| ObjectiveThin endometrial infertility is common in clinic,and its adverse pregnancy outcome is particularly prominent in assisted reproductive technology.Stem cell therapy is one of the most promising treatments at present,and more and more clinical cases have been reported to confirm the effectiveness of stem cells in the treatment of thin endometrium.However,a series of problems remain to be solved,such as how the stem cells implanted into the uterine cavity differentiate into endometrial epithelial cells,what is the mechanism,and whether traditional Chinese medicine can affect this differentiation process.The previous experiments of our group suggested that after intrauterine injection of bone marrow mesenchymal stem cell(BMSCs)into thin endometrial model SD rats,intragastric administration of Guishen Pill decoction could effectively promote the transformation of BMSCs into endometrial epithelial cells,and suggested that Guishen Pill could increase endometrial receptivity by regulating HOXA10.Therefore,in this experiment,the water extract and estrogen of Guishen pill were directly acted on BMSCs,to detect the effects of water extract and estrogen of Guishen pill on the proliferation and differentiation of BMSCs.And down-regulate the expression of HOXA10 gene to verify the regulatory effect of HOXA10 on PTEN/PI3K/AKT signal pathway,so as to verify the possible molecular mechanism of Guishen pill water extract in regulating the proliferation and differentiation of BMSCs.Methods1.The whole bone marrow of 3-week-old SD rats was obtained and purified by whole bone marrow adherent culture.2.The phenotype of P3 cells was identified by flow cytometry,the drug concentration gradient of traditional Chinese medicine and estrogen was set,and the best drug concentration was screened by CCK-8 kit.3.The specific target of HOXA10 gene was found on the bioinformatics software SnapgeneViewer4.2.3,and three HOXA10 genes were constructed and transfected into bone marrow mesenchymal stem cells.WesternBlot and qRT-PCR techniques were used to detect the knockdown efficiency of HOXA10,and the lentiviral vector with the highest knockdown rate was selected.4.The third generation of bone marrow mesenchymal stem cells were divided into the following two groups,six groups:the first group(non-transfection group):group 1.control group,10%FBS t DMEM/F12;group 2.Guishen pill group:10%FBS t DMEM/F12+5mg/L Guishen pill water extract;group 3.strogen group,10%FBS t DMEM/F12,1×10-7mol/L 17 β estradiol;the second largest group(transfection group):group 4.transfection control group,10%FBS t DMEM/F12;group 5.etransfection+Guishen pill group:10%FBS t DMEM/F12+5mg/L Guishen pill water extract,group 6.transfection+estrogen,10%FBS t DMEM/F12,1× 10-7 mol/L 17 β estradiol.5.After 3 days of drug intervention,the expression of ITG αvβ3 in the supernatant of the six groups was detected by ELISA,and the expression of wave protein,keratin,LIF and VEGF was detected by immunofluorescence.6.Western Blot technique was used to detect the expression of HOXA10,PTEN,PI3K,AKT,p-PI3K,p-AKT and other proteins related to PTEN/PI3K/AKT pathway in each group.7.QRT-PCR technique was used to detect the expression of genes related to HOXA10,PTEN,PI3K,AKT and other PTEN/PI3K/AKT pathway.The cell cycle distribution and apoptosis rate of bone marrow mesenchymal stem cells in each group were detected by flow cytometry,and the effects of lentivirus,water extract of Guishen pill and 17β estradiol on the cycle and apoptosis of bone marrow mesenchymal stem cells were detected.8.The cell cycle distribution and apoptosis rate of bone marrow mesenchymal stem cells in each group were detected by flow cytometry,and the effects of lentivirus,water extract of Guishen pill and 17β estradiol on the cycle and apoptosis of bone marrow mesenchymal stem cells were detected.Results1.The third generation of cells were arranged regularly and their morphology was unified.The phenotype was identified by flow cytometry,and the results showed that CD34 was negative,and the double positive rate of CD44 and CD90 was 91.5%,which accorded with the characteristics of bone marrow mesenchymal stem cells and could be used in follow-up experiments.2.The best concentration of traditional Chinese medicine selected by CCK-8 to promote cell proliferation was 80mg/L,17 β estradiol which had no obvious effect on cell proliferation(P>0.05).3.The lentivirus vector with the best knock-down effect is 3#:pLVSHG01-scramble shRNA.Its knock-down efficiency is up to 80%,which is significantly