【摘要】：第一部分椎間盤(pán)脫細(xì)胞方法及效果評(píng)估目的：評(píng)估多種新西蘭白兔椎間盤(pán)脫細(xì)胞方法其脫細(xì)胞效果以及新西蘭白兔椎間盤(pán)經(jīng)脫細(xì)胞后生物化學(xué)性質(zhì)里力學(xué)性質(zhì)方法：我們從4月齡新西蘭白兔中收集了45例胸椎及腰椎椎間盤(pán)標(biāo)本,用作體外實(shí)驗(yàn)。新西蘭白兔椎間盤(pán)周?chē)募∪饨M織及骨片被剔除干凈,切去軟骨終板保留髓核和纖維環(huán),并在PBS中清洗以洗去多余的血液。處理后的椎間盤(pán)標(biāo)本立刻凍存于液氮中以備后用。將標(biāo)本分為不作脫細(xì)胞處理的陰性對(duì)照組和行脫細(xì)胞組的A組以及B組。A組脫細(xì)胞方法：在37攝氏度水浴和液氮中反復(fù)溶凍5次,在2%Triton X-100中浸泡24小時(shí),在1%SDS溶液中浸泡24小時(shí),后以200U/mlDNA酶處理2小時(shí),處理后在PBS中清洗以洗去殘余試劑；B組脫細(xì)胞方法：在37攝氏度水浴和液氮中反復(fù)溶凍5次,在3%Triton X-100中浸泡24小時(shí),在2%SDS溶液中浸泡24小時(shí),處理后在PBS中清洗以洗去殘余試劑.所有標(biāo)本均以HE和Alican藍(lán)染色,以免疫組織化學(xué)檢測(cè)椎間盤(pán)中Collagen type Ⅰ, Collagen type Ⅱ以及Aggrecan保存量,以DNeasy Blood Kit試劑盒按生產(chǎn)商說(shuō)明書(shū)處理提取DNA,用NanoDrop8000檢測(cè)DNA含量,以吸光度比色法檢測(cè)HYP含量,根據(jù)以上結(jié)果確定適宜脫細(xì)胞方案,排除不適宜組,以?xún)龈煞ㄊ箻?biāo)本脫水,以質(zhì)量變化推測(cè)含水量。以SEM及TEM檢測(cè)對(duì)照組及適宜脫細(xì)胞法組的微觀結(jié)構(gòu)。將脫細(xì)胞材料分為纖維環(huán)及髓核和兩部分,適用電腦控制檢測(cè)裝置以檢測(cè)纖維環(huán)彈性模量,最大張力以及最大壓力,檢測(cè)髓核壓縮模量。結(jié)果：A組和對(duì)照組在HE和Alican藍(lán)染色,各項(xiàng)免疫組織化學(xué)結(jié)果中無(wú)明顯差異,B組則與對(duì)照組之間存在明顯差異；A組中HYP含量最高,對(duì)照組次之,B組中HYP含量最低,且三者存在明顯差異,A組方法為適宜椎間盤(pán)脫細(xì)胞方法。A組含水量明顯高于對(duì)照組,兩者SEM以及TEM結(jié)果無(wú)明顯差異；A組與對(duì)照組的彈性模量,壓力模量,最大壓力以及最大延長(zhǎng)度均無(wú)明顯差異。結(jié)論：在37攝氏度水浴和液氮中反復(fù)溶凍5次,在2%Triton X-100中浸泡24小時(shí),在1%SDS溶液中浸泡24小時(shí),后以200U/mlDNA酶處理2小時(shí),處理后在PBS中清洗以洗去殘余試劑是新西蘭白兔適宜的椎間盤(pán)脫細(xì)胞方法。這種方法可以最大程度上的保留椎間盤(pán)原有的生物化學(xué)特點(diǎn),微觀結(jié)構(gòu)以及力學(xué)特點(diǎn)。第二部分同種去細(xì)胞椎間盤(pán)材料安全性評(píng)估目的：評(píng)估同種去細(xì)胞椎間盤(pán)材料分別在體內(nèi)及體外的安全性。方法：為評(píng)估同種去細(xì)胞椎間盤(pán)材料分別在體內(nèi)及體外的安全性,我們將新西蘭白兔椎間盤(pán)標(biāo)本分為實(shí)驗(yàn)組及對(duì)照組,實(shí)驗(yàn)組按第一部分中得出的適宜的椎間盤(pán)脫細(xì)胞方法進(jìn)行處理,對(duì)照組不予處理,分別植入新西蘭白兔脊柱兩側(cè)皮下,1月后去除實(shí)驗(yàn)組及對(duì)照組,所有標(biāo)本均以HE染色觀察中性粒細(xì)胞分布,以免疫組織化學(xué)檢測(cè)MAC387及CD8水平觀察巨噬細(xì)胞及細(xì)胞毒T細(xì)胞水平。以梯度離心法收集新西蘭白兔MSC,將去細(xì)胞椎間盤(pán)材料置于DMEM-HG中,放置于培養(yǎng)箱48小時(shí),收集浸提液,將MSC培養(yǎng)于含F(xiàn)BS10%的DEME-HG(對(duì)照組),25%浸提液+DMEM-HG,50%浸提液+DMEM-HG和100%浸提液中培養(yǎng),以活死細(xì)胞染色觀察存活細(xì)胞,以CCK-8試劑盒及吸光度比色計(jì)觀察細(xì)胞活力。將MSC種植按第一部分方法脫細(xì)胞的新西蘭白兔椎間盤(pán)材料上,分別在第3天,第7天,第14天,第21天以活死細(xì)胞染色觀察存活細(xì)胞,以HE染色觀測(cè)材料結(jié)構(gòu)以及MSC分布。結(jié)果：經(jīng)第一部分適宜脫細(xì)胞方法處理后的新西蘭白兔椎間盤(pán)材料在植入同種白兔皮下后未見(jiàn)明顯中性粒細(xì)胞,巨噬細(xì)胞,細(xì)胞毒性T細(xì)胞浸潤(rùn)；未經(jīng)第一部分適宜脫細(xì)胞方法處理后的新西蘭白兔椎間盤(pán)材料在植入同種白兔皮下后出現(xiàn)明顯中性粒細(xì)胞,巨噬細(xì)胞,細(xì)胞毒性T細(xì)胞浸潤(rùn)并出現(xiàn)明顯血管生長(zhǎng)；FBS10%的DEME-HG(對(duì)照組),25%浸提液+DMEM-HG,50%浸提液+DMEM-HG和100%浸提液這四組在活細(xì)胞數(shù)計(jì)數(shù)以及細(xì)胞活性無(wú)明顯差異。MSC能在經(jīng)第一部分適宜脫細(xì)胞方法處理后的新西蘭白兔椎間盤(pán)材料生長(zhǎng),擴(kuò)增以及遷移。結(jié)論：經(jīng)第一部分適宜脫細(xì)胞方法處理后的新西蘭白兔椎間盤(pán)材料不會(huì)引起宿主產(chǎn)生明顯的炎癥反應(yīng)以及細(xì)胞介導(dǎo)的免疫反應(yīng),無(wú)體內(nèi)毒性。材料浸出液及材料對(duì)MSC無(wú)明顯毒性,不影響MSC增殖及遷移,不影響MSC細(xì)胞活力。第三部分驗(yàn)證脫細(xì)胞椎間盤(pán)材料的修復(fù)作用目的：驗(yàn)證脫細(xì)胞椎間盤(pán)能在體外誘導(dǎo)MSC向椎間盤(pán)樣細(xì)胞分化,并且能夠在體內(nèi)試驗(yàn)中有效阻止椎間盤(pán)的退變方法：通過(guò)掃描電鏡(SEM)觀察于體外植入脫細(xì)胞椎間盤(pán)材料后MSC的形態(tài)變化,并且用RT-PCR的方法測(cè)定MSC中椎間盤(pán)細(xì)胞相關(guān)基因(包括Col Ⅱ, Col Ⅰ,SOX-9,GPC3,AGN)的表達(dá)情況,來(lái)判斷MSC是否向椎間盤(pán)樣細(xì)胞分化。體外實(shí)驗(yàn)部分使用新西蘭大白兔,用針穿刺椎間盤(pán)制造椎間盤(pán)退變模型。L3-L4為陰性對(duì)照組,造模后注入生理鹽水；IA-L5為實(shí)驗(yàn)組,遣模后植入脫細(xì)胞椎間盤(pán)顆粒；L5-L6為空白對(duì)照組。然后分別觀察0、1、2、3月份的椎間盤(pán)含水量以及椎間盤(pán)高度。