| Part I growth-inhibitory effects of LBP and IFN a2b on murine renal carcinoma cell lines renca in vitroObjective:To investigate the possibility of the growth-inhitory effects of explore the ptimal concentration and the time of LBP and IFN a2b on murine renal carcinoma cell lines Renca, explore the optimal concentration and the time of LBP and IFN a2b to explain the mechanism of action.Methods:1. The changes of Renca cells viability were detected after treating with different concentrations of LBP (50,100,200 and 400μg/ml) IFN a2b (1000, 2,000,4,000 and 8,000 IU/ml) for 24h,48h or 72h by MTT assay.2. The changes of Renca cell line viability were detected after the treating with LBP (400μg/ml) and IFN a2b (4,000 IU/ml) for 24h,48h or 72h by MTT assay to explore the optimal time.3. The effects of LBP and IFN a2b on Renca cells apoptosis for 48h were analyzed by flow cytometry.4. The effects of 1 LBP and IFN a2b on Renca cells cycle distribution for 48h were analyzed by flow cytometry.5. Western Blot assay was used to compare the expression level of protein Bcl-2、Bax、cyclin D1 and c-Myc after treating with LBP and IFN a2b on Renca cells for 48h.Results:1. MTT assay showed that the monotherapy of LBP (200μg/ml) and IFN a2b (4,0O0IU/ml) for 48h could significantly decrease the viability of Renca cells. Within the range of concentration and tune, LBP and IFN a2b could decrease the viability of Renca cells in a dose-and time-dependent manner.2. MTT assay showed that the combination of LBP and IFN a2b on Renca cells for 48h could significantly decrease the viability of cells in a time-dependent manner. Within 48h, LBP and IFN a2b could decrease the viability of Renca cells in a time-dependent manner.3. Compared with the percentage of apoptosis in Renca cells in the control group, the LBP group and the IFN a2b group (5.42%±0.86%, 34.21%±1.43%,23.15%±1.56%),the proportion in the combination group of LBP and IFN a2b (56.21%±1.32%) were increased significantly, and mainly for late apoptosis, the difference was statistically significant (P <0 .01).4. Compared with the percentage of G0/G1 phase arrest in Renca cells in the control group, the LBP group and the IFN a2b group (35.44%±0.97%,47.61%±0.36%,57.09%±0.29%), the proportion in the combination group of LBP and IFN a2b (67.81%±0.50%)) were increased significantly, the difference was statistically significant (P<0.05). The percentage of S phase arrest (21.10%±0.46%) in Renca cells were decreased significantly by the action of LBP and IFN a2b.5. Compared with the control group, the monotherapy or the combination LBP and IFN a2b, western blot assay showed the expression level of pro-apoptotic protein Bax in Renca cells was significantly increased (P<0.01), the expression levels of Bcl-2 and c-Myc were significantly decreased (P<0.01).Compared with the IFN a2b group, the expression levels of Bcl-2, cyclin D1and c-Myc down-regulation (P <0.01), Bax up-regulation (P<0.01). The difference was no statistically significant between the IFN a2b group and the LBP group in the expression level of Bcl-2 and Bax (P>0.05), but the difference was statistically significant between the IFN a2b group and the LBP group in the expression level of c-Myc, cyclin D1 (P<0.05)Conclusion:1.MTT assay showed that LBP (200μg/ml)and IFN a2b(4,000IU/ml) on Renca cells for 48h could significantly decrease the cell viability.2. The combination of LBP and IFN a2b could induce cell apoptosis, induce G0/G1 phase arrest and decrease the S phase arrest in Renca cells.3. The combination group of LBP and IFN a2b could inhibit c-Myc, cyclin D1 and Bcl-2 protein expression in Renca cells, and induce Bax protein expression.Part II growth-inhibitory effects of LBP and IFN a2b on murine renal carcinoma cell lines renca in vivoObjective:To investigate the growth-inhitory effects of LBP and IFN a2b on murine renal carcinoma cells Renca in vivo.Methods:1. Renca cells (1.5* 106/mouse) were subcutaneously inoculated into the left ribs of 36 six-week-old male BALB/c mice for tumorigenesis experiments.Monitoring the body weight and