better than that of 1#and 3#.and the corresponding lentivirus sequence is TCTGCTCCCTTCGCCAAATTACTCGAGTAATTTGGCGAAGGGAGCAGA.4.The results of ELISA showed that the expression of ITG αvβ3 in BMSCs was weakly positive in all six groups,and there was no significant difference between the six groups(P>0.05).The immunofluorescence results showed that the expression of wave protein in blank control group and transfection control group was positive,while that of keratin,LIF and VEGF was negative.The expression of wave protein,keratin,LIF and VEGF in Guishen pill group and estrogen group were strongly positive,while the expression of wave protein,keratin,LIF and VEGF in transfection+Guishen pill group and transfection+estrogen group were weakly positive.5.WesternBlot assay showed that the expression of HOXA10,PI3K,AKT,p-PI3K and p-AKT in BMSCs of transfection group was lower than that of non-transfection group,while the expression of PTEN protein was higher than that of non-transfection group.The protein expression of HOXA10,PI3K and AKT in the traditional Chinese medicine group was the highest in the non-transfection group,which was significantly higher than that in the blank control group,and there was no significant difference between the estrogen group and the estrogen group(P>0.05).There was no significant difference in the protein expression between the estrogen group and the blank control group(P>0.05).There was no significant difference in the protein expression of each group in the),;transfection group.6.The results of qRT-PCR assay showed that the expression of HOXA10,PI3K and AKT genes in BMSCs in the transfection group was lower than that in the non-transfection group(P<0.05),while the expression of PTEN gene in the transfection group was higher than that in the non-transfection group(P<0.05).The gene expression of HOXA10,PI3K and AKT in the Guishen pill group was the highest in the non-transfection group,which was significantly higher than that in the blank control group(P<0.05),but there was no significant difference between the blank control group and the estrogen group in the non-transfection group(P>0.05).There was no significant difference in the expression of HOXA10,PTEN,PI3K and AKT genes in the transfection group(P>0.05).7.Flow results show the apoptosis rate in the transfection group was significantly higher than that in the non-transfection group,and the apoptosis rate in the Guishen pill group in the non-transfection group was significantly lower than that in the blank control group and estrogen group.The number of GO/GI phase cells in the transfection group was significantly higher than that in the non-transfection group(P<0.05).The number of S phase cells in the transfection group was significantly lower than that in the non-transfection group.There was no significant difference between G2/M phase transfection group and non-transfection group(P>0.05).ConclusionThe aqueous extract of Guishen Pill directly acts on bone marrow mesenchymal stem cells and has the effect of promoting proliferation and inducing differentiation,but the drug concentration of promoting proliferation and inducing differentiation is different.When the concentration is 10mg/L--100mg/L,it can promote the proliferation of BMSCs,when the concentration is lower than 10mg/L,it can induce BMSCs to differentiate into endometrial epithelial cells.The PI3K and AKT protein expression of BMSCs in the Guishen pill water extract group was significantly higher than that in the blank control group,suggesting that the water extract of Guishen pill has a certain regulatory effect on PI3K/AKT signal pathway.The HOXA10 gene expression of BMSCs in Guishen pill water extract group was significantly higher than that in blank group and estrogen group.After transfection with knock-down HOXA10 lentivirus,there was no significant difference in relative gene expression.It is speculated that the molecular mechanism of BMSCs differentiation induced by Guishen pill water extract may be related to its up-regulation of HOXA10 gene expression and regulation of PTEN/PI3K/AKT pathway.There is no direct evidence to prove that estrogen directly acts on bone marrow mesenchymal stem cells to promote proliferation and differentiation.At the same time,Guishen Pill water extract group could increase S phase cells,reduce GO/GI phase cells,and inhibit cells to enter the quiescent phase.Therefore,it can effectively inhibit cell apoptosis and promote cell growth at the same time. |
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