本文選題：17β-雌二醇 + 淫羊藿苷��； 參考：《河北北方學(xué)院》2017年碩士論文

【摘要】：因創(chuàng)傷、腫瘤、骨質(zhì)疏松等造成的骨不連、骨缺損等一直是骨科醫(yī)師需要解決的難題,骨組織工程學(xué)的長足發(fā)展有望在未來攻克這一問題。任何骨組織工程骨構(gòu)建均由種子細(xì)胞、培養(yǎng)環(huán)境與支架材料組成。骨髓間充質(zhì)干細(xì)胞(BMSCs),是來源于中胚層的一類干細(xì)胞,其在體外具有較大的可增殖能力和可多方向分化潛能,存在于骨髓內(nèi)并具有明顯的貼壁能力,可分化為成骨、心肌、脂肪等多種細(xì)胞。其在骨代謝調(diào)節(jié)中起著較為關(guān)鍵的作用。機(jī)體增加和維持骨量的細(xì)胞來源主要為BMSCs分化成的成骨細(xì)胞,是人體骨骼維持健康的重要因素。BMSCs比較容易獲得,體外擴(kuò)增后仍無免疫原性,且易轉(zhuǎn)染外緣性基因并能長期傳代表達(dá)。另外,應(yīng)用BMSCs進(jìn)行自體的移植實(shí)驗(yàn)和治療時幾乎不受倫理學(xué)限制。體外BMSCs的增殖分化可以受多因素的準(zhǔn)確調(diào)控,因此它一致被視為移植實(shí)驗(yàn)和治療首選的干細(xì)胞。絕經(jīng)后婦女骨質(zhì)疏松發(fā)病率明顯升高,絕經(jīng)后機(jī)體內(nèi)雌激素水平迅速下降是主要原因,激素替代療法(HRT)是該病最經(jīng)典的治療方案,17β-雌二醇能促進(jìn)BMSCs成骨分化,且體外濃度在10~(-9) mol/L時作用最為顯著。隨著祖國醫(yī)學(xué)的不斷發(fā)展進(jìn)步,由于“補(bǔ)腎壯骨”中藥在臨床治療骨質(zhì)疏松、骨不連等有明顯的療效。淫羊藿苷可提高堿性磷酸酶(ALP)活性,增加鈣化結(jié)節(jié)和堿性磷酸酶陽性克隆數(shù)(CFU-FALP)數(shù)量,促進(jìn)成纖維細(xì)胞生長因子(b FGF),類胰島素一號增長因子(IGF-1)的表達(dá),且淫羊藿苷可雙向調(diào)節(jié)對BMSCs的分化,即促進(jìn)BMSCs成骨性分化的同時又抑制了BMSCs成脂分化。但長期應(yīng)用雌激素的副作用較為顯著以及淫羊藿等中藥的作用又相對遲緩,因此本實(shí)驗(yàn)通過研究西藥成分17β-雌二醇聯(lián)合中藥淫羊藿提取物淫羊藿苷對大鼠骨髓間充質(zhì)干細(xì)胞體外成骨分化誘導(dǎo)的作用是否有協(xié)同作用,在降低藥量的同時提高骨髓間充質(zhì)干細(xì)胞向成骨細(xì)胞的分化效率,為臨床用藥提供理論依據(jù)。本實(shí)驗(yàn)所需的BMSCs均提取于3周齡大小的清潔級SD大鼠的長骨骨髓,運(yùn)用全骨髓貼壁法進(jìn)行培養(yǎng)。鏡下可見初分離的大鼠骨髓懸液細(xì)胞密集,12 h后細(xì)胞開始貼壁,部分細(xì)胞形態(tài)伸展、體積增大,貼附于瓶壁,72 h可見部分細(xì)胞伸出突起,突起圓鈍而光滑,細(xì)胞呈梭形、菱形或多邊形,核大而飽滿,外觀似成纖維細(xì)胞樣。培養(yǎng)14d后,細(xì)胞融合成片,分界變得較為模糊,胞體增大,形態(tài)則變?yōu)槎趟笮魏筒灰?guī)則形,部分細(xì)胞聚集成灶,21 d天后觀察誘導(dǎo)細(xì)胞部分出現(xiàn)散在的礦化結(jié)節(jié),其中心透光度較差。既有研究表明:BMSCs可表達(dá)CD29、CD50、CD90等間充質(zhì)細(xì)胞系及內(nèi)皮細(xì)胞系的表面抗原,而不表達(dá)CD14、CD45等造血細(xì)胞系表面抗原。本研究運(yùn)用流式細(xì)胞儀對BMSCs聯(lián)合表面抗原進(jìn)行鑒定,鑒定結(jié)果顯示,細(xì)胞表面抗原CD45陽性表達(dá)率為0.5%,細(xì)胞表面抗原CD29陽性表達(dá)率99.6%,細(xì)胞表面抗原CD90陽性表達(dá)率99.7%。其結(jié)果符合大鼠骨髓間充質(zhì)干細(xì)胞特征。取第4代BMSCs,消化后調(diào)整細(xì)胞密度至1×10~4/ml,接種于25ml培養(yǎng)瓶中,將培養(yǎng)細(xì)胞分為對照組(C、組)、17β-雌二醇作用組(A組)、淫羊藿苷組(B組)、聯(lián)合誘導(dǎo)組(A+B組)。第4代BMSCs培養(yǎng)24h后進(jìn)行定向誘導(dǎo),C組加入基礎(chǔ)誘導(dǎo)劑(地塞米松1×10~(-8)mol/L、β-甘油磷酸鈉1×10~(-2)mol/L、抗壞血酸5×10~(-2)g/L),A組加入基礎(chǔ)成骨誘導(dǎo)劑和17β-雌二醇1×10~(-9)mol/L,B組加入基礎(chǔ)成骨誘導(dǎo)劑及淫羊藿苷1×10~(-4)mol/L,A+B組加入基礎(chǔ)成骨誘導(dǎo)劑、17β-雌二醇1×10~(-9)mol/L及淫羊藿苷1×10~(-4)mol/L。持續(xù)誘導(dǎo)21天,隔天半量換液,7天傳代。將BMSCs以1×10~4個/ml接種到培養(yǎng)瓶中,按上述實(shí)驗(yàn)分組進(jìn)行成骨誘導(dǎo),各組細(xì)胞于成骨誘導(dǎo)第7天進(jìn)行ALP活性測定,各組細(xì)胞于成骨誘導(dǎo)第14天進(jìn)行COL-1相對濃度測定,各組細(xì)胞于成骨誘導(dǎo)第21天進(jìn)行BGP濃度測定,各組細(xì)胞分別于成骨誘導(dǎo)第21天進(jìn)行茜素紅鈣結(jié)節(jié)染色。結(jié)果顯示分別檢測到ALP、COL-1、BGP相對濃度為聯(lián)合組大于單因素誘導(dǎo)組,且單因素誘導(dǎo)組大于對照組,鈣結(jié)節(jié)計數(shù)為聯(lián)合組大于各單因素誘導(dǎo)組,各單因素誘導(dǎo)組均大于對照組,差別有統(tǒng)計學(xué)意義經(jīng)本實(shí)驗(yàn),能夠說明:第一、17β-雌二醇和中藥有效成分淫羊藿苷均可單獨(dú)誘導(dǎo)BMSCs體外分化為骨樣細(xì)胞;第二、二者聯(lián)合誘導(dǎo)的效果優(yōu)于各自單獨(dú)誘導(dǎo)。