tumor volume of mice twice a week.2. Thirty-six BALB/c tumor-bearing mice were randomly divided into four groups (n= 9):the control group, the LBP group, the IFN a2b group, and the combination group of LBP and IFN a2b, when the xenografts were 40-50 mm3.The sterile PBS100μl were injected into the intraperitoneal of each mouse in the control group twice a week, sterile PBS100μl gavaged once a day.The IFN a2b 200 IU were injected into the intraperitoneal of each mouse in the IFN a2b group twice a week, sterile PBS100μl gavaged once a day. The sterile PBS100μl were injected into the intraperitoneal of eacn mouse in the LBP group twice a week, LBP 20μg gavagc aday The IFN a2b 200IU were injected into the intraperitoneal of each mouse in the combination group of LBP and IFN a2b twice a week, LBP 20|ig gavaged once a day. Then monitoring the body weight and tumor volumes of mice twice a week for two weeks.3. The mice were killed after treated with LBP and IFN a2b for 2 weeks, the xenografts were cut for HE staining to identify the model is successful.4. The effects of LBP and IFN a2b on the protein Bcl-2 in the the xenografts of tumor-bearing mice by immunohistochemistry.Results:1. HE staining showed that tumor cells were uneven size, shape varies, derangement, nuclei increasing, the disorder proportion of nuclear and cytoplasm, nuclear stained, abnormal mitotic figures in xenograft, indic-ating the xenograft models of BALB/c mice were established successfully with murine renal carcinoma cells Renca.2. Compared with the control group, the LBP group and the IFN a2b group, the tumor volume (P<0.01) and weight (P<0.01) were significantly reduced in the combination group of LBP and IFN a2b, but the difference was no statistically significant between the monotherapy group of LBP or IFNa2b (P>0.05)3.The immunohistochemistry showed the average percentage of Bcl-2-positive renal carcinoma cells in each field from five high power fields, the control group (67.9%±4.2%), the IFNα2b group (68.7%±3.3%), the LBP group (66.1%±2.4%), the combination group of LBP and IFN a2b (65.2%±1.9%), the difference of anti-apoptotic protein Bcl-2 expression was no statistically significant among the groups (P>0.05)Conclusion:1. The combination of LBP and IFN a2b could inhibit the growth of renal carcinoma cells Renca in vivo.2. The effects of LBP and IFN a2b on the anti-apoptotic protein Bcl-2 expression in Renca cells were no significant. Part III the effects of LBP and IFNa 2b on the quantity and theexpression of proteins of exosomes derived from murine renalcarcinoma cells in vivo Objective:To investigate the effects of LBP and IFNa2b on the quantity of exosomes derived from murine renal carcinoma cells Renca and the protein expression in exosomes. Methods:1. Rewarming the supernatants collected after Renca cells were treated with LBP and IFNa2b according to the programs in part one.2. The exosomes in Renca cells supernatant were extracted by ultrafiltration and sucrose density gradient centrifugation.3. The morphology and quantity of exosomes were observed by transmission electron-after Renca cells were treated with the combination or monotherapy of lycium barbarum polysaccharide and interferon,a2b.4. The expressions of protein HSP70 and G250 in exosomes were analyzed by western blot assay.Results:1. The quantity of exosomes derived from the Renca cells treated with the combination of LBP and IFN a2b were significantly decreased than the control group and monotherapy group (P<0.05).2. The morphological changes of Renca-derived exosomes were not obvious by transmission electron microscopy after Renca cells were treated with monotherapy or the combination of LBP and IFN a2b (P>0.05).3. The protein expressions of HSP70 and G250 in exosomes derived from the Renca cells of combination of LBP and IFN a2b were significantly decreased than control group and monotherapy group (P<0.05).Conclusion:1. The quantity of exosomes derived from the Renca cells treated with the combination of LBP and IFN a2b were decreased, but he morphological changes were not obvious.2. The