CN108310466B - 一种天然组织来源的髓核脱细胞材料的制备方法 - Google Patents
一种天然组织来源的髓核脱细胞材料的制备方法 Download PDFInfo
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Abstract
本发明公开了一种天然组织来源的髓核脱细胞材料的制备方法,将哺乳动物任意大小的髓核组织经含蛋白酶抑制剂的PBS、含NaCl的PBS缓冲液、含Triton X的PBS缓冲液、含SDS的PBS缓冲液、含DNA酶的PBS缓冲液处理,获得天然组织来源的髓核脱细胞材料。本发明能对组织上的细胞进行完全去细胞化处理,且条件温和、无ECM损害、快速稳定。所得的材料,不仅最大限度的模拟了天然的髓核结构、较好的保留了细胞外基质成分、还具有极低的免疫原性等优点,可用于修复临床上各类病因造成的椎间盘损伤、髓核退变相关疾病。
Description
技术领域
本发明主要用于椎间盘髓核的修复及再生的医学领域。具体涉及一种天然组织来源的髓核脱细胞材料的制备方法。
背景技术
椎间盘退变是导致腰椎间盘突出的主要因素,给社会保障和经济带来巨大的负担。目前针对腰椎间盘突出的手术方式主要是椎体融合术,其并不能恢复正常椎间盘的功能。组织工程则力主恢复正常的椎间盘生理结构和力学功能,为未来椎间盘病变的治愈提供了一种可能的方式。椎间盘退变的组织学特征是髓核含水量降低,I型胶原增加而II型胶原减少,同时糖胺聚糖含量也降低,导致髓核缺乏弹性,容易引起椎间盘突出。因此,消除髓核退变,再生正常髓核,是组织工程治疗椎间盘退变的一种重要策略。目前,在组织工程领域,相比人工髓核材料,脱细胞材料在去除细胞的同时保留了原组织的三维结构,生长因子和力学特性。这些共同构成了髓核细胞生长的微环境,提供了机体干细胞分化为髓核细胞的信号。已有实验证明,脱细胞髓核材料可以诱导人来源的脂肪干细胞分化为髓核细胞,并成功分泌出髓核相关的糖胺聚糖和胶原II细胞外基质。这些预示了脱细胞髓核材料有足够的前景作为一种退变髓核再生的替代材料。
脱细胞技术是获得脱细胞材料的手段。其最重要评价指标是能够彻底地去除组织中的细胞,同时完好地保留细胞外基质,并且在脱细胞处理过程后不残留毒性物质。目前性能优良的髓核脱细胞支架还未见报道。因此本发明通过创新的脱细胞技术制备了一种天然的髓核脱细胞支架,在去除细胞的同时,最大化模拟了天然的髓核结构,证明了该支架具有极低的免疫原性,较好的保留了天然髓核的细胞外基质组分,并且证实该支架在体内能很好的再生退变髓核,在椎间盘退变再生组织工程中有极大的意义。
发明内容
本发明目的在于填补现有技术中,尚无脱细胞髓核支架制备的报道,提供一种具有极低免疫原性的,较好保留细胞外基质组分的,并能促进髓核再生的脱细胞髓核支架的制备方法。
一种天然哺乳动物或人组织来源的髓核脱细胞材料的制备方法,包括以下步骤:
(1)取宰杀12h以内任意哺乳动物的胸5到腰6脊椎,用刀分离出椎间盘,然后切开纤维环取出髓核。用含蛋白酶抑制剂的PBS缓冲液漂洗髓核3次,每次30min,去除血液,贴附的脂肪碎片和其他杂质。
(2)在含10K IU/ml蛋白酶抑制剂的PBS缓冲液中,低温(4℃)摇床100rpm震荡24-48小时;然后用液氮冻融3个循环(-80℃/37℃)
(3)在含NaCl的PBS缓冲液中,加入混合抗菌液,低温(4℃)摇床100rpm震荡24-48小时;
(4)在含Triton X的PBS缓冲液中,加入混合抗菌液,低温(4℃)摇床100rpm震荡24-48小时;
(5)在含SDS的PBS缓冲液中,加入混合抗菌液,低温(4℃)摇床100rpm震荡24-48小时;
(6)在含DNA酶的PBS缓冲液中,加入混合抗菌液,37℃摇床120rpm震荡12-24小时;
步骤(2)-(6)中,每个步骤完成后均用生理盐水冲洗8小时。
作为优选,所述含蛋白酶抑制剂的PBS缓冲液,蛋白酶抑制剂浓度为10-20K IU/ml;
作为优选,所述的含NaCl的PBS缓冲液为浓度1M-1.5M的NaCl的PBS缓冲液;
作为优选,所述的含Triton X的PBS缓冲液为浓度0.05%-0.1%的Triton X-100或Triton X-200的PBS缓冲液;
作为优选,所述的含SDS的PBS缓冲液为浓度0.1%-0.5%的SDS的PBS缓冲液;
作为优选,所述的含DNA酶的PBS缓冲液为浓度为1-2mg/mL DNA酶PBS缓冲液;
作为优选,所述的混合抗菌液中青霉素和链霉素的浓度分别为20K IU/ml,20g/ml;青霉素和链霉素的比例为1:1,加入的混合抗菌液与原溶液体积比为1:1。
