Initial Research On The FUT1 Expression Changes Which Were Artificially Induced By Yinchenhao Decoction

ObjectiveConstruct cell model of grafts accommodation induced artificially by Yinchenhao Decoction, search artificially induced technology of Tranditional Chinese Medicine on grafts accommodation,verify the effect of TCM;establish the TCM interventing and regulating technology on ABO-incompatible grafts accommodation, exert the advantage of TCM;lay experimental foundation for developing FUT1 gene as targe of graft accommodation. Apply siRNA and locked nucleic acid technology artificially intervene FUT1 gene, further clarify the role and participation mechanism of FUT1 gene in ABO-incompatible grafts accommodation. Search the mechanism of ABO-incompatible grafts accommation.Method1. Observe the cell morphology and benzidine staining positive rate of Hemin induced K562 cells D1-7 days under the microscope; compare changes of benzidine staining positive rate between D5-7 continuous induction and not induced group;detect FUT1-mRNA expression amount changes of erythroid differentiation K562 cells from 1 to 7 days, select the best experimental time.2. Inspect proliferation inhibition of erythroid differentiation K562 cell by different concentrations Yinchenhao Decoction intervening 24h,48h, and 72h. Calculate IC50, select the most appropriate drug concentration and optimum effect time.3. Detect apoptosis of erythroid differention K562 cell in different experimental groups （blank group, Yinchenhao Decoction group, anti-H group, anti-H+Yinchenhao Decoction group） by Annexin V/PI method, flow cytometry.4. Examine the FUT1-mRNA relative expression volume changes of K562 cells induced by Hemin for 4days in different traditional Chinese medicine experimental groups.5. Apply siRNA and locked nucleic acid technology to silence （inhibit） FUT1gene, detect the expression changes of FUT1 by RT-qPCR, assess the effect of siRNA and LNA.6. Western Blot detect FUT1 protein expression changes of erythroid differentiation K562 cells intervented by TCM groups, siRNA group, and LNA group.Result1. Persistent Hemin induced K562 cells for 7days, benzidine staining positive rate reach peak on D4, then decline gradually; no significant difference between the positive rate of 3,4, and 5 days（F=28.55, P>0.05）。 Benzidine staining positive rate was statistically significant, between D5-7 continuous induction and no induction groups （F=22.001, P<0.05）2. FUT1 gene mRNA relative expression quantity of K562 cells induced by Hemin for seven days were respectively 0.677,0.618,0.633,1.1095,0.663, 0.792, and 0.68 times of control group.3. Rearch of CCK-8 detect Yinchenhao Decoction inhibiting erythroid induced K562 cells proliferation:Different concentration Yinchenhao Decoction effect different time can inhibit the proliferation of K562 cells, and inhibition increased with Yinchenhao Decoction concentration raised and time prolonged, in a dose and time dependent relationship. 100mg/ml concentration Yinchenhao Decoction effect 72 hours inhibit strongest. Set 0. 001mg/ml as control group, different concentration （0. 01mg/ml,0. 1mg/ml, 1mg/ml, 10mg/ml, 100mg/ml） effect 24 hours proliferation inhibition rates were （24.43±3.74）%, （27.37±1.07）%, （39.53±2.39）%, （54.60±1.15）%�'� （57.90±0.50）%, respectively. IC50 （mg/ml） values of 24h,48h, and 72h were 9.983,4.957, and 3.477, respectively.4. Research on flow cytometric test apoptosis of K562 cells in different traditional Chinese medicine groups:Yinchenhao Decoction group effect on K562 cell early apoptosis rate, late apoptosis rate, total apoptosis rate were the lowest, respectively （1.570 ±0.101）%、（2.480±0.062）%　and （4.050 ± 0.066）%　;The anti-H group （anti-H concentration was 1:16） effect on K562 cell early apoptosis rate, late apoptosis rate, total apoptosis rate were （10.860±0.062）%、（6.420±0.061）% and （17.380±0.176）%　respectively; In anti-H+Yinchenhao Decoction group, the effects on K562 cell early apoptosis rate, late apoptosis rate, and total apoptosis rate were （9.960±0.085）%, （1.930±1.139）%　and （11.890+0.137）%, respectively. In Yinchenhao Decoction group, the early apoptosis rate and total apoptosis rate were the lowest, but in anti-H group, that was the highest; In anti-H+Yinchenhao Decoction group, the early apoptosis rate, late apoptosis rate, and total apoptosis rate were the lower than that in anti-H group （F＝3035.543, P<0.05）, late apoptosis rate is lowest and lower than in Yinchenhao Decoction group （F=1735.360, P<0.05）5. RT-qPCR detect FUT1-mRNA expression changes of K562 cells intervented by TCM groups:Compared with blank control group, the expression of FUT1-mRNA 1, FUT1 gene mRNA relative expression amount of Yinchenhao Decoction group minimum, is （0.089±0.005） times of the control group; anti-H group, anti-H+Yinchenhao Decoction group FUT1 gene mRNA expression was increased, are the control group （1.964±0.034） times, （1.993±0.127） times, no statistical significance between the two groups （F=620.529, P>0.05）.6. Western Blot detect FUT1 protein expression changes