鹿茸归经靶向调控骨关节炎TGF-β/Smads信号通路机制研究
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摘要
目的：
“归经”理论是中医药学理论体系中的重要组成部分,也是我们认识疾病和临证运药的基础,在“归经”理论的指导下,目前治疗骨关节炎多运用归肝、肾经的药物组方,为了进一步研究“归经”的本质,及“归经”理论与现代生物分子信号通路的相关性,选取归肝、肾经的代表中药“鹿茸”,对其“归经”本质进行相关研究,并探讨该药调控骨关节炎软骨中TGF-β/Smads信号通路的机制。
方法：
1文献研究
借助“电子版《中华医典》数据库”,分别以骨痹、膝痛、膝痹、痛痹、鹤膝风、关节疼痛、屈伸不利等为关键词检索,并用Excel表格建立数据库,用SPSS18.0软件进行统计,通过对方剂中药物的功效和归经进行频数分析,从而分析不同种类药物的运用情况,同时,采用系统聚类分析对药物进行样本聚类统计,并运用中医药理论对结果进行分析。
2归经研究
采用Hulth方法建立大鼠骨关节炎模型,随机分为鹿茸组、模型组和正常组,采用鹿茸水溶液进行灌胃,基于环核苷酸观测和受体观测理论,采用ELISA法检测不同组软骨中cAMP、cGMP含量,荧光定量PCR和蛋白免疫印迹杂交方法检测大鼠软骨TGF-βI型、TGF-βII型受体基因和蛋白的表达。
3软骨组织总RNA提取研究
选取3月龄SD大鼠9只,采用Rneasy Lipid Tissue Kit、RNAout试剂盒和经典Trizol三种方法对大鼠软骨组织进行总RNA提取,通过RNA琼脂糖凝胶电泳检测RNA的完整性,并进行普通PCR扩增,以研究软骨总RNA最佳提取方案。4鹿茸靶向调控TGF-β/Smads信号通路的研究
选取3月龄SPF级健康雌性SD大鼠100只,按体重分层随机选取20只作为正常组,余者行Hulth方法造模,病理组织切片验证造模成功后,按体重分层随机数字表法分成4组：鹿茸低剂量组、鹿茸高剂量组、盐水组、模型组,于灌胃后2周、4周、6周,分别取各组大鼠膝关节软骨,采用免疫组织化学法检测软骨II型胶原的表达,荧光定量PCR和蛋白免疫印迹杂交方法分别检钡TGF-β1、Smad2、 Smad3、 Smad4基因和蛋白的表达。
结果：
1药物功效使用频率分析结果显示,补虚药、解表药、活血化瘀药、祛风湿药、温里药和祛湿药的总使用频率达到79.2%,是构成治疗骨痹的常用药物；归经药物使用频率分析结果显示,入肾经的药物最多,频率为21.4%,其次是归肝经和脾经的药物分别占17.9%；根据高频数使用药物的样本聚类分析结果,并结合其功效,可以将这些药物聚为6类,多以滋补肝肾、活血通络、温里散寒等为法进行药物组合。
2ELISA法检测软骨组织中cAMP、cGMP结果显示,鹿茸组cAMP和cGMP值均高于模型组,模型组cAMP和cGMP值低于正常组,三组cAMP (F=10351.408, P=0.000)、cGMP (F=63032.016,P=0.000)、cAMP/cGMP (F=6530.879,P=O.000)之间比较均有显著性差异(P<0.01)；荧光定量-PCR和蛋白免疫印迹杂交方法检测各组大鼠软骨TGF-βI型、TGF-βII型受体表达结果显示,鹿茸低剂量、高剂量组在灌胃后随时间的延长,TGF-β RImRNA, TGF-β RIImRNA和蛋白的表达量逐步增加,经重复测量方差分析,三个时间段间总体差异有显著性意义(F=161.672,P=0.000)。
3Rneasy Lipid Tissue Kit法获得的总RNA纯度高、完整性和稳定性好,能成功逆转录合成双链cDNA,普通PCR扩增能够得到我们所需要的基因产物,而RNAout试剂盒和经典Trizol法获得的总RNA纯度低,不能满足后续PCR实验需要。
