基于Nanog基因的表达与调控探讨缩泉丸“补肾缩尿”的机理
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摘要
补肾缩尿法是改善尿频、多尿、遗尿、尿失禁等下尿路症状的重要治法。缩泉丸是一首通过温补肾阳、温化肾气而达到治疗尿量过多、小便频数或遗尿的经典古方。其功效可用四个字概括为“补肾缩尿”,补肾治其本,缩尿缓其标。肾为先天之本,能促进人体的生长、发育和生殖,为人体生命之本原。胚胎干细胞与肾精无论在来源或功能上均十分相似,用胚胎干细胞可以解释肾精的有关功能。因此,肾中精气与胚胎干细胞必然有着某种联系。
目的：
从胚胎干细胞角度入手探讨缩泉丸“补肾缩尿”的科学内涵,运用传统的理论研究与现代的分子生物技术相结合的方法,通过研究缩泉丸对胚胎干细胞增殖、细胞周期、凋亡、分化等基本生命活动的影响与干预,以及缩泉丸对胚胎干细胞功能的关键决定基因Nanog在转录、表达及功能方面的调节,从微观角度和干细胞水平深层次揭示缩泉丸“补肾缩尿”的科学内涵。
方法：
一、小鼠胚胎干细胞的培养
实验所用胚胎干细胞(embryonic stem cells, ESCs)是来源于中山大学干细胞与组织工程研究中心,采用的培养方法是ESCs的复苏采用饲养层法,传代过程采用无饲养层法并且添加PD0325901和Chir99021两种小分子抑制剂以维持ESCs的形态。
二、以胚胎干细胞作为肾精实质在细胞水平体现的模型,采用血清药理学的方法检测缩泉丸含药血清对胚胎干细胞活动的影响
设置组别：缩泉丸低、中、高剂量含药血清组,金匮肾气丸含药血清组,以正常青年大鼠等体积血清为对照组。
分别采用MTT比色法和CFSE标记法测缩泉丸含药血清对ESCs增殖的影响；采用流式细胞术法测定缩泉丸含药血清对ESCs细胞周期的影响；采用磷脂结合蛋白V/碘化丙啶(Annexin V/PI)流式细胞分析法测定缩泉丸含药血清对H202诱导的ESCs早期凋亡的影响；采用倒置相差显微镜观察缩泉丸含药血清对DMSO诱导的ESCs的分化情况。
三、观察缩泉丸对胚胎干细胞关键因子Nanog mRNA及蛋白表达的影响
设置组别：缩泉丸低、中、高剂量含药血清组,金匮肾气丸含药血清组,以正常青年大鼠等体积血清为对照组。
采用荧光定量PCR的方法测定各组ESCs中Nanog mRNA的表达；分别采用流式细胞术和Western印迹法测定各组ESCs中Nanog蛋白的表达。
四、采用RNA干扰方法观察缩泉丸对胚胎干细胞关键因子Nanog mRNA及蛋白表达改变的影响
设置组别：正常对照组、干扰阴性组、siRNA干扰组、缩泉丸低、中、高剂量含药血清组,金匮肾气丸含药血清组。
用siRNA特异性地降低胚胎干细胞中Nanog的表达后,观察各组形态学差异并采用荧光定量PCR和Western印迹法测定缩泉丸对胚胎干细胞Nanog mRNA及蛋白表达的影响。
结果：
一、ESCs的生长情况
培养的ESCs形态学特征为：细胞成集落状生长,集落立体感强,呈圆形或椭圆形,边缘光滑清楚,折光性强,细胞体积较小,细胞核大。采用无饲养层法传代培养的ESCs和饲养层法复苏的ESCs在形态上无差别,都具有ESCs的典型形态学特征。
二、缩泉丸含药血清对胚胎干细胞活动的影响
MTT法结果表明,缩泉丸中、高剂量组含药血清可使OD值提高,增强ESCs的增殖能力,CFSE染色法结果显示,随着缩泉丸给药剂量的增加,其荧光强度逐渐降低,表明缩泉丸含药血清有促进ESCs增殖的能力；流式法结果表明缩泉丸中、高剂量组含药血清可减少处于G0/G1期的细胞百分比,增加处于G2/M期的百分比,提高ESCs的增殖指数,促进ESCs由相对静止时相(G0/G1期)进入细胞增殖活跃时相(S+G2/M期)；Annexin V/PI双染法结果显示缩泉丸高剂量组含药血清可减少早期凋亡的细胞数目,能明显降低H202诱导的胚胎干细胞的早期凋亡率；倒置相差显微镜显示缩泉丸含药血清能够促进DMSO诱导的胚胎干细胞进行分化。
