本文選題：同種異體 + 組織工程骨��； 參考：《北京協(xié)和醫(yī)學(xué)院》2013年博士論文

【摘要】：目的： 組織工程技術(shù)為臨床骨缺損的治療帶來(lái)了新的希望,應(yīng)用自體骨髓間充質(zhì)干細(xì)胞(autogenic BMSCs, Auto-BMSCs)在體內(nèi)構(gòu)建組織工程骨修復(fù)骨缺損的方法已基本成熟。然而對(duì)于同種異體骨髓間充質(zhì)干細(xì)胞(allogeneic BMSCs, Allo-BMSCs),是否能成功構(gòu)建組織工程骨,及移植后的免疫原性如何,仍然有很多爭(zhēng)論。本課題擬應(yīng)用同種異體BMSCs與可降解支架復(fù)合構(gòu)建組織工程骨(Tissue engineered bone, TEB),并植入犬背部皮下非受力部位,探索同種異體組織工程骨的異位成骨能力,并檢測(cè)同種異體BMSCs移植的免疫原性。之后進(jìn)一步構(gòu)建犬顱骨臨界骨缺損模型,評(píng)估同種異體BMSCs復(fù)合β-磷酸三鈣(β-TCP)修復(fù)大型哺乳動(dòng)物顱骨缺損的可行性。 方法： 1.從犬骨髓血中分離純化BMSCs,體外向成骨、成軟骨、成脂三系誘導(dǎo)分化、鑒定。用CM-DiI對(duì)成骨誘導(dǎo)至第2代的BMSCs標(biāo)記后進(jìn)行體內(nèi)示蹤研究。MTT比色法測(cè)定標(biāo)記前后BMSCs的增殖狀況。RT-PCR檢測(cè)標(biāo)記細(xì)胞中Ⅰ型膠原、骨粘連蛋白、骨形態(tài)發(fā)生蛋白-2、骨鈣素的表達(dá)。將標(biāo)記CM-DiI后的BMSCs復(fù)合β-TCP后植入犬背部皮下,8周后取材,熒光顯微鏡下觀察BMSCs體內(nèi)轉(zhuǎn)歸,組織學(xué)觀察標(biāo)記BMSCs-TCP復(fù)合物異位成骨情況。 2.同種異體BMSCs體內(nèi)構(gòu)建異位組織工程骨：成骨誘導(dǎo)至第2代犬BMSCs復(fù)合β-TCP分別植入同種異體、自體犬背部皮下,分別作為同種異體組織工程骨組(Allo-TEB組),自體組織工程骨組(Auto-TEB組),單純?chǔ)?TCP作為材料對(duì)照組(Control組)。手術(shù)前及術(shù)后3、7、14、28、56天通過(guò)流式細(xì)胞學(xué)分別檢測(cè)三組的外周血T淋巴細(xì)胞亞群的變化,評(píng)估全身免疫反應(yīng)情況。術(shù)后24周取材并行HE染色,通過(guò)組織學(xué)計(jì)量分析,量化比較三組的成骨情況。 3.同種異體BMSCs體內(nèi)構(gòu)建原位組織工程骨：構(gòu)建犬雙側(cè)顱骨全層臨界骨缺損模型,應(yīng)用同種異體、自體BMSCs-TCP復(fù)合物原位移植修復(fù)犬顱骨臨界骨缺損,分別作分Allo-TEB組,Auto-TEB組,單純?chǔ)?TCP作為材料對(duì)照組。術(shù)后1、3、6、9月通過(guò)影像學(xué)量化比較三組的顱骨缺損修復(fù)情況。術(shù)后9月通過(guò)大體觀察、micro-CT、生物力學(xué)和組織學(xué)檢查,分別評(píng)估三組的顱骨缺損修復(fù)質(zhì)量。 結(jié)果： 1.犬骨髓血中分離得到的BMSCs可以向成骨、成軟骨、成脂細(xì)胞方向分化,CM-DiI標(biāo)記BMSCs前后的細(xì)胞形態(tài)基本一致,兩組間細(xì)胞的增殖率無(wú)顯著性差異(P0.05)；標(biāo)記后RT-PCR可檢測(cè)到Col-I、BMP-2、BGLAP、SPARC的表達(dá),顯示標(biāo)記對(duì)成骨分化無(wú)明顯影響。標(biāo)記細(xì)胞構(gòu)建的組織工程骨植入犬皮下8周后取材,標(biāo)記細(xì)胞仍能激發(fā)紅色熒光,且HE染色證實(shí)標(biāo)記BMSCs構(gòu)建的組織工程骨可在體內(nèi)異位成骨。 2.同種異體BMSCs體內(nèi)構(gòu)建異位組織工程骨：同種異體、自體BMSCs-TCP復(fù)合物均可異位成骨,24周時(shí)Allo-TEB組與Auto-TEB組比較,其成骨百分比無(wú)顯著性差異(P0.05),均顯著高于單純?chǔ)?TCP對(duì)照組(P0.001)。流式細(xì)胞學(xué)檢測(cè)顯示Allo-TEB組植入第3天、7天時(shí)的組內(nèi)CD4+T淋巴細(xì)胞及CD4+/CD8+T高于術(shù)前及之后的其他時(shí)間點(diǎn)(P0.05),隨著時(shí)間的延長(zhǎng),Allo-TEB組、Auto-TEB組CD4+/CD8+T細(xì)胞的百分比呈先升高后降低的曲線。但Allo-TEB組、Auto-TEB組及Control組組間的CD4+T細(xì)胞計(jì)數(shù)、CD8+T細(xì)胞計(jì)數(shù)、CD4+/CD8+T淋巴細(xì)胞的百分比無(wú)顯著性差異(P0.05)。 3.同種異體BMSCs體內(nèi)構(gòu)建原位組織工程骨修復(fù)犬顱骨臨界骨缺損：術(shù)后CT三維重建及計(jì)量分析顯示Allo-TEB組、Auto-TEB組的組織工程骨骨密度在術(shù)后1、3月時(shí)有所降低,但在術(shù)后6、9月時(shí)保持穩(wěn)定,兩組間骨密度無(wú)顯著性差異(P0.05),材料對(duì)照組骨密度隨時(shí)間延長(zhǎng)逐漸降低,在3、6、9月時(shí)明顯低于上述組織工程骨組(P0.001)。9月時(shí)標(biāo)本大體觀察及micro-CT顯示同種異體、自體組織工程骨仍能保持顱骨的完整性,抗壓能力檢測(cè)顯示Allo-TEB組、Auto-TEB組的組織工程骨之間無(wú)顯著性差異(P0.05)。組織學(xué)檢測(cè)顯示,Allo-TEB組、Auto-TEB組的組織工程骨的類(lèi)骨質(zhì)中有大量骨細(xì)胞、骨陷窩,在缺損邊緣與正常骨之間形成了骨性連接,單純TCP材料大部分降解,缺損區(qū)由纖維組織填充。 結(jié)論： 1.犬骨髓血中分離得到的BMSCs具備向成骨、成軟骨、成脂方向分化的潛能。CM-Dil標(biāo)記對(duì)BMSCs的生長(zhǎng)增殖、成骨分化無(wú)明顯影響。體內(nèi)示蹤實(shí)驗(yàn)證實(shí),BMSCs可以在體內(nèi)至少存活8周,且8周時(shí)BMSCs在體內(nèi)異位構(gòu)建的組織工程骨的成骨過(guò)程中發(fā)揮了種子細(xì)胞作用。 2.同種異體BMSCs-TCP在體內(nèi)構(gòu)建的組織工程骨有異位成骨的作用。同種異體組及自體組的異位成骨能力在24周時(shí)無(wú)顯著性差異。術(shù)后兩組間的全身免疫反應(yīng)無(wú)顯著性差異。同種異體BMSCs沒(méi)有引起明顯的免疫排斥反應(yīng)。 3.同種異體BMSCs-TCP在體內(nèi)構(gòu)建組織工程骨能夠原位修復(fù)犬顱骨臨界骨缺損,成骨速率在早期(3月時(shí))慢于自體組,最終(9月時(shí))兩組間無(wú)顯著性差異。
[Abstract]:Objective:
