【作者】 郑景辉；

【导师】 袁肇凯；

【作者基本信息】 湖南中医药大学 ， 中医诊断学， 2010， 博士

【摘要】 目的：研究心血瘀阻证心肌微环境对骨髓间充质干细胞(bone mesenchymal stem cells BMSCs)向心肌样分化的作用及养心通脉有效部位方(active principle region of Yangxin Tongmai Formula apr-YTF)对其影响。方法：(1)理论探讨：通过对“微环境(microenvironment)"概念,发展历史,及当前学术趋势的检索,从理论上探讨微环境的概念、学术发展趋势、心血瘀阻证心肌微环境的变化特点及其对BMSCs移植的影响,并为从改善微环境的角度寻求中医药干预BMSCs向心肌分化提供理论依据。(2)实验研究：①建立大鼠急性心梗心血瘀阻证模型,随机分为A、B两部分,A部分取大鼠心肌组织,用ELISA方法观测其基质金属蛋白酶2(matrix metalloproteinase-2 MMP-2)、基质金属蛋白酶9 (matrix metalloproteinase-9 MMP-9)的表达；用Western-Blots方法检测细胞分化相关信号转导通路PI3K、p38中相关蛋白的变化。B部分动物模型在梗死心肌部位移植BMSCs观测其上述指标；同时用免疫组化双染的方法观测梗死心肌组织中结蛋白Desmin、肌球蛋白重链MHC的表达,用RT-PCR的方法检测心肌转录因子GATA-4 mRNA的表达,然后分析心肌分化指标和MMP-2、MMP-9,信号转导通路PI3K、p38相关蛋白表达的相关性。②建立大鼠急性心梗心血瘀阻证模型,在梗死心肌部位移植BMSCs;并用apr-YTF进行干预。用ELISA方法观测心肌组织中MMP-2、MMP-9的表达；用Western-Blots方法检测心肌组织中细胞分化相关信号转导通路PI3K、p38中相关蛋白的变化。同时用免疫组化双染的方法观测其心肌结蛋白Desmin、肌球蛋白重链MHC的表达,用RT-PCR的方法检测心肌转录因子GATA-4 mRNA的表达,然后分析上述心肌分化指标和MMP-2、MMP-9、信号转导通路PI3K、p38中相关蛋白的相关性。③体外模拟急性心梗心血瘀阻证微环境,在此微环境下培养BMSCs并用apr-YTF进行干预。用ELISA方法观测其培养液中MMP-2、MMP-9的表达；用Western-Blots方法检测细胞分化相关信号转导通路PI3K、p38中相关蛋白的变化。同时用免疫细胞化学方法观测其心肌结蛋白Desmin、肌球蛋白重链MHC的表达,用RT-PCR的方法检测心肌转录因子GATA-4 mRNA的表达,然后分析上述心肌分化指标和MMP-2、MMP-9,信号转导通路PI3K、p38中相关蛋白的相关性。结果：(1)接受BMSCs移植后可见心血瘀阻证心肌梗死边缘区有Desmin-Brdu、MHC-Brdu两种蛋白的同时表达,而在假手术组和健康对照组未见双染细胞表达。GATA-4 mRNA心血瘀阻证组高于假手术组(P＜0.05),而健康对照组的心肌中未检出表达。(2)未作BMSCs移植各部分MMP-2心血瘀阻证表达高于健康对照和假手术组(P＜0.05),接受BMSCs移植后大鼠心肌组织各组间未见统计学差异(P＞0.05)；相同组别内部比较心血瘀阻证组移植BMSCs部分表达低于未作BMSCs移植部分(P＜0.05)。未作BMSCs移植各部分MMP-9三组间均有差异,其中心血瘀阻证组最高,其次为假手术组、健康对照组(P＜0.05),接受BMSCs移植后心血瘀阻证组和假手术组低于健康对照组(P＜0.05),相同组别内部比较心血瘀阻证组移植BMSCs部分表达低于未作BMSCs移植部分(P＜0.05)。未作BMSCs移植各组大鼠心肌组织p-Akt统计学未见明显差异(P＞0.05),接受BMSCs移植心血瘀阻证表达高于健康对照和假手术组(P＜0.05),相同组别内部比较心血瘀阻证组和健康对照组中移植BMSCs部分表达高于未作BMSCs移植部分(P＜0.05)。未作BMSCs移植各组p-p38数据统计学未见明显差异(P>0.05),接受BMSCs移植后各组大鼠心肌组织,各组之间未见差别(P＞0.05),相同组别内部比较心血瘀阻证组中移植BMSCs部分表达高于未作BMSCs移植部分(P＜0.05)。各组标本GATA-4 mRNA与MMP-9、p-p38含量表达呈正相关,而与MMP-2、p-Akt未见显相关性。(3)apr-YTF干预BMSCs移植心血瘀阻证心肌后经心肌结蛋白Desmin-Brdu抗体染色后,其中(rhG-CSF)+BMSCs组和(apr-YTF)+BMSCs组阳性细胞计数均高于NS+BMSCs组(P＜0.05),前两者之间无差别(P＞0.05)。MHC-Brdu抗体染色后,(rhG-CSF)+BMSCs组和(apr-YTF)+BMSCs组阳性细胞计数与高于NS+BMSCs组(P＜0.05),前两者之间无差别(P＞0.05)。GATA-4 mRNA IOD值半定量分析NS+BMSCs组、(rhG-CSF)+BMSCs组和(apr-YTF)+BMSCs组均高于NS+DMEM组,(apr-YTF)+BMSCs组高于NS+BMSCs组(P＜0.05)。(4) apr-YTF干预BMSCs移植心血瘀阻证心肌酶联免疫吸附法检测显示MMP-2 (apr-YTF)+BMSCs组表达低于NS+DMEM组和NS+BMSCs组(P＜0.05和P＜0.01)。MMP-9各组间未见差异(P＞0.05)。经对Western-Blots法检测p-Akt (apr-YTF)+BMSCs组表达高于NS+DMEM组、NS+BMSCs组和(rhG-CSF)+BMSCs组(P＜0.05)。p-p38 (apr-YTF)+BMSCs组表达高于NS+DMEM (P＜0.05),其余各组间未见统计学差异(P＜0.05)。(5)体外模拟心血瘀阻证微环境对BMSCs诱导后,在正常对照组和DMEM组中未发现Desmin及MHC阳性细胞；心血瘀阻证组、apr-YTF组和心血瘀阻证+(apr-YTF)组Desmin及MHC免疫组化染色均可见弱阳性免疫复合物。Desmin的表达的IOD值在在心血瘀阻证组和心血瘀阻证+(apr-YTF)组均高于apr-YTF组(P＜0.05)。MHC的表达的OD值在在心血瘀阻证组和心血瘀阻证+(apr-YTF)组均高于apr-YTF组(P＜0.05),但是前两者之间无统计学差异。心血瘀阻证组和心血瘀阻证+(apr-YTF)组GATA-4 mRNA相对光密度值与正常对照组比较差异具有统计学意义(P＜0.05和P＜0.01)。(6)体外模拟心血瘀阻证微环境细胞培养上清液MMP-2的质量浓度以心血瘀阻证+(apr-YTF)组含量最高,与正常对照组、DMEM组和apr-YTF组比较有统计学差异(P＜0.05)。MMP-9的质量浓度心血瘀阻证组和心血瘀阻证+(apr-YTF)组两个组高于DMEM组和apr-YTF组(P＜0.05)。BMSCs诱导28d后,信号转导通路中p-Akt以心血瘀阻证+(apr-YTF)组含量最高,与DMEM组、心血瘀阻证组和apr-YTF组比较周统计学差异(P＜0.05)。而p-p38各组间含量表达未见统计学差异(P＜0.05)结论：(1)心梗心血瘀阻证心肌微环境可以促进BMSCs向心肌样细胞分化；apr-YTF可以起到促进作用。(2)心梗心血瘀阻证心肌微环境中MMP-2、MMP-9表达增高,促使BMSCs迁移分化。BMSCs移植后心血瘀阻证心肌微环境中MMP-2、MMP-9含量表达降低,从而减轻组织降解,瘢痕形成,控制心梗后向心衰的发展。apr-YTF可以降低心梗心血瘀阻证心肌组织中MMP-2、MMP-9表达。(3)心梗心血瘀阻证心肌微环境中PI3K信号转导通路激活。BMSCs移植后心肌微环境中心肌样细胞分化及与PI3K及p38信号转导通路激活有关。apr-YTF能促使移植的BMSCs向心肌样细胞分化,其分化机制与PI3K及p38信号转导通路激活有关。(4)体外模拟的心血瘀阻证微环境可以诱导BMSCs向心肌样细胞分化。apr-YTF在体外可以诱导BMSCs向心肌样细胞分化。(5)体外模拟的心血瘀阻证微环境MMP-2、MMP-9可以促使BMSCs向心肌样细胞分化。在体外模拟的心血瘀阻证微环境中BMSCs向心肌样细胞分化及apr-YTF的干预作用与PI3K信号转导通路激活有关。更多还原

