本文選題：股骨頭缺血壞死 + 羥基紅花黃色素A��； 參考：《福建中醫(yī)藥大學(xué)》2014年博士論文

【摘要】：目的 探討羥基紅花黃色素A(HSYA)促進(jìn)激素誘導(dǎo)的股骨頭缺血壞死模兔股骨頭髓內(nèi)及離體兔骨髓基質(zhì)細(xì)胞(BMSCs)成骨分化及其MAPK信號(hào)轉(zhuǎn)導(dǎo)通路的作用機(jī)制。 方法 1.健康成年新西蘭大白兔48只,抽簽法隨機(jī)分為空白組、模型組,模型組采用臀肌注射醋酸潑尼松龍(7.5mg/kg)結(jié)合強(qiáng)迫立位制備激素性股骨頭缺血壞死模型。造模成功后抽簽法將模型組兔隨機(jī)分為模型對(duì)照組、髓芯減壓組、生理鹽水+髓芯減壓組、HSYA+髓芯減壓組,髓芯減壓組僅行髓芯減壓術(shù),生理鹽水+髓芯減壓組采用髓芯減壓術(shù)配合髓腔內(nèi)注射生理鹽水,HSYA+髓芯減壓組采用髓芯減壓術(shù)配合髓腔內(nèi)注射HSYA,空白組和模型對(duì)照組不作治療。1周后兔麻醉,腹主動(dòng)脈采血,采用化學(xué)比色法、氧化酶法檢測(cè)各組兔血清中堿性磷酸酶、甘油三脂的含量；取出股骨頭,光學(xué)顯微鏡、透射電子顯微鏡觀察各組兔股骨頭的形態(tài)結(jié)構(gòu)；RT-PCR檢測(cè)各組兔股骨頭中Cbfal及Collagen-I mRNA的表達(dá)；Western Blotting檢測(cè)各組兔股骨頭中p-ERK. p-JNK和p-P38蛋白的表達(dá)。 2.健康成年新西蘭大白兔36只,無(wú)菌條件下于脛骨上端內(nèi)側(cè)骨髓腔穿刺抽取骨髓血,分離、體外培養(yǎng)BMSCs,流式細(xì)胞儀檢測(cè)細(xì)胞表面標(biāo)記物結(jié)合成骨、成脂誘導(dǎo)劑誘導(dǎo)對(duì)其鑒定。取第3代生長(zhǎng)良好的BMSCs,抽簽法隨機(jī)分為空白組,模型組及HSYA低、中、高劑量組。模型組用大劑量地塞米松誘導(dǎo)BMSCs成脂分化,HSYA低、中、高劑量組加入地塞米松的同時(shí)分別加入不同濃度的HSYA;空白組無(wú)特殊處理。6d后,透射電子顯微鏡觀察各組細(xì)胞的形態(tài)結(jié)構(gòu),檢測(cè)細(xì)胞內(nèi)堿性磷酸酶、甘油三脂的含量,RT-PCR檢測(cè)細(xì)胞中Cbfal及Collagen-I mRNA的表達(dá),Western Blotting檢測(cè)細(xì)胞中p-ERK、p-JNK和p-P38蛋白的表達(dá)。 結(jié)果 1. HSYA促進(jìn)激素誘導(dǎo)兔BMSCs成骨分化及其MAPK信號(hào)轉(zhuǎn)導(dǎo)機(jī)制的在體實(shí)驗(yàn)： (1)血清中成骨標(biāo)志物堿性磷酸酶、成脂標(biāo)志物甘油三脂含量的檢測(cè)：干預(yù)1周后,空白組、髓芯減壓組、生理鹽水+髓芯減壓組和HSYA+髓芯減壓組兔血清中堿性磷酸酶含量明顯高于模型對(duì)照組,甘油三脂含量明顯低于模型對(duì)照組(P0.05)。其中HSYA+髓芯減壓組堿性磷酸酶含量明顯高于髓芯減壓組和生理鹽水+髓芯減壓組(P0.05),甘油三脂含量明顯低于髓芯減壓組和生理鹽水+髓芯減壓組(P0.05)。 (2)光鏡觀察股骨頭組織的病理改變：空白組軟骨下骨小梁致密,排列規(guī)則整齊；骨細(xì)胞清晰,核位于中央,較大；髓腔內(nèi)可見(jiàn)增生活躍的骨髓組織,造血細(xì)胞豐富。模型對(duì)照組軟骨層變薄、結(jié)構(gòu)紊亂,骨小梁較稀疏,部分有斷裂現(xiàn)象,小梁中有骨細(xì)胞壞死；軟骨下區(qū)骨髓內(nèi)脂肪細(xì)胞增多,造血細(xì)胞數(shù)量減少。髓芯減壓組、生理鹽水+髓芯減壓組、HSYA+髓芯減壓組骨細(xì)胞結(jié)構(gòu)較清晰,骨小梁較多,壞死骨細(xì)胞較模型對(duì)照組少,其中尤以HSYA+髓芯減壓組最為明顯。 (3)透射電鏡觀察股骨頭組織的超微結(jié)構(gòu)：空白組軟骨細(xì)胞粗面內(nèi)質(zhì)網(wǎng)豐富,無(wú)明顯脫顆粒；線粒體較多,嵴結(jié)構(gòu)清晰,未見(jiàn)明顯斷裂；染色質(zhì)分布相對(duì)均勻,細(xì)胞表面突起較多。模型對(duì)照組空泡樣變較空白組明顯增多,異染色質(zhì)部分邊聚,胞內(nèi)可見(jiàn)較大脂滴或次級(jí)溶酶體。HSYA+髓芯減壓組空泡樣變明顯減少,次級(jí)溶酶體明顯減少,大部分線粒體嵴存在。 (4)股骨頭中成骨標(biāo)志物Cbfα1及Collagen-Ⅰ mRNA的表達(dá)：干預(yù)1周后，，空白組、髓芯減壓組、生理鹽水+髓芯減壓組和HSYA+髓芯減壓組明顯高于模型對(duì)照組(P0.05)。其中HSYA+髓芯減壓組明顯高于髓芯減壓組和生理鹽水+髓芯減壓組(P0.05)。 (5)股骨頭中與MAPK信號(hào)通路相關(guān)的p-ERK、p-JNK和p-P38蛋白的表達(dá)：干預(yù)1周后,空白組、髓芯減壓組、生理鹽水+髓芯減壓組和HSYA+髓芯減壓組p-ERK1、p-ERK2蛋白表達(dá)明顯高于模型對(duì)照組(P0.05),p-JNK、p-P38蛋白表達(dá)明顯低于模型對(duì)照組(P0.05)。其中HSYA+髓芯減壓組p-ERK1、p-ERK2蛋白表達(dá)明顯高于髓芯減壓組和生理鹽水+髓芯減壓組(P0.05),p-JNK、p-P38蛋白表達(dá)明顯低于髓芯減壓組和生理鹽水+髓芯減壓組(P0.05)。 2. HSYA促進(jìn)激素誘導(dǎo)兔BMSCs成骨分化及其MAPK信號(hào)轉(zhuǎn)導(dǎo)機(jī)制的離體實(shí)驗(yàn)： (1)油紅“O”染色結(jié)果：空白組未發(fā)現(xiàn)紅染的脂肪顆粒；模型組發(fā)現(xiàn)細(xì)胞的胞漿內(nèi)可見(jiàn)大量染成紅色的脂肪滴,呈誘導(dǎo)后的脂肪細(xì)胞形態(tài)；HSYA低、中、高劑量組發(fā)現(xiàn)有脂肪滴分布,三組紅染脂肪滴明顯少于模型組,HSYA高劑量組脂肪滴明顯少于模型組,少于中、低劑量組。 (2)透射電鏡觀察BMSCs的超微結(jié)構(gòu)：各組細(xì)胞干預(yù)6d后,空白組細(xì)胞呈橢圓形或不規(guī)則狀,細(xì)胞質(zhì)少,核大,未見(jiàn)明顯脫顆�，F(xiàn)象。模型組細(xì)胞呈長(zhǎng)梭形,未見(jiàn)明顯核仁；細(xì)胞器中線粒體較多呈空泡,可見(jiàn)較多次級(jí)溶酶體或脂滴。HSYA低劑量組表面空泡不太明顯,損傷的線粒體數(shù)量較模型組少,脂滴減少。HSYA中劑量組線粒體較低劑量組增多,空泡樣變化減少,次級(jí)溶酶體減少,未見(jiàn)明顯脂滴。HSYA高劑量組線粒體明顯增多,空泡樣變明顯減少,次級(jí)溶酶體明顯減少,未見(jiàn)明顯脂滴。 (3) BMSCs中成骨標(biāo)志物堿性磷酸酶、成脂標(biāo)志物甘油三脂含量的檢測(cè)：各組細(xì)胞干預(yù)6d后,模型組BMSCs內(nèi)堿性磷酸酶含量較空白組明顯下降(P0.05),而HSYA各組較模型組均有明顯升高(P0.05),且隨著劑量的加大,堿性磷酸酶含量呈上升趨勢(shì)。模型組BMSCs內(nèi)甘油三脂含量較空白組明顯升高(P0.05),而HSYA各組較模型組均有明顯降低(P0.05),且隨著HSYA劑量的加大,甘油三脂含量呈下降趨勢(shì)。 (4) BMSCs中成骨標(biāo)志物Cbfα1及Collagen-I mRNA的表達(dá)：各組細(xì)胞干預(yù)6d后,模型組細(xì)胞內(nèi)Cbfα1、Collagen-Ⅰ mRNA含量均明顯低于空白組(P0.05),而HSYA各組Cbfα1、Collagen-Ⅰ mRNA含量均明顯高于模型組(P0.05),且隨著HSYA劑量的增加,細(xì)胞內(nèi)Cbfα1、Collagen-Ⅰ mRNA含量呈上升趨勢(shì)。 (5) BMSCs中與MAPK信號(hào)通路相關(guān)的p-ERK、p-JNK和p-P38蛋白的表達(dá)：各組細(xì)胞干預(yù)6d后,模型組細(xì)胞內(nèi)p-ERK1、p-ERK2蛋白表達(dá)明顯低于空白組(P0.05),p-JNK、p-P38蛋白表達(dá)明顯高于空白組(P0.05),而HSYA各組p-ERK1、 P-ERK2蛋白表達(dá)明顯高于模型組(P0.05),p-JNK、p-P38蛋白表達(dá)明顯低于模型組(P0.05),且隨著HSYA劑量的增加,細(xì)胞內(nèi)p-ERK1、p-ERK2蛋白表達(dá)呈上升趨勢(shì),p-JNK、p-P38蛋白表達(dá)呈下降趨勢(shì)。 結(jié)論 1.羥基紅花黃色素A(HSYA)能促進(jìn)激素性股骨頭缺血壞死模兔股骨頭髓內(nèi)及離體兔BMSCs內(nèi)成骨標(biāo)志物堿性磷酸酶、Cbfα1、Collagen-Ⅰ的表達(dá),抑制成脂標(biāo)志物甘油三脂的表達(dá),抑制激素誘導(dǎo)股骨頭髓內(nèi)及離體兔BMSCs成脂分化,促使其成骨分化。 2. HSYA通過(guò)激活MAPK信號(hào)轉(zhuǎn)導(dǎo)通路,上調(diào)通路中與增殖關(guān)聯(lián)的p-ERK蛋白的表達(dá),下調(diào)凋亡相關(guān)聯(lián)的p-JNK、p-P38蛋白的表達(dá),促進(jìn)兔股骨頭髓內(nèi)及離體兔BMSCs增殖,抑制其凋亡,減輕激素誘導(dǎo)的BMSCs凋亡的影響。
[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.