Tissue engineering technology has brought new hope for the treatment of clinical bone defect. The method of constructing tissue engineered bone defect with autogenous bone marrow mesenchymal stem cells (autogenic BMSCs, Auto-BMSCs) has been basically mature. However, it is possible to construct allogeneic BMSCs (Allo-BMSCs) for allogeneic bone marrow mesenchymal stem cells (Allo-BMSCs). There are still many controversies on how to build tissue engineering bone and the immunogenicity after transplantation. This topic is to construct tissue engineering bone (Tissue engineered bone, TEB) with allogeneic BMSCs and biodegradable scaffold, and to implant the non stressed parts of the dog's back subcutaneous, and explore the heterotopic osteogenesis ability of allograft tissue engineering bone and detect the allograft difference. The immunogenicity of the body BMSCs transplantation was further constructed and the critical bone defect model of the canine skull was further constructed to evaluate the feasibility of the allograft BMSCs compound beta tricalcium phosphate (beta -TCP) for the repair of large mammal skull defects.
Method:
1. isolation and purification of BMSCs from bone marrow blood of dogs, differentiation into osteogenesis, chondrogenesis, and lipid three lines, identification. BMSCs markers induced by osteogenesis to second generations by CM-DiI were traced in vivo. The proliferation of BMSCs before and after the determination of BMSCs by.MTT colorimetric assay was used to detect the type I collagen, osteonectin, and bone morphogenetic eggs in the labeled cells. The expression of white -2, osteocalcin. After labeling the BMSCs compound beta -TCP after CM-DiI, it was implanted subcutaneously in the back of the dog. After 8 weeks, the material was harvested. The changes of BMSCs in vivo were observed under the fluorescence microscope, and the ectopic osteogenesis of the BMSCs-TCP complex was observed by histological observation.
2. the construction of heterotopic tissue engineering bone in BMSCs allograft: osteogenesis induced by osteogenesis to second generation canine compound beta -TCP and subcutaneous allograft in the back of autologous dog, as allograft tissue engineering bone group (group Allo-TEB), autologous tissue engineering bone group (Auto-TEB group), simple beta -TCP as the material control group (Group Control). Before and after operation, the operation and operation were performed. After 3,7,14,28,56 days, the changes of T lymphocyte subsets in the peripheral blood of three groups were detected by flow cytometry, and the whole body immune response was evaluated. 24 weeks after the operation, the three groups were collected in parallel with the HE staining, and the osteogenesis of the three groups was quantified by histologic analysis.
3. in situ tissue engineering bone was constructed in vivo of allogeneic BMSCs: Construction of a canine bilateral cranial critical bone defect model, using allogeneic and autologous BMSCs-TCP complex in situ to repair the critical bone defect of the canine skull, which were divided into Allo-TEB group, Auto-TEB group and simple beta -TCP as the material control group. The image was quantified after 1,3,6,9 months after operation. The three groups of cranium defect repair were compared. In September, three groups of cranial defects were evaluated by gross observation, micro-CT, biomechanics and histological examination.
Result:
1. BMSCs isolated from bone marrow blood could be osteogenic, cartilaginous, and adipocyte differentiation. The cell morphology of CM-DiI before and after BMSCs was basically the same, and there was no significant difference in the proliferation rate between the two groups (P0.05); RT-PCR could detect the expression of Col-I, BMP-2, BGLAP, SPARC, indicating that the markers had no obvious effect on the osteogenesis. The tissue engineered bone constructed by labeled cells was implanted 8 weeks after subcutaneous tissue, and the labeled cells could still stimulate red fluorescence, and HE staining showed that the tissue engineered bone marked by BMSCs could be ectopic osteogenesis in the body.