HE染色、阿爾新藍(lán)染色觀察椎間盤(pán)微觀結(jié)構(gòu)。結(jié)果：SEM顯示植入MSC的脫細(xì)胞椎間盤(pán)內(nèi)可觀察到椎間盤(pán)樣細(xì)胞,且RT-PCR結(jié)果顯示植入后的MSC較平板培養(yǎng)的MSC椎間盤(pán)細(xì)胞相關(guān)基因表達(dá)上調(diào),如II型膠原(col Ⅱ)、蛋白多糖(AGN)、SOX-9和GPC3,而軟骨相關(guān)基因I型膠原(Col Ⅰ)表達(dá)不增加。體內(nèi)試驗(yàn)部分,實(shí)驗(yàn)組與陰性對(duì)照組組相比保持更高的水化程度,MRI顯示實(shí)驗(yàn)組的椎間盤(pán)高度減小情況也略?xún)?yōu)于陰性對(duì)照組�？傮w來(lái)說(shuō),實(shí)驗(yàn)組的含水量指數(shù)和椎間盤(pán)高度的減少在一定程度上得到改善。雖然實(shí)驗(yàn)組HE染色顯示媵原蛋白的組織學(xué)形態(tài)與對(duì)照組相比無(wú)明顯的變化,但是陰性對(duì)照組,AF的內(nèi)層失去了同心層狀結(jié)構(gòu)并伴有裂縫,而NP也結(jié)構(gòu)紊亂。阿爾新藍(lán)染色還顯示陰性對(duì)照組的GAGs明顯減少,但在實(shí)驗(yàn)治療組這種減少不明顯。結(jié)論：脫細(xì)胞椎間盤(pán)可以阻止椎間盤(pán)變性。
[Abstract]:Part one method and evaluation of the effect of intervertebral disc deactivation: To evaluate the decellular effect of a variety of New Zealand white rabbit intervertebral disc decellular methods and the mechanical properties of the New Zealand white rabbit intervertebral disc after decellular biochemistry: We collected 45 specimens of thoracic and lumbar intervertebral discs from 4 month old New Zealand white rabbits. In vitro experiment, the muscle tissue and bone slices around the intervertebral disc of New Zealand white rabbits were removed clean, and the nucleus and fibrous ring were removed from the cartilage end plate and cleaned to remove the excess blood in PBS. The treated intervertebral disc specimens were immediately frozen in the liquid nitrogen for later use. Cell group A and group B.A decellular methods: 5 times in 37 degrees centigrade water bath and liquid nitrogen, soaked in 2%Triton X-100 for 24 hours, soaked in 1%SDS solution for 24 hours, then treated with 200U/mlDNA enzyme for 2 hours, then cleaned in PBS to remove residual reagents; B group decellular method: water bath and liquid nitrogen at 37 degrees Celsius They were frozen 5 times repeatedly, soaked in 3%Triton X-100 for 24 hours and soaked in 2%SDS solution for 24 hours. After treatment, the remnant reagents were washed in PBS. All specimens were stained with HE and Alican blue, and the Collagen type I, Collagen type II and Aggrecan preservation in the intervertebral disc were detected by immunohistochemistry. DNA was extracted according to the manufacturer's instructions, and the content of DNA was detected by NanoDrop8000. The content of HYP was detected by absorbance colorimetric method. According to the above results, the suitable desiccation scheme was determined, and the unsuitable group was excluded. The samples were dehydrated by freezing dry method and the water content was speculated with the quality change. The microstructures of the control group and the suitable dehydrated group were detected by SEM and TEM. The acellular materials were divided into fibrous ring, nucleus pulposus and two parts. The computer control detection device was applied to detect the modulus of elasticity, maximum tension and maximum pressure, and the compression modulus of the nucleus pulposus was detected. Results: A and control groups were stained with HE and Alican blue, and there were no significant differences in various immunohistochemical results. The B group was between the control group and the control group. In the group A, the content of HYP was the highest, the control group was the highest, the HYP content in the group B was