去细胞同种椎间盘：使退变椎间盘再生的天然生物材料
发布时间：2018-07-31 12:10
【摘要】：第一部分椎间盘脱细胞方法及效果评估目的：评估多种新西兰白兔椎间盘脱细胞方法其脱细胞效果以及新西兰白兔椎间盘经脱细胞后生物化学性质里力学性质方法：我们从4月龄新西兰白兔中收集了45例胸椎及腰椎椎间盘标本,用作体外实验。新西兰白兔椎间盘周围的肌肉组织及骨片被剔除干净,切去软骨终板保留髓核和纤维环,并在PBS中清洗以洗去多余的血液。处理后的椎间盘标本立刻冻存于液氮中以备后用。将标本分为不作脱细胞处理的阴性对照组和行脱细胞组的A组以及B组。A组脱细胞方法：在37摄氏度水浴和液氮中反复溶冻5次,在2%Triton X-100中浸泡24小时,在1%SDS溶液中浸泡24小时,后以200U/mlDNA酶处理2小时,处理后在PBS中清洗以洗去残余试剂；B组脱细胞方法：在37摄氏度水浴和液氮中反复溶冻5次,在3%Triton X-100中浸泡24小时,在2%SDS溶液中浸泡24小时,处理后在PBS中清洗以洗去残余试剂.所有标本均以HE和Alican蓝染色,以免疫组织化学检测椎间盘中Collagen type Ⅰ, Collagen type Ⅱ以及Aggrecan保存量,以DNeasy Blood Kit试剂盒按生产商说明书处理提取DNA,用NanoDrop8000检测DNA含量,以吸光度比色法检测HYP含量,根据以上结果确定适宜脱细胞方案,排除不适宜组,以冻干法使标本脱水,以质量变化推测含水量。以SEM及TEM检测对照组及适宜脱细胞法组的微观结构。将脱细胞材料分为纤维环及髓核和两部分,适用电脑控制检测装置以检测纤维环弹性模量,最大张力以及最大压力,检测髓核压缩模量。结果：A组和对照组在HE和Alican蓝染色,各项免疫组织化学结果中无明显差异,B组则与对照组之间存在明显差异；A组中HYP含量最高,对照组次之,B组中HYP含量最低,且三者存在明显差异,A组方法为适宜椎间盘脱细胞方法。A组含水量明显高于对照组,两者SEM以及TEM结果无明显差异；A组与对照组的弹性模量,压力模量,最大压力以及最大延长度均无明显差异。结论：在37摄氏度水浴和液氮中反复溶冻5次,在2%Triton X-100中浸泡24小时,在1%SDS溶液中浸泡24小时,后以200U/mlDNA酶处理2小时,处理后在PBS中清洗以洗去残余试剂是新西兰白兔适宜的椎间盘脱细胞方法。这种方法可以最大程度上的保留椎间盘原有的生物化学特点,微观结构以及力学特点。第二部分同种去细胞椎间盘材料安全性评估目的：评估同种去细胞椎间盘材料分别在体内及体外的安全性。方法：为评估同种去细胞椎间盘材料分别在体内及体外的安全性,我们将新西兰白兔椎间盘标本分为实验组及对照组,实验组按第一部分中得出的适宜的椎间盘脱细胞方法进行处理,对照组不予处理,分别植入新西兰白兔脊柱两侧皮下,1月后去除实验组及对照组,所有标本均以HE染色观察中性粒细胞分布,以免疫组织化学检测MAC387及CD8水平观察巨噬细胞及细胞毒T细胞水平。以梯度离心法收集新西兰白兔MSC,将去细胞椎间盘材料置于DMEM-HG中,放置于培养箱48小时,收集浸提液,将MSC培养于含FBS10%的DEME-HG(对照组),25%浸提液+DMEM-HG,50%浸提液+DMEM-HG和100%浸提液中培养,以活死细胞染色观察存活细胞,以CCK-8试剂盒及吸光度比色计观察细胞活力。将MSC种植按第一部分方法脱细胞的新西兰白兔椎间盘材料上,分别在第3天,第7天,第14天,第21天以活死细胞染色观察存活细胞,以HE染色观测材料结构以及MSC分布。结果：经第一部分适宜脱细胞方法处理后的新西兰白兔椎间盘材料在植入同种白兔皮下后未见明显中性粒细胞,巨噬细胞,细胞毒性T细胞浸润；未经第一部分适宜脱细胞方法处理后的新西兰白兔椎间盘材料在植入同种白兔皮下后出现明显中性粒细胞,巨噬细胞,细胞毒性T细胞浸润并出现明显血管生长；FBS10%的DEME-HG(对照组),25%浸提液+DMEM-HG,50%浸提液+DMEM-HG和100%浸提液这四组在活细胞数计数以及细胞活性无明显差异。MSC能在经第一部分适宜脱细胞方法处理后的新西兰白兔椎间盘材料生长,扩增以及迁移。结论：经第一部分适宜脱细胞方法处理后的新西兰白兔椎间盘材料不会引起宿主产生明显的炎症反应以及细胞介导的免疫反应,无体内毒性。材料浸出液及材料对MSC无明显毒性,不影响MSC增殖及迁移,不影响MSC细胞活力。第三部分验证脱细胞椎间盘材料的修复作用目的：验证脱细胞椎间盘能在体外诱导MSC向椎间盘样细胞分化,并且能够在体内试验中有效阻止椎间盘的退变方法：通过扫描电镜(SEM)观察于体外植入脱细胞椎间盘材料后MSC的形态变化,并且用RT-PCR的方法测定MSC中椎间盘细胞相关基因(包括Col Ⅱ, Col Ⅰ,SOX-9,GPC3,AGN)的表达情况,来判断MSC是否向椎间盘样细胞分化。体外实验部分使用新西兰大白兔,用针穿刺椎间盘制造椎间盘退变模型。L3-L4为阴性对照组,造模后注入生理盐水；IA-L5为实验组,遣模后植入脱细胞椎间盘颗粒；L5-L6为空白对照组。然后分别观察0、1、2、3月份的椎间盘含水量以及椎间盘高度。HE染色、阿尔新蓝染色观察椎间盘微观结构。结果：SEM显示植入MSC的脱细胞椎间盘内可观察到椎间盘样细胞,且RT-PCR结果显示植入后的MSC较平板培养的MSC椎间盘细胞相关基因表达上调,如II型胶原(col Ⅱ)、蛋白多糖(AGN)、SOX-9和GPC3,而软骨相关基因I型胶原(Col Ⅰ)表达不增加。体内试验部分,实验组与阴性对照组组相比保持更高的水化程度,MRI显示实验组的椎间盘高度减小情况也略优于阴性对照组。总体来说,实验组的含水量指数和椎间盘高度的减少在一定程度上得到改善。虽然实验组HE染色显示媵原蛋白的组织学形态与对照组相比无明显的变化,但是阴性对照组,AF的内层失去了同心层状结构并伴有裂缝,而NP也结构紊乱。阿尔新蓝染色还显示阴性对照组的GAGs明显减少,但在实验治疗组这种减少不明显。结论：脱细胞椎间盘可以阻止椎间盘变性。
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R318.08;R681.53
本文编号：2155544
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R318.08;R681.53
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