2. BMSCs allogenic tissue engineering bone was constructed in vivo: Allogenic and autologous BMSCs-TCP complex could be ectopic osteogenesis. At 24 weeks, there was no significant difference in the percentage of bone formation between the Allo-TEB group and the Auto-TEB group (P0.05), which was significantly higher than that in the simple beta -TCP control group (P0.001). The flow cytology test showed that the group of Allo-TEB was implanted for third days and 7 days. The CD4+T lymphocyte and CD4+/CD8+T in the group were higher than the other time points before and after the operation (P0.05). The percentage of CD4+/CD8+T cells in group Allo-TEB and Auto-TEB group increased first and then decreased with the prolongation of time. But the CD4+T cells count, CD8+T cell count, CD4+/CD8+T lymphocytes in group Allo-TEB, Auto-TEB and Control groups were counted. There was no significant difference in percentage (P0.05).
3. BMSCs in situ tissue engineering bone was constructed to repair the critical bone defect of the canine skull. After the operation, CT three-dimensional reconstruction and metrological analysis showed that the bone mineral density of group Auto-TEB decreased at 1,3 months after the operation, but remained stable at 6,9 months after the operation, and there was no significant difference in bone density between the groups (P0.05). The material control was not significant (P0.05). Group bone density gradually decreased with time. At 3,6,9 month, the specimens were significantly lower than that of the tissue engineering bone group (P0.001).9 months, and micro-CT showed the same allograft. Autologous tissue engineering bone still maintained the integrity of the skull. The test of compression ability showed Allo-TEB group, and there was no significant difference between the tissue engineering bone in group Auto-TEB (P0.). 05). The histological examination showed that there were a large number of bone cells and bone lacunae in the tissue engineering bone of group Allo-TEB and Auto-TEB, which formed a bone connection between the defect edge and the normal bone, and the most of the TCP materials were degraded, and the defect area was filled with fibrous tissue.
Conclusion:
1. the BMSCs isolated from the bone marrow blood of the dog has the potential for osteogenesis, cartilage, and adipogenic differentiation..CM-Dil markers have no significant effect on BMSCs growth and proliferation. In vivo tracer experiments confirmed that BMSCs could survive for at least 8 weeks in the body, and at 8 weeks, BMSCs played a role in the osteogenesis of tissue engineered bone constructed in the body. The effect of seed cells.
2. the tissue engineered bone constructed by allogeneic BMSCs-TCP had ectopic osteogenesis. The heterotopic osteogenesis ability of the allograft group and the autologous group had no significant difference at 24 weeks. There was no significant difference in the systemic immune response between the two groups after the operation. The allogenic BMSCs did not cause obvious immunization rejection.
The construction of tissue engineered bone in 3. allogeneic BMSCs-TCP in vivo can repair the critical bone defect of the canine skull in situ, and the rate of osteogenesis is slower in the early (March) than in the autologous group, and there is no significant difference between the two groups at the end of the year.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R318.08

