| Objective:The enzyme efficiency and specificity characteristics analysis of methylation enzyme and acetyltransferase series,analyze its related epigenetic characteristics.The essence of the theory from a new perspective to explore and verify the preliminary exploration of traditional Chinese medicine,traditional Chinese medicine by the evaluation system of objective standard.Methods: 1、Collection and preparation of drug-containing serum in each group SPF grade male SD rats which they were 6-8 weeks old were got randomly,at the same time we had ready for decoction of three Chinese herbal medicines that Tribuli fructus attributed to the liver meridian,Cnidii fructus attributed to the kidney meridian and Dipsaci radix attributed to the liver and kidney meridian.Rats were fed continuously for 7 days and two times daily.The abnormal state of the rats was observed after each feeding.After the last time,the blood was taken from the femoral artery in a certain period of time.The last 12 hours was fasting but water.Vacuum tube was placed at refrigerator of 4℃ for 2 hours.Centrifuge for 15 min at 3000rpm/min speed.Supernatant was aspirate,we placed in water bath of 56℃ for 30 min in order to inactivate alexin and prevent cell poison,and it was filtrated with 0.22μm syring-driven filter and sealed in minus 80℃ refrigerator.2、Isolation,culture and identification of bone marrow mesenchymal stem cells This study used the whole bone marrow culture method,SD rats was cervical dislocation and soak with 75% ethanol for 3-5 minutes.We used iodine cotton to sterilize the skin,complete bilateral femur and humerus were removed under sterile conditions and placed in culture dishes,we removed carefully the muscle tissue that it was adhere to the bone tissue,cut the bone and inserted into the bone marrow cavity to washed repeatedly with the syringe in which proper medium,until femur and humerus became white.The flushing fluid was collected directly in the centrifuge tube.Centrifuge for 5 min at 1500rpm/min speed.We discarded the supernatant,added fresh complete medium(containing 10% FBS,1/100 ml double antibiotic solution),removed in the culture dish,and placed at 37℃,5% CO2 incubator overnight.Cell medium was replaced half of the amount of liquid after 24 hours,and replaced total amount of liquid 3days,each 2-3 days for 1 times in later.When the cells were grown to about 80% fusion,they were digested by 0.25% trypsin solution,and gone down to posterity at a ratio of 1:1.The morphological changes were observed and the growth curve was drawn by continuous culture medium,the CD90 、CD34 and CD45 surface antigens were identified by flow cytometry.3、Effects of drug-containing serum on bone marrow mesenchymal stem cells The third generation of BMSCs was used to make cell suspension,and the cells were counted by blood counting plate.The cell density was adjusted to 1×106/ml,the cells seeded on the 24 hole plate for 500μl per hole.The cells were divided into four groups: blank group,Tribulus terrestris group,Cnidium group and Teasel group.The drug groups were divided into two groups: 24 hours and 72 hours.Each drug group was divided into high serum concentrations and low serum concentrations.The blank group was cultured with DMEM/F12 cell medium containing 10% fetal bovine serum.Each group of cells was cultured apart for 24 hours and 72 hours in 37℃,5% CO2 incubator.4、Effects of different the meridian tropism of Chinese herbal medicine on the expression of epigenetic modification of BMSCs related genes Q-RT-PCR method was used to detect the effects of Dnmt3 a,Camkmt,Kdm1 a,Kat2a and Hdac1 on the expression of 24 hours and 72 hours in m RNA treated with BMSCs.5、Effects of different the meridian tropism of Chinese herbal medicine on the expression of epigenetic modification of the tissue related genes Q-RT-PCR method was used to detect the effects of Dnmt3 a,Camkmt,Kdm1 a,Kat2a and Hdac1 on the expression of 24 hours and 72 hours in m RNA with the tissue of liver,kidney and spleen.Results: 1、During intragastric administration,three groups of rats were energetic,agile in moving,normal diet,well-weight gain,normal stool,and no death occurred in each group.2、The newly isolated primary cells were suspended in the culture medium,most of them were round or oval.After 24 hours,we could see that some of the cells had been adherent and appeared spindle shaped,polygonal and other forms.The third generation cells were identified by flow cytometry.The results showed that the expression of the cell surface marker CD90 was positive,the positive rate was 94%;The expression of CD34 and CD45 were negative.The expression rates were 14.2% and 1.1% respectively.The results showed that cultured and purified BMSCs with the whole bone marrow culture method reached the requirement in the laboratory.3、BMSCs were seeded in 24-well plates.The morphology of the cells was observed by using the above-mentioned methods.The morphology of the cells was observed to be shuttle,and fibroblast-like cells were observed.After 72 hours of treatment,the cell culture medium was slightly cloudy,see more suspended cells.4、After the drug group of BMSCs for 24 hours,m RNA expression of Dnmt3 a,Camkmt,Kdm1 a,Kat2a and Hdac1 in which Teasel group with high concentrations was significantly higher than other drug group(P<0.05),and Dnmt3 a,Camkmt and Hdac1 expression is greater than 1,it showed that the expression was up-regulated.After the drug group of BMSCs for 72 hours,m RNA expression of Dnmt3 a,Camkmt,Kdm1 a,Kat2a and Hdac1 in which Tribulus terrestris group with high concentrations was significantly higher than other drug group(P<0.05),and Camkmt and Kat2 a expression is greater than 1,it showed that the expression was up-regulated.5、In the tissue of liver,m RNA expression of Dnmt3 a,Camkmt,Kdm1 a,Kat2a and Hdac1 in which Tribulus terrestris group was significantly higher than other drug group(P<0.05),and Kdm1 a and Kat2 a expression is greater than 1,it showed that the expression was up-regulated in the tissue of liver.In the tissue of kidney,m RNA expression of Camkmt in which Cnidium group was significantly higher than other drug group(P<0.05),and Camkmt expression is greater than 1,it showed that the expression was up-regulated in the tissue of kidney.In the tissue of spleen,m RNA expression of Dnmt3 a,Camkmt,Kdm1 a,Kat2a and Hdac1 in which Teasel group was significantly higher than other drug group(P<0.05),and all expression is greater than 1,it showed that the expression was up-regulated in the tissue of spleen.Conclusions: 1、The experiment of bone marrow mesenchymal stem cells isolated and cultured in vitro,using a simple and easy and the most commonly used method which was whole bone marrow culture,through the continuous replacement of fresh medium to purify cells,cell surface markers were identified by flow cytometry.The results were consistent with those reported in many literatures,which showed that this method could be used to isolate and culture bone marrow mesenchymal stem cells.2、From the point of view of animal organs,whether methyltransferase,demethylase,acetylation or deacetylation,the relative m RNA expression levels of 4 kinds of epigenetic related enzymes in the liver for Tribuli fructus attributed to the liver meridian were significantly increased.This showed that the effect of drugs attributed to the liver meridian on the liver,it also proved the rationality of the meridian tropismtheory of Chinese herbal medicine.3、From the results of the study of the apparent enzyme,it had undergone a sensitive epigenetic expression change under the influence of different drugs,which indicated that it was susceptible to different drugs.it could be used as a candidate gene for the objective criteria of traditional Chinese medicine evaluation system. |
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