【Abstract】 Objective:To study the function about cardiac blood stasis syndrome cardiac microenvironment towards BMSCs cardiac differentiation as well as the influences about the active principle region of Yangxin Tongmai Formula(apr-YTF).Methods:(1)Theoretical discussion:Through revieve the concept, development history and current academic tendency of microenvironment, to explore its exact concept academic development tendency and change characteristics of cardiac blood stasis syndrome and its influences on the bone marrow mesenchyma stem cell(BMSCs) transplant. To provide theoretical basis for TCM interferences on BMSCs transplant into cardiac differentiation.(2)Experimental research:①Established the model of rats acute myocardial infarction (AMI) cardiac blood stasis syndrome, use ELISA method to observe the expression about matalloprotease-2 (MMP-2) and matalloprotease-9 (MMP-9);use Western-Blots method examine the change about the protein of cellular differentiation signal transduction path PI3K and p38, meanwhile use the immunohistochemistry double-dyed method to measure the Desmin and MHC;Use RT-PCR method to observe the expression about GATA-4 mRNA, and analyze the index of cellular differentiation as well as the MMP-2 and MMP-9 and associativity of signal transduction PI3K、p38.②Established the model of rats AMI cardiac blood stasis syndrome and transplanted BMSCs in infarctus cardiac muscule, use ELISA method to observe the expression about MMP-2 and MMP-9, use Western-Blots method examine the change about the protein of cellular differentiation signal transduction path PI3K and p38;meanwhile use the immunohistochemistry double-dyed method to measure the Desmin and MHC, use RT-PCR method to observe the expression about GATA-4 mRNA, and analyze the associativity between the index of cellular differentiation as well as the MMP-2 and MMP-9 and content of signal transduction PI3K、p38.③Simulated the environment about AMI cardiac blood stasis syndrome in vitro, to cultivate the BMSCs and interfere by apr-YTF, use ELISA method to observe the expression about MMP-2 and MMP-9, use Western-Blots method examine the change about the protein of cellular differentiation signal transduction path PI3K、p38, meanwhile use the immunohistochemistry double-dyed method to measure the Desmin and MHC;Use RT-PCR method to observe the expression about GATA-4 mRNA, and analyze the ssociativity between the index of cellular differentiation as well as the MMP-2 and MMP-9 and content of signal transduction PI3K、p38.Results:(1)After BMSCs transplant there was simultaneous expression of Desmin-Brdu、MHC-Brdu in the cardiac blood stasis infarctus edge area. There was no expression in the sham operation group and health control group. GATA-4 mRNA in the cardiac blood stasis group was higher than that in the sham operation group(P＜0.05),while there was no expression in the health control group.