| Melanoma is a kind of malignant tumor with high recurrence,high metastasis and poor prognosis.The incidence of melanoma is increasing rapidly in all regions of the world.The role of various miRNAs in melanoma has been defined.The up-regulation of the expression of tumor suppressor miRNAs or down-regulation of tumor promoting miRNAs have the therapeutic potential of melanoma.Although regulation of miRNAs is considered to be an important potential means of tumor therapy,there is currently a lack of techniques and methods to achieve the regulation of miRNAs expression in tumor cells and tumor tissues.Basic research has applied widely in liposome or lentiviral transfection methods due to the instability of the vector.In recent years,the construction of miRNAs carriers based on nanomaterials has brought hope to regulate the expression of miRNAs.Due to its metal properties,gold nanoparticles have the disadvantages of high biotoxicity.In recent years,the biosynthesis of the green gold nanoparticles are considered to have the advantages of low biotoxicity and high biocompatibility,which are the basic requirements of nanomaterials as tumor drug carriers.Biologically synthesized gold nanoparticles provide a new possibility for the treatment of melanoma,Based on this,we will be selected in the black adenoma with potential therapeutic value of mi R-664 as a genetic drugs,combined with biological synthesis of gold nanoparticles for the following research: 1)in the Siberian ginseng extract,for medium,through the biological synthesis of gold nanoparticles,characterization of the material characteristics of the stability of the gold nanoparticles cell toxicity and biological compatibility;2)To construct the gold nanoparticle mi R-664 vector and investigate its effects on cell proliferation,migration and invasion at the cell level;3)Establish the xenograft model of melanoma mice,investigate the inhibitory effect of gold nanoparticles mi R-664 on xenograft tumor,and investigate its feasibility as a drug for the treatment of melanoma.Part One Synthesiz gold nanoparticles from Siberian ginseng using the biosynthetic methodObjective: In the Siberian ginseng extract,for medium,through the biological synthesis of gold nanoparticles,characterization of the material characteristics,the stability of the gold nanoparticles,cell toxicity and biological compatibility.Methods: The water extract of Siberian ginseng was prepared,and gold nanoparticles(SG-GNPs)were prepared by mixed incubation of water extract and chloro-auric acid.In addition,traditional gold nanoparticles(GNPs)were prepared by chemical method.1)The material characteristics of nanomaterials were characterized by UV-Vis spectroscopy(UV-Vis)analysis,selected area diffraction(SAED)mode analysis,transmission electron microscopy(TEM)-XRD analysis and Fourier transform infrared(FTIR)spectroscopy analysis.2)Stability of SG-GNPs.SG-GNPs and GNPs were placed in Na Cl,Li Cl and HCl solutions,respectively,and the absorption peaks at 538 nm were analyzed by UV-Vis spectroscopy.3)Biological toxicity of SG-GNPs.SG-GNPs and GNPs were co-incubated with normal mouse melanocytes cells,respectively,and the cell survival rate at 24 h was detected.The absorption peaks at 538 nm were analyzed by UV-Vis spectroscopy in Na Cl,Li Cl and HCl solutions.4)Biocompatibility of SG-GNPs.SG-GNPs and GNPs were injected into the stomach of mice by gastric perfusion method.The venous blood of mice was extracted to detect AST and ALT levels to evaluate the effects of nanoparticles on liver function of mice.The serum creatinine and urea nitrogen levels of mice were detected,and the urine protein levels of mice were detected to evaluate the effect of nanoparticles on renal function of mice.Results: The synthesis of gold nanoparticles from Siberian ginseng: 1)UV-Vis spectrum analysis