三、缩泉丸对胚胎干细胞关键因子Nanog mRNA及蛋白表达的影响
荧光定量PCR结果表明缩泉丸低、高剂量可以显著提高ESCs中Nanog mRNA的表达；流式细胞术和WB结果表明,缩泉丸中、高剂量可以显著提高ESCs中Nanog蛋白的表达。
四、采用RNA干扰方法观察缩泉丸对胚胎干细胞关键因子Nanog mRNA及蛋白表达改变的影响
用siRNA特异性地降低胚胎干细胞中Nanog的表达后,各给药组与空白血清组从形态上观察无明显差异,荧光定量PCR和WB结果分别表明缩泉丸含药血清可以提高siRNA作用后胚胎干细胞Nanog mRNA的表达,对于其Nanog蛋白的表达有一定的上调作用,但作用不显著。
结论：
缩泉丸含药血清可以影响ESCs的基本活动,增加ESCs的增殖能力,促进其进入细胞增殖活跃时相,抑制H202诱导的ESCs的凋亡,促进DMSO诱导的胚胎干细胞进行分化。其可通过上调ESCs中Nanog的表达来促进细胞分裂、维持ESCs的全能性。因ESCs与肾精的相似性,提示缩泉丸可能是通过培育和充养肾中精气的作用发挥其温补肾阳、温化肾气的功效,从而治疗肾气虚寒而致的小便频数、遗尿、夜尿等病症。
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.
Objective
The 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.
Method
1. The cultivation of the mouse embryonic stem cells
The 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.
Result
l.The growth situation of ESCs
The 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.
Conclusion
SQW 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.
目的：
从胚胎干细胞角度入手探讨缩泉丸“补肾缩尿”的科学内涵,运用传统的理论研究与现代的分子生物技术相结合的方法,通过研究缩泉丸对胚胎干细胞增殖、细胞周期、凋亡、分化等基本生命活动的影响与干预,以及缩泉丸对胚胎干细胞功能的关键决定基因Nanog在转录、表达及功能方面的调节,从微观角度和干细胞水平深层次揭示缩泉丸“补肾缩尿”的科学内涵。
方法：
一、小鼠胚胎干细胞的培养
实验所用胚胎干细胞(embryonic stem cells, ESCs)是来源于中山大学干细胞与组织工程研究中心,采用的培养方法是ESCs的复苏采用饲养层法,传代过程采用无饲养层法并且添加PD0325901和Chir99021两种小分子抑制剂以维持ESCs的形态。
二、以胚胎干细胞作为肾精实质在细胞水平体现的模型,采用血清药理学的方法检测缩泉丸含药血清对胚胎干细胞活动的影响
设置组别：缩泉丸低、中、高剂量含药血清组,金匮肾气丸含药血清组,以正常青年大鼠等体积血清为对照组。
分别采用MTT比色法和CFSE标记法测缩泉丸含药血清对ESCs增殖的影响；采用流式细胞术法测定缩泉丸含药血清对ESCs细胞周期的影响；采用磷脂结合蛋白V/碘化丙啶(Annexin V/PI)流式细胞分析法测定缩泉丸含药血清对H202诱导的ESCs早期凋亡的影响；采用倒置相差显微镜观察缩泉丸含药血清对DMSO诱导的ESCs的分化情况。
三、观察缩泉丸对胚胎干细胞关键因子Nanog mRNA及蛋白表达的影响
设置组别：缩泉丸低、中、高剂量含药血清组,金匮肾气丸含药血清组,以正常青年大鼠等体积血清为对照组。
