本文選題：干細(xì)胞 + 股骨頭壞死　； 參考：《北京協(xié)和醫(yī)學(xué)院》2015年博士論文

【摘要】：研究背景：激素性股骨頭壞死是骨科常見(jiàn)疾病,其致殘率高,保守治療效果欠佳。激素導(dǎo)致骨髓間充質(zhì)干細(xì)胞成骨分化能力下降是其中的一個(gè)重要機(jī)制。課題組前期通過(guò)microRNA芯片技術(shù),篩選出激素性股骨頭壞死患者與正常健康人骨髓間充質(zhì)干細(xì)胞增殖分化過(guò)程中miRNA表達(dá)譜的差異特征,并將其與正常健康人骨髓間充質(zhì)干細(xì)胞經(jīng)大劑量激素刺激前后的miRNA表達(dá)差異譜進(jìn)行比較,找到表達(dá)變化一致的miRNAs,為microRNA-23a,并進(jìn)一步構(gòu)建microRNA-23a mimics和inhibitor,進(jìn)一步發(fā)現(xiàn)上調(diào)microRNA-23a可以導(dǎo)致骨髓間充質(zhì)干細(xì)胞成骨分化減弱,下調(diào)microRNA-23a能夠促進(jìn)BMSC成骨分化。利用生物信息學(xué)分析結(jié)合雙熒光素酶報(bào)告基因系統(tǒng)預(yù)測(cè)低密度脂蛋白相關(guān)蛋白5(low density lipoprotein receptor-related protein 5, LRP-5)為miRNA-23a的靶基因,使用shRNA沉默LRP5基因后可觀察到BMSC成骨分化減弱,與上調(diào)miRNA-23a類似。激素性股骨頭壞死壞死目前仍沒(méi)有特效藥物,研究發(fā)現(xiàn),間斷小劑量應(yīng)用甲狀旁腺激素能夠促進(jìn)成骨細(xì)胞生成,促進(jìn)干細(xì)胞向成骨方向分化。人重組甲狀旁腺激素[rhPTH (1-34)]是目前唯一批準(zhǔn)上市的具有促進(jìn)骨合成的抗骨質(zhì)疏松藥物,除治療骨質(zhì)疏松外,在促進(jìn)骨折愈合、促進(jìn)脊柱融合、促進(jìn)軟骨修復(fù)及下頜骨壞死都顯示出了良好治療效果。激素性股骨頭壞死的發(fā)病機(jī)制包括激素抑制干細(xì)胞成骨分化,我們推測(cè),人重組甲狀旁腺激素[rhPTH(1-34)]是否也可以通過(guò)促進(jìn)成骨細(xì)胞生成,促進(jìn)干細(xì)胞向成骨方向分化的作用來(lái)治療股骨頭壞死。研究目的：1.構(gòu)建microRNA-23a mimics和inhibitor,轉(zhuǎn)染大鼠骨髓間充質(zhì)于細(xì)胞。將轉(zhuǎn)染成功的大鼠骨髓間充質(zhì)干細(xì)胞注射至大鼠激素性股骨頭壞死模型體內(nèi)。評(píng)價(jià)microRNA-2 3a調(diào)控BMSC對(duì)大鼠股骨頭壞死的修復(fù)作用。2.研究目前唯一的骨合成類抗骨質(zhì)疏松藥物特立帕肽對(duì)大鼠股骨頭壞死的治療作用。研究方法：1.第一部分：全骨髓貼壁培養(yǎng)法分離并培養(yǎng)大鼠骨髓間充質(zhì)干細(xì)胞并進(jìn)行鑒定。采用慢病毒法攜帶microRNA-23a mimics,inhibitor,vector轉(zhuǎn)染大鼠骨髓間充質(zhì)干細(xì)胞。使用脂多糖(LPS,20μg/Kg×2)聯(lián)合大劑量甲強(qiáng)龍(40mg/Kg×3)的方法建立大鼠激素性股骨頭壞死動(dòng)物模型。將miRNA-23a mimics, inhibitor, vector轉(zhuǎn)染成功后的BMSC通過(guò)大鼠股骨髓腔注射的方法將干細(xì)胞懸液移植到大鼠激素性股骨頭壞死模型體內(nèi)。處死大鼠取大鼠股骨頭行micro CT掃描及病理切片,觀察股骨頭壞死情況。免疫組化檢測(cè)LRP-5在各組表達(dá)情況,觀察股骨頭壞死發(fā)生率和LRP5表達(dá)的差異。2.第二部分：首先應(yīng)用脂多糖(LPS,20μg/Kg×2)聯(lián)合大劑量甲強(qiáng)龍(40mg/Kg×3)的方法建立大鼠激素性股骨頭壞死動(dòng)物模型。大鼠造模成功后,實(shí)驗(yàn)組給予人重組甲狀旁腺激素[rhPTH (1-34)] 20μg/Kg每天皮下注射,對(duì)照組給予等量生理鹽水,治療4周處死大鼠,取大鼠靜脈血行血清骨轉(zhuǎn)換標(biāo)志物骨鈣素檢測(cè),取雙側(cè)股骨頭行micro CT檢查,觀察股骨頭壞死情況及分析骨小梁參數(shù),行病理切片觀察骨壞死情況。研究結(jié)果：1.采用全骨髓貼壁培養(yǎng)法可快速簡(jiǎn)便的分離培養(yǎng)大鼠骨髓間充質(zhì)干細(xì)胞,大鼠BMSC在體外可誘導(dǎo)成骨及成脂方向分化,流式細(xì)胞儀分析結(jié)果顯示表面CD29,CD44,CD105,CD106 陽(yáng)性, CD31,CD34,HLA-DR陰性。采用慢病毒法可成功轉(zhuǎn)染大鼠BMSC。脂多糖(LPS,20μg/Kg×2)聯(lián)合大劑量甲強(qiáng)龍(40mg/Kg×3)的方法可成功建立大鼠激素性股骨頭壞死動(dòng)物模型,造模成功率83%。Micro CT分析顯示模型組股骨頭出現(xiàn)囊變,骨小梁參數(shù)分析顯示模型組骨小梁體積、骨小梁厚度等參數(shù)明顯低于對(duì)照組。HE染色可觀察到空骨陷窩形成、骨髓壞死、骨小梁斷裂纖維組織修復(fù)。注射轉(zhuǎn)染microRNA23a inhibitor BMSC組BV/TV、Tb.Th明顯高于mimics組,而對(duì)照組介于兩者之間,骨小梁數(shù)量及骨皮質(zhì)厚度兩組差異沒(méi)有統(tǒng)計(jì)學(xué)意義。microPNA-23a mimics組股骨頭壞死發(fā)生率明顯高于inhibitor組(75%vs.18.2%,P=0.024),免疫組化顯示microRNA-23a mimics組LRP5表達(dá)低于inhibitor組。2.人重組甲狀旁腺激素[rhPTH(1-34)]20μg/Kg×28天治療組股骨頭壞死率為16.7%(2/12),對(duì)照組股骨頭壞死率為75%(9/12),兩組差異有統(tǒng)計(jì)學(xué)意義。(P0.05),骨小梁參數(shù)定量分析顯示BV/TV、Tb.Th、BMD治療組明顯高于對(duì)照組。結(jié)論：1.microRNA-23a在大鼠體內(nèi)能夠調(diào)控BMSC成骨分化能力,從而對(duì)大鼠激素性股骨頭壞死呈現(xiàn)不同程度的修復(fù)效應(yīng)。抑制microRNA-23a,股骨頭壞死發(fā)生率降低,骨小梁參數(shù)BV/TV、Tb.Th升高,LRP5表達(dá)升高,micRNA-23a表達(dá)升高則呈現(xiàn)相反的效應(yīng)。2.人重組甲狀旁腺激素[rhPTH (1-34)]20μg/Kg×28天皮下注射能夠降低大鼠激素性股骨頭壞死率,提高骨小梁體積等相關(guān)參數(shù)。
