羟基红花黄色素A促进激素诱导兔骨髓基质细胞成骨分化及其MAPK信号转导机制的研究

发布时间：2018-04-29 01:28

本文选题：股骨头缺血坏死 + 羟基红花黄色素A　；参考：《福建中医药大学》2014年博士论文

【摘要】：目的探讨羟基红花黄色素A(HSYA)促进激素诱导的股骨头缺血坏死模兔股骨头髓内及离体兔骨髓基质细胞(BMSCs)成骨分化及其MAPK信号转导通路的作用机制。方法 1.健康成年新西兰大白兔48只,抽签法随机分为空白组、模型组,模型组采用臀肌注射醋酸泼尼松龙(7.5mg/kg)结合强迫立位制备激素性股骨头缺血坏死模型。造模成功后抽签法将模型组兔随机分为模型对照组、髓芯减压组、生理盐水+髓芯减压组、HSYA+髓芯减压组,髓芯减压组仅行髓芯减压术,生理盐水+髓芯减压组采用髓芯减压术配合髓腔内注射生理盐水,HSYA+髓芯减压组采用髓芯减压术配合髓腔内注射HSYA,空白组和模型对照组不作治疗。1周后兔麻醉,腹主动脉采血,采用化学比色法、氧化酶法检测各组兔血清中碱性磷酸酶、甘油三脂的含量；取出股骨头,光学显微镜、透射电子显微镜观察各组兔股骨头的形态结构；RT-PCR检测各组兔股骨头中Cbfal及Collagen-I mRNA的表达；Western Blotting检测各组兔股骨头中p-ERK. p-JNK和p-P38蛋白的表达。 2.健康成年新西兰大白兔36只,无菌条件下于胫骨上端内侧骨髓腔穿刺抽取骨髓血,分离、体外培养BMSCs,流式细胞仪检测细胞表面标记物结合成骨、成脂诱导剂诱导对其鉴定。取第3代生长良好的BMSCs,抽签法随机分为空白组,模型组及HSYA低、中、高剂量组。模型组用大剂量地塞米松诱导BMSCs成脂分化,HSYA低、中、高剂量组加入地塞米松的同时分别加入不同浓度的HSYA;空白组无特殊处理。6d后,透射电子显微镜观察各组细胞的形态结构,检测细胞内碱性磷酸酶、甘油三脂的含量,RT-PCR检测细胞中Cbfal及Collagen-I mRNA的表达,Western Blotting检测细胞中p-ERK、p-JNK和p-P38蛋白的表达。结果 1. HSYA促进激素诱导兔BMSCs成骨分化及其MAPK信号转导机制的在体实验： (1)血清中成骨标志物碱性磷酸酶、成脂标志物甘油三脂含量的检测：干预1周后,空白组、髓芯减压组、生理盐水+髓芯减压组和HSYA+髓芯减压组兔血清中碱性磷酸酶含量明显高于模型对照组,甘油三脂含量明显低于模型对照组(P0.05)。其中HSYA+髓芯减压组碱性磷酸酶含量明显高于髓芯减压组和生理盐水+髓芯减压组(P0.05),甘油三脂含量明显低于髓芯减压组和生理盐水+髓芯减压组(P0.05)。 (2)光镜观察股骨头组织的病理改变：空白组软骨下骨小梁致密,排列规则整齐；骨细胞清晰,核位于中央,较大；髓腔内可见增生活跃的骨髓组织,造血细胞丰富。模型对照组软骨层变薄、结构紊乱,骨小梁较稀疏,部分有断裂现象,小梁中有骨细胞坏死；软骨下区骨髓内脂肪细胞增多,造血细胞数量减少。髓芯减压组、生理盐水+髓芯减压组、HSYA+髓芯减压组骨细胞结构较清晰,骨小梁较多,坏死骨细胞较模型对照组少,其中尤以HSYA+髓芯减压组最为明显。 (3)透射电镜观察股骨头组织的超微结构：空白组软骨细胞粗面内质网丰富,无明显脱颗粒；线粒体较多,嵴结构清晰,未见明显断裂；染色质分布相对均匀,细胞表面突起较多。模型对照组空泡样变较空白组明显增多,异染色质部分边聚,胞内可见较大脂滴或次级溶酶体。HSYA+髓芯减压组空泡样变明显减少,次级溶酶体明显减少,大部分线粒体嵴存在。 (4)股骨头中成骨标志物Cbfα1及Collagen-Ⅰ mRNA的表达：干预1周后，，空白组、髓芯减压组、生理盐水+髓芯减压组和HSYA+髓芯减压组明显高于模型对照组(P0.05)。其中HSYA+髓芯减压组明显高于髓芯减压组和生理盐水+髓芯减压组(P0.05)。 (5)股骨头中与MAPK信号通路相关的p-ERK、p-JNK和p-P38蛋白的表达：干预1周后,空白组、髓芯减压组、生理盐水+髓芯减压组和HSYA+髓芯减压组p-ERK1、p-ERK2蛋白表达明显高于模型对照组(P0.05),p-JNK、p-P38蛋白表达明显低于模型对照组(P0.05)。