4软骨组织病理学观察显示,模型组2周时,光镜下可见表层软骨细胞肿胀变圆,扁平的软骨细胞减少,软骨细胞簇集,4周时,表层及中层细胞收缩,细胞数量弥漫性增多,出现大量簇集细胞团,6周后,大部分软骨的表层细胞消失,软骨表面出现溃烂缺损,软骨细胞数量减少,可见坏死崩解细胞,潮线紊乱甚至消失；灌胃6周后,软骨切片Mankin's评分结果显示,鹿茸高剂量、低剂量组Mankin's得分小于模型组和盐水组,单因素方差分析显示,组间比较差异显著,有统计学意义(F=87.840,P=0.000)；免疫组织化学检测软骨II型胶原蛋白表达,可见鹿茸低剂量和高剂量组的软骨层有广泛的棕褐色细胞,数量明显高于盐水组和模型组；荧光定量PCR和蛋白免疫印迹杂交方法分别检测TGF-β1、Smad2、 Smad3、Smad4表达结果显示,TGF-β1mRNA和蛋白表达量随灌胃时间延长逐步增加,且表达量均高于其它组,经重复测量方差分析,三个时间段间总体差异有显著性意义(F=408.841, P=0.000), Smad2、Smad3mRNA和蛋白检测可见,鹿茸低剂量、高剂量组在灌胃后2、4周时,关节软骨组织中的Smad2.Smad3mRNA和蛋白的表达增高,6周时,其表达量没有继续增高反而降低,但与其他组比较,经重复测量方差分析,三个时间段间总体差异有显著性意义(F=240.511,P=0.000, F=1325.174, P=0.000), Smad4mRNA和蛋白的表达随灌胃时间的延长逐步增加,经重复测量方差分析,三个时间段间总体差异有显著性意义(F=197.035,P=0.000)。
结论：
1古人在临证运药治疗“骨痹”时,始终以肝肾亏虚为“骨痹”的发病根本原则为指导,多选用归肝、肾、脾经的药物,以滋阴补肾、活血化瘀、温里除湿、补虚活络等为法进行药物组合,在治疗“肝肾亏虚”之本时,兼以活血、温里、通络、祛湿以治其标。
2实验结果证实鹿茸能够影响软骨内环核苷酸的变化,而且鹿茸能够上调软骨细胞膜上TGF-βRI、TGF-RII受体的表达,由此推断软骨可能是鹿茸归经选择性作用的靶组织之一,并且软骨细胞膜上的TGF-β I型、TGF-βII受体可能是鹿茸归经选择性作用于软骨的靶点之一。
3基于软骨组织的特异性,Trizol法不适合用于大鼠关节软骨组织总RNA的提取,而采用RNeasy Lipid Tissue Mini Kit法提取的软骨总RNA纯度较高,能够保证后续实验的实施。
4鹿茸能够调控软骨组织中TGF-β1Smad2、 Smad3、 Smad4基因和蛋白的表达,而且软骨病理切片Mankin's评分和软骨II型胶原表达检测提示,鹿茸能够促进软骨细胞增殖、分化,延缓软骨的退变,由此推论,鹿茸提高了TGF-β/Smads信号通路中TGF-β1、Smad2、Smad3、Smad4分子的表达,从而促进该信号通路的转导,起到促进软骨形成的作用,而且,软骨细胞内的Smad2、Smad3、 Smad4基因和蛋白的变化可能是骨性关节炎发病的重要机制之一。
Objective:
The Chinese medicinal'channel tropism'theory is an important part of the theories of TCM, and is the basis of disease which we know and treat. Under the guidance of the'channel tropism'theory, we formulate prescription to treat osteoarthritis by applying to liver and kidney. In order to further research'channel tropism'nature, and the relevance of'channel tropism'theory and signaling pathways. We select pilos antler of applying to liver and kidney to research its nature as the representative of Chinese traditional medicine, to explore the mechanism of pilos antler regulate TGF beta/Smads signal pathway in the osteoarthritic cartilage.
