本文選題：骨髓間充質(zhì)干細(xì)胞 + 牛松質(zhì)骨��； 參考：《天津醫(yī)科大學(xué)》2013年博士論文

【摘要】：第一部分去抗原牛松質(zhì)骨的制備方法與結(jié)構(gòu)分析 目的：探討去抗原牛松質(zhì)骨材料的制備方法并分析所制備材料的結(jié)構(gòu)特點(diǎn)。 方法：選取新鮮小牛肱骨近端松質(zhì)骨鋸為8mm×5mm×5mm規(guī)格顆粒后溫水沖洗。分別以0.5%Triton X-100與10%氯化鈉脫細(xì)胞、甲醇與氯仿脫脂、30%過(guò)氧化氫脫蛋白以及0.4M氯化氫部分脫鈣處理。雙蒸水沖洗后烘干顆粒。鉆60輻照后-80℃低溫保存。對(duì)制備的去抗原松質(zhì)骨支架進(jìn)行大體結(jié)構(gòu)和掃描電鏡微觀結(jié)構(gòu)觀察分析以及孔徑大小與孔隙率測(cè)定。 結(jié)果：去抗原牛松質(zhì)骨材料為乳白色疏松多孔結(jié)構(gòu),孔洞間相互連通成立體網(wǎng)狀結(jié)構(gòu)。掃描電鏡觀察可見(jiàn)彼此相連相互溝通且孔隙大小較為規(guī)則的多孔狀固態(tài)結(jié)構(gòu)。松質(zhì)骨材料的孔徑平均值為(374.2±69.2)μm,孔隙率平均值為(58.1±11.4)%。 結(jié)論：去抗原牛松質(zhì)骨材料呈現(xiàn)彼此連通的多孔網(wǎng)狀三維立體結(jié)構(gòu),具有與骨組織工程要求相適應(yīng)的孔徑與孔隙率,具備作為骨組織工程適宜的支架材料應(yīng)有的基本結(jié)構(gòu)特征。 第二部分去抗原牛松質(zhì)骨載體的生物相容性研究 目的：觀察與評(píng)價(jià)經(jīng)過(guò)脫細(xì)胞、脫脂、脫蛋白與部分脫鈣處理的去抗原牛松質(zhì)骨支架材料的生物相容性。 方法：使用去抗原牛松質(zhì)骨材料進(jìn)行以下五個(gè)體內(nèi)實(shí)驗(yàn)。一、急性毒性實(shí)驗(yàn)：將去抗原牛松質(zhì)骨材料浸提液與生理鹽水分別注入小鼠腹腔,觀察記錄是否有不良反應(yīng)或死亡發(fā)生。二、熱源實(shí)驗(yàn)：將去抗原牛松質(zhì)骨浸提液與生理鹽水分別注入家兔耳緣靜脈后連續(xù)測(cè)體溫3次。三、溶血實(shí)驗(yàn)：將兔血混懸液分別加入去抗原牛松質(zhì)骨、碳酸鈉(陽(yáng)性對(duì)照)、生理鹽水(陰性對(duì)照)中。觀察有無(wú)溶血現(xiàn)象并計(jì)算溶血率。四、皮內(nèi)刺激實(shí)驗(yàn)：將去抗原牛松質(zhì)骨、20%乙醇生理鹽水分別注射于新西蘭白兔皮下,注射后即刻、6h、24h、48h、72h觀察記錄注射后狀況并評(píng)分計(jì)算局部原發(fā)刺激指數(shù)和平均原發(fā)刺激指數(shù)。五、肌袋實(shí)驗(yàn)：在大鼠大腿肌袋處植入去抗原牛松質(zhì)骨顆粒。術(shù)后每日觀察動(dòng)物狀態(tài)并于第7d取材進(jìn)行組織學(xué)分析。 結(jié)果：去抗原牛松質(zhì)骨材料植入動(dòng)物體內(nèi)后未引起毒性反應(yīng)、熱源反應(yīng)、溶血反應(yīng),未見(jiàn)皮內(nèi)明顯刺激反應(yīng)產(chǎn)生,且植入大鼠肌肉內(nèi)后未發(fā)生排斥反應(yīng)。 結(jié)論：去抗原牛松質(zhì)骨具有良好的生物相容性。 第三部分去抗原牛松質(zhì)骨載體與綠色熒光蛋白轉(zhuǎn)基因大鼠的骨髓間充質(zhì)干細(xì)胞的細(xì)胞相容性 目的：探討去抗原牛松質(zhì)骨材料與綠色熒光蛋白轉(zhuǎn)基因大鼠的骨髓間充質(zhì)干細(xì)胞的細(xì)胞相容性,以及構(gòu)建去抗原牛松質(zhì)骨材料與骨髓間充質(zhì)干細(xì)胞復(fù)合物的可行性。 方法：獲取綠色熒光蛋白轉(zhuǎn)基因SD大鼠骨髓,對(duì)其中的骨髓間充質(zhì)干細(xì)胞進(jìn)行分離、純化、培養(yǎng)、鑒定與多向誘導(dǎo)分化。取第三代骨髓間充質(zhì)干細(xì)胞消化后制備單細(xì)胞懸液緩慢滴加于去抗原牛松質(zhì)骨支架表面后連續(xù)培養(yǎng)12d。倒置相差顯微鏡和熒光顯微鏡下觀察材料表面的細(xì)胞形態(tài)及其黏附、生長(zhǎng)與增殖狀態(tài)。