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 李科成;常強(qiáng);魯峰;;三種示蹤技術(shù)標(biāo)記人脂肪組織來(lái)源干細(xì)胞的對(duì)照研究[J];南方醫(yī)科大學(xué)學(xué)報(bào);2011年04期

2 張衛(wèi)群;王宜人;巢永烈;;類(lèi)骨質(zhì)羥磷灰石和自體骨修復(fù)兔下頜骨臨界性骨缺損的研究[J];華西口腔醫(yī)學(xué)雜志;2010年02期

3 ;A rapid forming method and simulation on controllable-porosity coating[J];Science in China(Series E:Technological Sciences);2007年06期

4 胡幫友;張海鷗;王桂蘭;;一種孔隙率可控薄膜材料快速生長(zhǎng)成形方法及過(guò)程模擬[J];中國(guó)科學(xué)(E輯:技術(shù)科學(xué));2007年11期

5 紀(jì)玲;武延格;孫學(xué)峰;王正;楊林;;DiI熒光標(biāo)記的軟骨細(xì)胞與支架材料復(fù)合后的觀察[J];暨南大學(xué)學(xué)報(bào)(自然科學(xué)與醫(yī)學(xué)版);2009年06期

6 馬慧雨;肖苒;杜曉巖;;熒光活性染料(CM-Dil)對(duì)人骨髓間充質(zhì)干細(xì)胞增殖能力的影響[J];口腔頜面外科雜志;2011年04期

7 操石磊;張春禮;徐虎;陳輝;鄭佳鵬;;DiI熒光標(biāo)記示蹤兔皮膚成纖維細(xì)胞修復(fù)前交叉韌帶損傷的研究[J];科學(xué)技術(shù)與工程;2008年01期

8 南華;高建華;魯峰;;靜脈移植的脂肪來(lái)源干細(xì)胞在創(chuàng)傷鼠體內(nèi)的分布[J];中國(guó)美容醫(yī)學(xué);2009年06期

9 趙明東;尹望平;董健;;血管束植入治療骨缺損[J];復(fù)旦學(xué)報(bào)(醫(yī)學(xué)版);2007年04期

10 李偉民;傅祖植;;核心結(jié)合因子α_1基因修飾MSCs對(duì)骨質(zhì)疏松大鼠骨形成作用研究[J];陜西醫(yī)學(xué)雜志;2012年03期