[Abstract]:Bone nonunion and bone defect caused by trauma, tumor and osteoporosis have always been a difficult problem for Department of orthopedics physicians to solve. The rapid development of bone tissue engineering is expected to overcome this problem in the future. Any bone tissue engineering bone construction is composed of seed cells, culture environment and scaffold materials. Bone marrow mesenchymal stem cells (BMSCs) are the source of bone tissue engineering. A class of stem cells in the mesoderm, which have large proliferative and multidirectional differentiation potential in vitro, exist in the bone marrow and have obvious adhesion to the wall, and can differentiate into bone, myocardium, and fat cells. It plays a key role in the regulation of bone metabolism. The main source of cells to increase and maintain bone mass is the main source. Osteoblast differentiated from BMSCs is an important factor for maintaining the health of human skeleton,.BMSCs is easy to obtain. After amplification in vitro, there is still no immunogenicity, and it is easy to transfect the external gene and can be expressed for a long time. In addition, the use of BMSCs for autologous transplantation and treatment is not subject to ethical restriction. In vitro, the proliferation and differentiation of BMSCs in vitro It can be regulated by multiple factors, so it is considered to be the first choice of stem cells for transplantation experiments and treatment. The incidence of osteoporosis in postmenopausal women is significantly higher, and the rapid decline in estrogen levels in postmenopausal women is the main reason. Hormone replacement therapy (HRT) is the most classical treatment for the disease, and 17 beta estradiol can promote BMSCs osteogenesis. Differentiation, and in vitro concentration in 10~ (-9) mol/L, the effect is most significant. With the continuous development and progress of Chinese medicine, the Chinese medicine of "kidney tonifying and strong bone" has obvious effect on the clinical treatment of osteoporosis and bone nonunion. Icariin can increase the activity of alkaline phosphatase (ALP), increase the number of calcified nodules and alkaline phosphatase positive clones (CFU-FALP). Quantity, promoting the expression of fibroblast growth factor (B FGF) and insulin like growth factor (IGF-1), and icariin can regulate the differentiation of BMSCs by two way, that is to promote the differentiation of BMSCs into osteogenic differentiation while inhibiting the lipid differentiation of BMSCs, but the side effect of the long-term use of estrogen is more significant and the effect of epimedium and other Chinese herbs is also phase In this experiment, the effect of 17 beta estradiol and epimedium extract of Western medicine and epimedium extract of Epimedium extract on bone differentiation induction in vitro of rat bone marrow mesenchymal stem cells was studied. Theoretical basis. The BMSCs needed in this experiment was extracted from the bone marrow of the long bone of the clean grade SD rats of 3 weeks old. It was found that the bone marrow suspension cells of the first separated rats were dense, and the cells began to stick to the wall after 12 h. Some cells were extended, the volume was enlarged, the cells were attached to the bottle wall, and the 72 h visible part of the cells protruded out. The protuberance is blunt and smooth, the cells are spindle shaped, rhombic