【摘要】：背景骨折愈合是復(fù)雜的生物學(xué)修復(fù)過程,臨床上骨折延遲愈合／不愈合的間題仍未解決,傳統(tǒng)的中西藥物干預(yù)治療時間長,效果不理想。目前,促進(jìn)骨折愈合最好細(xì)胞因子是骨形態(tài)發(fā)生蛋白。實(shí)驗(yàn)研究發(fā)現(xiàn)骨形成蛋白具有誘導(dǎo)骨髓基質(zhì)細(xì)胞向成骨細(xì)胞分化的作用。海藻酸鈉(sodium alginate,SA)是一種從褐藻中提取的天然多糖聚合物,若能使SA部分糖醛酸單元的羥基轉(zhuǎn)變成醛基,即羧甲基海藻酸鈉(dicarboxmethyl trisodium alignate,DCMTSA),可改善SA的生物相容性,可作為誘導(dǎo)骨形態(tài)發(fā)生蛋白的有效藥物。目的改善SA的生物學(xué)特性,探討DCMTSA對骨形成蛋白表達(dá)的影響,明確DCMTSA對骨髓基質(zhì)細(xì)胞向成骨細(xì)胞分化促進(jìn)作用,提供一種制備方法簡單、原料來源廣泛的治療和預(yù)防骨折的藥物,為應(yīng)用于臨床預(yù)防和治療骨折等疾病提供實(shí)驗(yàn)室依據(jù)。方法1制備DCMTSA:SA經(jīng)過氧化氫溶液或高錳酸鉀部分氧化,部分糖醛酸單元的羥基轉(zhuǎn)變成醛基,改善SA的生物相容性。2動物模型制備及分組：45只體重(261±12)g,2-3月齡SD大鼠,制成創(chuàng)傷性股骨頭壞死模型后,以數(shù)字隨機(jī)法平均分為三組,每組15只。DCMTSA組,大鼠每日以DCMTSA 0.1 g.kg-1.d-1灌胃；SA組,大鼠每日以SA 0.1g.kg-1.d-1灌胃模型組；對照組,大鼠每天0.1g.kg-1.d-1生理鹽水灌胃。3實(shí)驗(yàn)方法：每組大鼠分別于術(shù)后7d、14d、21d各處死5只。取大鼠骨折股骨分為兩份：一份經(jīng)10%甲醛固定后脫鈣,HE染色光鏡下觀察各組骨折處骨痂生長組織結(jié)構(gòu),免疫組化SP法檢測骨形成蛋白一2(bone morphogenetie protein-2,BMP-2)骨形成蛋白-7(bone morphogenetie protein-7,BMP-7)的表達(dá)情況；一份脫鈣軟化后組織勻漿,以RT-PCR檢測BMP-2 mRNA和BMP-7 mRNA的表達(dá)。結(jié)果1 HE染色結(jié)果：術(shù)后7d、14d、21d,DCMTSA組大鼠骨折斷端骨組織的纖維組織增生,骨小梁形成,骨基質(zhì)形成及骨結(jié)構(gòu)的形成情況明顯優(yōu)于SA組,SA組優(yōu)于對照組。2免疫組化實(shí)驗(yàn)結(jié)果：術(shù)后7d、14d、21d,三組大鼠BMP-2和BMP-7蛋白表達(dá)DCMTSA組高于SA組,SA組高于對照組。3 RT-PCR檢測結(jié)果：術(shù)后21d,BMP-2和BMP-7蛋白mRNA的表達(dá)DCMTSA組高于SA組,SA組高于對照組。4股骨骨組織計量學(xué)結(jié)果：術(shù)后21d大鼠平均骨小梁數(shù)、平均骨小梁厚度和平均骨小梁面積DCMTSA組大于SA組,SA組大于對照組。且對照組的松骨質(zhì)和密骨質(zhì)容積明顯降低,骨小梁數(shù)目明顯減少,骨髓腔明顯擴(kuò)大,骨小梁厚度和骨皮質(zhì)變薄。結(jié)論DCMTSA對骨組織細(xì)胞釋放BMP-2和BMP-7的促進(jìn)作用大于SA,促進(jìn)成骨細(xì)胞分化,抑制破骨細(xì)胞造成的骨吸收,有利于創(chuàng)傷性股骨頭壞死大鼠的骨愈合。
[Abstract]:Background fracture healing is a complex biological repair process, the clinical delayed healing / non-union problem remains unresolved, the traditional Chinese and western medicine intervention treatment time is long, the effect is not ideal. At present, bone morphogenetic protein is the best cytokine to promote fracture healing. It was found that bone morphogenetic protein could induce bone marrow stromal cells to differentiate into osteoblasts. Sodium alginate (sodium alginate,SA) is a kind of natural polysaccharide polymer extracted from brown algae. The biocompatibility of SA can be improved if the hydroxyl group of part of SA uronic acid unit is converted into aldehyde group, that is, carboxymethyl alginate sodium (dicarboxmethyl trisodium alignate,DCMTSA. It can be used as an effective drug to induce bone morphogenetic protein. Objective to improve the biological characteristics of SA, to investigate the effect of DCMTSA on the expression of bone morphogenetic protein, to clarify the effect of DCMTSA on the differentiation of bone marrow stromal cells into osteoblasts, and to provide a simple preparation method. A wide range of raw materials for the treatment and prevention of fracture drugs, for clinical prevention and treatment of fracture and