本文選題：藥物復(fù)合聚甲基丙烯酸甲酯骨水泥　切入點(diǎn)：甲氨蝶呤　出處：《武漢大學(xué)》2016年博士論文

【摘要】：目的：篩選出熱穩(wěn)定性佳且可被復(fù)合入聚甲基丙烯酸甲酯(PMMA)骨水泥的化療藥；觀察甲氨蝶呤(MTX)和鹽酸表柔比星(EPI)分別或者聯(lián)合與PMMA骨水泥以不同質(zhì)量比例混合制成的復(fù)合化療藥PMMA骨水泥的機(jī)械強(qiáng)度和顯微結(jié)構(gòu),探索MTX和EPI可復(fù)合入PMMA骨水泥的較大添加量；觀察MTX和EPI復(fù)合PMMA骨水泥的藥物體外模擬釋放特性和釋放藥物活性,并對比釋放前后骨水泥機(jī)械強(qiáng)度和顯微結(jié)構(gòu)的變化；為制作可臨床應(yīng)用的MTX和EPI復(fù)合PMMA骨水泥提供理論依據(jù)。方法：1、將不同濃度的實(shí)驗(yàn)組MTX (10、20、40、80、160μg/ml)和EPI(1.25、2.5、5、10、20μg/ml)溶液在75℃的條件下熱處理10min之后,同時對照組在室溫下靜置10min之后,分別將上述藥品溶液加入MG63和U2OS兩種細(xì)胞中培養(yǎng)24、48h后使用CCK8試劑盒進(jìn)行細(xì)胞毒性檢測。2、分別將MTX與PMMA骨水泥以1%、2%、4%的質(zhì)量比復(fù)合；EPI與PMMA骨水泥以0.1%、0.2%、0.4%的質(zhì)量比復(fù)合； MTX聯(lián)合EPI兩種藥物與PMMA骨水泥以1%MTX+O.1%EPI、2%MTX+0.2%EPI、4%MTX+0.4%EPI的質(zhì)量比復(fù)合,以及未復(fù)合藥品的骨水泥分別制成骨水泥標(biāo)準(zhǔn)試件分別檢測計(jì)算其機(jī)械性能(抗壓強(qiáng)度、抗彎強(qiáng)度和抗彎模量)及觀察電鏡下微結(jié)構(gòu)的改變。3、分別將MTX與PMMA骨水泥以2%的質(zhì)量比復(fù)合、EPI與PMMA骨水泥以0.1%的質(zhì)量比復(fù)合的PMMA骨水泥制成圓柱形骨水泥測試標(biāo)準(zhǔn)試件各5個,稱量并記錄每一個標(biāo)準(zhǔn)試件的質(zhì)量。將每個標(biāo)準(zhǔn)試件放入10ml的離心管中,加入10ml的37℃生理鹽水,每隔24h換液,分別將第1、2、3、5、7、10、15、20、30天時的釋放洗脫液置入-20℃冰箱中保存。使用高效液相色譜儀對置換出來的液體進(jìn)行定性及定量測試。4、將定量定性測試后的藥物釋放洗脫液加入143b細(xì)胞系中培養(yǎng)24h,同對照組作對比,采用CCK8試劑盒檢測其藥物活性。5、分別將MTX與PMMA骨水泥以2%的質(zhì)量比復(fù)合、EPI與PMMA骨水泥以0.1%的質(zhì)量比復(fù)合的PMMA骨水泥制成圓柱體和長方體骨水泥測試標(biāo)準(zhǔn)試件各5個。將每個標(biāo)準(zhǔn)試件放入10ml的離心管中,加入10ml的37℃生理鹽水,并且每隔24h換液,持續(xù)5個月后檢測并計(jì)算其機(jī)械性能(抗壓強(qiáng)度、抗彎強(qiáng)度和抗彎模量)及電鏡下微結(jié)構(gòu)的改變。結(jié)果：1、不同濃度的兩種化療藥在進(jìn)行75℃處理后,分別于24h和48h采用CCK8試劑盒測試,方差分析其結(jié)果顯示各組MTX和EPI熱處理后細(xì)胞毒性實(shí)驗(yàn)結(jié)果同對照組差異無統(tǒng)計(jì)學(xué)意義(P0.05)。表明MTX和EPI在75℃下處理10分鐘后其活性均無明顯降低。2、MTX和EPI分別或聯(lián)合與PMMA骨水泥以質(zhì)量比不高于4%和0.4%復(fù)合后,復(fù)合骨水泥各項(xiàng)機(jī)械性能指標(biāo)均高于國際參考值(抗壓強(qiáng)度70MPa,抗彎強(qiáng)度50MPa,≥1800MPa)。以抗壓強(qiáng)度為因變量,采用回歸分析發(fā)現(xiàn),MTX和EPI與PMMA骨水泥復(fù)合質(zhì)量比對抗壓強(qiáng)度無顯著影響(P=0.120,P=0.951)。以抗彎強(qiáng)度為因變量,采用回歸分析發(fā)現(xiàn)MTX與PMMA骨水泥復(fù)合質(zhì)量比對抗彎強(qiáng)度無顯著影響(P=0.912)；EPI與PMMA復(fù)合質(zhì)量比對抗彎強(qiáng)度有顯著影響(P=0.029,13=2315.305),EPI在一定程度上可加強(qiáng)PMMA骨水泥的抗彎強(qiáng)度,以抗彎模量為因變量,采用回歸分析發(fā)現(xiàn),MTX和EPI與PMMA骨水泥復(fù)合質(zhì)量比對抗壓強(qiáng)度及抗彎模量無顯著影響(P=0.947,P=0.556)。電鏡觀察下未復(fù)合化療藥的PMMA骨水泥表面或斷面上分布著大量不規(guī)則孔隙；復(fù)合入MTX后的PMMA骨水泥表面或斷面上的不規(guī)則孔隙變淺變少;復(fù)合入EPI后的骨水泥表面或斷面上的不規(guī)則孔隙明顯變淺變少;隨著藥品量的增加,不規(guī)則空隙會進(jìn)一步變淺變少。3、MTX和EPI均可以從骨水泥中釋放洗脫出來,其中MTX在第一天釋放峰值出現(xiàn)之后便急劇下降后趨于平緩穩(wěn)定,EPI則始終較為穩(wěn)定,未有較高峰值出現(xiàn)。4、MTX和EPI在第三十天的釋放洗脫液加入143b培養(yǎng)細(xì)胞中培養(yǎng)24h,t檢驗(yàn)分析其結(jié)果顯示實(shí)驗(yàn)組和對照組細(xì)胞存活率差異有顯著統(tǒng)計(jì)學(xué)意義。說明骨水泥釋放藥物具有長期抗腫瘤作用。5、MTX與PMMA骨水泥以2%的質(zhì)量比復(fù)合、EPI與PMMA骨水泥以0.1%的質(zhì)量比復(fù)合的PMMA骨水泥在藥物釋放前后,復(fù)合骨水泥各項(xiàng)機(jī)械強(qiáng)度性能指標(biāo)均高于國際參考值(抗壓強(qiáng)度70MPa,抗彎強(qiáng)度50MPa,≥1800MPa)。同對照組相比較藥物復(fù)合骨水泥在藥物釋放洗脫后表面孔隙中的藥物顆粒明顯減少。結(jié)論：甲氨蝶呤(MTX)和鹽酸表柔比星(EPI)在PMMA骨水泥聚合時產(chǎn)生的長時高溫環(huán)境下生物活性不會明顯降低,可以被復(fù)合入PMMA骨水泥來制作化療藥復(fù)合骨水泥；分別或者聯(lián)合,MTX與PMMA骨水泥以不超過4%的質(zhì)量比復(fù)合,EPI與PMMA骨水泥以不超過0.4%的質(zhì)量比復(fù)合,不會對PMMA骨水泥的機(jī)械強(qiáng)度產(chǎn)生不利影響；骨水泥中的MTX和EPI可以長期穩(wěn)定地從骨水泥中釋放出來并具備抗腫瘤效果；PMMA骨水泥可作為藥品的良好載體。
