| Objective:To investigate the effect of kidney-tonifying herbs on the develop-ent and differentiation of human embryonic stem cells into follicle-like structures in vitro.Method:Human embryonic stem cells was selected as the experimental object which was induced to form an embryonic body(EB).There are 6 medicated serum:22#,23#,24#,25#,26#and27#at 3%final concentration.Medium group which has no serum addition was the control group.The factor group with 100pg/ml BMP4 as the final concentration was a positive control.Complete culture solution without BMP4 was used as a blank control.Do microscopic dynamic observation of H9 morphological changes in the growth and differentiation of the embryonic bodies;The expression of germ cell on different days were measured by RT-qPCR;The expression of marker protein in germ cell differentiation was detected by immunofluorescence confocal technique;Eventually,The content of E2 in the late culture medium of the pseudo embryo differentiation was determined by ELISA.All the experimental data are processed by SPSS 19.0 statistical software.If the data is satisfied with the normal distribution and homogeneity of variance,then the x±S is used to express the data,and the paired T-test is used to detect the data.Results:1.Serum containing kidney-tonifying herbs induces the development and differentiation of H9 EB1.1 EB cultured by serum with herbs appeared similar early differentiation characteristics to the positive control on 3rd day:a large number of cells escaped from the EB,gradually narrowing with irregular in shape.At the same time collect EB and dissociated cells detected in embryo stem cell differentiation markers Oct-4 gene mRNA expression.The results showed that before adding the medicated serum,the Oct-4 gene expression of Oct-4 gene is significant,but on the third day of medicated serum addition,Oct-4 gene was significantly reduced,indicating that serum containing kidney tonifying herbs induced EB into differentiation stage.1.2 To detect the expression and reproductive development of related genes,different medicated serum groups showed different results:among which 23#medicated serum is better than BMP4 in terms of inducing the expression of GDF9,ZP1 and SCP3.This experiment further applies immunofluorescence technique to detect the expression of GDF9 protein of the embryoid body in serum 23#on the fifth day.The result is consistent with that of mRNA level.Because of the self-differentiat-ion feature of the negative control group,the expression of GDF9 is less evident.BMP4 and serum 23#play much better roles in inducing the expression of GDF9.Serum 23#is more effective than BMP4.2.Serum containing kidney-tonifying herbs induced hESCs reproductive specific differentiation of tissues and cells.2.1 Culturing for about 2 weeks,23#,25#,26#,27#medicated serum group the differentiation cell morphology were similar to BMP4 positive control group,and the major cell types included epithelial cells,fibroblasts,endothelial cells,multinucleated cells,the fat cells,neuron like cells,follicular or egg structure and hematopoietic cells.By counting follicular structure,we discovered that this type cells appeared most in the BMP-4 positive group and 23#,25#,26#medicated serum group,and the number increased with the prolongation of the culture time;moreover,the differentiation of this kind of cells were from the initial polygon gradually to circle,the diameter increased with increasing incubation time,granular substances gradually formed on the edge of the cell with time increased.2.2Selected 23#group which had the largest number of follicular morphology and analyzed the expression and distribution of follicular structure of reproductive differentiation associated markers,the results showed that various reproductive differentiation associated genes expression up-regulated among follicular structure.2.3Collected 23#group culture supernatants,levels of E2 were quantitatively measured,and the results showed that 23#medicated serum in inducing differentiation of follicular structure has the function of estrogen secretion.2.Serums with kidney-tonifying herbs induce embryonic stem cell to differentiate and become reproductive specific tissue cells2.1 Serums with herbs induce differentiation for 5 to 7 days.A different kind of cells,which are larger than other differentiated cells,can be observed in both BMP-4 positive control group and serums 23#,25#and 26#groups.The diameter of those cells is larger than 50μm,and its cell morphology is just like oocyte cell.Continue to induce the differentiation to 7 to 10 days,the diameter of the oocyte-like cell increases and some fine particles begin to form around the cell.Continue the differentiation to 11 to 16 days.the particle layer gradually increases and thickens,with the diameter of the cell exceeding 100μm.Some particle layers exhibit flat structures.This type of particle-surrounded cell is presumed to be follicle-like structures(FLSs).In order to verify the FLSs induced by serum with herbs.23#serum with herbs is chosen to induce the differentiation and the process continues for 16 days.The dynamic change of the FLSs formation is observed with microscope.Analysis results show that serum with herbs and BMP4 can induce H9 embryonic stem cell to differentiate into at least three FLSs structures,including the development process from primordial follicles to primary follicles and then to secondary follicles.2.2 Identification of molecular makers of FLSs induced by kidney-tonifying herbs2.2.1 Analysis of molecular makers of primordial folliclesIn order to further verify the formation of FLSs.Serum 23#with herbs is chosen to cultivate H9 ESC for 3 to 7 days.When primordial follicles form in large quantity,RT-qPCR is