(2)Every part of no BMSCs transplant of MMP-2 cardiac blood stasis syndrome was higher that of sham operation group and health control group. After BMSCs transplant there was statistical difference of rats cardiac tissues between each groups (P＜0.05). The transplant group’s expression was higher than that no BMSCs transplant of cardiac blood stasis in the same group(P＜0.05). Every part of no BMSCs transplant of MMP-9 had statistical differences between each group. Cardiac blood stasis syndrome group was the highest, sham operation group was the next one and health control group was the last one. After BMSCs transplant, the cardiac blood stasis syndrome group was lower than that of health control group and sham operation group. The transplant group’s expression was higher than that no BMSCs transplant of cardiac blood stasis in the same group(P＜0.05). Every part of no BMSCs transplant of p-Akt had no statistical differences between each group.After BMSCs transplant, the cardiac blood stasis syndrome group was lower than that of health control group and sham operation group, the expression of transplant group’s was higher than that no BMSCs transplant of cardiac blood stasis in the same group(P＜0.05).Every part of no BMSCs transplant of p-p38 had no statistical differences between each group. There was no difference between each group’s rats cardiac tissues after BMSCs transplant. The transplant group’s expression was higher than that no BMSCs transplant of cardiac blood stasis in the same group(P＜0.05). The expression of GATA-4 mRNA,MMP-9 and p-p38 was positive correlation. The incidence coefficient were 0.49and 0.605 while there was no obvious associativity of MMP-2、p-Akt.(3)Apr-YTF interfered in BMSCs transplant of cardiac blood stasis of Desmin-Brdu, the (rhG-CSF)+BMSCs group’s positive cell counting were higher than that of NS+BMSCs group. There was no difference between (rhG-CSF)+BMSCs group and NS+BMSCs group. Through MHC-Brdu antibody dyeing, (rhG-CSF)+BMSCs and (apr-YTF)+BMSCs group’s positive cell counting were higher than that of NS+BMSCs group. The semi-quantitative IOD of GATA-4 mRNA, NS+BMSCs group as well as (rhG-CSF)+BMSCs and (apr-YTF)+BMSCs group were higher than that of NS+DMEM group;while the (apr-YTF)+BMSCs group was higher than that of NS+BMSCs.(4)Apr-YTF interfered in BMSCs transplant of cardiac blood stasis of MMP-2 and MMP-9, ELISA test showed that the MMP-2 (apr-YTF)+BMSCs group’s expression was lower than that of NS+BMSCs group and NS+BMSCs. There was no difference between each group of MMP-9. Through p-Akt Western-Blot, (apr-YTF)+BMSCs’s expression was higher than that of in NS+DMEM group NS+BMSCs group as well as (rhG-CSF)+BMSCs group. The p-p38 (apr-YTF)+BMSCs group’s expression was higher than that of NS+DMEM group. There was no difference between each other group.