其中HSYA+髓芯减压组p-ERK1、p-ERK2蛋白表达明显高于髓芯减压组和生理盐水+髓芯减压组(P0.05),p-JNK、p-P38蛋白表达明显低于髓芯减压组和生理盐水+髓芯减压组(P0.05)。 2. HSYA促进激素诱导兔BMSCs成骨分化及其MAPK信号转导机制的离体实验： (1)油红“O”染色结果：空白组未发现红染的脂肪颗粒；模型组发现细胞的胞浆内可见大量染成红色的脂肪滴,呈诱导后的脂肪细胞形态；HSYA低、中、高剂量组发现有脂肪滴分布,三组红染脂肪滴明显少于模型组,HSYA高剂量组脂肪滴明显少于模型组,少于中、低剂量组。 (2)透射电镜观察BMSCs的超微结构：各组细胞干预6d后,空白组细胞呈椭圆形或不规则状,细胞质少,核大,未见明显脱颗粒现象。模型组细胞呈长梭形,未见明显核仁；细胞器中线粒体较多呈空泡,可见较多次级溶酶体或脂滴。HSYA低剂量组表面空泡不太明显,损伤的线粒体数量较模型组少,脂滴减少。HSYA中剂量组线粒体较低剂量组增多,空泡样变化减少,次级溶酶体减少,未见明显脂滴。HSYA高剂量组线粒体明显增多,空泡样变明显减少,次级溶酶体明显减少,未见明显脂滴。 (3) BMSCs中成骨标志物碱性磷酸酶、成脂标志物甘油三脂含量的检测：各组细胞干预6d后,模型组BMSCs内碱性磷酸酶含量较空白组明显下降(P0.05),而HSYA各组较模型组均有明显升高(P0.05),且随着剂量的加大,碱性磷酸酶含量呈上升趋势。模型组BMSCs内甘油三脂含量较空白组明显升高(P0.05),而HSYA各组较模型组均有明显降低(P0.05),且随着HSYA剂量的加大,甘油三脂含量呈下降趋势。 (4) BMSCs中成骨标志物Cbfα1及Collagen-I mRNA的表达：各组细胞干预6d后,模型组细胞内Cbfα1、Collagen-Ⅰ mRNA含量均明显低于空白组(P0.05),而HSYA各组Cbfα1、Collagen-Ⅰ mRNA含量均明显高于模型组(P0.05),且随着HSYA剂量的增加,细胞内Cbfα1、Collagen-Ⅰ mRNA含量呈上升趋势。 (5) BMSCs中与MAPK信号通路相关的p-ERK、p-JNK和p-P38蛋白的表达：各组细胞干预6d后,模型组细胞内p-ERK1、p-ERK2蛋白表达明显低于空白组(P0.05),p-JNK、p-P38蛋白表达明显高于空白组(P0.05),而HSYA各组p-ERK1、 P-ERK2蛋白表达明显高于模型组(P0.05),p-JNK、p-P38蛋白表达明显低于模型组(P0.05),且随着HSYA剂量的增加,细胞内p-ERK1、p-ERK2蛋白表达呈上升趋势,p-JNK、p-P38蛋白表达呈下降趋势。结论 1.羟基红花黄色素A(HSYA)能促进激素性股骨头缺血坏死模兔股骨头髓内及离体兔BMSCs内成骨标志物碱性磷酸酶、Cbfα1、Collagen-Ⅰ的表达,抑制成脂标志物甘油三脂的表达,抑制激素诱导股骨头髓内及离体兔BMSCs成脂分化,促使其成骨分化。 2. HSYA通过激活MAPK信号转导通路,上调通路中与增殖关联的p-ERK蛋白的表达,下调凋亡相关联的p-JNK、p-P38蛋白的表达,促进兔股骨头髓内及离体兔BMSCs增殖,抑制其凋亡,减轻激素诱导的BMSCs凋亡的影响。
[Abstract]:objective
To investigate the effect of hydroxysafflor yellow A (HSYA) on the osteogenesis of femoral head and rabbit bone marrow stromal cells (BMSCs) induced by hormone induced ischemic necrosis of the femoral head and the mechanism of MAPK signal transduction pathway in rabbit bone marrow stromal cells (BMSCs).