the lowest, and the three had the obvious difference. The A group method was a suitable method for the intervertebral disc dehydration, the water content of the.A group was significantly higher than that of the control group. There was no significant difference between the SEM and TEM results of the two groups, and the modulus of elasticity, pressure modulus, maximum pressure and maximum of the A group and the control group. Conclusion: 37 degrees centigrade water bath and liquid nitrogen are repeatedly frozen 5 times, soaked in 2%Triton X-100 for 24 hours, soaked in 1%SDS solution for 24 hours, and then treated with 200U/mlDNA enzyme for 2 hours. After treatment, cleaning the remnant reagents in PBS to remove the reagents of New Zealand white rabbits. This method is suitable for New Zealand white rabbits. The original biochemical, microstructural and mechanical characteristics of the retained intervertebral disc to the maximum extent. Second part of the safety assessment of the allogeneic intervertebral disc material Objective: To evaluate the safety of the allogeneic disks in vivo and in vitro. Methods: To evaluate the allogeneic disks in the body, respectively. And in vitro safety, we divided the specimens of New Zealand white rabbit disc into the experimental group and the control group. The experimental group was treated according to the suitable method of intervertebral discs removal in the first part. The control group was not treated, and the rabbits were implanted subcutaneously on both sides of the spine of new Zealand white rabbits. After January, the experimental group and the control group were removed. All the specimens were stained with HE. The distribution of neutrophils was observed by color. The level of macrophages and cytotoxic T cells was observed by immunohistochemical detection of MAC387 and CD8. The New Zealand white rabbit MSC was collected by gradient centrifugation. The material of the degenerated intervertebral disc was placed in DMEM-HG and placed in the incubator for 48 hours, and the extracts were collected, and MSC was cultured in DEME-HG containing FBS10% (control group), 25% The extracts were cultured in +DMEM-HG, 50% extract +DMEM-HG and 100% extract, and live dead cells were observed with live dead cells. The cell vitality was observed by CCK-8 kit and absorbance colorimeter. The dead cells of New Zealand white rabbits were planted on the first part of the cell by the first part of the method. The living dead cells were on third days, seventh days, fourteenth days and twenty-first days respectively. The structure of the material and the distribution of MSC were observed with HE staining. Results: the intervertebral disc materials of New Zealand white rabbits treated with the first part of the cells were not subcutaneously implanted with the same white rabbit, and no apparent neutrophils, macrophages, and cytotoxic T cells were infiltrated. After implantation of the rabbit's intervertebral disc material in the rabbits, there were obvious neutrophils, macrophages, cytotoxic T cells infiltration and obvious vascular growth, FBS10% DEME-HG (control group), 25% extract +DMEM-HG, 50% extract +DMEM-HG and 100% extract solution, the number of living cells count and cell activity There is no obvious difference