【學(xué)位授予單位】：福建中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R285.5

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 劉峰;潘小紅;張尊勝;;紅花注射液治療慢性腰腿痛的實(shí)驗(yàn)研究[J];按摩與導(dǎo)引;2005年12期

2 陳浩;中醫(yī)內(nèi)外治結(jié)合治療股骨頭缺血性壞死35例[J];成都中醫(yī)藥大學(xué)學(xué)報(bào);1999年04期

3 張弛;何洪陽(yáng);楊志偉;陳日高;彭正剛;紀(jì)向輝;;活血通絡(luò)法防治激素性股骨頭壞死的作用及機(jī)制研究[J];成都中醫(yī)藥大學(xué)學(xué)報(bào);2007年01期

4 李炳鉆;陳長(zhǎng)賢;鐘黎娟;王建嗣;蔡秀英;宮偉;陳群華;;西紅花在發(fā)育性髖關(guān)節(jié)脫位治療中預(yù)防股骨頭缺血性壞死的療效觀察[J];當(dāng)代醫(yī)學(xué);2010年21期

5 黃少慧;張娟;李娟;蔡紅兵;;骨靈片通過(guò)p38 MAPK信號(hào)通路影響成骨細(xì)胞功能的機(jī)制[J];南方醫(yī)科大學(xué)學(xué)報(bào);2008年07期

6 齊振熙;康靖東;;激素性股骨頭缺血性壞死模型的建立與評(píng)價(jià)[J];福建中醫(yī)學(xué)院學(xué)報(bào);2008年05期

7 齊振熙;康靖東;李樹(shù)強(qiáng);;桃紅四物湯對(duì)激素性股骨頭缺血壞死模鼠TGF-β_1 mRNA轉(zhuǎn)錄的影響[J];福建中醫(yī)學(xué)院學(xué)報(bào);2009年01期

8 李樹(shù)強(qiáng);齊振熙;仲衛(wèi)紅;于濤;;活血化瘀中藥對(duì)激素誘導(dǎo)骨間充質(zhì)干細(xì)胞Ⅰ型膠原mRNA表達(dá)的影響[J];福建中醫(yī)學(xué)院學(xué)報(bào);2009年04期

9 齊振熙;李樹(shù)強(qiáng);于濤;;土鱉蟲(chóng)對(duì)激素誘導(dǎo)骨髓間充質(zhì)干細(xì)胞PPARγ和aP2mRNA表達(dá)的影響[J];福建中醫(yī)學(xué)院學(xué)報(bào);2010年04期

10 于濤;齊振熙;余航;張占勇;仲衛(wèi)紅;;羥基紅花黃色素A對(duì)激素誘導(dǎo)骨髓間充質(zhì)干細(xì)胞成脂分化的干預(yù)作用[J];福建中醫(yī)藥大學(xué)學(xué)報(bào);2011年06期

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