本文關(guān)鍵詞： 2型糖尿病 骨質(zhì)疏松癥 胰島素信號(hào)通路 骨髓脂肪組織 皮質(zhì)骨 脊柱骨折 中草藥　出處：《河北醫(yī)科大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：2型糖尿病(Type 2 diabetes mellitus,T2DM)和骨質(zhì)疏松癥(Osteoporosis,OP)作為老年人的常見病、多發(fā)病,其發(fā)病率呈逐年升高趨勢(shì),嚴(yán)重影響人們的生活質(zhì)量。國內(nèi)外各項(xiàng)研究對(duì)1型糖尿病與骨質(zhì)疏松癥的關(guān)系已經(jīng)得出較為肯定的結(jié)論,即1型糖尿病是骨質(zhì)疏松癥的高風(fēng)險(xiǎn)因素,并可由此導(dǎo)致病理性骨折,其原因可能與胰島素分泌絕對(duì)不足引發(fā)糖類、脂類及蛋白質(zhì)代謝異常有關(guān),而糖類、脂類、蛋白質(zhì)代謝異�？蓪�(dǎo)致機(jī)體鈣磷代謝失衡,從而減低骨量;而2型糖尿病患者骨量變化情況尚未得到較為一致的結(jié)論,二者發(fā)病機(jī)制是否存在共通之處亦不甚明確。近年來研究發(fā)現(xiàn),胰島素信號(hào)通路可能與2型糖尿病和骨質(zhì)疏松癥的發(fā)病機(jī)制有關(guān)。胰島素信號(hào)通路包括:胰島素受體底物(insulin receptor substrate,IRS)的磷酸化,磷脂酰肌醇3激酶(phosphatidylinositol 3-kinase,PI3K)的活化,Akt(即蛋白激酶B)活化,雷帕霉素靶蛋白(molecular target of rapamycin,m TOR)活化,核糖體蛋白S6激酶(p70 S6 kinase,p70 S6 k)活化等。正常情況下,胰島素或胰島素樣生長(zhǎng)因子(insulin-like growth factor-1,IGF-1)在細(xì)胞表面與胰島素受體結(jié)合,誘導(dǎo)胰島素受體發(fā)生自身磷酸化,酪氨酸激酶被激活,活化的酪氨酸激酶使胰島素受體底物(insulin receptor substrate,IRS)的酪氨酸磷酸化,該底物被激活�；罨腎RS在細(xì)胞膜處與磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)的p85-p110亞基結(jié)合,PI3K被激活,后者生成磷脂酰肌醇(3、4)二磷酸(PIP2)和磷脂酰肌醇(3、4、5)三磷酸(PIP3),并與磷脂酰肌醇依賴性激酶(phosphoinositide dependent kinase,PDK)-1結(jié)合,進(jìn)一步使下游通路中蛋白激酶B(protein kinase B,PKB)絲氨酸殘基磷酸化,最終導(dǎo)致葡萄糖轉(zhuǎn)運(yùn)蛋白-4(glucose transporter 4,GLUT-4)移位到細(xì)胞膜而發(fā)揮降糖作用。T2DM時(shí),外周組織對(duì)胰島素的敏感性降低,細(xì)胞對(duì)葡萄糖的攝取能力下降,葡萄糖調(diào)節(jié)受損,血糖升高。近來研究發(fā)現(xiàn),胰島素信號(hào)通路不僅是糖代謝的關(guān)鍵信號(hào)通路之一,其各因子也參與骨代謝的調(diào)節(jié)。PI3K參與成骨細(xì)胞和破骨細(xì)胞的分化過程,在骨生成和骨重建方面發(fā)揮重要作用。實(shí)驗(yàn)發(fā)現(xiàn),PI3K信號(hào)通路的活化在成骨前體細(xì)胞MC3T3-E1的分化過程中是必需的,阻斷該信號(hào)通路在成骨細(xì)胞中的活化將嚴(yán)重抑制成骨細(xì)胞的正常分化。Akt是PI3K信號(hào)通路的主要下游分子。在成骨細(xì)胞和破骨細(xì)胞上,Aktl的表達(dá)占絕對(duì)優(yōu)勢(shì),敲除小鼠Akt1基因發(fā)現(xiàn)其骨量減少,骨組織形態(tài)測(cè)量顯示骨形成和骨吸收活性均明顯下降,二次骨化中心形成延遲;Akt1-/-野生型小鼠成骨細(xì)胞分化指標(biāo)(如ALP、I型膠原、骨鈣素表達(dá)等)均降低。此外,Akt與骨形態(tài)發(fā)生蛋白2共同調(diào)節(jié)間充質(zhì)細(xì)胞向成骨細(xì)胞的分化。骨質(zhì)疏松癥不僅與體重和身體總脂肪有關(guān),與骨髓脂肪細(xì)胞也密切相關(guān)。骨髓間充質(zhì)干細(xì)胞(Mesenchymal stem cells,MSCs)具有多向分化潛能。若MSCs向脂肪細(xì)胞方向分化增加,會(huì)導(dǎo)致其向成骨細(xì)胞分化減少,故骨髓成骨細(xì)胞與脂肪細(xì)胞呈競(jìng)爭(zhēng)性負(fù)相關(guān)關(guān)系。激素、脂肪因子,PPARγ以及機(jī)械性刺激均可影響MSCs向成骨或成脂方向轉(zhuǎn)化。抑制成骨細(xì)胞內(nèi)信號(hào)通路可促使MSCs向脂肪細(xì)胞方向轉(zhuǎn)化。由骨質(zhì)疏松癥所導(dǎo)致的椎體壓縮性骨折(OVCFs)十分常見,嚴(yán)重影響患者的生活質(zhì)量,給家庭和社會(huì)都帶來沉重的經(jīng)濟(jì)負(fù)擔(dān)。過去的十年中,制藥公司已經(jīng)研發(fā)出一系列可提高骨量的骨質(zhì)疏松治療藥物。這些藥物主要在促進(jìn)骨形成(如特立帕肽,鍶鹽等)或抑制骨吸收(如雙磷酸鹽類)方面發(fā)揮治療骨質(zhì)疏松癥的作用。雖然這些藥物對(duì)骨質(zhì)疏松癥療效明顯,但發(fā)熱、骨痛加重等副反應(yīng)也伴之而來。中草藥可與已有的藥物聯(lián)合應(yīng)用于治療由骨質(zhì)疏松癥所導(dǎo)致的脊柱壓縮性骨折。