protein expressions of HSP70 and G250 in exosomes derived from the Renca cells of combination of LBP and IFN a2b were significantly decreased.Part IV the effects of LBP and IFN a2b on the proportions of MDSCs and CD4/CD8 T lymphocyte in renal cell carcinoma tumor-beari-ng mice in vivoObjective:To investigate the effects of LBP and IFNa2b on the proportion of MDSCs and CD4/CD8 T lymphocyte in tumor-bearing mice of renal cell carcinoma in vivo.Methods:1. The mice were killed after treated with LBP and IFN a2b for 2 weeks in part two, and the bone marrow cells of bilateral femur/tibia of each mouse were extracted, MDSCs proportions of mice bone marrow in each group were detected by flow cytometry.2. The proportions of CD4/CD8 T lymphocyte in mice spleen from each group were detected by flow cytometry.Results:1. The percentage of MDSCs in the LBP group (37.1%±0.41%),the IFN a2b group (37.7%±0.50%) and the combination group of LBP and IFN a2b (30.1%±1.01%) were significantly reduced compared with the control group (47.65%±0.25%) (P<0.05), especially in the combination group of LBP and IFN a2b (P＜0.01).Compared with the LBP group and the IFN a2b group, the percentage of MDSCs in the combination group were significantly reduced (P<0.05).The difference was no statistically sig-nificant between the monotherapy group of LBP or IFN a2b (.P>0.05)2. The percentages of CD4/CD8 T lymphocyte in the LBP group (1.95%±0.21%),the IFNa2b group (1.87%±0.14%) and the combination group of LBP and IFN a2b (2.21%±0.23%) were significantly increased compared with the control group (1.25%±0.12%) (P<0.05), especially in the combination group of LBP and IFN a2b (P<0.01). The difference was no statistically significant between the monotherapy group of LBP or IFN a2b (P>0.05)Conclusion:1. The combination of LBP and IFN a2b could significantly decrease the proportion of MDSCs in the bone marrow of tumor-bearing mice, which may be the mechanism of the combination of LBP and IFN a2b reducing the immunosuppression of MDSCs and inhibiting the growth of Renca cells.2. The combination of LBP and IFN a2b could significantly increase the proportion of CD4/CD8 T lymphocyte in the spleens of tumor-bearing mice, which may be the mechanism of the combination of LBP and IFN a2b enhancing immune function of lymphocyte, inhibiting the growth of Renca cells.Part V the activation of MDSCs by exosomes derived from murine renal carcinoma cells in vivoObjective:To investigate the activation of MDSCs by exosomes derived from murine renal carcinoma cells in vivo.Methods:1. Fifty-four BALB/c mice were randomly divided into three groups (n = 18):the NS group (control group), the exosomes group, and the Renca cell group. Normal saline 100μl were subcutaneously inoculated into the left ribs of each mouse in the NS group. The exosomes 100μg drived from murine renal carcinoma cells Renca were injected into the tail vein in the exosomes group. Renca cellsl.5×106 were subcutaneously inoculated into the left ribs of each mouse in the Renca cell group.Monitoring the body weight and tumor volume of mice in the Renca cell group twice a week.2. Six BALB/c mice in the NS group, the exosomes group and the Renca cell group were killed in the 10d,20d and 30d respectively. Bone marrow cells were dealed according to the methods of Part four, the percentage of MDSCs of mice bone marrow in each group were detected by flow cytometry.3. The xenografts in the Renca cell group were cut for HE staining.Results:1. HE staining showed the experimental models of BALB/c mice were established successfully.2. Six BALB/c mice in group were killed on the 10d, compared with the percentage of MDSCs in the NS group (22.31%±1.15%), the exosomes group (31.15%±0.83%) and the Renca cell group (32.85%±1.32%) were significantly increased (P<,0.05). The difference was no statistically significant between the exosomes groupand the Renca cell group (P>0.05).On the 20th day, compared with the percentage of MDSCs in the NS group (25.56%±0.36%), the exosomes group (40.15%±0.26%) and the Renca cell group (41.85%±0.56%) were