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1 孫蘭軍;徐強(qiáng);趙英強(qiáng);李艷芬;;復(fù)方丹參滴丸干預(yù)骨髓間充質(zhì)干細(xì)胞移植治療AMI的實(shí)驗(yàn)研究[A];第一屆全國(guó)中西醫(yī)結(jié)合心血管病中青年醫(yī)師論壇論文匯編[C];2008年

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1 李彪;普伐他汀對(duì)BMSCs復(fù)合FS移植治療兔早期激素性股骨頭壞死的干預(yù)研究[D];昆明醫(yī)學(xué)院;2011年

2 李康杰;脫細(xì)胞骨基質(zhì)聯(lián)合骨髓間充質(zhì)干細(xì)胞修復(fù)動(dòng)物骨缺損實(shí)驗(yàn)研究[D];延邊大學(xué);2011年

3 廖云君;應(yīng)用去分化脂肪細(xì)胞構(gòu)建工程化脂肪組織的實(shí)驗(yàn)研究[D];南方醫(yī)科大學(xué);2011年

4 董世武;Cbfa1基因修飾間充質(zhì)干細(xì)胞治療骨缺損的實(shí)驗(yàn)研究[D];第三軍醫(yī)大學(xué);2004年

5 宋克東;生物反應(yīng)器內(nèi)成骨細(xì)胞的擴(kuò)增和組織工程骨的構(gòu)建[D];大連理工大學(xué);2006年

6 程立華;反義寡核苷酸肝靶向制劑的研究[D];沈陽(yáng)藥科大學(xué);2005年

7 陶春生;體外三維培養(yǎng)條件下人重組骨形態(tài)發(fā)生蛋白-2納米緩釋系統(tǒng)對(duì)骨髓間充質(zhì)干細(xì)胞增殖和分化影響的實(shí)驗(yàn)研究[D];第二軍醫(yī)大學(xué);2007年

8 張?jiān)扑?人脂肪來(lái)源干細(xì)胞構(gòu)建組織工程化脂肪組織的體內(nèi)外實(shí)驗(yàn)研究[D];第一軍醫(yī)大學(xué);2007年

9 馮衛(wèi);豬異種骨移植靶抗原分布及除抗原處理的研究[D];四川大學(xué);2007年

10 鄧興力;神經(jīng)干細(xì)胞聯(lián)合多巴胺神經(jīng)元移植治療帕金森病的實(shí)驗(yàn)研究[D];昆明醫(yī)學(xué)院;2008年

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1 潘明利;重組膠原蛋白調(diào)控鈦表面仿生礦化的研究[D];浙江理工大學(xué);2010年

2 禹娜娜;人工骨支架內(nèi)孔系結(jié)構(gòu)設(shè)計(jì)及內(nèi)部流場(chǎng)的CFD分析[D];燕山大學(xué);2011年

3 鄧平平;骨組織工程支架內(nèi)微流體及其變形的數(shù)值仿真分析[D];燕山大學(xué);2011年

4 苗宗寧;骨髓間充質(zhì)干細(xì)胞向骨細(xì)胞誘導(dǎo)分化及其組織工程化骨組織的實(shí)驗(yàn)研究[D];蘇州大學(xué);2005年

5 王興宇;表達(dá)人骨形態(tài)發(fā)生蛋白-4的非復(fù)制型腺病毒的構(gòu)建與鑒定[D];安徽醫(yī)科大學(xué);2006年

6 唐浩;小鼠骨髓衍生肝干細(xì)胞的篩選及其分化潛能的研究[D];第一軍醫(yī)大學(xué);2006年

7 郭華;大鼠延髓巨細(xì)胞網(wǎng)狀核與腦神經(jīng)運(yùn)動(dòng)核的纖維聯(lián)系[D];鄭州大學(xué);2007年

8 佘厚德;羥基磷灰石及組織工程用聚己內(nèi)酯復(fù)合支架的制備和研究[D];福建師范大學(xué);2007年

9 潘廣生;工程骨低溫保存中CPA的導(dǎo)入過(guò)程研究[D];大連理工大學(xué);2007年

10 黃凱;納米脫鈣骨基質(zhì)作為骨移植替代物的實(shí)驗(yàn)研究[D];第二軍醫(yī)大學(xué);2008年

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