(5)After the induction of BMSCs simulated the environment about AMI cardiac blood stasis syndrome in vitro, there was no MHC positive cell been detected in the normal control group and DMEM group, while the weakly positive immunocomplex had been detected in the cardiac blood stasis syndrome group, apr-YTF group, cardiac blood stasis syndrome+apr-YTF in the Desmin and MHC immunohistochemistry dyeing. The IOD of Desmin expressionin the cardiac blood stasis syndrome group and cardiac blood stasis syndrome+(apr-YTF)group were higher than in the apr-YTF group, but there was no statistical difference between the former two ones。The GATA-4 mRNA IOD in cardiac blood stasis syndrome group and cardiac blood stasis syndrome+(apr-YTF)group had statistical differences compared with the normal control group.(6)The MMP-2’s mass concentration in the cell culture of simulated the environment of cardiac blood stasis syndrome+apr-YTF group was the highest which had the statistical difference compared with the normal control group DMEM and apr-YTF group. The mass concentration of MMP-9 in the two cardiac blood stasis syndrome group, and cardiac blood stasis syndrome+apr-YTF group were higher than in the DMEM and apr-YTF group.After 28 days of BMSCs’ induction, the p-Akt in the signal transduction path was much higher in the cardiac blood stasis syndrome+apr-YTF group than that in the DMEM, apr-YTF and cardiac blood stasis syndrome group. The p-p38’s expression in each group had no statistical difference.Conclusion:(1)The microenvironment of myocardial infarction cardiac blood stasis syndrome’s cardiac cell can promote the BMSCs cardiac cell cytodifferentiation. Apr-YTF can promote it.(2)The microenvironment of myocardial infarction cardiac blood stasis syndrome’s cardiac cell can increase the expression of MMP-2 and MMP-9, promote the BMSCs migration and differentiation. After BMSCs transplant cardiac blood stasis syndrome’s cardiac cell microenvironment’s content expression lower and thus mitigate the tissure degradation cicatrization and control the deterioration of MI. Apr-YTF can lower the MMP’s expression of AMI cardiac blood stasis syndrome in cardian tissure. (3)PI3K signal transduction path in cardiac blood stasis syndrome’s cardiac cell microenvironment has been activated. After BMSCs transplant cardiac blood stasis syndrome’s cardiac cell microenvironment’s cardiac cell differentiation related to the activation of PI3K and p38 signal transduction. Apr-YTF can promote the BMSCs transplant into cardiac cell differentiation and the activation of PI3K and p38 signal transduction.(4)Simulated the microenvironment about cardiac blood stasis syndrome in vitro can induce the BMSCs cardiac cell differentiation. Apr-YTF can induce the BMSCs cardiac cell differentiation in vitro.(5)Simulated the microenvironment about cardiac blood stasis syndrome in vitro, MMP-2 and MMP-9 can promote the BMSCs cardiac cell differentiation. BMSCs differentiation to cardiac cell and apr-YTF’s interferences has related to the activation of PI3K signal transduction path.更多还原