of erythroid differentiation K562 cells intervented by TCM groups:Compared with blank control group was 1, anti-H group, Yinchenhao Decoction group, anti-H+Yinchenhao Decoction group group FUT1 protein expression respectively was 1.4555,0.5237,0.3557 times of blank group.7. RT-qPCR detect FUT1-mRNA expression changes of K562-cells intervented by siRNA and LNA groupsSynthesis of FUT1-siRNA sequence and negative control sequence （NC）, concentration of 100nmol/L,200nmol/L,300nmol/L,400nmol/L, application of Lipofectamine transfection,24 hours after the inverted fluorescence microscope to detect the transfection efficiency, the transfection efficiency of 200moml/L group was （48.00±7.94）%; Two comparison of 200nmmol/L, 300nmol/L,400nmol/L groups, no significant difference （P>0.05）, with the increase of concentration, without transfection efficiency increasing; the replacement of RNAimax transfection reagent, transfection efficiency （24.33 ±6.11）%; electric transfection rate is the lowest, and the apoptosis rate reach to 64.9%. low transfection efficiency maybe with the relevant of suspension cells transfected difficulty. Choose Lipofectamine transfect K562 cells which induced by Hemin. RT-qPCR results showed that siRNA-FUT1₁438 in the concentration of 200nM interfere efficiency above 60%. WB detect 200nM-siRNA-FUT1₁438 interfere efficiency, the results show that it can down regulate the expression of FUT1 gene protein.Locked nucleic acid:modified design and synthetize FUT1₁438 siRNA sequence for locked nucleic acid, and design NC sequence, RT-qPCR detect interference efficiency, set up NC100nmol/L group as the control group, the intervention effect of 100nmol/L,200nmol/L concentration of locked nucleic acid on FUT1-mRNA were 1.074、0.824 times of control group, the interference efficiency is low, respectively, up 7.4%　and down 17.6%.Conclusion　1.K562 cells induced by Hemin for fourth days the benzidine staining positive rate is the highest, the largest quantity of hemoglobin synthesis, then benzidine staining positive rate continued to decline.不 enzidine staining positive rate of K562 cells is higher than the D5-7 no induced K562 cells. benzidine staining positive rates of Sustained induced for 3、 4、5days are high and no statistic difference. Select hemin-induced for three days K562 cells to do experiment, drugs act on 24-48 hours, the content of hemoglobin stability, be beneficial to the consistency of experiment.2. FUT1-mRNA expression of Hemin-induced-seven-days-K562 cells reach peak at the fourth day; continuous induction group FUT1 gene mRNA relative expression quantity is higher than D5-7 days without induction group.3. Yinchenhao Decoction on K562 cell proliferation inhibition showed a positive correlation withconcentration, time of action. According to the IC50 value, in combination with benzidinestaining positive rate, selection of lmg/ml concentrations of Yinchenhao Decoction for 24 hours experiment.4. The cytotoxicity of Yinchenhao Decoction act on K562 cells is small, the rate of apoptosis is low. Yinchenhao Decoction can resist the toxicity of anti-H antibody, cell survive. Play a great role in late apoptosis rate, and little effect on on early apoptosis rate, which may be related to the action time, resist the toxic effects of anti-H and enhanced with the time increase5. Yinchenhao Decoction group, anti-H, anti-H+ group of Yinchenhao Decoction group act on Hemin-induced-K562-cells, there was significant difference between each group about intervention effect. Yinchenhao Decoction almost silence FUT1-mRNA expression, but Yinchenhao Decoction can not resist /neutralizing the toxicity role of anti-H on mRNA expression level.6. Yinchenhao Decoction can down regulate the expression of FUT1 protein, anti-H+ Yinchenhao Decoction group FUT1 protein express in the lowest, down-regulation effect is better than pureYinchenhao Decoction group. Yinchenhao Decoction can resist the toxic effects of anti-H on protein expression, and these two have Synergistic effect.7. SiRNA can down regulate the expression of FUTl gene mRNA and protein in K562 cells, but the inhibition efficiency is low, the present studyshowed that it is difficult to achieve the effect of gene silencing（inhibition）. FUT1 gene mRNA relative expression levels of K562 cells intervened by LNA is not obvious, need to be further in-depth study.In a word, cell culture experiments prove that Yinchenhao Decoction can down-regulate FUTl gene mRNA and protein expression, reduce the expression of ABH antigen, resist/neutralize anti-H antibody toxicity effect, then induce ABO-incompatible grafts accommodation. SiRNA can inhibit the expression of FUT1 gene mRNA and protein, non silencing effect, effects of locked nucleic acid is not clear and low transfection efficiency, need to be further studied. It is helpful to to reveal the incompatible graft antigenicity, to provide new ideas for hyperacute rejection of incompatible organ transplantation, facilitate new strategies for inducing accommodation of graft, improving therapy of PRCA.