Method
1. healthy adult New Zealand white rabbits were randomly divided into blank group, model group, model group and model group by gluteal injection of prednisolone acetate (7.5mg/kg) combined with compulsive erect to prepare the model of glucocorticoid avascular necrosis of the femoral head. The model rabbits were randomly divided into model control group, pulp core decompression group, saline + pith core after the model group was successful. The decompression group, the HSYA+ core decompression group, the core decompression group only performed the pulp core decompression, the physiological saline + core decompression group was treated with intramedullary decompression and intramedullary injection of saline, and the HSYA+ core decompression group was treated with intramedullary decompression and intramedullary injection of HSYA. The blank group and the model control group were not treated for.1 weeks after the rabbit anesthesia, abdominal aorta blood sampling, extraction and extraction. The chemical colorimetry was used to detect the content of alkaline phosphatase and glycerol three fat in the serum of rabbits in each group, the femoral head, optical microscope, and transmission electron microscope were taken to observe the morphological structure of the femoral head of rabbits in each group; the expression of Cbfal and Collagen-I mRNA in the femoral head of rabbits in each group was detected by RT-PCR; Western Blotting was used to detect the femoral head in each group of rabbits. Expression of p-ERK. p-JNK and p-P38 protein.
2. healthy adult New Zealand white rabbits (36 rabbits), bone marrow blood was extracted from the medial tibial medial bone marrow cavity under aseptic conditions, separated and cultured in vitro, and BMSCs was cultured in vitro. Flow cytometry was used to detect the cell surface markers combined with osteogenesis, and a fat inducer was used to identify them. The third generation of good BMSCs were selected and randomly divided into blank group, model group and HSYA In the low, medium, high dose group, the model group induced BMSCs fat differentiation with high dose dexamethasone, HSYA low, middle, and high dose group adding dexamethasone with different concentrations of HSYA respectively. The blank group had no special treatment.6d, the morphological structure of each cell was observed by transmission electron microscope, and the content of alkaline phosphatase and glycerol three fat was detected by the transmission electron microscope. RT-PCR was used to detect the expression of Cbfal and Collagen-I mRNA in the cells. Western Blotting was used to detect the expression of p-ERK, p-JNK and p-P38 protein in the cells.
Result
1. HSYA promotes hormone induced osteogenesis in rabbit BMSCs and its MAPK signal transduction mechanism in vivo:
(1) detection of alkaline phosphatase of bone markers in serum and glycerol three fat content of lipid marker: after 1 weeks of intervention, the content of alkaline phosphatase in rabbit serum of blank group, core decompression group, saline + core decompression group and HSYA+ core decompression group was significantly higher than that of model control group, and the content of glycerol three fat was significantly lower than that of model control group (P0.05). The content of alkaline phosphatase in the HSYA+ core decompression group was significantly higher than that of the core decompression group and the physiological saline + core decompression group (P0.05). The content of glycerol three fat was significantly lower than that of the core decompression group and the saline + core decompression group (P0.05).
(2) the pathological changes of the femoral head tissue were observed by light microscope: the subchondral bone trabecula in the blank group was dense and arranged regularly, the bone cells were clear, the nucleus was located in the central area, and the myeloid tissue was abundant in the medullary cavity, and the hematopoietic cells were rich. The cartilage layer of the model control group was thinner, the structure was disorganized, the trabecula was thinner, and some fracture, trabeculae were broken. Osteonecrosis, intramedullary adipose cells in subchondral bone marrow increased, and the number of hematopoietic cells decreased. Core decompression group, physiological saline + core decompression group, HSYA+ core decompression group had a clear bone cell structure, more bone trabecula and less necrotic bone cells than model control group, especially HSYA+ core decompression group was the most obvious.
(3) the ultrastructure of the femoral head tissue was observed by transmission electron microscope: in the blank group, the rough endoplasmic reticulum of the cartilage cells in the blank group was rich and there was no obvious degranulation, the mitochondria were more, the ridge structure was clear, no obvious fracture was found, the chromatin distribution was relatively uniform and the surface of the cell appeared more prominently. In the cytoplasm, larger lipid droplets or secondary lysosomes were found in.HSYA+. Vacuolar changes in the core decompression group were significantly reduced, secondary lysosomes were significantly reduced, and most mitochondrial crest existed.