Methods:
1Literature resaerch
By means of'The Chinese medical database',Some medicine cases about Arthralgia Syndrome will be analyzed and imported into database, and using of herbs will be analyzed through mathematics and physics Statistieal methods of SPSS18.0saftware. We will concluded the regulation of different kinds of drugs by analysis frequency of efficacy and'channel tropism'of drugs. At the same time, the system clustering analysis is adopted to clustering statistics on drugs, and using traditional Chinese medicine theory to analyze the results.
2Channel tropism research
Hulth method is adopted to establish the rat model of osteoarthritis. Twenty four healthy female SD rats were selected and randomly divided into3groups:group of the pilose antler, model group and control group. Animals in each of group after treatment, the expressions of cAMP, cGMP, TGF-βRI, TGF-β RII were measured by ELISA, Quantitative PCR, Western blot to observe the curative effects.
3Explore the different methods to extract total RNA from cartilage tissue of SD rats
Select9SD rats of3-month-old, respectively Rneasy Lipid Tissue Kit, RNAout kit and classic Trizol method of total RNA was extracted, and Agarose gel electrophoresis was included to detect RNA integrity and quality. In order to explore best extraction scheme of total RNA.
4Research mechanism of Hairy Deerhorn regulate TGF-beta/Smad signalling pathways
One hundred healthy female SD rats of3months old were selected and subjected to classic Hulth modeling. Knees were harvested to observe the pathologic change of cartilage macroscopically and microscopically, and randomly were divided into5groups:low-dose group of the pilose antler, high-dose group of the pilose antler, saline control group, model group and control group. Animals in each of group at2,4,6weeks after treatment, the expressions of collagen type II, TGF-β1, Smad2, Smad3, Smad4were measured by Immunohistochemistry, Quantitative PCR, Western blot to observe the curative effects.
Rusult:
1According to the results of frequency analysis, Chinese herb reaches79.2%which are for restoratives Chinese Medicinal Herbs. Diaphoretics or exterior syndrome relieving Chinese Medicinal Herbs, Chinese Medicinal Herbs for invigorating the blood and removing blood stasis, Antirheumatic Chinese Medicinal Herbs and Warming the internal are the main treatment drugs for osteoarthritis. Frequency of kidney is21.4%which is more than others, Frequency of liver and spleen is17.9%which account for second. According to the results of hierarchical cluster analysis, the herbs for treatment could be divided into six cluster,with nourishing liver and kidney, promoting blood circulation to remove meridian obstruction, temperature in the cold,etc as a method for drug combination.
2According to the results ELISA method to detect cAMP and cGMP in cartilage tissue, group of Velvet antler of cAMP and cGMP were higher than model group,model group of cAMP and cGMP were lower than normal group. Comparison of three groups had significant difference(cAMP F=10351.408, P=0.000, cGMP F=63032.016, P=0.000, cAMP/cGMP F=6530.879, P=0.000). The expression of TGF-β RImRNA, TGF-β RIImRNA and proteins were increased gradually in low-dose group and high dose group by quantitative PCR and Western blot hybridization assay. By repeated measures analysis of variance, the difference is significant compare to other groups (F=161.672, P=0.000)
3Total RNA of Rneasy Lipid Tissue Kit was obtained high purity, integrity and stability, and can successfully reverse transcriptase synthesize double-stranded cDNA. PCR amplification can get gene product what we need. Methods of RNAout kit and classic Trizol can not meet the need.
4We prepared successfully osteoarthritis model in the athology. After two week, the surface of chondrocyte were swelling, the flat chondrocyte reduced and clusted through optical microscope. After four week, the surface and middle cell were shrinkage, diffuse, cluster to group. After six week, the most of the surface layer of chondrocyte were disappear, decay defects, chondrocytes disintegration, necrosis, and tidal line disorder or even disappear. According to the results of Mankin's score of cartilage slice, Mankin's score of low-dose and high-dose group of the pilose antler was less than the model group and saline group, the difference is significant compare to other groups(P=87.840, P=0.000). The expression of collagen type II by detection of immunohistochemical, chondrocyte layer of low-dose and high-dose group of the pilose antler had a wide range of tan cells, the number was significantly higher than that of saline group and model group, the expression by quantitative PCR and Western blot hybridization assay of TGF-β1was increased gradually in low-dose group and high dose group, the difference is significant compare to other groups by repeated measures analysis of
variance(F=408.841,P=0.000).Smad2,Smad3gene and proteins were increased gradually after2,4weeks, and reduced after6weeks, the difference is significant compare to other groups by repeated measures analysis of variance(F=240.511, P=0.000,F=1325.174, P=0.000). The expression of Smad4was increased gradually in low-dose group and high dose group, the difference is significant compare to other groups by repeated measures analysis of variance (F=197.035, P=0.000).