[Abstract]:Objective: to screen a better thermal stability and can be mixed into polymethylmethacrylate (PMMA) bone cement chemotherapy; observation of methotrexate (MTX) and epirubicin hydrochloride (EPI) mechanical strength and microstructure of bone cement composite chemotherapy combined with PMMA or PMMA respectively with different ratio of bone cement mixed, exploration a large amount of MTX and EPI can be combined into PMMA bone cement; observation of MTX and EPI composite PMMA bone cement in vitro drug release characteristics and release of drug activity, and to compare the changes of bone cement mechanical strength and microstructure before and after release; for the production can provide a theoretical basis for the clinical application of MTX and EPI composite PMMA bone cement. Methods: 1, the experimental groups with different concentrations of MTX (10,20,40,80160 g/ml) and EPI (1.25,2.5,5,10,20 g/ml) solution after heat treatment 10min the temperature of 75 DEG C, and the control group at room temperature static The 10min, respectively, the drug solution was added to two kinds of MG63 and U2OS in cultured cells after 24,48h using CCK8 kit to detect the cytotoxic.2, respectively MTX and PMMA bone cement with 1%, 2%, 4%, the mass ratio of EPI and PMMA composite; bone cement with 0.1%, 0.2%, 0.4% mass ratio of the composite; MTX combined with EPI two kinds of drugs and PMMA bone cement with 1%MTX+O.1%EPI, 2%MTX+0.2%EPI, the mass ratio of 4%MTX+0.4%EPI composite bone cement composite, and no drugs were made from bone cement standard specimens were measured to calculate the mechanical properties (compressive strength, flexural strength and flexural modulus of.3 micro structure change) and observed under electron microscope, respectively. The MTX and PMMA bone cement with 2% mass ratio of EPI and PMMA composite bone cement with 0.1% of the mass ratio of PMMA bone cement composite cylindrical bone cement test standard test piece 5, weighing and recording every standard specimen quality. Each standard specimen in the 10ml centrifugal tube, 37 DEG C with saline 10ml, every 24h in liquid, respectively, the first 1,2,3,5,7,10,15,20,30 days of the release of elution preservation solution into the -20 C refrigerator. Using HPLC qualitative and quantitative test of.4 replacement out of the liquid, the qualitative and quantitative test of drugs the release of the eluent culture 24h joined the 143B cell line, compared with the control group, using CCK8 kit to detect the activity of drugs.5, MTX and PMMA respectively, with 2% of the mass ratio of bone cement composite, EPI and PMMA of bone cement with 0.1% of the mass ratio of PMMA bone cement composite made of cylindrical and rectangular bone cement standard test specimen of the 5. Each standard specimen in the 10ml centrifugal tube, 37 DEG C with saline 10ml, and every 24h was changed, continued 5 months after the test and calculate the mechanical properties (compressive strength, flexural strength And the flexural modulus) and change the micro structure under electron microscope. Results: 1. Two kinds of chemotherapeutic drugs at different concentrations of 75 DEG C after treatment, using CCK8 kit to test 24h and 48h respectively, the variance analysis results of each group of MTX and EPI after heat treatment, cell toxicity test results showed no difference with the control group meaning (P0.05). The results indicated that MTX and EPI at 75 DEG C for 10 minutes after the activity did not significantly decrease.2, MTX and EPI respectively or combined