四甲基偶氮唑鹽比色法檢測(cè)去抗原牛松質(zhì)骨材料浸提液對(duì)大鼠骨髓間充質(zhì)干細(xì)胞的毒性作用。 結(jié)果：綠色熒光蛋白轉(zhuǎn)基因大鼠骨髓間充質(zhì)干細(xì)胞能粘附于去抗原牛松質(zhì)骨材料表面上,細(xì)胞具有良好形態(tài)并發(fā)出明亮綠色熒光,生長(zhǎng)與增殖狀態(tài)良好。去抗原牛松質(zhì)骨與大鼠骨髓間充質(zhì)干細(xì)胞復(fù)合培養(yǎng)的12d內(nèi),OD值與對(duì)照組比較,無(wú)顯著性差異。去抗原牛松質(zhì)骨材料浸提液對(duì)綠色熒光蛋白轉(zhuǎn)基因大鼠骨髓間充質(zhì)干細(xì)胞的細(xì)胞毒性為0～1級(jí)。 結(jié)論：去抗原牛松質(zhì)骨材料對(duì)綠色熒光蛋白轉(zhuǎn)基因大鼠骨髓間充質(zhì)干細(xì)胞無(wú)細(xì)胞毒性作用,具有良好的細(xì)胞相容性；去抗原牛松質(zhì)骨材料可作為理想支架用于構(gòu)建細(xì)胞-材料復(fù)合物。 第四部分去抗原牛松質(zhì)骨復(fù)合骨髓間充質(zhì)干細(xì)胞重建大鼠節(jié)段性眶骨缺損的實(shí)驗(yàn)研究 目的：評(píng)價(jià)去抗原牛松質(zhì)骨支架復(fù)合骨髓間充質(zhì)干細(xì)胞重建大鼠的節(jié)段性眶骨缺損的效果。 方法：選取經(jīng)過(guò)脫細(xì)胞、脫脂、脫蛋白與半脫鈣處理后制備的去抗原牛松質(zhì)骨材料作為支架材料。將綠色熒光蛋白轉(zhuǎn)基因SD大鼠骨髓中分離、提純、擴(kuò)增后獲取的骨髓間充質(zhì)干細(xì)胞接種于去抗原牛松質(zhì)骨載體上成功構(gòu)建細(xì)胞-材料復(fù)合體,體外成骨誘導(dǎo)培養(yǎng)5天后移植入SD大鼠右側(cè)8mm長(zhǎng)的眶骨缺損區(qū)域內(nèi)建立經(jīng)成骨誘導(dǎo)BMSCs/BCB組；分別設(shè)置未經(jīng)成骨誘導(dǎo)BMSCs/BCB組、單純BCB組和空白對(duì)照組。觀察四組動(dòng)物的傷口愈合與術(shù)后一般情況,分別于移植后第2、4、8、12周進(jìn)行眼眶螺旋CT掃描并進(jìn)行三維立體眶重建。移植后12周處死動(dòng)物并收集眶骨移植區(qū)域標(biāo)本制備病理切片,進(jìn)行眶骨缺損修復(fù)的病理學(xué)評(píng)價(jià)與病理形態(tài)學(xué)分析。 結(jié)果：細(xì)胞-支架復(fù)合體移植手術(shù)后所有大鼠一般情況良好,未見(jiàn)傷口感染或移植物脫出或排斥反應(yīng),局部外觀與功能未見(jiàn)異常。經(jīng)誘導(dǎo)骨髓間充質(zhì)干細(xì)胞復(fù)合去抗原牛松質(zhì)骨移植后第12周,螺旋CT與3D眶重建結(jié)果顯示植入物能夠有效地修復(fù)眶緣缺損區(qū)域并恢復(fù)眶緣的正常完整形態(tài)。病理學(xué)分析顯示去抗原牛松質(zhì)骨顆粒幾乎完全降解,缺損區(qū)內(nèi)大量新骨形成并發(fā)生新生骨與原有骨斷端之間的骨性連接。病理形態(tài)學(xué)分析顯示移植物植入后缺損區(qū)的新骨形成率為(57.12±6.28)%。 結(jié)論：去抗原牛松質(zhì)材料具有適宜眶缺損修復(fù)的機(jī)械強(qiáng)度和骨傳導(dǎo)性；去抗原牛松質(zhì)骨載體復(fù)合經(jīng)誘導(dǎo)的骨髓間充質(zhì)干細(xì)胞能夠完整地修復(fù)大鼠的節(jié)段性眶骨缺損,是一種利用組織工程學(xué)方法修復(fù)重建眶缺損的有效治療方案。
[Abstract]:Preparation method and structure analysis of first partial antigen bovine cancellous bone

Objective : To study the preparation method of acellular bovine cancellous bone material and to analyze the structural characteristics of the prepared material .

