| Objective: Fuhe Preparation Decoction is a national famous and old Chinese doctor Professor Rong Yuanming who has accumulated many years of experience in the treatment.The basic prescription for the treatment of liver cancer is to invigorate the spleen,regulate the liver,promote dampness and dissipate blood stasis,and has a good effect in the clinical treatment of liver cancer.This study aims to isolate CD133+Hep G2 liver cancer stem cells by immunomagnetic bead method after stem cell induction,use flow cytometry to identify the positive rate of CD133,and co-culture the serum containing Fuhe Beihua prescription with CD133+Hep G2 cells.q RT-PCR and Western blot were used to detect the m RNA and protein expression levels of related genes in the Wnt/β-catenin signaling pathway;at the same time,RNA interference technology was used to construct RNA interference plasmids targeting β-catenin gene,and stable CD133+ was obtained after cell transfection Hep G2 liver cancer stem cell positive cell line,the m RNA and protein expression levels of each group of stem transcription factors SOX2,NANOG,OCT4 were detected by q RT-PCR and Western blo,so as to verify that Fu and Beihua can regulate the Wnt/β-catenin signaling pathway The expression levels of related genes and proteins can affect the differentiation of liver cancer stem cells,thereby achieving the therapeutic effect on CD133+liver cancer stem cells.Provide new ideas for Chinese medicine in the field of liver cancer stem cell research and clinical treatment of liver cancer.Method:Experiment 1: isolation and purification of liver cancer stem cells(CD133 +Hep G2): CD133 +Hep G2 liver cancer stem cells were selected by immunomagnetic beads method,and identified by flow cytometry.When the positive rate was more than 90%,CD133 + Hep G2 liver cancer stem cells could be used for follow-up experiments.Experiment 2: Take the CD133+Hep G2 cell suspension in the logarithmic growth phase and inoculate it into a 6-well plate,set up a serum control group,an experimental group,a positive control group,and a blank control group.The serum control group was added with normal rat serum culture medium,the experimental group was added with 16% volume fraction of Fuhe Beihua prescription medicine-containing serum,the positive control group was added with 1μmol/L XAV939 medium,and the blank control group was added with the same dose of normal medium.After 6 days of drug action,q RT-PCR and Western blot were used to detect the m RNA and protein expression levels of related genes in the Wnt/β-catenin signaling pathway.Experiment 3: Using RNA interference technology to construct an RNA interference plasmid targeting β-catenin gene,after plasmid extraction and cell transfection,a stable interfering CD133+Hep G2 liver cancer stem cell positive cell line was obtained,and the cell lines were divided into empty cell groups,KD-nc group and KD group,after 24 hours of culture,the cell pellets of each group were collected,m RNA and protein were extracted,and the m RNA and protein expression levels of stem transcription factors SOX2,NANOG,OCT4 in each group were detected by q RT-PCR and Western blot.Result:Experiment 1: Isolation and purification of CD133+Hep G2 liver cancer stem cells: CD133+Hep G2 liver cancer stem cells were successfully sorted out by the immunomagnetic bead method and cultured.The cells grew in good condition and the cell structure was complete under the microscope.Flow cytometry was used to identify CD133+ The positive rate of Hep G2 cells reached 94.9%,which can be used for follow-up experiments.Experiment two:(1)q RT-PCR detects the expression level of related genes in the Wnt/β-catenin signaling pathway: Compared with the blank control group and the serum control group,the experimental group and the positive control group can down-regulate the pathway-related factor Wnt1,β-catenin,PI3 K and AKT m RNA levels(P<0.05).The positive control group has the best inhibitory effect on Wnt1 and AKT(P<0.05),and the experimental group has the best inhibitory effect on β-catenin(P<0.05).0.05);(2)Western blo detects the expression level of related proteins in the Wnt/β-catenin signaling pathway: Compared with the blank control group and the serum control group,the experimental group and the positive control group can down-regulate the pathway-related factors Wnt1,β-The protein levels of catenin,PI3 K and AKT(P<0.05),the positive control group had the best inhibitory effect on Wnt1(P<0.05),and the experimental group had the best inhibitory effect onβ-catenin(P<0.05).Experiment 3: The p Lent-U6-GFP-Puro-sh RNA-β-catenin RNA interference plasmid targeting β-catenin gene was successfully constructed,which was identified as the target plasmid by restriction enzyme digestion and sequencing.After transfection of CD133+Hep G2 cells,β-catenin interference plasmid effectively reduced the m RNA and protein expression levels of stem transcription factors NANOG,SOX2,OCT4 in liver cancer stem cells.Conclusion1.The medicated serum of Fuhe Preparation Decoction can effectively reduce the m RNA and protein expression levels of Wnt/β-catenin pathway related factors Wnt1,β-catenin,PI3 K,AKT,and reduce the embryos of liver cancer stem cells through Wnt/β-catenin signaling pathway At the lineage level,it may be possible to induce liver cancer stem cells to differentiate into normal cells.2.After transfection with β-catenin interference plasmid,the m RNA and protein expression levels of CD133+Hep G2 liver cancer stem cell stem cells NANOG,SOX2,OCT4 were significantly reduced,indicating that the Wnt/β-catenin signaling pathway can affect CD133+Hep G2 liver cancer stem cells The germline stem cell markers have important regulatory effects,and knocking out the β-catenin gene can greatly reduce the expression level of germline stem cell markers,reduce the stemness of liver cancer stem cells,and achieve the effect of primary liver cancer CD133+Hep G2 liver cancer stem cells Therapeutic effect.The occurrence and development of liver cancer are closely related to the Wnt/β-catenin signaling pathway,and abnormal activation of β-catenin is often found in patients with liver cancer.Therefore,β-catenin is an ideal therapeutic target for liver cancer.At the same time,this study also provides an experimental basis for studying the regulation of Wnt/β-catenin signaling pathway related genes and protein expression levels in traditional Chinese medicine,so as to regulate the differentiation level of liver cancer stem cells. |
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