with PMMA bone cement in the ratio is not higher than 4% and 0.4% compound, the mechanical properties of bone cement composite index was higher than the international reference value (anti compressive strength 70MPa the bending strength is 50MPa, or 1800MPa). The dependent variable by compressive strength is found by regression analysis, no significant effect of MTX and EPI and PMMA composite bone cement ratio on compressive strength (P=0.120, P=0.951). The dependent variable bending strength was found, MTX and PMMA bone by regression analysis Composite cement mass ratio on flexural strength had no significant effect (P=0.912); EPI and PMMA composite quality have significant effect on the bending strength ratio (P=0.029,13=2315.305), EPI can enhance the flexural strength of PMMA bone cement in a certain extent, with the dependent variable bending modulus is found by regression analysis, no significant effect on MTX and EPI with PMMA bone cement ratio on the compressive strength and flexural modulus (P=0.947, P=0.556). Electron microscope is not combined with chemotherapy of PMMA bone cement surface or on the cross section of the distribution of a large number of irregular pores; composite bone cement into the PMMA section on the surface or irregular pores after MTX shallower and less into the composite; the cement surface or section of irregular pore after EPI significantly shallower and less; with the increase of drug consumption, irregular gap will further shallower and less.3, MTX and EPI were eluted can be released from bone cement, in which MTX After the first day of release peak decreased rapidly after gently stable, EPI remained stable, no high peak of.4, MTX and EPI on the thirtieth day of the release of the eluent into 143B in cultured 24h cells, t analysis showed statistically significant for the experimental group and the control group difference in survival rate. Bone cement release drugs have long-term antitumor effects of.5, MTX and PMMA to 2% mass ratio of bone cement composite, EPI and PMMA to 0.1% mass ratio of bone cement composite PMMA bone cement in drug release and the mechanical strength of the composite bone cement performance index are higher than the international reference value (anti compressive strength 70MPa the bending strength is 50MPa, or 1800MPa). Compared with the control group of drug particles in the surface pore drug release after elution of the drug composite bone cement was significantly reduced. Conclusion: Methotrexate (MTX) and epirubicin acid salt Star (EPI) in the PMMA bone cement polymerization long without biological activity under high temperature significantly reduced, can be incorporated into PMMA bone cement to make chemotherapy combined with bone cement; separately or in combination, MTX and PMMA of bone cement to no more than 4% of the mass ratio of EPI and PMMA composite bone cement with no more than 0.4% the mass ratio of composite, does not adversely affect the mechanical strength of PMMA bone cement; bone cement in MTX and EPI can be stably for a long period of time and released from the bone cement have anti-tumor effect; PMMA bone cement can be used as a good drug carrier.

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R943

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