第一部分胰島素下游信號(hào)因子PI3K、Akt1、Akt2及炎癥因子NFκB在2型糖尿病骨質(zhì)疏松大鼠肝、腎及骨骼肌組織中表達(dá)目的:探討胰島素信號(hào)通路下游PI3K、Akt1、Akt2及炎癥因子NFκB表達(dá)變化是否影響2型糖尿病合并骨質(zhì)疏松癥的發(fā)生、發(fā)展。方法:選取100只健康雌性Wistar大鼠(合格證書編號(hào)905105,2.5-3月齡)適應(yīng)性喂養(yǎng)1周后隨機(jī)分為正常對(duì)照組(NS組,N=24),單純?nèi)ヂ殉步M(NOVX組,N=26),2型糖尿病(DS組,N=24),2型糖尿病去卵巢組(DOVX組,N=26)。采用高糖高脂飼料喂養(yǎng)聯(lián)合小劑量STZ腹腔注射法制作T2DM大鼠模型,雙側(cè)卵巢切除法制作OP大鼠模型。去卵巢后12周麻醉處死大鼠,分離雙側(cè)肝臟、腎臟及右側(cè)股四頭肌組織,分別制作各組織石蠟切片和提取RNA和總蛋白,應(yīng)用免疫組織化學(xué)和western blot方法檢測(cè)NS組、NOVX組、DS組、DOVX組各組織PI3K、Akt1、Akt2、NFκB蛋白表達(dá),反轉(zhuǎn)錄聚合酶鏈反應(yīng)(RT-PCR)方法檢測(cè)PI3K、Akt1、Akt2、NFκB m RNA的表達(dá)。應(yīng)用SPSS13.0統(tǒng)計(jì)軟件處理實(shí)驗(yàn)數(shù)據(jù),所有實(shí)驗(yàn)數(shù)據(jù)均采用均數(shù)±標(biāo)準(zhǔn)差(x±s)表示,計(jì)量資料多組間比較采用方差分析,P0.05認(rèn)為有統(tǒng)計(jì)學(xué)意義。結(jié)果:應(yīng)用免疫組化方法觀察大鼠肝、腎、骨骼肌組織PI3K、Akt1、Akt2、NFκB分別在大鼠肝細(xì)胞胞漿、腎小管上皮細(xì)胞胞漿及骨骼肌細(xì)胞胞漿中表達(dá)。各組織免疫組化結(jié)果顯示,與NS組相比,NOVX、DS和DOVX的PI3K、Akt1和Akt2均降低,以DOVX組降低最明顯(P0.05)。NFκB趨勢(shì)則相反,12周時(shí)NOVX、DS和DOVX的NFκB均升高,以DOVX組升高最明顯(P0.05)。肝組織RT-PCR結(jié)果顯示,DOVX組PI3K、Akt1和Akt2降低最為明顯(P0.05),NFκB升高最明顯(P0.05)。DS組和DOVX組Akt2無明顯差別(P0.05),NOVX組、DS組和DOVX組NFκB無明顯差別(P0.05)。腎組織RT-PCR結(jié)果顯示,DOVX組PI3K、Akt1和Akt2降低最為明顯(P0.05),NFκB升高最明顯(P0.05)。NOVX組和DS組PI3K無明顯差別(P0.05),NOVX組、DS組和DOVX組Akt2表達(dá)無明顯變化(P0.05)。骨骼肌RT-PCR結(jié)果顯示,DOVX組PI3K、Akt1和Akt2降低最為明顯(P0.05),NFκB升高最明顯(P0.05)。NOVX組、DS組和DOVX組PI3K、Akt1、NFκB無明顯差別(P0.05)。肝組織western blot結(jié)果顯示,以DOVX組PI3K、Akt1和Akt2降低最為明顯(P0.05),NOVX組和DS組PI3K無明顯差別(P0.05)。NFκB以DOVX組升高最明顯(P0.05),NOVX組和DS組NFκB無明顯差別(P0.05)。腎組織western blot結(jié)果顯示,DOVX組PI3K、Akt1和Akt2降低最明顯(P0.05),NFκB升高最明顯(P0.05)。NOVX組和DS組PI3K、NFκB無明顯差別(P0.05),DS組與DOVX組Akt2無明顯差別(P0.05)。骨骼肌western blot結(jié)果顯示,DOVX組PI3K、Akt1和Akt2降低最明顯(P0.05),NFκB升高最明顯(P0.05)。NOVX組和DS組Akt1、NFκB表達(dá)無明顯差別(P0.05)。結(jié)論:胰島素信號(hào)通路中PI3K、Akt1、Akt2、NFκB表達(dá)受抑制可能與2型糖尿病合并骨質(zhì)疏松癥的發(fā)病有關(guān)。第二部分絕經(jīng)后骨質(zhì)疏松患者骨髓脂肪組織與皮質(zhì)骨區(qū)關(guān)系的研究目的:探討絕經(jīng)后骨質(zhì)疏松癥患者骨髓脂肪組織與皮質(zhì)骨區(qū)是否存在負(fù)相關(guān)關(guān)系。方法:收集2013年1月-2013年12月在河北醫(yī)科大學(xué)第三醫(yī)院內(nèi)分泌二科住院的2型糖尿病女性患者30例。采用核磁共振技術(shù)測(cè)量患者右側(cè)股骨BMA,右側(cè)股骨CBA,右側(cè)股骨皮下脂肪組織(SAT)及右側(cè)股骨骨骼肌,并將圖像用Slice Omatic分析軟件進(jìn)行數(shù)據(jù)分析。采用SPSS13.0統(tǒng)計(jì)分析軟件進(jìn)行統(tǒng)計(jì)分析,描述性指標(biāo)以均數(shù)±標(biāo)準(zhǔn)差表示。采用Pearson相關(guān)系數(shù)計(jì)算CBA、BMA、體重、BMI百分比、年齡、SAT和骨骼肌之間的關(guān)系。采用多元線性回歸進(jìn)行相關(guān)分析。結(jié)果:Pearson相關(guān)分析顯示,右側(cè)股骨BMA和右側(cè)股骨CBA都與體重、BMI、年齡、右側(cè)股骨皮下脂肪組織(SAT)及右側(cè)股骨骨骼肌呈顯著正相關(guān)(分別為r=0.081,r=-0.215,r=0.175,r=-0.252,r=0.317;r=0.385,r=0.153,r=-0.298,r=-0.087,r=0.650;P0.05)。結(jié)論:BMA與CBA之間呈負(fù)相關(guān)關(guān)系。這可能與骨髓間充質(zhì)干細(xì)胞優(yōu)先向脂肪細(xì)胞分化而向成骨細(xì)胞分化減少有關(guān),為骨質(zhì)疏松癥提供新的治療方向。