| Objective: To investigate the influence of CD4~+CD25~+Foxp3~+Treg andTGF-β1in the tumor microenvironment of melanoma model mice with thetotal body radiation (TBI) method; To observe the impact of TBI on the tumorimmunosuppressive microenvironment; To provide new theoretical reason andexperimental data for the cellular adoptive immunotherapy based ondisturbing tumor microenvironment.Methods: To establish the tumor-bearing mice models by subcutaneousinoculation on back of C57BL/6J mice with melanoma cells (B16).Aftersuccessful establishing of the tumor-bearing mice models, they were randomlydivided into8groups with8mice in each group, at the same time, put thenormal mice as healthy controls, one of the eight groups was the tumor controlgroup, and the other7groups a total body irradiation (TBI) with a Co-60radioactive source with7Gy were given respectively. The spleens, plasma andtumor tissues were obtained on days1st,3rd,5th,7th,9th,11thand13thafter theTBI. The proportion of the CD4~+CD25~+Foxp3~+Tregs were detected by flowcytometry, TGF-β1levels in plasma were detected by ELISA, and theexpression of TGF-β1mRNA in the tumor tissues were detected by Real-TimePCR, respectively.Results:1. The proportion of CD4~+CD25~+Foxp3~+/CD4~+in each group detected byflow cytometry were obtained as:①The proportion of CD4~+CD25~+Foxp3~+/CD4~+in the tumor control group (15.57±0.36%) was significantly higherthan the healthy controls (3.47±0.17%)(P<0.05).②The proportion ofCD4~+CD25~+Foxp3~+/CD4~+in tumor-bearing mice was significantly decreasedas time went on after TBI, and on the9thday went to the lowest level and kept for several days, and then gradually went up. The proportion in the9thday(3.70±0.20%) was significantly lower than the tumor control group (15.57±0.36%)(P<0.05). Compared with the healthy control group, the proportion ofCD4~+CD25~+Foxp3~+/CD4~+in5d (4.25±0.20%),7d (4.00±0.19%) and9d(3.70±0.20%) had no significant difference(P>0.05).③The proportion ofCD4~+CD25~+Foxp3~+/CD4~+among the groups of5d (4.25±0.20%),7d (4.00±0.19%),9d (3.70±0.20%) had no significant difference (P>0.05),which wereall in low level.2. The content of TGF-β1in plasma was detected by ELISA and theresults shows as:①The content of TGF-β1in the tumor control group(139.78±2.55ng/ml)was significantly higher than the healthy controls (63.60±2.12ng/ml)(P <0.05).②The content of TGF-β1in tumor-bearing mice afterTBI was gradually decreased significantly with the days passed, kept lowerlevel several days, and in the11thday (18.15±0.39ng/ml)was significantlylower than the tumor control group(139.78±2.55ng/ml)(P<0.05).③Thecontent of TGF-β1in tumor-bearing mice after TBI was significantly lowerthan the tumor control group(P<0.05).④The content of TGF-β1between5d(38.15±1.10ng/ml) and7d(35.43±1.61ng/ml) had no statistical difference(P>0.05).⑤The content of TGF-β1among the groups of9d (19.09±0.58ng/ml),11d (18.15±0.39ng/ml), and13d(18.65±0.36ng/ml)had nosignificant difference(P>0.05),which were all in low level, and showed thelowest level in the11thday.3. The expression of TGF-β1mRNA in the tumor tissues were detected byReal-Time PCR.The results showed:①Compared with the tumor controlgroup, the expression of TGF-β1mRNA in the tumor-bearing mice after TBIwas all lower than the tumor control group(P<0.05).②In tumor-bearing miceunderwent TBI, the levels of TGF-β1mRNA in tumor tissues were graduallydecreased as time went on, and in the11thday was significantly lower than thetumor control group (P<0.05).③The expression of TGF-β1mRNA between1d(0.708±0.064)and3d(0.651±0.059)had no statistical difference(P>0.05)④The expression of TGF-β1mRNA among7d (3.44±0.026),9d (0.234± 0.043),11d (0.203±0.035), and13d (0.254±0.019) had no significantdifference between each other (P>0.05), which were all in low level, andshowed the lowest level in the11thday.4. There was no mice dead underwent TBI in each groups during theexperimental process.Conclusions：1. The proportion of CD4~+CD25~+Foxp3~+/CD4~+in spleen and the content ofTGF-β1in plasma in the tumor-bearing mice were significantly higher thanthe healthy mice, which showed that there was a tumor immunosuppressivemicroenvironment. This is similar with previous study.2. The method of total body irradiation could effectively impact the tumormicroenvironment of the tumor-bearing mice, and there was no mice deadunderwent TBI with the7Gy irradiation in each groups, which couldobviously reduce the proportion of CD4~+CD25~+Foxp3~+/CD4~+in spleen andthe level of TGF-β1in plasma and tumor tissue.3. Flow cytometry technology could exactly reflect the real level ofCD4~+CD25~+Foxp3~+Treg in the spleen. This study indicated that the level ofCD4~+CD25~+Foxp3~+Treg went to decrease on the1stday after TBI, and keptlow level from the5thto9thday, which had no significant difference comparedwith the healthy mice, however, it went up in11thday. It could not onlyinterference the tumor immunosuppressive microenvironment, but also did notmake the level of CD4~+CD25~+Foxp3~+Treg too low to cause AutoimmuneReaction. It also provided the perfect time to doping cells for cellular adoptiveimmunotherapy.4. The content of TGF-β1in plasma were detected by ELISA,at the sametime used Real-Time PCR to test the expression of TGF-β1mRNA in thetumor tissues, respectively on the protein level and gene level to reveal thechange trend of TGF-β1.The results of the two methods indicated that thechange trend were consistent. The expression of TGF-β1in the tumor-bearingmice after TBI was lowest on the eleventh day. It could hold about5days. Itcould not only interference the tumor microenvironment, but also provided the sufficient response time for activated killer cells in the cellular adoptiveimmunotherapy. |
PDF Full Text Request