采用荧光定量PCR的方法测定各组ESCs中Nanog mRNA的表达；分别采用流式细胞术和Western印迹法测定各组ESCs中Nanog蛋白的表达。
四、采用RNA干扰方法观察缩泉丸对胚胎干细胞关键因子Nanog mRNA及蛋白表达改变的影响
设置组别：正常对照组、干扰阴性组、siRNA干扰组、缩泉丸低、中、高剂量含药血清组,金匮肾气丸含药血清组。
用siRNA特异性地降低胚胎干细胞中Nanog的表达后,观察各组形态学差异并采用荧光定量PCR和Western印迹法测定缩泉丸对胚胎干细胞Nanog mRNA及蛋白表达的影响。
结果：
一、ESCs的生长情况
培养的ESCs形态学特征为：细胞成集落状生长,集落立体感强,呈圆形或椭圆形,边缘光滑清楚,折光性强,细胞体积较小,细胞核大。采用无饲养层法传代培养的ESCs和饲养层法复苏的ESCs在形态上无差别,都具有ESCs的典型形态学特征。
二、缩泉丸含药血清对胚胎干细胞活动的影响
MTT法结果表明,缩泉丸中、高剂量组含药血清可使OD值提高,增强ESCs的增殖能力,CFSE染色法结果显示,随着缩泉丸给药剂量的增加,其荧光强度逐渐降低,表明缩泉丸含药血清有促进ESCs增殖的能力；流式法结果表明缩泉丸中、高剂量组含药血清可减少处于G0/G1期的细胞百分比,增加处于G2/M期的百分比,提高ESCs的增殖指数,促进ESCs由相对静止时相(G0/G1期)进入细胞增殖活跃时相(S+G2/M期)；Annexin V/PI双染法结果显示缩泉丸高剂量组含药血清可减少早期凋亡的细胞数目,能明显降低H202诱导的胚胎干细胞的早期凋亡率；倒置相差显微镜显示缩泉丸含药血清能够促进DMSO诱导的胚胎干细胞进行分化。
三、缩泉丸对胚胎干细胞关键因子Nanog mRNA及蛋白表达的影响
荧光定量PCR结果表明缩泉丸低、高剂量可以显著提高ESCs中Nanog mRNA的表达；流式细胞术和WB结果表明,缩泉丸中、高剂量可以显著提高ESCs中Nanog蛋白的表达。
四、采用RNA干扰方法观察缩泉丸对胚胎干细胞关键因子Nanog mRNA及蛋白表达改变的影响
用siRNA特异性地降低胚胎干细胞中Nanog的表达后,各给药组与空白血清组从形态上观察无明显差异,荧光定量PCR和WB结果分别表明缩泉丸含药血清可以提高siRNA作用后胚胎干细胞Nanog mRNA的表达,对于其Nanog蛋白的表达有一定的上调作用,但作用不显著。
结论：
缩泉丸含药血清可以影响ESCs的基本活动,增加ESCs的增殖能力,促进其进入细胞增殖活跃时相,抑制H202诱导的ESCs的凋亡,促进DMSO诱导的胚胎干细胞进行分化。其可通过上调ESCs中Nanog的表达来促进细胞分裂、维持ESCs的全能性。因ESCs与肾精的相似性,提示缩泉丸可能是通过培育和充养肾中精气的作用发挥其温补肾阳、温化肾气的功效,从而治疗肾气虚寒而致的小便频数、遗尿、夜尿等病症。
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.
Objective
The 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.
Method
1. The cultivation of the mouse embryonic stem cells
The 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.
Result
l.The growth situation of ESCs
The 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.
Conclusion
SQW 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.
引文
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