第三部分傳統(tǒng)中藥治療骨質(zhì)疏松性椎體壓縮性骨折的系統(tǒng)性回顧研究目的:系統(tǒng)評(píng)價(jià)中草藥治療在骨質(zhì)疏松性椎體壓縮性骨折的臨床效果。方法:計(jì)算機(jī)檢索三個(gè)電子數(shù)據(jù)庫(中國國家知識(shí)基礎(chǔ)設(shè)施工程,中國科技期刊數(shù)據(jù)庫(VIP)和萬方數(shù)據(jù)庫),檢索時(shí)間從1999年至2013年2月,搜索的中文關(guān)鍵字是中國草藥、草藥、骨質(zhì)疏松性椎體壓縮性骨折。本研究還使用了以下關(guān)鍵字的任意組合:一)壓縮性骨折、腰椎骨折、胸椎骨折;和b)傳統(tǒng)中國醫(yī)藥或者中國傳統(tǒng)藥物。搜索的英文關(guān)鍵詞包括Chinese herb、herbal、osteoporotic vertebral compression fracture、compression fracture、vertebra fracture、lumbar vertebrae fracture、thoracic vertebrae fracture、traditional Chinese medicine、traditional Chinese drug。實(shí)驗(yàn)類型為隨機(jī)對(duì)照實(shí)驗(yàn)。根據(jù)入排標(biāo)準(zhǔn)對(duì)入選文章進(jìn)行嚴(yán)格篩選、評(píng)估質(zhì)量、提取數(shù)據(jù),采用Rev Man 5.1軟件進(jìn)行數(shù)據(jù)分析。結(jié)果:第三部分:最終納入11項(xiàng)隨機(jī)對(duì)照試驗(yàn),共953例受試者。結(jié)果顯示,與安慰劑組相比,使用中草藥治療的患者其骨密度明顯升高。此外,與安慰劑組相比,聯(lián)合中藥治療的患者其骨密度提高更明顯。敏感性分析證實(shí)了這一點(diǎn)。結(jié)論:使用中草藥治療可以增加脊柱骨密度。中草藥可作為一種創(chuàng)傷小、成本低的治療骨質(zhì)疏松癥導(dǎo)致的脊柱壓縮性骨折的方法。
[Abstract]:Type 2 diabetes mellitus (Type 2 diabetes mellitus, T2DM) and osteoporosis (Osteoporosis, OP) is a common disease, the elderly disease, its incidence is increasing year by year, seriously affecting people's quality of life at home and abroad. The research on the relationship between type 1 diabetes and osteoporosis has drawn more positive conclusion: type 1 diabetes is a high risk factor for osteoporosis, and thus lead to pathological fracture, the reason may be related to insulin secretion absolutely caused by insufficient carbohydrate, lipid and protein metabolism, abnormal lipid and carbohydrate, protein, metabolic abnormalities can lead to imbalance of calcium and phosphorus metabolism, thereby reducing bone mass changes; the situation of bone mass in patients with type 2 diabetes has not been consistent conclusions, the pathogenesis of the existence of two common is not very clear. Recent studies have found that the Shima Sonobu pathway may be related to type 2 diabetes The pathogenesis of diabetes and osteoporosis. The insulin signaling pathway including insulin receptor substrate-1 (insulin receptor, substrate, IRS) phosphorylation of phosphatidylinositol 3 kinase (phosphatidylinositol, 3-kinase, PI3K) activation, Akt (i.e. protein kinase B) activation, rapamycin (molecular target of rapamycin, m TOR) activation of ribosomal protein S6 kinase (p70 S6 kinase, p70 S6 K) activation. Under normal circumstances, insulin or insulin-like growth factor (insulin-like growth, factor-1, IGF-1) on the cell surface binding with the insulin receptor, insulin receptor induced autophosphorylation and tyrosine kinase activated tyrosine kinase activation to insulin (insulin receptor substrate receptor tyrosine