Probe Into Connotation Of Interdependence Between Storing And Discharging Of Liver And Kidney Based On Proliferation And Differentiation Of Neural Stem Cells In Vitro

ObjectiveTo observe the effects of Sini Powder, the basic prescription for soothing liver and regulating qi, and Liuwei Rehumannia Pill, the classical prescription for tonifying Kidney and replenishing essence, on the proliferation and the neuronal differentiation rate of hippocampal neural stem cells (NSCs) in vitro, and probe into theirs mechanism under the guidance of the basic theory of traditional Chinese medicine. In order to provide theories and experimental gist for the treatment of difficult and complicated diseases with the method of soothing liver or the method of tonifying Kidney combined with stem cell technology, to probe into the theoretical connotation of liver governing conveyance and dispersion and kidney storing essence.Methods1. The research of the basic theory of traditional Chinese medicineThis paper systematically analyzes and summarizes the relationship between liver governing conveyance and dispersion and innate essence as well as that between kidney storing essence and innate essence, the internal connection between innate essence and stem cells and the effects of the prescriptions for soothing liver and the prescriptions for tonifying Kidney on stem cells. Using the comparing methods, this paper analyzes and discusses the correlation between liver governing conveyance and dispersion and stem cells as well as that between kidney storing essence and innate essence and stem cells.2. The experimental research2.1The cultivation and identification of hippocampal neural stem cells of SD rats in vitro The single-cell suspensions of neural stem cells extracted from hippocampus tissues, isolated by simple mechanical agitation from10SD rats within24hours after birth without gender limitation, were cultured in DMEM/F12(1:1) containing20ng/ml of bFGF and EGF,2%of B27,1%of double antibody and1%of L-glutamine with the density of1×10°. The cells were cultivated in the incubator with5%CO2and95%humidity at37℃and repeatedly passaged every4days. The culture medium was changed30%every other day.(1)The ability of proliferating and forming neurospheres of cells were observed morphologically under the inverted microscope.(2)The expression of Nestin and SOX2was detected by immunofluorescence cytochemistry technique.(3)The proliferation of cells were detected by MTT test.(4)The multi-directional differentiation of cells induced with serum were observed morphologically under inverted microscope. The differentiated cells from neural stem cells were detected by immunofluorescence cytochemistry technique. Neurons, astrocytes and oligodendrocytes were identified respectiv-ely with the monoclonal antibody of NSE, GFAP and MBP.2.2The research of the expression of c-myc mRNA and CyclinDl mRNA and the proliferation of Neural stem cells induced by Sini Powder and Liuwei Rehumannia PillNeural stem cells were isolated, cultivated and identified as above. After inoculation, cells were passaged repeatedly every4days. The cultured NSCs of3rd generation were divided into five groups into the normal control group, the low doses of Sini Powder group and the high doses of Sini Powder group, the low doses of Liuwei Rehumannia Pill group and the high doses of Liuwei Rehumannia Pill group. All NSCs in five groups were cultivated with DMEM/F12(1:1) medium for4days. The culture medium was changed30%every other day. The low concentration of Sini Powder decoction with0.133g crude drug per milliliter, the high concentration of Sini Powder decoction with0.267g crude drug per milliliter, the low concentration of Liuwei Rehumannia Pill decoction with0.208g crude drug per milliliter and the high concentration of Liuwei Rehumannia Pill decoction with0.416g crude drug per milliliter were added respectively into the medium in the low doses of Sini Powder group, the high doses of Sini Powder group, the low doses of Liuwei Rehumannia Pill group and the high doses of Liuwei Rehumannia Pill group. The proliferation of cells was detected by BrdU labeling fluorescence immunocytochemistry every day. The relative expression of c-myc mRNA and CyclinDl mRNA were detected by fluorescence real-time quantitative PCR in each group.2.3The research of the protein and gene expression of Wntl, Wnt3a and β-catenin and the differentiation of Neural stem cells into neurons induced by Sini Powder and Liuwei Rehumannia PillThe cultured NSCs was divided into five groups into normal control group, the low doses of Sini Powder group, the high doses of Sini Powder group, the low doses of Liuwei Rehumannia Pill group and the high doses of Liuwei Rehumannia Pill group. All cells in five groups were cultivated with DMEM/F12medium containing10%fetal bovine serum for7days. The culture medium was changed every other day. The low concentration of Sini Powder decoction with0.133g crude drug per milliliter, the high concentration of Sini Powder decoction with0.267g crude drug per milliliter, the low concentration of Liuwei Rehumannia Pill decoction with0.208g crude drug per milliliter and the high concentration of Liuwei Rehumannia Pill decoction with0.416g crude drug per milliliter