showed that there was a surface plasmon resonance peak at 538 nm,and it was time stable.HR-TEM,XRD and SAED test results confirmed the spherical shape,crystal properties and size of the synthesized gold nanoparticles.The results of infrared spectrum showed that the biological components in the Siberian ginseng reduced the gold ions to gold nanoparticles.2)The absorbances of SG-GNPs mixed with Na Cl,Li Cl and HCl at 538 nm are 0.624±0.039,0.518±0.034 and 0.616±0.026,respectively.After mixed with Na Cl,Li Cl and HCl,the absorbances at 538 nm were0.216±0.014,0.194±0.019 and 0.208±0.017,respectively.The absorbance of SG-GNPS was significantly higher than that of GNPS,and the difference was statistically significant(P < 0.05).3)The 24 h survival rates of 5,10 and 20?g/ml SG-GNPs cells were 92.74±5.192,89.64±4.167 and 84.12±4.197,respectively.GNPS cells were 82.14±5.021,73.16±3.182 and 65.21±6.142,respectively.The survival rate of SG-GNPS cells was significantly higher than that of GNPS cells,and the difference was statistically significant(P < 0.05).4)The levels of ALT and AST in control group were 55.4±4.62 and91.06±7.19,respectively.ALT and AST levels in SG-GNPs mice were72.7±8.31 and 126.7±11.2,respectively.The levels of GNPS mice were278.9±26.8 and 318.7±28.8,respectively.The levels of ALT and AST in SG-GNPS mice and GNPS mice were significantly higher than those in control group,with statistical significance(P < 0.05).The levels of ALT and AST in SG-GNPS mice were significantly lower than those in GNPS mice,with statistical significance(P < 0.05).5)The levels of urea nitrogen,creatinine and uric acid in control group were 5.76±0.257,respectively;24.28±2.184 and 56.66±4.98;The levels of urea nitrogen,creatinine and uric acid in SG-GNPs mice were 10.33±1.42,37.18±2.048 and 92.56±8.842,respectively.The levels of urea nitrogen,creatinine and uric acid in GNPS mice were 23.84±1.99,52.25±4.121 and 128.5±10.58,respectively.The levels of urea nitrogen,creatinine and uric acid in SG-GNPS mice and GNPS mice were significantly higher than those in control group,with statistical significance(P < 0.05).The levels of creatinine,urea nitrogen and urinary protein in SG-GNPS mice were significantly lower than those in GNPS mice,with statistical significance(P < 0.05).Conclusions: The gold nanoparticles synthesized by biological method from Siberian ginseng water extract have the characteristics of good stability,low biotoxicity and good biocompatibility.Part Two Extra-vivo evaluation of the construction of mi R-664 vector based on SG-GNP for the treatment of melanomaObjective: To construct the gold nanoparticle mi R-664 vector and investigate its effects on cell proliferation,migration and invasion at the cell level.Methods: PAMAM modified gold nanoparticles(Au-PAMAM)were prepared by co-incubation of Pa MAM and SG-GNPs,and Au-PAMAM was further incubated with mi R-664 to prepare Au-PAMAM /mi R-664.B16 cells were treated with Au-PAMAM /mi R-664.1)The expression level of mi R-664 in cells was detected by RT-PCR.2)CCK-8 assay was used to detect cell proliferation.3)The invasion ability of cells was detected by scratch test;4)The migration ability of cells was detected by Transwell assay.5)The expression levels of apoptosis-related proteins Caspase-9,Caspase-3,Bax,Bcl-2 and Bid were detected by Western blot.Results: B16 cells were transfected with Au-PAMAM /mi R-664.1)The level of mi R-664 in Au-PAMAM /mi R-664 group was significantly higher than that in blank control group(4.393±0.172 vs1.000±0.000),and the difference was statistically significant(P < 0.05).2)The cell proliferation rates of Au-PAMAM /mi R-664 group at 12,24 and 48 h were 116.5±5.48,148.9±5.48 and 178.7±6.37,respectively;In blank control group were169.7±10.28,212.4±9.18 and 246.1±10.9,respectively;The cell proliferation rate of