in the growth, amplification and migration of.MSC in the first part of the New Zealand white rabbit intervertebral disc. Conclusion: the intervertebral disc material of New Zealand white rabbit after the first part of the suitable decellular method does not cause the host to produce obvious inflammatory reaction and cell mediated immune response. In vivo toxicity. Material leach and materials have no obvious toxicity to MSC, does not affect the proliferation and migration of MSC, and does not affect the vitality of MSC cells. Third the purpose of verifying the repair effect of the decellular disc material is to verify that the decellular disc can induce MSC to differentiate into the intervertebral disc cells in vitro, and can effectively prevent the intervertebral body in the body test. Disc degeneration: the morphological changes of MSC after implantation of acellular intervertebral disc materials in vitro were observed by scanning electron microscopy (SEM), and the expression of intervertebral disc cell related genes in MSC (including Col II, Col I, SOX-9, GPC3, AGN) in MSC was measured by RT-PCR to determine whether MSC to differentiate into intervertebral disc cells. In vitro experimental part was used. New Zealand white rabbits were treated with needle puncture disc to make disc degeneration model.L3-L4 as negative control group and injected physiological saline after modeling. IA-L5 was used as experimental group. After sending mold, the intervertebral disc particles were implanted. L5-L6 was a blank control group. Then, the water content of intervertebral disc and the height of.HE in the month of 0,1,2,3 were observed, and alxin was observed respectively. The microstructures of the intervertebral disc were observed by blue staining. Results: SEM showed that intervertebral disc cells could be observed in the MSC intervertebral disc, and the RT-PCR results showed that the MSC of the MSC intervertebral disc cells after the implantation was up to up, such as II type collagen (Col II), proteoglycan (AGN), SOX-9 and GPC3, and I gum of cartilage related genes. The expression of Col I did not increase. In the experimental part, the experimental group maintained a higher degree of hydration compared with the negative control group. MRI showed that the height reduction of the intervertebral disc in the experimental group was slightly better than that in the negative control group. In general, the water content index and the decrease of the disc height in the experimental group were improved to a certain extent. Although the experiment was to some extent, the experiment group improved the degree of the decrease of the intervertebral disc height. Group HE staining showed that the histologic morphology of the protein had no significant changes compared with the control group, but in the negative control group, the inner layer of AF lost the concentric layer structure with the cracks, and the NP structure was disorganized. Alnew blue staining also showed that the GAGs in the negative control group decreased obviously, but the decrease in the experimental group was not obvious. Conclusion: take off. The cell disc can prevent the degeneration of the intervertebral disc.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R318.08;R681.53