[Abstract]:Background: steroid induced osteonecrosis of the femoral head is a common disease in the Department of orthopedics. The rate of disability is high and the effect of conservative treatment is not good. The decrease of osteogenic differentiation of bone marrow mesenchymal stem cells is an important mechanism. In the earlier period, the microRNA chip technology was used to screen out the bone marrow of the patients with irritable necrosis of the femoral head and the normal healthy human bone marrow. The differential characteristics of miRNA expression profiles during the proliferation and differentiation of mesenchymal stem cells were compared with the miRNA expression profiles of normal healthy human bone marrow mesenchymal stem cells before and after the large dose of hormone stimulation, to find the miRNAs which was consistent with the expression of microRNA-23a, and to construct microRNA-23a mimics and inhibitor further. Up regulation of microRNA-23a can lead to osteogenic differentiation in bone marrow mesenchymal stem cells, and down regulation of microRNA-23a to promote BMSC osteogenesis. Using bioinformatics analysis and double luciferase reporter gene system to predict low density lipoprotein related protein 5 (low density lipoprotein receptor-related protein 5, LRP-5) is miRNA-23a The target gene, after shRNA silencing the LRP5 gene, can be observed that the osteogenic differentiation of BMSC is weakened and is similar to the up regulation of miRNA-23a. There is still no special drug for the necrosis and necrosis of the femoral head. The study found that intermittent small dose of parathyroid hormone can promote osteoblast formation and promote the differentiation of stem cells into osteogenic direction. Adenohormone [rhPTH (1-34)] is the only approved anti osteoporosis drug to promote bone synthesis at present. Except for the treatment of osteoporosis, it has shown good therapeutic effect in promoting fracture healing, promoting spinal fusion, promoting cartilage repair and mandibular necrosis. The pathogenesis of steroid induced osteonecrosis includes steroid suppression. We speculate whether recombinant parathyroid hormone [rhPTH (1-34)] can also promote osteonecrosis of the femoral head by promoting osteoblast formation and promoting osteonecrosis of stem cells to osteogenesis. Purpose: 1. to construct microRNA-23a mimics and inhibitor, and to transfer the bone marrow mesenchymal to cells. Rat bone marrow mesenchymal stem cells injected into the rat model of steroid necrosis of the femoral head. Evaluation of the effect of microRNA-2 3A on the repair of the femoral head necrosis in rats..2. research is the only therapeutic effect of the bone synthetic anti osteoporosis drug stranmpeptide on the necrosis of the femoral head in rats. 1. part 1: the first part: the whole bone marrow The rat bone marrow mesenchymal stem cells were isolated and cultured with adherent culture method. The rat bone marrow mesenchymal stem cells were transfected with microRNA-23a mimics, inhibitor and vector with lentivirus method. The rat model of steroid femoral head necrosis was established by using lipopolysaccharide (LPS, 20 u g/Kg x 2) combined with large dose of methylprednisolone (40mg/Kg x 3). After transfection of miRNA-23a mimics, inhibitor and vector, BMSC was injected into the rat femoral head necrosis model in rat femoral bone marrow cavity. The rat femoral head was killed by micro CT scan and pathological section, and the necrosis of the femoral head was observed. Immunohistochemistry was used to detect the expression of LRP-5 in each group. The difference between the incidence of osteonecrosis of the femoral head and the difference of LRP5 expression was observed