applied to detect the dynamic changes of the expression of three molecular makers during the formation process.On the 3rd,5th and 7th day,the expression of GDF9 and SCP3 are increased,exhibiting time-dependent feature.Serum 23#with herbs also induces the expression of VASA,but on the 7th day of differentiation,the expression decreases.The time-dependent feature of GDF9 and SCP3 are consistent with that of primordial follicles differentiating in vitro.VASA gene expression decreases on the 7th day of the induction,which shows that the expression of VASA is related to the primordial follicles’ development.In addition,the primordial follicles induced by serum 23#with herbs on the 7th day is chosen to detect its GDF9 protein expression.The result shows that it is consistent with the gene expression level.Strong fluorescence signal can be found in every single FLS.2.2.2Analysis of molecular markers growth follicular phaseChoose cells that have differentiated for 7 to 16 days(the number of growth folliculars are markedly increased)and detect the dynamic changes of the expression of three molecular makers during the follicle growing process.Serum 23#with herbs induces differentiation for 7,9 and 11 days.The expressions of ZP1,ZP2 and ZP3 are all increased and show time-dependent feature.Growing follicles induced by serum 23#with herbs on the 11th day are chosen to detect its ZP1 protein expression.Results show that there is strong fluorescence signal between particle layer and oocyte of an growing follicle.2.3The morphological feature of oocytes induced by kidney-tonifying herbsPrimordial follicles and oocytes of follicular phase are both primary oocytes.They have all the characteristics of meiosis I.In order to prove that the primary oocytes induced by serum with herbs also have those characteristics,this experiment uses immunofluorescence technique,β-actin staining cytoskeleton and Hoechst33258 to dye cell nucleus of every meiosis phase.By doing so,oocyte-like cells wrapped by particle layers can be detected.Results show that oocyte-like cells induced by serum 23#for 7 to 16 days include oogonium and primary oocyte of leptotene stage,zygotene stage and pachytene stage.During the differentiation process,oocyte of diplotene stage can also be spotted.From the perspective of the morphological feature of different meiosis,those results further prove that kidney-tonifying herbs can induce FLSs.2.4 Molecular makers and functional assessment of kidney-tonifying herbs induced granulosa-like cells2.4.1 Analysis of granulosa-like cells’ molecular makerDifferentiated cells on the 11th day are chosen for analyzing.The expression of Granulosa’s molecular maker FSHR is detected.FSHr gene expression under the influence of serum 23#with herbs is much higher than that of BMP4 positive control group.Therefore,growing follicles induced by serum 23#with herbs on the 11th day are singled out to detect the expression of Aromatase protein.Results show that there is strong fluorescence signal in the particle layer of an growing follicle structure.2.4.2 Functional assessment of granulosa-like cellsAdd testosterone(50ng/ml)to every group;take some culture fluid on the 9th and 11th day of the induced differentiation and detect the expression of E2 in different culture fluid.The results are as follows:on the 9th day of the differentiation.concentration of E2 from the culture fluid with serum 23#containing herbs is much higher than that of negative control group but slightly lower than BMP4 group.On the 11th day of the differentiation,concentration of E2 from the culture fluid with serum 23#containing herbs is increased remarkably and much higher than that of BMP4 group.On the 11th day of the differentiation,add FSH(0.11U/mL)to every group.After 48 hours’ cultivation,the concentration of E2 of every group is much higher compared with the former value when no FSH is added.The concentration of E2 of serum 23#containing herbs group is much higher than that of BMP4 group.It indicates that FLSs induced by BMP4 and serum 23#with herbs has certain functions.Conclusions:1.In this study.the cultured embryos were undifferentiated and pluripotent.Rat medicated serum containing kidney-tonifying herbs have obvious potential to induce the development and differentiation of Human embryonic stem cells in vitro on the 3rd day of culture,indicating that kidney-tonifying herbs can quickly induce the differentiation of human embryonic stem cells in vitro.2.Rat medicated serum containing kidney-tonifying herb can promote Human embryonic stem cells into follicle-like structures in vitro.Including its development:primordial follicles→primary follicles→secondary follicles.But under the condition of this experiment.we can’t observe the formation of mature follicles.It indicates rat medicated serum containing kidney-tonifying herb can induce Human embryonic stem cells into follicle-like structures,but the development of FLSs has stopped in the stage of preantral follicles.3.The follicle oocytes in the follicle-like structureshave the morphological characteristics of meiosis I.oogonium and primary oocyte of leptotene stage,zygotene stage and pachytene stage.4.The granulosa-like cells in follicle-like structures have the function of estrogen synthesis,and estrogen levels rises after FSH stimulation.Rat medicated serum containing kidney-tonifying herbs can promote the human embryonic stem cells to differentiate into follicle-like structures in vitro,and have the function of estrogen synthesis.It is the first finding that human embryonic stem cells successfully induced to differentiate into follicle-like structures,which laying a good foundation for extensive research of idney-tonifying herbs differentiate into follicle-like structures。... |