Conclusion:
1The ancients treat'GUBI'under the onset of liver and kidney deficiency as the fundamental principle in clinical, they choose drugs to liver, kidney, spleen. Enriching yin and nourishing kidney, promoting blood circulation to remove blood stasis, etc. is a method for drug combination. In the treatment of liver and kidney deficiency, invigorate the circulation, temperature, Tongluo, dampness to treat the symptoms.
2Experimental results demonstrate that pilos antler can influence the changes of cAMP and cGMP, pilos antler can increase the expression of TGF-beta RI,TGF-beta RII on the chondrocyte membrane,so we consider the cartilage may be one of the target tissue which pilos antler select, and TGF-betaRI, TGF-RII of chondrocyte membrane may be one of the targets which pilos antler via selective effect on cartilage.
3Based on the specificity of the cartilage, we think Trizol method is not suitable for total RNA extraction of articular cartilage tissue in rat. While using RNeasy Lipid Tissue Mini Kit can guarantee the implementation of the follow-up study.
4Pilos antler can influence the expression of TGF-betal, Smad2,3,4genes and proteins in cartilage, and promote chondrocyte proliferation, differentiation, delay the degeneration of cartilage. So we can concluded that pilos antler can regulate the expression of difference molecularof TGF-beta/Smad signaling pathways, thus promotes the signal transduction pathways, play the role of promoting chondrogenesis.And the changes of Smad2,3,4may is one of the important mechanisms of the pathogenesis of osteoarthritis.
“归经”理论是中医药学理论体系中的重要组成部分,也是我们认识疾病和临证运药的基础,在“归经”理论的指导下,目前治疗骨关节炎多运用归肝、肾经的药物组方,为了进一步研究“归经”的本质,及“归经”理论与现代生物分子信号通路的相关性,选取归肝、肾经的代表中药“鹿茸”,对其“归经”本质进行相关研究,并探讨该药调控骨关节炎软骨中TGF-β/Smads信号通路的机制。
方法：
1文献研究
借助“电子版《中华医典》数据库”,分别以骨痹、膝痛、膝痹、痛痹、鹤膝风、关节疼痛、屈伸不利等为关键词检索,并用Excel表格建立数据库,用SPSS18.0软件进行统计,通过对方剂中药物的功效和归经进行频数分析,从而分析不同种类药物的运用情况,同时,采用系统聚类分析对药物进行样本聚类统计,并运用中医药理论对结果进行分析。
2归经研究
采用Hulth方法建立大鼠骨关节炎模型,随机分为鹿茸组、模型组和正常组,采用鹿茸水溶液进行灌胃,基于环核苷酸观测和受体观测理论,采用ELISA法检测不同组软骨中cAMP、cGMP含量,荧光定量PCR和蛋白免疫印迹杂交方法检测大鼠软骨TGF-βI型、TGF-βII型受体基因和蛋白的表达。
3软骨组织总RNA提取研究
选取3月龄SD大鼠9只,采用Rneasy Lipid Tissue Kit、RNAout试剂盒和经典Trizol三种方法对大鼠软骨组织进行总RNA提取,通过RNA琼脂糖凝胶电泳检测RNA的完整性,并进行普通PCR扩增,以研究软骨总RNA最佳提取方案。4鹿茸靶向调控TGF-β/Smads信号通路的研究
选取3月龄SPF级健康雌性SD大鼠100只,按体重分层随机选取20只作为正常组,余者行Hulth方法造模,病理组织切片验证造模成功后,按体重分层随机数字表法分成4组：鹿茸低剂量组、鹿茸高剂量组、盐水组、模型组,于灌胃后2周、4周、6周,分别取各组大鼠膝关节软骨,采用免疫组织化学法检测软骨II型胶原的表达,荧光定量PCR和蛋白免疫印迹杂交方法分别检钡TGF-β1、Smad2、 Smad3、 Smad4基因和蛋白的表达。
结果：
1药物功效使用频率分析结果显示,补虚药、解表药、活血化瘀药、祛风湿药、温里药和祛湿药的总使用频率达到79.2%,是构成治疗骨痹的常用药物；归经药物使用频率分析结果显示,入肾经的药物最多,频率为21.4%,其次是归肝经和脾经的药物分别占17.9%；根据高频数使用药物的样本聚类分析结果,并结合其功效,可以将这些药物聚为6类,多以滋补肝肾、活血通络、温里散寒等为法进行药物组合。