phosphorylation, IRS), the substrate is activated. The activation of IRS kinase and phosphatidylinositol 3- at the cell membrane (phosphatidylinositol 3-kinase, PI3K) p85-p110 subunit binding, PI3K is activated, the generation of phosphatidylinositol (3,4) two phosphoric acid (PIP2) and phosphatidylinositol (3,4,5) three (PIP3), and phosphate dependent kinase and phosphatidylinositol (phosphoinositide dependent, kinase, PDK) -1 binding protein kinase, further downstream pathways B (protein kinase B, PKB) phosphorylated serine residues, resulting in glucose transporter -4 (glucose transporter 4, GLUT-4) shift to the cell membrane and play the hypoglycemic effect of.T2DM, reduce the sensitivity of peripheral tissues to insulin, decreased ability of glucose uptake by cells, impaired glucose regulation, blood glucose increased recently. The study found that insulin signaling pathway is not only one of the most important signal pathway of glucose metabolism, the factors are also involved in the regulation of bone metabolism of.PI3K in osteoblast and osteoclast differentiation in bone formation and bone remodeling. The surface play an important role. The experimental results revealed that the activation of PI3K signaling pathway in the differentiation of osteoblastic progenitor cells in MC3T3-E1 is necessary, to block the signal pathway in activation of bone cells in severely inhibited the normal differentiation of.Akt into osteoblasts is the main downstream PI3K signaling pathway molecules in osteoblasts and. Osteoclasts, the expression of Aktl dominant Akt1 gene knockout mice found reduced bone mass, bone tissue morphometry showed bone formation and bone resorption activity were significantly decreased, two secondary ossification center formation delay; Akt1-/- wild type mouse osteoblastic cell differentiation index (such as ALP, type I collagen, osteocalcin expression etc.) were reduced. In addition, Akt and bone morphogenetic protein 2 co regulation of mesenchymal cells to differentiate into bone cells. Osteoporosis is not only related with body weight and total body fat, and also closely related to bone marrow mesenchymal cells. Mesenchymal stem cells (Mesenchymal stem cells, MSCs) have the potential of multi-directional differentiation. If MSCs differentiation into adipocytes increased, will lead to the reduction of osteoblastic differentiation, the bone marrow cells and fat cells showed competitive negative correlation. The fat factor hormone, PPAR, gamma stimulation can affect the osteogenic or MSCs adipogenic transformation and Mechanical properties. The inhibition of osteoblasts in signaling pathway