| ObjectiveBy means of lentiviral system,we aim to construct cell models of adenomyosis with PTEN overexpression and PTEN interference,and to detect the PI3K/Akt/HIF-1α/VEGF signaling pathway-related factors regulated by PTEN gene in Jiawei Shaoyao Gancao Decoction in vitro.The role of the purpose of clarifying the deep mechanism of Jiawei Shaoyao Gancao Decoction in the treatment of adenomyosis provides a basis for clinical treatment.Methods1.The primary culture of human adenomyosis cells was performed by collagenase digestion method,and the expression levels of smooth muscle protein,vimentin and keratin were identified by immunocytochemistry to identify whether it was adenomyosis cells.2.Firstly,three shRNA sequences were designed to be ligated to the interference vector pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO,and an h-PTEN sequence was designed to be ligated into the overexpression vector pHBLV-CMV-MCS-3flag-EF1-ZSgreen.On the-puro,a higher titer of the PTEN shRNA lentiviral vector carrying the green fluorescent protein and puromycin resistance,and the PTEN overexpressing lentiviral vector were obtained.After 48 hours of lentivirus transfection,the green fluorescence expression of the cells was observed by fluorescence microscopy to confirm the effectiveness of transfection.Then,the interference model and the overexpression model were quantitatively identified by RT-qPCR and Western blot.3.Puromycin was used to screen PTEN-interfering and PTEN-overexpressing adenomyosis cells.4.CCK-8 technique was used to detect the proliferation of adenomyosis cells after different drug interventions.RT-qPCR was used to detect PTEN mRNA,PI3K mRNA,AKT mRNA,TSC2 mRNA,mTOR mRNA and HIF in adenomyosis cells.The expression of-1α mRNA and VEGF mRNA and the expression changes after drug intervention were detected by Western blot.The expressions of PTEN,p-AKT,p-TSC2,HIF-1 a and VEGF in adenomyosis cells were detected by Western blot.Results1.A total of four patient specimens were collected,and the primary cultures were successful.The primary cells were identified by immunocytochemistry,and the expression of smooth muscle protein,vimentin and keratin was significant,which was consistent with the characteristics of adenomyosis cells.2.The number of lentiviral infections in human adenomyosis cells screened in the preliminary experiment was 50.After 48 hours of transfection,the expression of green fluorescence was more than 80%,which was effective in transfection.After transfection with PTEN overexpressing lentiviral vector,the expression of PTEN and PTEN in cells increased significantly after RT-qPCR and WB assay.After transfection of three PTEN shRNA lentiviral vectors,intracellular PTEN gene and PTEN were detected.The expression of protein was significantly decreased,and the interference efficiency of PTEN shRNA3 lentiviral vector was the highest(92.98%).Therefore,PTEN shRNA3 lentiviral vector was selected as PTEN interference group for subsequent drug intervention experiments.3.The concentration of puromycin in human adenomyosis cells screened in the pre-experiment was 2 μ g/mL.Successfully transfected cells were screened in a medium containing 2 μ g/mL puromycin after lentiviral transfection,and cell passage and subsequent experiments were performed.4.CCK-8 technology detects the proliferation of adenomyosis cells4.1 In the non-transfected adenomyosis cells,the flavored peony licorice soup,mifepristone,paeoniflorin and glycyrrhizin can significantly inhibit the cell proliferation activity,and the inhibitory effect of the flavored peony licorice soup is the most obvious.The inhibition of the traditional Chinese medicine monomer is slightly weaker than the flavored peony licorice soup and mifepristone.4.2 PTEN overexpression inhibits the proliferation of adenomyosis cells,and PTEN interference promotes the proliferation of adenomyosis cells.4.3 In the overexpressing PTEN adenomyosis cells,the added peony licorice soup,mifepristone,paeoniflorin and glycyrrhizin can significantly inhibit the cell proliferation activity,and the scented peony licorice soup has the best inhibitory effect.The inhibitory effect of the monomer was slightly weaker than that of the medicinal herb licorice;in the interference with PTEN adenomyosis cells,the four drug interventions could significantly inhibit the cell proliferation activity.4.4 Comparison of proliferation inhibition rate:After over-expressing PTEN,the inhibition rate of agglutinin cell proliferation was significantly increased after adding Piao medicinal herbs,mifepristone,paeoniflorin and glycyrrhizin;after interfering with PTEN,adding peony licorice soup The inhibitory rates of mifepristone,paeoniflorin and glycyrrhizin on the proliferation of adenomyosis cells were significantly reduced.5.Detection of PTEN mRNA,PI3K mRNA,AKT mRNA,TSC2 mRNA,mTOR mRNA,HIF-1α mRNA and VEGF mRNA expression in adenomyosis cells by RT-qPCR5.1 In non-transfected adenomyosis cells:1 Addition of peony licorice and mifepristone can effectively up-regulate the expression of PTEN mRNA and TSC2 mRNA,down-regulate PI3K mRNA,AKT mRNA,mTOR mRNA,HIF-1 α mRNA,Expression of VEGF mRNA.2 Paeoniflorin and glycyrrhizin can effectively up-regulate the expression of PTEN mRNA and TSC2 mRNA,down-regulate the expression of PI3K mRNA,mTOR mRNA,HIF-1α mRNA and VEGF mRNA,but down-regulate the AKT mRNA but not statistically significant.3 The four drugs have the most obvious effect of adding peony licorice soup.5.2 Overexpression of PTEN in adenomyosis