2ELISA法检测软骨组织中cAMP、cGMP结果显示,鹿茸组cAMP和cGMP值均高于模型组,模型组cAMP和cGMP值低于正常组,三组cAMP (F=10351.408, P=0.000)、cGMP (F=63032.016,P=0.000)、cAMP/cGMP (F=6530.879,P=O.000)之间比较均有显著性差异(P<0.01)；荧光定量-PCR和蛋白免疫印迹杂交方法检测各组大鼠软骨TGF-βI型、TGF-βII型受体表达结果显示,鹿茸低剂量、高剂量组在灌胃后随时间的延长,TGF-β RImRNA, TGF-β RIImRNA和蛋白的表达量逐步增加,经重复测量方差分析,三个时间段间总体差异有显著性意义(F=161.672,P=0.000)。
3Rneasy Lipid Tissue Kit法获得的总RNA纯度高、完整性和稳定性好,能成功逆转录合成双链cDNA,普通PCR扩增能够得到我们所需要的基因产物,而RNAout试剂盒和经典Trizol法获得的总RNA纯度低,不能满足后续PCR实验需要。
4软骨组织病理学观察显示,模型组2周时,光镜下可见表层软骨细胞肿胀变圆,扁平的软骨细胞减少,软骨细胞簇集,4周时,表层及中层细胞收缩,细胞数量弥漫性增多,出现大量簇集细胞团,6周后,大部分软骨的表层细胞消失,软骨表面出现溃烂缺损,软骨细胞数量减少,可见坏死崩解细胞,潮线紊乱甚至消失；灌胃6周后,软骨切片Mankin's评分结果显示,鹿茸高剂量、低剂量组Mankin's得分小于模型组和盐水组,单因素方差分析显示,组间比较差异显著,有统计学意义(F=87.840,P=0.000)；免疫组织化学检测软骨II型胶原蛋白表达,可见鹿茸低剂量和高剂量组的软骨层有广泛的棕褐色细胞,数量明显高于盐水组和模型组；荧光定量PCR和蛋白免疫印迹杂交方法分别检测TGF-β1、Smad2、 Smad3、Smad4表达结果显示,TGF-β1mRNA和蛋白表达量随灌胃时间延长逐步增加,且表达量均高于其它组,经重复测量方差分析,三个时间段间总体差异有显著性意义(F=408.841, P=0.000), Smad2、Smad3mRNA和蛋白检测可见,鹿茸低剂量、高剂量组在灌胃后2、4周时,关节软骨组织中的Smad2.Smad3mRNA和蛋白的表达增高,6周时,其表达量没有继续增高反而降低,但与其他组比较,经重复测量方差分析,三个时间段间总体差异有显著性意义(F=240.511,P=0.000, F=1325.174, P=0.000), Smad4mRNA和蛋白的表达随灌胃时间的延长逐步增加,经重复测量方差分析,三个时间段间总体差异有显著性意义(F=197.035,P=0.000)。
结论：
1古人在临证运药治疗“骨痹”时,始终以肝肾亏虚为“骨痹”的发病根本原则为指导,多选用归肝、肾、脾经的药物,以滋阴补肾、活血化瘀、温里除湿、补虚活络等为法进行药物组合,在治疗“肝肾亏虚”之本时,兼以活血、温里、通络、祛湿以治其标。
2实验结果证实鹿茸能够影响软骨内环核苷酸的变化,而且鹿茸能够上调软骨细胞膜上TGF-βRI、TGF-RII受体的表达,由此推断软骨可能是鹿茸归经选择性作用的靶组织之一,并且软骨细胞膜上的TGF-β I型、TGF-βII受体可能是鹿茸归经选择性作用于软骨的靶点之一。
3基于软骨组织的特异性,Trizol法不适合用于大鼠关节软骨组织总RNA的提取,而采用RNeasy Lipid Tissue Mini Kit法提取的软骨总RNA纯度较高,能够保证后续实验的实施。
4鹿茸能够调控软骨组织中TGF-β1Smad2、 Smad3、 Smad4基因和蛋白的表达,而且软骨病理切片Mankin's评分和软骨II型胶原表达检测提示,鹿茸能够促进软骨细胞增殖、分化,延缓软骨的退变,由此推论,鹿茸提高了TGF-β/Smads信号通路中TGF-β1、Smad2、Smad3、Smad4分子的表达,从而促进该信号通路的转导,起到促进软骨形成的作用,而且,软骨细胞内的Smad2、Smad3、 Smad4基因和蛋白的变化可能是骨性关节炎发病的重要机制之一。
Objective:
The Chinese medicinal'channel tropism'theory is an important part of the theories of TCM, and is the basis of disease which we know and treat. Under the guidance of the'channel tropism'theory, we formulate prescription to treat osteoarthritis by applying to liver and kidney. In order to further research'channel tropism'nature, and the relevance of'channel tropism'theory and signaling pathways. We select pilos antler of applying to liver and kidney to research its nature as the representative of Chinese traditional medicine, to explore the mechanism of pilos antler regulate TGF beta/Smads signal pathway in the osteoarthritic cartilage.
Methods:
1Literature resaerch