or polygonal, the nucleus is large and full, and the appearance is like fibroblast like. After 14d, the cells fuse into pieces, the boundary becomes more blurred, the cell body increases, the morphology becomes short shuttle and irregular, some cells gather together, and 21 d days after the observation of the scattered mineralization of the induced cells. BMSCs can express the surface antigens of CD29, CD50, CD90 and other mesenchymal cells and endothelial cells, but not the surface antigens of CD14, CD45 and other hematopoietic cells. This study uses flow cytometry to identify the joint surface antigen of BMSCs, and the identification results show that the cell surface antigen CD45 Yang The expression rate was 0.5%, the positive expression rate of cell surface antigen CD29 was 99.6%, and the positive expression rate of CD90 of cell surface antigen was 99.7%.. The result accorded with the characteristics of rat bone marrow mesenchymal stem cells. Take fourth generation of BMSCs, and adjust the cell density to 1 x 10~4/ml after digestion and inoculated in the 25ml culture bottle. The cultured cells were divided into the control group (C, group), and 17 beta estradiol. Group (group A), icariin group (group B), combined induction group (group A+B). After the fourth generation of BMSCs culture, 24h was directed, and C group added basic inducer (dexamethasone 1 x 10~ (-8) mol/L, beta glycerol sodium 1 x 10~ (-2) mol/L, 5 x of ascorbic acid), and the group joined the basic osteogenic inducer and 17 beta estradiol 1 x. The bone inducer and icariin 1 x 10~ (-4) mol/L, A+B group added basic osteogenic inducer, 17 beta estradiol 1 x 10~ (-9) mol/L and icariin 1 x 10~ (-4) mol/L. continued to be induced for 21 days, half a half amount of liquid and 7 days to be passed. The BMSCs was inoculated into the culture bottle by 1 * 10~4, and the cells were induced by the above-mentioned experiments. The cells in each group were induced by osteogenesis. The activity of ALP was measured at seventh days. The relative concentration of COL-1 was measured at fourteenth days after osteogenesis induction. The cells in each group were measured at twenty-first days of osteogenesis induction. The cells of each group were stained with alizarin red calcium nodules on twenty-first days of osteogenesis induction. The results showed that the relative concentration of ALP, COL-1, and BGP was greater than the single cause, respectively. The single factor induction group was larger than the control group, and the calcium nodule count was larger than the single factor induction group, and the single factor induction group was larger than the control group. The difference has statistical significance. The first, 17 beta estradiol and the effective component of Chinese medicine can induce BMSCs to differentiate into bone like cells in vitro. The combined effect of the second, second methods is better than that of the separate induction.
【學(xué)位授予單位】：河北北方學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R68

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