本文選題：磷酸鈣 + 碳酸鈷�。� 參考：《南方醫(yī)科大學(xué)》2017年碩士論文

【摘要】：實(shí)驗(yàn)?zāi)康?低氧誘導(dǎo)因子(HIF-1α)是調(diào)控組織和細(xì)胞適應(yīng)低氧環(huán)境的核心轉(zhuǎn)錄因子,二價鈷離子作為低氧誘導(dǎo)劑可以明顯促進(jìn)其表達(dá)。本實(shí)驗(yàn)將微量元素鈷負(fù)載于磷酸鈣骨水泥材料中,以持久模擬細(xì)胞缺氧狀態(tài),誘導(dǎo)細(xì)胞自主產(chǎn)生促血管因子,如:VEGF-A等,可望研制出具有超長誘導(dǎo)血管生成活性的可降解骨修復(fù)材料,促進(jìn)材料周圍組織的血管大量形成,進(jìn)而有利于新骨的生成及改建,加快骨缺損修復(fù)的進(jìn)程。實(shí)驗(yàn)方法:1、制備并表征該材料的可注射性、凝固時間、降解性能及鈷離子緩釋效果。2、評價材料的細(xì)胞毒性與粘附性及其對細(xì)胞形態(tài)及骨架的影響3、實(shí)時熒光定量PCR檢測MC3T3-E1細(xì)胞中HIF-1α的表達(dá)情況,ELISA檢測VEGF-A的表達(dá)情況,觀察HUVEC成管效果。4、通過組織病理學(xué)和免疫組化方法評估體內(nèi)成血管成骨能力。實(shí)驗(yàn)結(jié)果:1、骨水泥早期可注射性未受到明顯的影響,推注時間超過3min時,隨著碳酸鈷含量的增加,可注射性逐漸變差且水泥初凝時間逐漸縮短,終凝時間無明顯變化;骨水泥降解率比較:0%1%5%10%;摻鈷的骨水泥均能夠緩釋鈷離子,且緩釋長達(dá)56天。2、0%、1%、5%及10%組細(xì)胞毒性依次為:100.4%、123.2%、96.7%、83.1%;所有細(xì)胞均能粘附,生長,不同濃度鈷離子促進(jìn)細(xì)胞增殖的增殖速率比較:1%0%5%10%;0%、1%、5%的細(xì)胞形態(tài)無明顯改變,10%組的細(xì)胞質(zhì)萎縮,突觸明顯減少,細(xì)胞形態(tài)變化較大。3、摻鈷磷酸鈣能夠模擬細(xì)胞缺氧狀態(tài);隨著碳酸鈷含量的提高,細(xì)胞表達(dá)VEGE-A的量逐漸增加;4h時的各組血管標(biāo)志物表達(dá)情況:空白對照組≈0%1%5%,6h后各組成管效果差距更加明顯,空白對照組0%1%5%。4、皮下免疫組化結(jié)果:1%組出現(xiàn)大量毛細(xì)血管,5%組血管密度最高,但管徑略小于1%組,其余各組血管很少;骨組織免疫組化結(jié)果:術(shù)后2月,空白組和0%組未見明顯毛細(xì)血管,1%組和5%組在新生骨質(zhì)內(nèi)可見明顯血管狀結(jié)構(gòu),5%組的毛細(xì)血管狀結(jié)構(gòu)密度最高。術(shù)后3個月,血管狀結(jié)構(gòu)退化,1%和5%組新生骨生長充分,此外,5%組可見相當(dāng)成熟的哈弗氏管;骨組織HE染色:成骨效果比較可見,空白對照組0%1%5%組;骨組織Masson染色:空白對照和0%組骨膠原稀疏,1%和5%組骨膠原大量充滿缺損處,其中5%組新生的骨膠原更加密實(shí)、成熟,已可見明顯的哈弗氏管。結(jié)論:1、采用微量金屬元素鈷元素提高磷酸鈣材料的生物學(xué)功能具有一定的可行性。2、少量的碳酸鈷(≤1%)有利于細(xì)胞的增殖粘附,明顯提高材料的生物相容性;但是,大量的碳酸鈷(≥10%)反而增加該水泥的細(xì)胞毒性,抑制細(xì)胞增殖粘附。3、摻鈷的磷酸鈣骨水泥均能夠明顯促進(jìn)細(xì)胞分泌VEGF-A并直接作用于血管內(nèi)皮細(xì)胞,促進(jìn)其增殖、遷移、分化及芽生毛細(xì)血管。4、在動物體內(nèi),摻鈷的磷酸鈣骨水泥能夠明顯促進(jìn)局部毛細(xì)血管再生及骨膠原的合成,加快骨痂的生成、改建和重塑。
[Abstract]:Objective: hypoxia inducible factor (HIF-1 偽) is a core transcription factor regulating the adaptation of tissues and cells to hypoxia. In this experiment, the trace element cobalt was loaded into calcium phosphate cement material to simulate the anoxic state of cells and induce the spontaneous production of vascular stimulating factors, such as: VEGF-A, et al. It is expected that a biodegradable bone repair material with super-long angiogenic activity will be developed, which can promote the formation of blood vessels in the surrounding tissues of the material, thus facilitate the formation and remodeling of new bone and accelerate the process of bone defect repair. Experimental method: 1, preparation and characterization of the injectability, solidification time of the material, Biodegradability and sustained release effect of cobalt ion. The cytotoxicity and adhesion of the materials and their effects on cell morphology and cytoskeleton were evaluated. The expression of HIF-1 偽 in MC3T3-E1 cells was detected by real-time fluorescence quantitative PCR and the expression of VEGF-A in MC3T3-E1 cells was detected by Elisa. To observe the effect of HUVEC tube formation. 4. To evaluate the ability of vascular formation by histopathology and immunohistochemistry. The results showed that the early injectability of bone cement was not affected obviously. When the injection time was longer than 3min, the injectability became worse with the increase of cobalt carbonate content, and the initial setting time of cement decreased gradually, but the final setting time did not change obviously. The degradation rate of bone cement was 1: 015. The bone cement doped with cobalt was able to release cobalt ion slowly, and the cytotoxicity of 5% and 10% groups was 100.4% and 123.2% respectively. All the cells were able to adhere and grow. Comparison of proliferation rates of different concentrations of cobalt ions on cell proliferation; the cell morphology of 5% of the cells in the 10% group did not change significantly, the synapses decreased significantly, and the morphology of the cells changed significantly. 3. Calcium cobalt phosphate could mimic the anoxic state of the cells. With the increase of cobalt carbonate content, the expression of VEGE-A in the cells increased gradually at 4 h. The difference in the effect of each component tube was more obvious in the blank control group 鈮，

本文編號：2058497