were added respectively into the medium in the low doses of Sini Powder group and the high doses of Sini Powder group, the low doses of Liuwei Rehumannia Pill group and the high doses of Liuwei Rehumannia Pill group. The neuronal differentiation rate of cells reckoned by the counting method of NSE positive cells in each group was detected by fluorescence immunocytochemistry. The gene expression and the protein expression of Wntl, Wnt3a and β-catenin were detected respectively by fluorescence real-time quantitative PCR and Western blot in each group.Results1. Research of the basic theory of traditional Chinese medicineThe normal physiological functions of innate essence depends upon the synergistic regulation of kidney storing essence and liver governing conveyance and dispersion. There are many similarities between innate essence of traditional Chinese medicine and the sources, function and distribution of stem cells. The prescriptions for soothing liver and the prescriptions for tonifying Kidney have certain interventional effect on stem cells.2. The experimental research2.1The cultivation and identification of hippocampal neural stem cells of SD rats in vitro 2.1.1The morphological observation of neural stem cells cultured in vitroAfter24hours of culturing, the inoculated primary neural stem cells were observed under inverted microscope. The cell body was round with the high transparency. Most of cells grew in suspension Two or more cells got together. The number of cells increased remarkably. After2-3days, NSCs congregated with time and many suspended neural spheres were formed. With neural spheres size increase gradually, the amount of individual sporadic suspended cells reduced obviously. After4days of cultivating, the Diameter of most neural spheres were around100μm. After5-7days, the Diameter of most neural spheres was more than150μm. The subdued light at the centre of neural spheres indicated nutritional deficiencies of central cells in bad growth status. Oversize neural spheres were harmful to the growth of cells.2.1.2The identification result of the expression of neuronal specific marker Nestin and SOX2The expression of Nestin and SOX2was positive.2.1.3Results of the proliferation of cells detected by MTT assayWithin4days after inoculation, the number of cells which were in the status of proliferation increased. The number of cell proliferation was double as that before inoculation after the3rd day of inoculation. On the4th day after inoculation, the number of cell proliferation reached the peak value. The number of cell proliferation reduced after the5th day of inoculation.2.1.4The identification result of the multiple differential potential capacity of neural stem cells(1)The morphological observed result of the multiple differential potential capacity of neural stem cellsAfter culturing for2hours, neural spheres induced with fetal calf serum began to grow adhering to the wall. After48hours, the border cells of neural spheres began to migrate to outside. There were some longer protrusion in peripheral parts of neural spheres. The differentiation were gradually remarkable over time. After7days, NSCs almost completely differentiated into all kinds of difformity cells.(2)The identification result of the multiple differential potential capacity of neural stem cells detected by fluorescence immunocytochemistryThe expression of NSE, GFAP and MBP were positive. Neural stem cell could differentiate into neure, astrocyte and oligodendrocyte. 2.2The research results of the proliferation of neural stem cells induced by Sini Powder and Liuwei Rehumannia Pill and the expression of c-myc and CyclinDl mRNA2.2.1The results of BrdU labeling fluorescence immunocytochemistryThe proliferation of cells in the low and high doses of Liuwei Rehumannia Pill group was more obviously than that in the low doses of Sini Powder group and the high doses of Sini Powder group (P<0.01). There was no obvious difference between the proliferation of cells, among the low doses of Sini Powder group, the high doses of Sini Powder group and normal control group (P>0.05).2.2.2Results of fluorescence real-time quantitative PCRThe relative expression of c-myc mRNA and CyclinDl mRNA in the low doses of Liuwei Rehumannia Pill group and the high doses of Liuwei Rehumannia Pill group was higher obviously than that in the low doses of Sini Powder group and the high doses of Sini Powder group(P<0.01). There was no obvious difference between the relative expression of c-myc mRNA and CyclinDl mRNA among the low doses of Sini Powder group, the high doses of Sini Powder group and normal control group (P>0.05).2.3The research results of the differentiation of Neural stem cells into neurons induced by Sini Powder and Liuwei Rehumannia Pill and the protein and gene expression of Wntl, Wnt3a and β-catenin2.3.1The morphological observed result of the differentiation of neural stem cellsNeural spheres induced with fetal calf serum began to grow adhering to the wall under inverted microscope2hours after induced differentiation. After3days, the border cells of neural spheres were migrating to outside continuously. After7days, neural stem cells almost completely differentiated into all kinds of difformity cells.2.3.2The results of