| 13kinds of TLRs has found in the mammals who can identify a variety ofmicroorganisms and their products PAMP. Among of them, TLR4is the most widelyexpress on the tumor cells, and the expression is abundant, thus the TLR4isconcerning in the occurrence and development of tumor. This paper is basis on theprevious studies, via observing the proliferation, colony formation, apoptosis, cellcycle, and the impact of migration of the exposed high expression and low expressionof TLR4in tumor cells, at the same time co-cultured the Lewis lung carcinoma cellswith lymphocytes, observed tumor the effect of interaction of local immune cells andtumor cells to the CD4+CD25+Treg cell number. The results of this study willprovide new experimental evidence of the impact of TLR4on the radiosensitivity oftumor cells and tumor immune escape mechanisms.1Influence of TLR4on the radiation sensitivity of tumor cells1.1Select high and low expression of TLR4in tumor cells after irradiationDetected the expression of TLR4after irradiation by flow cytometry, the resultsshow: compared with the negative control group (normal mouse tissue sourcesNIH3T3cells), after24h of5Gy X-ray irradiation of mouse tumor cells, the murineleukemia RAW264.7, than before irradiation were higher, but only the TLR4expression levels of Lewis cell were significantly higher than before irradiation (P<0.01); the TLR4expression levels of the gastric cancer cells MFC reduced afterirradiation, and there is a statistically significant (P <0.01).1.2Effect of proliferation activity of TLR4on Lewis and MFC cellBy using the CCK8kit to detect the proliferation of Lewis and MFC cells, resultsshowed that: after5Gy irradiation, the proliferation activity of Lewis wassignificantly lower than that before irradiation (P<0.01),after the TAK242treatmentof the blocker of TLR4the proliferation activity decreased significantly (P<0.05),afterthe LPS treatment of the TLR4agonist, the proliferation activity was increased(P<0.05). The proliferation activity of MFC cell was significantly lower than thatbefore5Gy irradiation (P<0.01), but compared with Lewis cells (51.1%), the proliferation of MFC cell was small decline (42.9%), and after TAK242blockingTLR4and give irradiation, the proliferation activity was significantly higher than thatof the irradiated group (P<0.05), but was significantly lower than that beforeirradiation block group (P<0.05). After the treatment of LPS, the proliferation activitywere higher in the groups (P<0.01).1.3Influence of cells clone formation rate of TLR4on irradiated Lewis and MFCThe effects of radiation sensitivity of TLR4to the Lewis and MFC detected bycloning formation test, the results showed that after5Gy irradiation, the survival rateof Lewis cell was significantly lower than that before irradiation (P<0.01), afterTAK242treatment of the blocker of TLR4, the cell survival fraction decreasedsignificantly (P<0.01), and use of TLR4agonists of LPS, the cell survival fractionincreased significantly (P<0.01).after5Gy irradiation, the cell survival fraction ofMFC cells was significantly lower than that before irradiation (P<0.01), after TAK242treatment of the blocker of TLR4, the cell survival fraction decreased significantly(P<0.05), and after treatment of the TLR4agonist LPS, the cell survival fractionincreased significantly (P<0.05). The experimental results show that the blocker ofTLR4, the survival fraction of MFC and Lewis cells were decreased irradiated, buthave greater range. But after TLR4stimulation in irradiated Lewis and MFC cellswere increased on survival fraction, and MFC cell survival fraction increased evenmore sharply, illustrate TLR4may be related with the radiosensitivity of tumor cells.1.4Influence of apoptosis rate of TLR4to the irradiated Lewis and MFC cellsDetect the apoptosis rate of before and after irradiated Lewis and MFC cell byflow cytometry, the results showed that Lewis cells, TAK242blocked TLR4, theapoptosis rate was significantly increased (P<0.05), and the rate of apoptosis afterLPS stimulation was significantly inhibited (P<0.05). In the5Gy radiation condition,TAK242blocked the apoptosis rate was significantly higher than that of the irradiatedgroup (P<0.05), and apoptosis was higher than that of pure TAK242blocked group,the apoptosis of LPS cells excited group was obviously lower than that of theirradiated group (P<0.05). MFC cells, TAK242blocked TLR4, the apoptosis rate wassignificantly increased (P<0.05), and the rate of apoptosis after LPS stimulation wassignificantly inhibited (P<0.05). In the5Gy radiation condition, TAK242blockedgroup and LPS stimulation group, the apoptosis rate of MFC cells were lower than theradiation group (P<0.05), and group TAK242apoptosis significantly. The experimental results show that, after5Gy irradiation, TAK242blocked TLR4Lewisapoptosis rate was obviously increased after stimulation with LPS apoptosis rate wasobviously inhibited.Show that TLR4associated with apoptosis of tumor cells inducedradiation.1.5Effect of TLR4on irradiated Lewis and MFC cell cycle progressionDetect the cell cycle changes of before and after exposure Lewis and MFC byflow cytometry: the results display that compared with the control group, Lewis cellsfrom G2/M phase cells increased significantly after irradiation (P<0.01), while G0/G1and S phase cells were significantly reduced (P<0.01); with the changes afterblockage of TAK242in each cycle and only after irradiation, and stimulated with LPSafter irradiation of Lewis cells, S phase cells did not change significantly, G0/G1phase and G2/M phase cells decreased and increased (P<0.01). MFC cells by G2/Mcells after irradiation significantly more than control group (P<0.01), G0/G1and Sphase cells were reduced, with statistical significance (P<0.01); MFC cell cyclechange process with TAK242block and LPS after stimulation similar with irradiatedgroup,show that the high expressed TLR4of Lewis isn’t related its cycle progression.1.6Effect of TLR4on irradiated Lewis and MFC cells migrate changesDetected the changes of migration of Lewis and MFC cells by of scratch testmethod, the experimental results show that: blocking group and radiation groupcompared with5Gy irradiation by Lewis and MFC cells after TAK242, Lewis andMFC cell scratch width becomes wider, and compared the two, Lewis cell scratchwidth greater than MFC cells, and excited agent LPS, Lewis and MFC cells wassignificantly reduced, scratch width become wider, and compared with MFC cells,scratch width narrower. The experimental results show that the change of themigration rate of tumor cells may be related with the high expression level of TLR4after irradiation.2. Effect of TLR4on tumor immune toleranceFlow cytometry was used to detect the changes the number of Treg cells ofco-cultured with Lewis lung cancer cells and lymphocytes system, the experimentalresults show that: after5Gy irradiation the number of Treg cells of the co-culturesystem was significantly lower than that before irradiation (P<0.05), but when treatwith TLR4antagonists TAK242and agonists LPS after irradiated, the number of Tregcells significantly decreased and increased respectively(P<0.05). And after5Gy irradiation and irradiation alone group compared with the TAK242, blocking and afterstimulation by LPS, the number of Treg cells significantly decreased and increased(P<0.05), and after irradiation compared with before irradiation, the Treg cells of noirradiated group change even more sharply, showed that TLR4can promote thenumber of Treg cell in the co-culture of Lewis lung cancer cell and lymphocytesystem, which display that this may be related with the high expression of TLR4ofLewis cells. |
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