【摘要】：目的：本實(shí)驗(yàn)選用體外分離培養(yǎng)的蒙古羊骨髓間充質(zhì)干細(xì)胞接種貼附到同種異體骨上形成載體復(fù)合物,植入蒙古羊體內(nèi)脛骨缺損處,觀察細(xì)胞-異體骨復(fù)合物修復(fù)蒙古羊脛骨缺損的療效,為骨組織工程治療骨缺損的臨床應(yīng)用提供實(shí)驗(yàn)依據(jù)。 方法：2只1月齡的蒙古羊,在嚴(yán)格無(wú)菌條件下穿刺收集股骨骨髓5m1,采用聯(lián)合培養(yǎng)法分離純化骨髓間充質(zhì)細(xì)胞,系統(tǒng)地研究其生物學(xué)特性以及體外成骨能力。將骨髓間充質(zhì)干細(xì)胞與處理為凍干骨的同種異體骨聯(lián)合培養(yǎng)24h,制成細(xì)胞-異體骨復(fù)合物后備用。 取9只蒙古羊,體重在50-60kg,用速眠新注射液(0.1m1/kg)麻醉后固定,脛骨處脫毛、碘酒消毒、鋪無(wú)菌單。手術(shù)制造雙側(cè)脛骨1cm長(zhǎng)的骨一骨膜缺損區(qū),隨機(jī)選擇一側(cè)作為實(shí)驗(yàn)組,植入骨髓間充質(zhì)干細(xì)胞與同種異體骨復(fù)合物；另一側(cè)為對(duì)照組移植單純同種異體骨。于術(shù)后4、8、12周隨機(jī)選取3只蒙古羊處死做微-CT檢查及組織學(xué)觀察。 結(jié)果：聯(lián)合培養(yǎng)法分離得到較高純度的骨髓間充質(zhì)干細(xì)胞,且細(xì)胞形態(tài)均一,呈成纖維細(xì)胞樣,融合后形成明顯的細(xì)胞生長(zhǎng)紋路。骨髓間充質(zhì)干細(xì)胞經(jīng)地塞米松等可以誘導(dǎo)分化為成骨細(xì)胞,堿性磷酸酶、茜素紅染色均為陽(yáng)性。 同種異體骨與骨髓間充質(zhì)干細(xì)胞在體外分別聯(lián)合培養(yǎng)2h、12h、24h、48h,經(jīng)掃描電鏡觀察顯示異體骨表面及其中心均有細(xì)胞貼附生長(zhǎng),且形態(tài)多樣。證明同種異體骨與骨髓間充質(zhì)干細(xì)胞之間具有良好的相容性。 將9只蒙古羊隨機(jī)分為三組,術(shù)后4、8、12周隨機(jī)抽取一組宰殺取骨,進(jìn)行微-CT檢查及組織學(xué)觀察。結(jié)果顯示,各個(gè)時(shí)期,實(shí)驗(yàn)組骨缺損修復(fù)效果均優(yōu)于對(duì)照組,骨痂形成量明顯多于對(duì)照組。這些結(jié)果均表明細(xì)胞-異體骨復(fù)合材料可以治療蒙古羊脛骨缺損且效果優(yōu)于單純異體骨移植。 結(jié)論： 1.通過(guò)密度梯度離心法與貼壁培養(yǎng)法可以分離純化BMSCs,BMSCs可以作為種子細(xì)胞應(yīng)用到骨組織工程學(xué)中。 2. BMSCs與同種異體骨具有良好的生物相容性,二者聯(lián)合培養(yǎng)可以作為臨床治療脛骨缺損的實(shí)驗(yàn)依據(jù)。
[Abstract]:Objective: in this experiment, Mongolian sheep bone marrow mesenchymal stem cells isolated and cultured in vitro were inoculated and attached to allogenic bone to form carrier complex and implanted into the tibia defect of Mongolian sheep, and the curative effect of cell-allogenic bone complex in repairing Mongolian sheep tibia defect was observed, so as to provide experimental basis for the clinical application of bone tissue engineering in the treatment of bone defect. Methods: bone marrow 5M1 was collected from 2 1-month-old Mongolian sheep under strict aseptic conditions. Bone marrow mesenchymal cells were isolated and purified by co-culture, and their biological characteristics and osteogenic ability in vitro were systematically studied. Bone marrow mesenchymal stem cells (BMSCs) were co-cultured with allogenic bone treated as freeze-dried bone for 24 hours to make cell-allogenic bone complex and set aside. Nine Mongolian sheep weighing 50 kg and 60 kg were fixed with 0.1m1/kg injection, hair removal in tibia, disinfection with iodine wine and aseptic sheet. The osseous periosteal defect area of bilateral tibia 1cm was made by operation. One side of the experimental group was randomly selected as the experimental group, and the bone marrow mesenchymal stem cells and allogenic bone complex were implanted on the other side, and the control group was treated with simple allogenic bone transplantation. At 4, 8 and 12 weeks after operation, 3 Mongolian sheep were randomly selected for micro-CT examination and histology observation. Results: high purity bone marrow mesenchymal stem cells were isolated by co-culture method, and the cells were uniform in morphology and fibroblasts, and obvious cell growth patterns were formed after fusion. Bone marrow mesenchymal stem cells can be induced to differentiate into osteoblasts by dexamethasone and so on. Alkaline phosphatase and alizalin red staining are positive. Allogenic bone and bone marrow mesenchymal stem cells were co-cultured in vitro for 2 h, 12 h, 24 h and 48 h, respectively. scanning electron microscope (SEM) showed that the allogenic bone surface and its center had cell attachment growth, and the morphology was diverse. It is proved that allogenic bone has good compatibility with bone marrow mesenchymal stem cells. Nine Mongolian sheep were randomly divided into three groups. 4, 8, 12 weeks after operation, a group of slaughtered bones was randomly selected for microCT examination and histology observation. The results showed that the effect of bone defect repair in the experimental group was better than that in the control group, and the amount of eschar formation in the experimental group was significantly higher than that in the control group. These results show that the cell-allogenic bone composite can treat Mongolian sheep tibia defect and the effect is better than that of allogenic bone transplantation alone. Conclusion: 1. BMSCs,BMSCs can be isolated and purified by density gradient centrifugation and adherent culture, which can be used as seed cells in bone tissue engineering. 2. BMSCs has good biocompatibility with allogenic bone, and their co-culture can be used as an experimental basis for clinical treatment of tibia defects.
【學(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R681;Q813

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