(4) the expression of Cbf alpha 1 and Collagen- I mRNA of the bone marker in the femoral head: after 1 weeks, the blank group, the core decompression group, the saline + core decompression group and the HSYA+ core decompression group were significantly higher than those of the model control group (P0.05). The HSYA+ core decompression group was significantly higher than the pulp core reduction group and the physiological saline + core decompression group (P0.05).
(5) the expression of p-ERK, p-JNK and p-P38 protein associated with the MAPK signaling pathway in the femoral head: after 1 weeks, the blank group, the core decompression group, the saline + core decompression group and the HSYA+ core decompression group p-ERK1, the expression of p-ERK2 protein was significantly higher than that of the model control group (P0.05), and the expression of p-JNK, p-P38 protein was significantly lower than that of the model control group (P0.05). The expression of p-ERK1, p-ERK2 protein in the core decompression group was significantly higher than that of the core decompression group and the physiological saline + core decompression group (P0.05), and the expression of p-JNK, p-P38 protein was significantly lower than that of the core decompression group and the physiological saline + core decompression group (P0.05).
2. HSYA in vitro promotes the osteogenesis of rabbit BMSCs induced by steroid and the mechanism of MAPK signal transduction.
(1) the result of oil red "O" staining: no red dye was found in the blank group; in the model group, a large number of red fat drops were found in the cytoplasm of the cells, and the form of adipocyte after induction was found. HSYA was low, middle, high dose group found the distribution of fat droplets, and the three groups of red dye drops were obviously less than the model group, and the high dose group of HSYA fat drops Less than the model group, less than the low dose group.
(2) the ultrastructure of BMSCs was observed by transmission electron microscope: after the intervention of 6D, the cells in the blank group were oval or irregular, the cytoplasm was small, the nucleus was large and no obvious degranulation was found. The cells in the model group showed long spindle shape and no obvious nucleolus. There were many vacuoles in the organelles, and many secondary lysosomes or lipid droplet.HSYA low dose groups were found. The number of damaged mitochondria was less than that of the model group. The number of mitochondria in the.HSYA medium dose group increased, the change of vacuoles decreased and the secondary lysosome decreased. There was no obvious increase in mitochondria in the high dose group of lipid droplet.HSYA, the decrease of vacuoles like brightness, the decrease of the secondary lysosome and no obvious lipid droplets.
(3) detection of alkaline phosphatase and glycerol three fat content of lipid markers in BMSCs: after 6D, the content of alkaline phosphatase in the model group was significantly lower than that in the blank group (P0.05), and all HSYA groups were significantly higher than those in the model group (P0.05), and the alkaline phosphatase content increased with the increase of dosage. The content of glycerol three fat in the BMSCs group was significantly higher than that in the blank group (P0.05), while the HSYA groups were significantly lower than those in the model group (P0.05), and the content of glycerol and glycerol decreased with the increase of HSYA dosage.
(4) the expression of Cbf alpha 1 and Collagen-I mRNA in BMSCs: after 6D, the content of Cbf alpha 1 and Collagen- I mRNA in the model group were significantly lower than that in the blank group (P0.05), while Cbf alpha 1 in HSYA groups, Collagen- I content was significantly higher than that of the model group. The content of mRNA is on the rise.
(5) the expression of p-ERK, p-JNK and p-P38 protein related to MAPK signaling pathway in BMSCs: after each group of cells intervened 6D, the expression of p-ERK1, p-ERK2 protein in the model group was significantly lower than that in the blank group (P0.05), and the expression of p-JNK and p-P38 protein was significantly higher than that of the blank group (P0.05). The expression of p-P38 protein was significantly lower than that of the model group (P0.05), and with the increase of HSYA dose, the expression of p-ERK1, p-ERK2 protein in the cells showed an upward trend, and the expression of p-JNK and p-P38 protein showed a declining trend.
conclusion
1. hydroxy safflor yellow A (HSYA) can promote the expression of alkaline phosphatase, Cbf alpha 1, Collagen- I in the intramedullary and BMSCs intramedullary osteogenic markers of femoral head ischemic necrosis of the rabbit femoral head, inhibit the expression of glycerol three fat, inhibit the formation of lipid differentiation in the intramedullary and isolated rabbit BMSCs of the femoral head, and promote its osteogenesis differentiation.
2. HSYA up-regulated the expression of p-ERK protein associated with proliferation in the pathway by activating the MAPK signal transduction pathway, downregulating the expression of p-JNK and p-P38 protein associated with apoptosis, promoting the proliferation of BMSCs in rabbit femoral head and in vitro, inhibiting its apoptosis and alleviating the effect of hormone induced BMSCs apoptosis.

【学位授予单位】：福建中医药大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R285.5

【参考文献】