other diseases to provide laboratory basis. Methods (1) DCMTSA:SA was partially oxidized by hydrogen peroxide solution or potassium permanganate, and the hydroxyl groups of some uronic acid units were transformed into aldehydes, which improved the biocompatibility of SA. SD rats aged 2-3 months were made into traumatic femoral head necrosis model and were randomly divided into three groups with 15 rats in each group. The rats in the DCMTSA group were given DCMTSA 0.1 g.kg-1.d-1 daily. SA group, rats were fed with SA 0.1g.kg-1.d-1 daily; Rats in control group were treated with 0.1g.kg-1.d-1 normal saline daily. 3 Experimental methods: 5 rats in each group died at 14 days and 21 days after operation respectively. The femur of rat fracture was divided into two parts: one was decalcified after 10% formaldehyde fixation, the structure of callus growth was observed under light microscope with HE staining, and bone morphogenetic protein-2 (bone morphogenetie protein-2, was detected by immunohistochemical SP method. The expression of bone morphogenetic protein-7 (bone morphogenetie protein-7,BMP-7 (BMP-2); After decalcification and softening, the expression of BMP-2 mRNA and BMP-7 mRNA was detected by RT-PCR. Results 1 the results of HE staining showed that the fibrous tissue proliferation, trabecular formation, bone matrix formation and bone structure formation in the DCMTSA group were significantly better than those in the SA group. SA group was superior to control group. 2 Immunohistochemical results showed that the expression of BMP-2 and BMP-7 protein in DCMTSA group was higher than that in SA group, and that in SA group was higher than that in SA group. 3 the results of RT-PCR detection in SA group were higher than those in control group: 21 days after operation, 21 days after operation, the expression of BMP-2 and BMP-7 protein in three groups was higher than in SA group. The expression of BMP-2 and BMP-7 protein mRNA in DCMTSA group was higher than that in SA group, and that in SA group was higher than that in control group. 4 the average trabecular bone count, mean trabecular thickness and mean trabecular area in DCMTSA group were higher than those in SA group 21 days after operation. The SA group was larger than the control group. In the control group, the volume of cancellous bone and dense bone decreased significantly, the number of trabecular bone decreased, the medullary cavity enlarged, and the thickness of trabecular bone and cortical bone thinned. Conclusion DCMTSA can promote the release of BMP-2 and BMP-7 from bone tissue cells more than SA, can promote osteoblast differentiation and inhibit bone resorption caused by osteoclasts, which is beneficial to bone healing in rats with traumatic osteonecrosis of femoral head.
【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R683

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