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3 馮建華;;椎間盤(pán)突出的調(diào)養(yǎng)與康復(fù)[A];中華醫(yī)學(xué)會(huì)第八次全國(guó)物理醫(yī)學(xué)與康復(fù)學(xué)學(xué)術(shù)會(huì)議論文匯編[C];2006年

4 吳毅文;吳紹偉;王斌;;突出的椎間盤(pán)能還納嗎?[A];中國(guó)康復(fù)醫(yī)學(xué)會(huì)頸椎病專(zhuān)業(yè)委員會(huì)第十次學(xué)術(shù)年會(huì)論文匯編[C];2008年

5 童國(guó)海;陸勇;丁曉毅;顏凌;陳克敏;;半導(dǎo)體激光氣化椎間盤(pán)突出的臨床應(yīng)用評(píng)價(jià)[A];中華醫(yī)學(xué)會(huì)第十三屆全國(guó)放射學(xué)大會(huì)論文匯編（下冊(cè)）[C];2006年

6 姜宏;劉錦濤;王擁軍;;破裂型椎間盤(pán)突出重吸收機(jī)理的研究[A];中國(guó)康復(fù)醫(yī)學(xué)會(huì)頸椎病專(zhuān)業(yè)委員會(huì)第十次學(xué)術(shù)年會(huì)論文匯編[C];2008年

7 姜宏;劉錦濤;;大鼠破裂型椎間盤(pán)突出模型的建立及其重吸收機(jī)理的研究[A];中國(guó)康復(fù)醫(yī)學(xué)會(huì)第十一次全國(guó)頸椎病學(xué)術(shù)會(huì)議論文集[C];2009年

8 李旭輝;呂有望;王喜彬;;經(jīng)皮自動(dòng)式椎間盤(pán)初級(jí)吸儀治療腰椎間盤(pán)突出癥附20例報(bào)告[A];全國(guó)第六屆骨科微創(chuàng)手術(shù)與多種針刀手術(shù)學(xué)術(shù)會(huì)議論文集[C];2008年

9 柳健;;探討椎間盤(pán)微創(chuàng)治療的臨床體會(huì)[A];首屆全國(guó)中西醫(yī)結(jié)合骨科微創(chuàng)學(xué)術(shù)交流會(huì)暨專(zhuān)業(yè)委員會(huì)成立大會(huì)論文匯編[C];2011年

10 徐云強(qiáng);馬信龍;張義修;王沛;鄭永發(fā);馮世慶;;椎間盤(pán)造影術(shù)與腰椎間盤(pán)突出癥病理學(xué)分型的研究[A];第十三屆全國(guó)中西醫(yī)結(jié)合骨傷科學(xué)術(shù)研討會(huì)論文集[C];2005年

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5 劉燕玲;“別亂動(dòng)我的椎間盤(pán)”[N];健康報(bào);2007年

6 通訊員　 那偉 高樹(shù)灼;第一醫(yī)院推出“椎間盤(pán)超市”[N];廈門(mén)日?qǐng)?bào);2006年

7 記者 馮立中;椎間盤(pán)突出治療應(yīng)首選絕對(duì)臥床[N];健康報(bào);2010年

8 通訊員 李春梅;椎間盤(pán)突出切莫盲目手術(shù)[N];家庭醫(yī)生報(bào);2009年

9 記者 牟春麗;創(chuàng)新技術(shù) 立足椎間盤(pán)醫(yī)學(xué)前沿[N];西安日?qǐng)?bào);2006年

10 記者 牟春麗;創(chuàng)新技術(shù) 立足椎間盤(pán)醫(yī)學(xué)前沿[N];西安日?qǐng)?bào);2007年

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2 方向前;去細(xì)胞同種椎間盤(pán)：使退變椎間盤(pán)再生的天然生物材料[D];浙江大學(xué);2016年

3 張宏軍;神經(jīng)生長(zhǎng)相關(guān)蛋白-43在大鼠椎間盤(pán)炎癥模型中的表達(dá)及意義[D];浙江大學(xué);2007年

4 趙新剛;重組人骨形態(tài)發(fā)生蛋白-7基因腺病毒載體轉(zhuǎn)染對(duì)兔受損椎間盤(pán)影響的體內(nèi)研究[D];第二軍醫(yī)大學(xué);2006年

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7 辛洪奎;組織工程化同種異體椎間盤(pán)移植的體外及動(dòng)物實(shí)驗(yàn)研究[D];第四軍醫(yī)大學(xué);2012年

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9 劉志恒;FasL在椎間盤(pán)免疫赦免缺失調(diào)控中作用的實(shí)驗(yàn)研究[D];第四軍醫(yī)大學(xué);2013年

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