Methods : After washing with 0.5 % Triton X - 100 and 10 % sodium chloride , methanol and chloroform were degreased , 30 % hydrogen peroxide and 0.4 M hydrogen chloride were removed .

Results : The cancellous bone material of the acellular bovine cancellous bone was a porous structure with a porous structure , and the pores were communicated with each other . The average pore size of cancellous bone material was ( 374.2 鹵 69.2 ) 渭m and the average porosity was ( 58.1 鹵 11.4 ) % .

Conclusion : The anti - antigen bovine cancellous bone material presents a three - dimensional porous network structure which is in communication with each other , and has the pore size and the porosity which are compatible with the requirements of the bone tissue engineering , and has the basic structural characteristics as a suitable scaffold material for bone tissue engineering .

Biocompatibility of the second partial antigen bovine cancellous bone carrier

Objective : To observe and evaluate the biocompatibility of acellular bovine cancellous bone scaffold material subjected to dedifferentiation , degreasing , deprotein and partial decalcium treatment .

Methods : In order to observe whether there was an adverse effect or a death occurred in the rabbit ' s ear vein after injection of the antigen bovine cancellous bone extract and physiological saline into the abdominal cavity of the rabbits . The results of the experiment were as follows : the antigen bovine cancellous bone , sodium carbonate ( positive control ) and normal saline ( negative control ) were injected into the rabbits .

Results : The antigen bovine cancellous bone material did not induce toxic reaction , heat source reaction , hemolytic reaction , no obvious irritation reaction in skin , and no rejection after implantation in the muscle of rats .

Conclusion : Anti - antigen bovine cancellous bone has good biocompatibility .

Cell Compatibility of Bone Marrow Mesenchymal Stem Cells Derived from the Third Part of Anti - Antigen Bovine cancellous Bone Vector and Green Fluorescent Protein Transgenic Rats

Objective : To investigate the cell compatibility of bone marrow mesenchymal stem cells ( MSCs ) with acellular bovine cancellous bone ( GFP ) and green fluorescent protein ( GFP ) transgenic rats , and to investigate the feasibility of constructing acellular bovine cancellous bone material and bone marrow mesenchymal stem cell complexes .

Methods : The bone marrow of bone marrow mesenchymal stem cells were isolated , purified , cultured , identified and multi - directionally induced by green fluorescent protein transgenic SD rat bone marrow . After the third generation of bone marrow mesenchymal stem cells were digested , the cell morphology and its adhesion , growth and proliferation were observed under the reversed phase contrast microscope and fluorescence microscope .

Results : The green fluorescent protein transgenic rat bone marrow mesenchymal stem cells were able to adhere to the surface of the acellular bovine cancellous bone material . The cells had good morphology and bright green fluorescence , and the growth and proliferation were good . The OD value was not significantly different from that of the control group . The cytotoxicity of the acellular bovine cancellous bone material extract on the bone marrow mesenchymal stem cells of the green fluorescent protein transgenic rats was 0 - 1 .

Conclusion : The anti - antigen bovine cancellous bone material has no cytotoxicity on the bone marrow mesenchymal stem cells of the green fluorescent protein transgenic rat , and has good cell compatibility ;
Anti - antigen bovine cancellous bone material can be used as an ideal scaffold for the construction of cell - material complexes .

Experimental study on the reconstruction of segmental orbital bone defects in rat segmental bone marrow mesenchymal stem cells with the fourth part of acellular bovine cancellous bone

Objective : To evaluate the effect of acellular bovine cancellous bone scaffold composite bone marrow mesenchymal stem cells on segmental orbital bone defects in rats .

Methods : The bone marrow mesenchymal stem cells were isolated , purified and amplified from bone marrow of transgenic SD rats .
After transplantation , orbital spiral CT scanning was performed and three - dimensional orbital reconstruction was performed on the 2nd , 4th , 8th , 12th week after transplantation . The animals were sacrificed at 12 weeks after transplantation and orbital bone graft area specimens were collected to prepare pathological sections .

Results : All the rats were generally in good condition after transplantation of the cell - scaffold complex . There was no abnormal wound infection or graft rejection or rejection , and the local appearance and function were not abnormal . After the induction of bone marrow mesenchymal stem cells , the results of spiral CT and 3D orbital reconstruction showed that the implants were almost completely degraded , and the bony connection between the new bone and the original bone fracture occurred in the defect area .

Conclusion : Anti - antigen bovine cancellous bone material has the mechanical strength and osteoconductivity suitable for orbital defect repair .
Bone marrow mesenchymal stem cells ( MSCs ) induced by acellular bovine cancellous bone can completely repair segmental orbital bone defects in rats , and is an effective treatment scheme for repairing orbital defects by tissue engineering method .

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R777.5

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