-phycocyanin from the level of protein expression.The main content of the in vivo experiment was to study the anti-tumor effect of C-phycocyanin mediated photodynamic therapy on mouse SW1990 cell tumor.To investigate the anti-tumor effect of C-phycocyanin in vivo,and the anti-tumor effect was preliminarily discussed from the perspective of immunomodulation of organism.The anti-tumor effect of C-phycocyanin of Spirulina platensis in vitro and in vivo was detected by cell culture experiment in vitro and tumor-bearing experiment in nude mice,and the anti-tumor mechanism of C-phycocyanin of Spirulina platensis was preliminally discussed,which provide experimental basis for the anti-tumor of C-phycocyanin of Spirulina platensis in clinic.Methods: This paper mainly including three aspects,and its discussed the anti-tumor effect and its mechanism of C-phycocyanin of Spirulina platensis.(1)The reserach of in vitro anti-tumor effect of C-phycocyanin of Spirulina platensis.Be eight kinds of tumor cells(SK-MES-1,Tca-8113,SGC-7901,EJ,Eca-109,A2780,MHCC97-H and SW1990)were used as in vitro model for the preliminary screening test of anti-tumor activity,to study whether C-phycocyanin has a direct inhibitory effect on the growth of tumor cells.According to the preliminary screening test results,C-phycocyanin has the strongest inhibitory effect on MHCC97-H and SW1990.Subsequently,the paper was determined C-phycocyanin on the proliferation of these 2 tumor cells and 1 normal liver cell(L-O2)in vitro.The inhibition effect of C-phycocyanin and the relationship on tumor cells between dose-effect and time-effect were preliminaryinvestigated.The changes of cell morphology induced by C-phycocyanin apoptosis in two tumor cells were observed by inverted microscope.(2)Anti-tumor effect of C-phycocyanin mediated photodynamic therapy on SW1990 cell tumor in mice.SW1990 was inoculated into BALB/c nude mice to establish for the animal model of xenograft tumor.Combined with photodynamic therapy,the anti-tumor effect in vivo of C-phycocyanin was preliminary discussion,and to study its anti-tumor effect and mechanism from the perspective of whether it can improve the immune regulatory function of the body.(3)Study on the mechanism of anti-tumor of C-phycocyanin of Spirulina platensis.To further explore how C-phycocyanin inhibits the growth and promotes the apoptosis of tumor cells.In this paper,flow cytometry was used to detect the changes of apoptosis,and the technology of Western Blot was used to detect the expression levels of apoptosis-related proteins(Bcl-2,Bcl-xl,Bax,Bad).The migration ability in vitro of the tumor cells treated with C-phycocyanin was preliminarily detected by scratch repair experiment in vitro.Results:(1)In vitro culture conditions,MTT assay was used to detect the effect of different concentrations of C-phycocyanin(the ultimate concentrations were 10,20,40,80,160,320 μg/mL)on the growth of tumor cells.The preliminary screening results showed that C-phycocyanin had different effects on the growth of eight kinds of tumor cells in different degrees in comparison with the control group,and the inhibitory effects showed a significant concentration-dose-dependent effect with the extension of the concentration and time of action.Among,C-phycocyanin has the strongest inhibitory effect on MHCC97-H cells and SW1990 cells.Subsequently,the effect of C-phycocyanin on the proliferation of two tumor cells and one normal cell in vitro was detected.The results showed that C-phycocyanin had significant relationship between dose-effect and time-effect on both tumor cells,but had no inhibitory effect on L-O2 in normal cells.The optimal concentration range of C-phycocyanin was set according to the results of the preliminary screening experiment,and then the concentration of C-phycocyanin was uniformly adjusted to three representative concentrations(80,160,320 μg/mL)in the verification of experiment of anti-tumor activity.The influence of C-phycocyanin on the morphology of two tumor cells was observed under an inverted microscope.It was found that C-phycocyanin could make the surface of MHCC97-H cells wrinkle,the tail edge elongated,and the intercellular space becames larger.Its can make SW1990 cells from adherent state to free state,and the cell margins are irregular and puckered.Morphological observation showed that C-phycocyanin could cause abnormal morphological changes of two tumor cells,and it was prelimatively believed that C-phycocyanin could promote the apoptosis of tumor cells.(2)The results of analysis in vivo showed that the tumor inhibition rates of Laser irradiation group,C-phycocyanin group and C-phycocyanin+Laser irradiation group were up to 39.90 %,45.12 % and75.51 %,respectively.Therefore,both Laser irradiation and C-phycocyanin can significantly inhibits tumor growth,and the anti-tumor effect is more obvious after the combination of the two.After Laser and C-phycocyanin treatment can promote the growth and development of spleen in mouse,and increase the index of spleen organ in mouse,and the combined treatment of Laser and C-phycocyanin is more significant(P<0.01).The expression level of TNF-α in spleen cell culture medium and serum of mice,and the expression level of IL-6 in serum of mice were determined by ELISA.The results showed that both Laser and C-phycocyanin could increase the expression of TNF-α and IL-6 compared with the control group,which indicated that the Laser and C-phycocyanin could enhance the immunity of cancer in mice and thus play a role in inhibiting tumor growth.(3)Flow cytometry was used to detect the changes of apoptosis of tumor cells treated with C-phycocyanin.The results showed that the proportion of early and late apoptosis of tumor cells increased with the increase of C-phycocyanin concentration.To further verify the regulatory mechanism of C-phycocyanin on apoptotic proteins,the optimal concentration of apoptosis was 320 μg/mL for Western Blot detection.The results showed that C-phycocyanin could up-regulate the expression levels of pro-apoptotic proteins(Bax and Bad)and down-regulate the expression levels of anti-apoptotic proteins(Bcl-2 and Bcl-xl).These results indicated that C-phycocyanin could induce apoptosis by regulating the expression of apoptotic protein.Furthermore,according to the migration characteristics of tumor cells,To investigate the effect of C-phycocyanin on the migration ability of tumor cells in vitro.The results showed that the cell migration rate decreased with the increase of C-phycocyanin concentration,which indicated that C-phycocyanin could inhibit the migration of the two tumor cells in vitro.Subsequently,the expression of migratory proteins(MMP9 and MMP2)associated with the two tumor cells was further detected,and the results showed that C-phycocyanin could inhibit cell migration in vitro by down-regulating to the expression levels of MMP9 and MMP2 proteins.Conclusion: C-phycocyanin of Spirulina platensis showed a broad spectrum of anti-tumor activity to different tumor cell lines in vitro.It plays an anti-tumor mechanism by inducing apoptosis proteins and promoting migration proteins of tumor cells.The main content of in vivo experiment is the study of C-phycocyanin mediated photodynamic therapy for SW1990 cell tumor in mice.To investigate the effect of C-phycocyanin on tumor growth by enhancing immune system function. |
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