By means of'The Chinese medical database',Some medicine cases about Arthralgia Syndrome will be analyzed and imported into database, and using of herbs will be analyzed through mathematics and physics Statistieal methods of SPSS18.0saftware. We will concluded the regulation of different kinds of drugs by analysis frequency of efficacy and'channel tropism'of drugs. At the same time, the system clustering analysis is adopted to clustering statistics on drugs, and using traditional Chinese medicine theory to analyze the results.
2Channel tropism research
Hulth method is adopted to establish the rat model of osteoarthritis. Twenty four healthy female SD rats were selected and randomly divided into3groups:group of the pilose antler, model group and control group. Animals in each of group after treatment, the expressions of cAMP, cGMP, TGF-βRI, TGF-β RII were measured by ELISA, Quantitative PCR, Western blot to observe the curative effects.
3Explore the different methods to extract total RNA from cartilage tissue of SD rats
Select9SD rats of3-month-old, respectively Rneasy Lipid Tissue Kit, RNAout kit and classic Trizol method of total RNA was extracted, and Agarose gel electrophoresis was included to detect RNA integrity and quality. In order to explore best extraction scheme of total RNA.
4Research mechanism of Hairy Deerhorn regulate TGF-beta/Smad signalling pathways
One hundred healthy female SD rats of3months old were selected and subjected to classic Hulth modeling. Knees were harvested to observe the pathologic change of cartilage macroscopically and microscopically, and randomly were divided into5groups:low-dose group of the pilose antler, high-dose group of the pilose antler, saline control group, model group and control group. Animals in each of group at2,4,6weeks after treatment, the expressions of collagen type II, TGF-β1, Smad2, Smad3, Smad4were measured by Immunohistochemistry, Quantitative PCR, Western blot to observe the curative effects.
Rusult:
1According to the results of frequency analysis, Chinese herb reaches79.2%which are for restoratives Chinese Medicinal Herbs. Diaphoretics or exterior syndrome relieving Chinese Medicinal Herbs, Chinese Medicinal Herbs for invigorating the blood and removing blood stasis, Antirheumatic Chinese Medicinal Herbs and Warming the internal are the main treatment drugs for osteoarthritis. Frequency of kidney is21.4%which is more than others, Frequency of liver and spleen is17.9%which account for second. According to the results of hierarchical cluster analysis, the herbs for treatment could be divided into six cluster,with nourishing liver and kidney, promoting blood circulation to remove meridian obstruction, temperature in the cold,etc as a method for drug combination.