can promote the conversion of MSCs into adipocytes. Caused by osteoporosis vertebral compression fracture (OVCFs) is very common, seriously affecting the quality of life of patients has brought heavy economic burden to family and society over the past ten years. In the pharmaceutical company has developed a series of osteoporosis drugs can increase bone mass. These drugs mainly in promoting bone formation (such as teriparatide, strontium salts) or inhibit bone resorption (such as bisphosphonates) play therapy Treatment of osteoporosis. Although these drugs significantly, the curative effect of osteoporosis but fever, pain exacerbated side reactions are coming. Chinese herbal medicine can be used together with existing drugs in the treatment of osteoporosis caused by vertebral compression fractures. The first part of the insulin downstream signal factor PI3K, Akt1 Akt2 NF and inflammatory factor kappa B in type 2 diabetic osteoporosis rat liver, kidney and skeletal muscle tissue expression objective: To explore the insulin signaling pathway downstream of PI3K, Akt1, Akt2 and the expression of inflammatory cytokines NF kappa B whether changes in type 2 diabetic patients with osteoporosis. Methods: 100 healthy female Wistar rats (certificate number 905105,2.5-3 months) after 1 weeks of feeding were randomly divided into normal control group (group NS, N=24), ovariectomized group (NOVX group, N=26), type 2 diabetes (DS group, N=24), ovariectomized group (type 2 diabetes D Group OVX, N=26). The T2DM rat model by high-fat diet and low dose STZ intraperitoneal injection, OP rats were bilaterally ovariectomized. Rats were sacrificed 12 weeks after ovariectomy, anesthesia, bilateral liver, kidney and muscle tissues of the right femoral head four, paraffin section and RNA extraction and the total protein of tissue were made, using immunohistochemical method to detect the NS and western of blot group, NOVX group, DS group, DOVX group PI3K, Akt1, Akt2, expression of NF kappa B protein, reverse transcription polymerase chain reaction (RT-PCR) method for detection of PI3K, Akt1, Akt2, m RNA expression of NF kappa B to deal with the experimental data. The application of SPSS13.0 statistical software, all experimental data were used to mean standard deviation (x + s) said that the measurement data were compared by analysis of variance, P0.05 was considered statistically significant. Results: immunohistochemistry of rat liver, kidney, skeletal muscle PI3K, Akt1, Akt2 ,NF魏B鍒嗗埆鍦ㄥぇ榧犺倽緇嗚優(yōu)鑳?yōu)娴?鑲懼皬綆′笂鐨粏鑳?yōu)鑳?yōu)嫻嗗強(qiáng)楠ㄩ鑲岀粏鑳?yōu)鑳?yōu)嫻嗕腑琛ㄨ揪.鍚勭粍緇囧厤鐤粍鍖栫粨鏋滄樉紺，

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