cells up-regulated the expression of PTEN mRNA in cells,and up-regulated the expression of TSC2 mRNA,but the difference was not statistically significant;PI3K mRNA,AKT mRNA,HIF-1α mRNA,VEGF were down-regulated.mRNA expression,while down-regulating the expression of mTOR mRNA,but the difference was not statistically significant.5.3 PTEN interference in adenomyosis cells down-regulated the expression of PTEN mRNA and TSC2 mRNA in cells;up-regulated the expression of PI3K mRNA,AKT mRNA,HIF-1α mRNA and VEGF mRNA,and up-regulated the expression of mTOR mRNA,but the difference was not has statistical significane.5.4 In the overexpressed PTEN and interfering PTEN adenomyosis cell lines,the four drug interventions can significantly up-regulate the expression of PTEN mRNA,down-regulate the expression of PI3K mRNA,Akt mRNA and mTOR mRNA;overexpressing PTEN uterus In the adenomyosis cell line,the four drug interventions can significantly up-regulate the expression of TSC2 mRNA in cells.The addition of peony licorice and mifepristone can down-regulate the expression of HIF-1 α mRNA and VEGF mRNA in cells;In the myosin cell line,the four drug interventions could significantly down-regulate the expression of HIF-1α mRNA and VEGF mRNA,and mifepristone and paeoniflorin could significantly up-regulate the expression of TSC2 mRNA.5.5 Comparison of three kinds of cells:1 The intervention method was the same as the medicated medicinal herb Gancao Decoction.The expression of PTEN mRNA and TSC2 mRNA in the overexpressing group were higher than those in the non-transfected group,PI3K mRNA,AKT mRNA,mTOR mRNA and HIF-1α mRNA.The expression of VEGF mRNA was lower than that of non-transfected cells.2 The intervention method was the same as the medicinal herb Gancao Decoction.The expression of PTEN mRNA and TSC2 mRNA in the interference group was lower than that in the non-transfected group,PI3K mRNA,Akt mRNA,The expressions of mTOR mRNA,HIF-1α mRNA and VEGF mRNA were higher than those in non-transfected cells.6.Western blot analysis of PTEN,p-AKT,p-TSC2,HIF-1 α,VEGF expression in adenomyosis cells6.1 In the non-transfected adenomyosis cells:1 The expression of PTEN protein and p-TSC2 protein in the cells increased significantly after the intervention of Jiawei Shaoyao Gancao Decoction,and the expression of p-Akt protein,HIF-1α protein and VEGF protein The amount is significantly reduced.After 2 mifepristone treatment,the expression of PTEN protein in the cells increased significantly,and the expression levels of p-Akt protein,HIF-1αprotein and VEGF protein decreased significantly.The expression of p-TSC2 protein increased,but the difference was not statistical.Learning meaning.After the intervention of paeoniflorin and glycyrrhizin,the expression of PTEN protein in the cells increased significantly,and the expression of p-Akt protein and VEGF protein decreased significantly.The expression of p-TSC2 protein increased,but the difference was not statistically significant.The expression level of HIF-1 α protein was decreased,but the difference was not statistically significant.6.2 In the overexpressing PTEN adenomyosis cells:1 After the intervention of Jiawei Shaoyao Gancao Decoction for 24 h,the expression of PTEN protein and p-TSC2 protein in PTEN overexpressing adenomyosis cells increased significantly,p-Akt protein The expression levels of HIF-1 α protein and VEGF protein were significantly reduced.After treatment with 2 mifepristone,paeoniflorin and glycyrrhizin for 24 h,the expression of PTEN protein in PTEN overexpressing adenomyosis cells increased significantly,and the expression of p-Akt protein,HIF-1α protein and VEGF protein were significantly increased.Both were significantly reduced;there was no significant change in the expression level of p-TSC2 protein.6.3 In the adenomyosis cells interfering with PTEN:After the intervention of the medicinal herbs licorice,mifepristone,paeoniflorin and glycyrrhizin for 24 h,the expression of PTEN protein in the cells of the adenomyosis caused by PTEN increased significantly.The expression levels of p-Akt protein,HIF-1 α protein and VEGF protein were significantly decreased.The expression of p-TSC2 protein was increased,but the difference was not statistically significant.6.4 Comparison of three kinds of cells:1 In each drug intervention group,the expression of PTEN protein and p-TSC2 protein in overexpressing group were higher than those in non-transfected group,p-Akt protein,HIF-1 α protein and VEGF protein.The expression of PTEN protein and p-TSC2 protein in the interference group was lower than that in the non-transfection group,p-Akt protein,HIF-1α protein and VEGF.Protein expression was higher than that of non-transfected cells.Conclusion1.In this study,PTEN shRNA lentiviral vector and PTEN overexpression lentiviral vector were successfully constructed,and human adenomyosis cells were effectively transfected in vitro,and stable cell lines with PTEN knockdown and PTEN overexpression were established.2.Jiawei Shaoyao Gancao Decoction and its monomer may inhibit the expression of downstream PI3K/Akt/HIF-1 α/VEGF signaling pathway-related factors by up-regulating PTEN,thereby inhibiting the proliferation of adenomyosis cells.3.Overexpression of PTEN can effectively improve the inhibitory effect of Jiawei Shaoyao Gancao Decoction and its monomer on adenomyosis cells,which may become a new idea to enhance the efficacy of traditional Chinese medicine compound or monomer. |
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