2According to the results ELISA method to detect cAMP and cGMP in cartilage tissue, group of Velvet antler of cAMP and cGMP were higher than model group,model group of cAMP and cGMP were lower than normal group. Comparison of three groups had significant difference(cAMP F=10351.408, P=0.000, cGMP F=63032.016, P=0.000, cAMP/cGMP F=6530.879, P=0.000). The expression of TGF-β RImRNA, TGF-β RIImRNA and proteins were increased gradually in low-dose group and high dose group by quantitative PCR and Western blot hybridization assay. By repeated measures analysis of variance, the difference is significant compare to other groups (F=161.672, P=0.000)
3Total RNA of Rneasy Lipid Tissue Kit was obtained high purity, integrity and stability, and can successfully reverse transcriptase synthesize double-stranded cDNA. PCR amplification can get gene product what we need. Methods of RNAout kit and classic Trizol can not meet the need.
4We prepared successfully osteoarthritis model in the athology. After two week, the surface of chondrocyte were swelling, the flat chondrocyte reduced and clusted through optical microscope. After four week, the surface and middle cell were shrinkage, diffuse, cluster to group. After six week, the most of the surface layer of chondrocyte were disappear, decay defects, chondrocytes disintegration, necrosis, and tidal line disorder or even disappear. According to the results of Mankin's score of cartilage slice, Mankin's score of low-dose and high-dose group of the pilose antler was less than the model group and saline group, the difference is significant compare to other groups(P=87.840, P=0.000). The expression of collagen type II by detection of immunohistochemical, chondrocyte layer of low-dose and high-dose group of the pilose antler had a wide range of tan cells, the number was significantly higher than that of saline group and model group, the expression by quantitative PCR and Western blot hybridization assay of TGF-β1was increased gradually in low-dose group and high dose group, the difference is significant compare to other groups by repeated measures analysis of
variance(F=408.841,P=0.000).Smad2,Smad3gene and proteins were increased gradually after2,4weeks, and reduced after6weeks, the difference is significant compare to other groups by repeated measures analysis of variance(F=240.511, P=0.000,F=1325.174, P=0.000). The expression of Smad4was increased gradually in low-dose group and high dose group, the difference is significant compare to other groups by repeated measures analysis of variance (F=197.035, P=0.000).
Conclusion:
1The ancients treat'GUBI'under the onset of liver and kidney deficiency as the fundamental principle in clinical, they choose drugs to liver, kidney, spleen. Enriching yin and nourishing kidney, promoting blood circulation to remove blood stasis, etc. is a method for drug combination. In the treatment of liver and kidney deficiency, invigorate the circulation, temperature, Tongluo, dampness to treat the symptoms.
2Experimental results demonstrate that pilos antler can influence the changes of cAMP and cGMP, pilos antler can increase the expression of TGF-beta RI,TGF-beta RII on the chondrocyte membrane,so we consider the cartilage may be one of the target tissue which pilos antler select, and TGF-betaRI, TGF-RII of chondrocyte membrane may be one of the targets which pilos antler via selective effect on cartilage.
3Based on the specificity of the cartilage, we think Trizol method is not suitable for total RNA extraction of articular cartilage tissue in rat. While using RNeasy Lipid Tissue Mini Kit can guarantee the implementation of the follow-up study.
4Pilos antler can influence the expression of TGF-betal, Smad2,3,4genes and proteins in cartilage, and promote chondrocyte proliferation, differentiation, delay the degeneration of cartilage. So we can concluded that pilos antler can regulate the expression of difference molecularof TGF-beta/Smad signaling pathways, thus promotes the signal transduction pathways, play the role of promoting chondrogenesis.And the changes of Smad2,3,4may is one of the important mechanisms of the pathogenesis of osteoarthritis.
引文
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