neural stem cells differentiating into NeuresNeural stem cells in each group could differentiate into neure. The expression quantity of NSE in the low doses of Liuwei Rehumannia Pill group, the high doses of Liuwei Rehumannia Pill group, the low doses of Sini Powder group, the high doses of Sini Powder group and normal control group were respectively12.15±2.32%,12.58±1.70%,12.28±1.01%,11.84±1.01%and6.16±1.42%. There was no obvious difference of the expression quantity of NSE in the low doses of Liuwei Rehumannia Pill group, the high doses of Liuwei Rehumannia Pill group, the low doses of Sini Powder group and the high doses of Sini Powder group (P>0.05). The expression quantity of NSE in normal control group was lower than that in the other groups (P<0.01).2.3.3The result of Western blot(1)The expression of WntlThe expression of Wntl in normal control group had a significantly difference from that in the other groups (P<0.01). The expression of Wntl in the high doses of Liuwei Rehumannia Pill group had a significantly difference from that in the low doses of Sini Powder group and the high doses of Sini Powder group (P<0.05).(2)The expression of Wnt3aThe expression of Wnt3a in normal control group had a significantly difference from that in the other groups (P<0.01).(3)The expression of β-cateninThe expression of β-catenin in normal control group had a significantly difference from that in the other groups (P<0.01). The expression of β-catenin in the low doses of Liuwei Rehumannia Pill group had a significantly difference from that in the low doses of Sini Powder group and the high doses of Sini Powder group (P<0.05, P<0.01). There was a significantly difference from the expression of β-catenin in the high doses of Liuwei Rehumannia Pill group and the high doses of Sini Powder group(P<0.01). There was a significantly difference from the expression of β-catenin in the low doses of Sini Powder group and the high doses of Sini Powder group(P<0.05).2.3.4The results of fluorescence real-time quantitative PCR(1)The expression of WntlThe expression of Wntl in normal control group had a significantly difference from that in the other groups (P<0.01).(2)The expression of Wnt3aThe expression of Wnt3a in normal control group had a significantly difference from that in the other groups (P<0.01).(3)The expression of β-cateninThe expression of β-catenin in normal control group had a significantly difference from that in the other groups (P<0.01).Conelusion 1.The research of the basic theory of traditional Chinese medicineInnate essence of traditional Chinese medicine and the sources, function and distribution of stem cells have something in common. The functions of liver governing conveyance and dispersion and kidney storing essence not only are closely to innate essence but also are intimately associated with stem cells.2.The experimental research2.1The cultivation and identification of hippocampal neural stem cells of SD rats in vitroThe cultured cells in this research were neural stem cells with the multiple differential potential capacity. To avoid the damage from trypsin and the interference of serum, vigorous cells with strong proliferative ability were isolated by simple mechanical agitation. The cultured cells met with the standard of research. The best passage of cultured cells time were4days after inoculation. This method of culturing neural stem cells is feasible and economizes time and resource.2.2Comparing with Liuwei Rehumannia Pill, Sini Powder had no significant stimulative effect on the proliferation of neural stem cells or the expression of c-myc mRNA and CyclinDl mRNA. Liuwei Rehumannia Pill could promote the proliferation of neural stem cells and raised the expression of c-myc mRNA and CyclinDl mRNA. The latter might be the action mechanism of the former.2.3Both of Sini Powder and Liuwei Rehumannia Pill could promote the differentiation of neural stem cells into neure and raise the protein and gene expression of Wnt1、 Wnt3a and β-catenin. The action mechanism of promoting the differentiation of neural stem cells into neure might be relevant for rising the expression of Wnt1、 Wnt3a and β-catenin and activating the wnt/β-catenin signal pathway.In conclusion, liver governing conveyance and dispersion is displayed principally by the promotion of the differentiation of neural stem cells into neure in vitro. Kidney storing essence is displayed principally by the promotion of the proliferation of neural stem cells and the differentiation of neural stem cells into neure in vitro. The regulation mechanism of Sini Powder, the basic prescription for soothing liver and regulating qi, on the differentiation of neural stem cells into neure might be relevant for activating the wnt/β-catenin signal pathway. The regulation mechanism of Liuwei Rehumannia Pill, the classical prescription for tonifying Kidney and replenishing essence, on the proliferation of neural stem cells might be relevant for rising the expression of c-myc mRNA and CyclinDl mRNA. The regulation mechanism of Liuwei Rehumannia Pill, the classical prescription for tonifying Kidney and replenishing essence, on the differentiation of neural stem cells into neure might be relevant for activating the Wnt/β-catenin signal pathway.