significantly increased (P<0.05). On the 30th day, compared with the percentage of MDSCs in the NS group (25.67±0.41%), the exosomes group (41.23%±0.30%) and the Renca cell group (41.95%±0.42%) were significantly increased (P<0.05). compared with the percentage of MDSCs on 10d in the exosomes group or the Renca cell group, the percentage of MDSCs on 20d and 30d were increased (P<0.05).There was no significant difference in the NS group among 10d,20d, and 30d (P>0.05)Conclusion:1. The murine renal cell carcinoma Renca cells-drived exosomes could activate MDSCs, and promote MDSCs proliferation in vivo.Part VI the activation and activation mechanism of exosomes drived from murine renal carcinoma cells activate MDSCs in vitroObjective:To investigate the activation and activation mechanism of exosomes drived from murine renal carcinoma cells activate MDSCs in vitro.Methods:1. The bone marrow cells of bilateral femur/tibia in the exosomes group nd the Renca cell group mice were extracted,in which MDSCs were sorted by Flow cytometry or MACS according to instructions.2. MDSCs sored were centrifugated according to 2,000 rpm for 7min, resuspended with DMEM/F-12 medium (containing 10%fetal bovine serum imports,100 U/ml penicillin/streptomycin), inoculated in culture dishes coated with 0.1 mg/ml poly-lysine and cultured at 37℃,5% CO2 conditions. Medium was changed every other day.The morphology of MDSCs were observed, digesed and passaged with 0.25% trypsin when the cells were fused to the bottom 90%.3. MDSCs were identiflcated by immunofluorescence and confocal laser technology.4.The MDSCs in primary culture were divided into four groups: MDSCs group, MDSCs and exosomes group, MDSCs and exosomes plus HSP70 inhibitor group,MDSC and exosomes plus anti-TLR2 antibody group.MDSCs group were cultured with primary Medium.MDSCs and exosomes group were co-cultured with 20μg exosomes. MDSCs and exosomes plus HSP70 inhibitor group were co-cultured with 20μg exosomes and 4μg Pifithrin-μ..MDSC and exosomes plus anti-TLR2 antibody group were co-cultured with 20μg exosomess and 3μg anti-TLR2 antibody. MDSCs in each group were cultured for 24h.Western Blot assay was used to compare the expression level of protein TLR2, MyD88, TRAF6, p38, p-p38 and AP-1 in MDSCs.Results:1. MACS could elevate the positive rate of MDSCs than flow cytometry sorting.MACS was selected for MDSCs in subsequent expe-riments.2. Immunofluorescence shown MDSCs had adherent growth, confocal laser technology shown MDSCs had suspended growth.The differentiation of MDSCs occured in primary culture.The number of Gr-1+CD1 lb+MDSCs was reduced after passed three generations.3. Compared with MDSCs group, the expression level of protein TLR2,MyD88,TRAF6,p38,p-p38 and AP-1 in MDSCs and exosome group were significantly upregulated (P<0.05). Compared with MDSCs and exosomes group, the expression level of protein MyD88,TRAF6,p38,p-p38 andAP-1 in MDSCs and exosomes plus HSP70 inhibitor group were significantly downregulated (P<0.05), the expression level of protein MyD88,TRAF6 and p-p38 in MDSCs and exosomes plus anti-TLR2 antibody group were significantly downregulated (P<0.05)Conclusion:1. MACS could elevate the positive rate of MDSCs than flow cytometry sorting.2. The growth modes of MDSCs are adherent growth and suspended growth during primary culture, and the differentiation of MDSCs occured in primary culture.3. The exosomes membrane-bound HSP70 drived from renal cell carcinoma Renca cells could activate MDSCs.The mechanism may be that HSP70 could activate MDSCs through TLR2/MyD88/TRAF6-/P38/AP-1 signaling pathway, promote MDSCs proliferation.And because of the complexity of exosomes component, it is possible that MDSCs could be activated by other signaling pathways.Therefore,LBP in combination with IFN a2b coud reduce exosomes drived from renal cell carcinoma Renca cell,decrease the protein expression of HSP70 in exosomes, reduce the actviation of MDSCs and the immunosuppression, inhibiting the growth of renal cell arcinoma. |
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