Based On The Expression And Accommodation Of The Key Gene Nanog To Investigate The Supp Lementing Kidney And Shrinkaging Urination Mechanism Of SQW

The method of supplementing kidney and shrinkaging urination was the main therapy for improving the urinary tract symptoms of frequent micturition, dieresis, enuresis, uroclepsia and so on. SuoQuanWan was a classic prescription for curing dieresis, frequent micturition or enuresis through supplementing kidney-yang and warming kidney-Qi. Its effect summarized as four words for supplementing kidney and shrinkaging urination, the base of therapy was supplement kidney and shrinkaging urination just relaxed the symptom. The kidney was the root of earlier heaven and human body’s life that improve the growth, development and reproduction of human body. Embryonic stem cells was very similar with kidney essence in source or function, it can explain most of the function of kidney essence. Thus, the essence in kidney has some connection with embryonic stem cells.ObjectiveThe scientific connotation of SuoQuanWan for supplementing kidney and shrinkaging urination was discussed through the aspects of embryonic stem cells, the method of traditional theory research combined with modern molecular biology technology were used to research the effect and intervention of SuoQuanWan on the proliferation, polarization, cell cycle, apoptosis of embryonic stem cells’s life movement and the accommodation of SuoQuanWan on the transcription, express and function of the key gene Nanog in embryonic stem cells, it can explain the scientific connotation of SuoQuanWan in supplementing kidney and shrinkaging urination from deep levels of the micro angle and stem cells’s level. Method1. The cultivation of the mouse embryonic stem cellsThe embryonic stem cells that we use is actually come from the research center of stem cells and tissue engineering which is in Sun Yat-sen University. We Recovery the ESCs on feeding layer, than we adopt no feed layer method but add PD0325901and Chir99021in the culture medium during subculture to keep the culture of the embryonic stem cells.2. The model was established as the reflection of the kidney essence in cell level, we adopt the method of serum pharmacology to detect the influence of serum of SuoQuanWan to ESCs.We set the categories:the high, middle, low dose groups containing serum of SuoQuanWan and the group containing serum of Jinkuishenqiwan were prepared, the isopyknic serum of normal rats as control group.The effect of SuoQuanWan serum on proliferation for ESCs by the method of MTT and CFSE; the effect of SuoQuanWan serum on cell cycle for ESCs by the method of flow cytometry; The influence of SQW on the induced by H202was detected with Annexin V/PI flow cytometry analysis. The effect of SQW on ESCs differentiation induced by DMSO were observed by inverted phase contrast microscope.3. Research the influence of serum of SuoQuanWan to the expression of Nanog mRNA and protein in ESCs.We set the categories:the high, middle, low dose groups containing serum of SuoQuanWan and the group containing serum of Jinkuishenqiwan were prepared, the isopyknic serum of normal rats as control group.The expression of Nanog mRNA in ESCs was detected by the fluorescence quantitative PCR and the expression of Nanog protein in ESCs was determined by flow cytometry analysis and Western blotting.4. We use RNA interference method to research the influence of serum of SuoQuanWan to the expression of Nanog mRNA and protein in ESCs.We set the categories:normal control group, interfere with the negative group, interference with the siRNA group, the high, middle, low dose groups containing serum of SuoQuanWan and the group containing serum of Jinkuishenqiwan.The expression of Nanog mRNA and protein in ESCs was determined by fluorescence quantitative PCR and Western blotting after the expression of Nanog in ESCs decrease specifically due to the siRNA.Resultl.The growth situation of ESCsThe Morphologic Characters of ESCs are as follows:cells links to grow into a colony, the stereo sense is strong, the shape is usually roundness or oval, the edge is smooth and clear and had high refraction, cells are small but the nucleus is large. The two cultural methods are borh fit to ESCs.2. The influence of serum of SuoQuanWan to the basic activity of ESCs.The result of MTT demonstrated that the high, middle, low dose serum of SQW can improve the OD value and the multiplication capacity of ESCs; As the increase of the SQW dose, the fluorescence intensity decreases gradually, the result of CFSE proved that the serum of SQW can improve the multiplication capacity of ESCs; the flow cytometry result show that the serum of SQW can reduce the percentage of cell in G0/G, phase, increase the percentage of cell in G2/M phase, increase the proliferation index of ESCs, promote the ESCs from G0/d phase to S+G2/M phase; the result of Annexin V/PI show that the number of early ESCs apoptosis can be reduced when serum of high-dose SQW was added and the rate of early embryonic stem cell apoptosis induced by H202can be decreased obviously, the result of inverted phase contrast microscope show that the drug serum can promote differentiation of ESCs induced by DMSO.3. The influence of serum of SuoQuanWan to the expression of Nanog mRNA and protein in ESCs.The result of fluorescence quantitative PCR show that the low and high dose of SQW can improve the express of Nanog mRNA in ESCs signally; the result of flow cytometry analysis and Western bloting show that the high and middle dose of SQW can improve the express of Nanog protein in ESCs signally.4. The influence of serum of SuoQuanWan to the expression of Nanog mRNA and protein in ESCs after using RNA interference method.The result of fluorescence quantitative PCR and Western blotting show that the serum of SQW can improve the expression of Nanog mRNA in ESCs after the effect of siRNA, the effect on the expression of protein was effective but not signally.ConclusionSQW can increase the multiplication capacity of ESCs, promote ESCs into the phase of cell proliferation, inhibit the apoptosis of ESCs induce by H2O2, promote the polarization of ESCs induce by DMSO. SQW can improve cell division and maintain the totipotency of ESCs by improving the express of Nanog in ESCs. Those result show that SQW can cure frequent micturition, dieresis, enuresis by supplementing and culturing the essence in kidney so as to express the effect of warming Yang and Qi in kidney.