in.2. second part: first, a rat model of steroid necrosis of the femoral head was established by using lipopolysaccharide (LPS, 20 mu g/Kg x 2) combined with large dose of methylprednisolone (40mg/Kg * 3). After the rat model was successful, the experimental group was given a recombinant parathyroid hormone [rhPTH (1-34)] 20 u g/Kg each. In the control group, the control group was given the same amount of normal saline, and the rats were sacrificed for 4 weeks, and the blood serum bone conversion markers were detected for 4 weeks. The bilateral femoral head was examined by the osteonecrosis of the femoral head and the parameters of the bone trabecula were observed. The results of pathological section were observed. 1. the results of the study were all bone marrow adherence. Rat bone marrow mesenchymal stem cells could be isolated and cultured quickly and easily. The rat BMSC could induce osteogenesis and lipid differentiation in vitro. Flow cytometry showed that the surface CD29, CD44, CD105, CD106 positive, CD31, CD34, HLA-DR negative. The rat BMSC. lipopolysaccharide (LPS, 20 mu g/Kg x 2) could be successfully transfected by the lentivirus method. The method of dose methylprednisolone (40mg/Kg x 3) could successfully establish the rat model of steroid femoral head necrosis. The success rate of the model group 83%.Micro CT analysis showed that the femoral head appeared cystic change in the model group. The bone trabecular parameter analysis showed that the size of the bone trabecular bone in the model group and the bone small Liang Houdu parameters were obviously lower than the control group.HE staining to observe the empty lacunae. Osteonecrosis, bone trabecular fracture and fibrous tissue repair. MicroRNA23a inhibitor BMSC group BV/TV, Tb.Th was significantly higher than group mimics, while the control group was between the two groups, the number of trabecular bone and the thickness of bone cortex of two groups were not statistically significant, the incidence of necrosis of the femoral head in.MicroPNA-23a mimics group was significantly higher than that of the inhibitor group (75%vs). .18.2%, P=0.024), immunohistochemical staining showed that the expression of LRP5 in group microRNA-23a mimics was lower than that of inhibitor group.2. human recombinant parathyroid hormone [rhPTH (1-34)]20 micron g/Kg x 28 days, the necrosis rate of femoral head was 16.7% (2/12), and the necrosis rate of the femoral head in the control group was 75% (9/12), and the quantitative analysis of the bone trabecular parameters showed that the two groups were statistically significant. TV, Tb.Th, BMD treatment group was significantly higher than the control group. Conclusion: 1.microRNA-23a can regulate the osteogenic differentiation ability of BMSC in rats, thus to the hormone induced osteonecrosis in rats to different degree of repair effect. Inhibition of microRNA-23a, the incidence of necrosis of the femoral head decreased, bone trabecular parameters BV/TV, Tb.Th, LRP5 expression increases, micRNA-23a, micRNA-23a. The increase in expression showed the opposite effect of.2. human recombinant parathyroid hormone [rhPTH (1-34)]20 mu g/Kg for 28 days, which could reduce the rate of steroid necrosis of the femoral head and improve the volume of trabecular bone.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R681.8
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本文編號(hào)：1856836