Au-PAMAM /mi R-664 group was significantly lower than that of blank control group,with statistical significance(P < 0.05).3)The scratch reduction rate of Au-PAMAM /mi R-664 group was significantly lower than that of blank control group(32.3±6.19 vs 55.8±4.54),and the difference was statistically significant(P<0.05).3)The number of cells passing through the compartment in Au-PAMAM /mi R-664 group was significantly lower than that in the blank control group(144.08±10.69 vs 387.62±17.0),and the difference was statistically significant(P<0.05).5)The levels of caspase-9,caspase-3,Bax,Bid and Bcl-2 in AU-PAMAM /mi R-664 group were 2.324±0.128,2.499±0.229,1.897±0.068,1.719±0.097 and 0.554±0.011,respectively.The levels of Caspase-9,Caspase-3,Bax,Bcl-2 and Bid in blank control group were1.006±0.018,1.022±0.017,1.015±0.004,1.035±0.056 and 1.004±0.028,respectively.The levels of Caspase-9,Caspase-3,Bax and Bid in AUPAMAM /mi R-664 group were significantly higher than those in blank control group,and the levels of Bcl-2 were significantly lower than those in blank control group,with statistical significance(P < 0.05).Conclusions: The Au-PAMAM /mi R-664 prepared in this study can regulate the expression level of mi R-664 in B16 cells,inhibit the proliferation,migration and invasion of melanoma cells at the cellular level,and negatively regulate the apoptotic-related proteins Part Three Construction of mi R-664 vector based on SG-GNP for trea-tment of melanoma in vivo evaluationObjective: Establish a mice melanoma xenograft model,detect the inhibitory effect of gold nanoparticle mi R-664 carrier on xenograft tumor,and study its feasibility as a drug for the treatment of melanoma.Methods: C57BL/6 mice were used to construct the xenograft animal model of melanoma,and the effect of Au-PAMAM /mi R-664 on the expression level of mi R-664 in xenograft was investigated by injecting Au-PAMAM /mi R-664 into xenograft.1)The growth rate of xenograft was observed.The graft volume was measured at 0,7 and 14 days after intratumoral injection.2)On the 14 st day,mice were sacrificed to obtain the transplanted tumor tissues,and the expression level of mi R-664 was detected by RT-PCR.3)The expression levels of apoptosis-related proteins Caspase-9,Caspase-3,Bax,Bcl-2 and Bid in transplanted tumors were determined by Western blot.Results: Au-PAMAM/mi R-664 was injected into mouse melanoma xenograft.1)Tumor volume of Au-PAMAM /mi R-664 group at 7d and 14 d was 146.7±10.36 and 622.7±85.9,respectively;Tumor volume of blank control group on day 7 and 14 was 297.8±21.52 and 936.4±104.2,respectively.The tumor growth rate of Au-PAMAM /mi R-664 group was significantly lower than that of blank control group,and the difference was statistically significant(P < 0.05).2)The expression level of mi R-664 in Au-PAMAM /mi R-664 group was significantly higher than that in blank control group(6.493±0.267 vs 2.456±0.133),and the difference was statistically significant(P < 0.05).3)The levels of caspase-9,caspase-3,Bax,Bid and Bcl-2 in AU-PAMAM/mi R-664 group were 2.542±0.198,2.267±0.123,1.885±0.141,1.448±0.129 and 0.539±0.043,respectively.The levels of Caspase-9,Caspase-3,Bax,Bid and Bcl-2 in blank control group were 1.065±0.057,1.033±0.028,1.057±0.034,1.369±0.094 and 1.138±0.056,respectively.The levels of Caspase-9,Caspase-3,Bax and Bid in AU-PAMAM /mi R-664 group were significantly higher than those in blank control group,and the levels of Bcl-2were significantly lower than those in blank control group,with statistical significance(P < 0.05).Conclusions: Au-PAMAM /mi R-664 prepared in this study can regulate the expression of mi R-664 in melanoma transplantation,inhibit the growth of melanoma at the level of mouse animal model,and negatively regulate apoptosis related proteins. |
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