Screening Of Antitumor Active Ingredients Of Scutellaria Barbata And The Preliminary Construction Of Cell Model With AhR Gene Defect

Approximately 80%to 90%of the body’s tumors are caused by chemical carcinogens.Chemical carcinogens into the body,to be subject by metabolism of various enzymes in vivo,Ⅱphase enzyme in the prevention and treatment of tumor occurrence and development plays a vital role.The flavonoids in Scutellaria barbata had better ability to scavenge hydroxyl radicals.The preliminary results showed that the flavonoids of Scutellaria barbata could induce the expression of Ⅱ phase metabolizing enzymes and extract the flavonoids,The role of each compound and its mechanism is still not clear.Therefore,we carried out a further study on the active constituents of flavonoids from Scutellaria barbata and its mechanism.Part Ⅰ Screening of Flavonoids of Scutellaria barbata by Hep G2-4ARE-TK-GFP cell modelObjective:To Screening the flavonoids(A,B,C,D,E,F,G)of Scutellaria barbata who has active ingredients by Hep G2-4ARE-TK-GFP cell model.Methods:Flavonoids(A,B,C,D,E,F,G)with a final concentration of 50 μg/ml and tBHQ with a final concentration of 100 μM were used to process Hep G2-4ARE-TK-GFP cell model.After 24 hours,we measure the relative luminous intensity of GFP in different group.And then we detected the dose effect of positive flavonoid G.Results:Flavonoids G showed positive results,relative fluorescence intensity of 1.97 times,and flavonoids A,B,C,D,E,F were negative.Flavonoid G concentration at 80μg/ml to achieve maximum activity.Conclusion:The flavonoids G of Scutellaria barbata was the main active ingredients.Part Ⅱ Preliminary construction of gene knockout Hep G2 cellsObjective:To verify the feasibility that the AhR gene in Hep G2 cells can be deleted by CRISPR-Cas9 gene targeting technique.To provide a basis for further construction of Hep G2-4ARE-TK-GFP cell model with AhR gene deletion.Methods:Three sgRNA sites were designed for AhR gene,and the corresponding oligo double-stranded DNA was synthesized.Then the oligo double-stranded DNA were connected with linear vector plasmid GV392 to construct AHR-sgRNA carriers.Then Hep G2 cells were transfected with AHR-sgRNA carriers who had be lentivirus packaged.The positive results were extracted and were amplified.Using mismatched enzyme method to detect the activity of sgRNA.Results:The sgRNA sequence was found in the sequencing results of AHR-sgRNA vector.The activity of sgRNA in Hep G2 cells was detected,and the activity of sgRAN3 was the most active fraction.Conclusion:The AhR gene in Hep G2 cells was successfully knocked out by CRISPR-Cas9 gene targeting.Hep G2 cells with AhR gene deletion were successfully constructed.The results shows that the using of CRISPR-Cas9 gene targeting to detect AhR gene in Hep G2 cells was feasible.And to provide a basis for the construction of a cell model for the single expression of Nrf2/Keap 1 signaling pathway induced II phase enzyme.