针对目前用于退变髓核修复和再生的材料及其制备方法存在的缺陷与不足,发明人建立了一种天然组织来源的髓核脱细胞材料的制备方法,该方法将哺乳动物任意大小的髓核组织经含蛋白酶抑制剂的PBS、含NaCl的PBS缓冲液、含Triton X的PBS缓冲液、含SDS的PBS缓冲液、含DNA酶的PBS缓冲液处理,获得天然组织来源的髓核脱细胞材料。本发明能对组织上的细胞进行完全去细胞化处理,且条件温和、无ECM损害、快速稳定。所得的材料,不仅最大限度的模拟了天然的髓核结构、较好的保留了细胞外基质成分、还具有极低的免疫原性等优点,可用于修复临床上各类病因造成的椎间盘损伤、髓核退变相关疾病。
相对于现有技术,本发明的显著进步在于:
(1)本发明通过脱细胞技术制备的髓核脱细胞材料在去除异种/异体细胞的同时,可较好保留原先ECM的完整性,可以最大限度的模拟正常髓核微观结构和细胞外基质成分。
(2)本发明材料不含细胞、细胞核、DNA等抗原性物质,具有极低的免疫排斥反应,具有很好的生物相容性。
(3)本发明材料在腰椎间盘退变的模型中能促进退变髓核再生,在髓核再生组织工程中有极大的意义。
附图说明
图1是本发明材料大体外观图
图2是本发明材料HE染色无细胞及无细胞核成分残留图
图3是本发明材料DNA定量检测几乎不含有DNA成分图
图4是本发明材料胶原成分定性(胶原1和胶原2免疫组化染色,A1-2,B1-2)以及定量检测保留大量胶原成分图(D);糖胺聚糖成分定性(alcian blue染色,C1-2)以及定量检测保留大量糖胺聚糖成分图(E)
图5是本发明材料扫描和透射电镜证明纤维排布以及空间立体结构保留图
图6是本发明材料用CCK8法证明无细胞毒性图
图7是本发明材料体外复细胞HE染色检测证明极好的生物相容性图
图8是本发明材料体外复细胞PCR定量检测间充质干细胞产生基因表达图
图9是本发明材料移植兔子退变椎间盘2周,4周,8周的HE染色和核磁共振成像,以及椎间盘高度指数百分比,共同证明了髓核脱细胞材料可促进退变髓核修复再生图
具体实施方案
下面结合附图及实施例对本发明进行详细描述,但本发明的实施不仅限于此。
实施例1:天然组织来源的髓核脱细胞材料的制备方法和再生修复研究
1.制备髓核脱细胞材料
(1)取材:取宰杀12h以内猪的胸5到腰6脊椎,用刀分离出椎间盘,然后切开纤维环取出髓核,髓核大小约为1.5*1.5*1cm。用含10K IU/ml蛋白酶抑制剂的PBS缓冲液漂洗髓核3次,每次30min,去除血液,贴附的脂肪碎片和其他杂质。
(2)脱细胞步骤如下:
①、在含20K IU/ml蛋白酶抑制剂的PBS缓冲液中,低温(4℃)摇床100rpm震荡24小时,然后用液氮冻融3个循环(-80℃/37℃);
②、在含1M NaCl的PBS缓冲液中,1:1加入青霉素和链霉素(20KIU/ml,20g/ml)混合抗菌液,低温(4℃)摇床100rpm震荡24小时;
③、在浓度为0.05%的Triton X-100的PBS缓冲液中,1:1加入青霉素和链霉素(20KIU/ml,20g/ml)混合抗菌液,低温(4℃)摇床100rpm震荡24小时;
④、在浓度为0.1%的含SDS的PBS缓冲液中,1:1加入青霉素和链霉素(20KIU/ml,20g/ml)混合抗菌液,低温(4℃)摇床100rpm震荡24小时;
⑤、在浓度为1mg/ml的含DNA酶的PBS缓冲液中,1:1加入青霉素和链霉素(20KIU/ml,20g/ml)混合抗菌液,37℃摇床100rpm震荡12小时,即可得到猪髓核脱细胞材料。
注:完成每一步骤后均用生理盐水冲洗8小时。
2.天然组织来源的髓核脱细胞材料细胞去除评定
图1是本发明材料的大体外观图(A1是正常髓核组织,A2是脱细胞处理后的髓核组织);图2是本发明材料HE染色无细胞及无细胞核成分残留图;图3是本发明材料DNA检测几乎不含有DNA成分图(其中A是DNA定量检测,B是DNA是否存在的定性检测)。
3.髓核脱细胞材料细胞外基质保留的评估
图4是本发明材料胶原,糖胺聚糖成分定性和定量检测图。其中A1-2是1型胶原的免疫组化染色,B1-2是2型胶原的免疫组化染色,C1-2是糖胺聚糖的alcian blue染色,D是胶原含量的定量测量,E是糖胺聚糖含量的定量测量,以上数据均说明了脱细胞后,髓核组织的细胞外基质成分保存完好;图5是本发明材料扫描电镜(D)和透射电镜(E)证明纤维排布以及空间立体结构保留图。
4.髓核脱细胞材料细胞相容性的评估
图6是本发明材料细胞毒性的检测,通过提取材料的浸提液,用CCK8法证明了材料对细胞无明显毒性;图7是本发明材料体外复细胞HE染色检测证明极好的细胞相容性图,可从图中见到细胞(黄色箭头所示)可以粘附到材料上并成功爬入材料使材料成功复细胞化;图8是本发明材料体外复细胞PCR定量检测间充质干细胞产生基因表达图,说明材料所营造的微环境适合干细胞向髓核细胞分化。
5.髓核脱细胞材料促进退变髓核的修复与再生
图9是本发明材料移植兔子退变椎间盘2周,4周,8周的HE染色和核磁共振成像,以及椎间盘高度指数百分比,共同证明了髓核脱细胞材料可促进退变髓核修复再生图
实施例2:脱细胞髓核材料的制备和再生修复研究
(1)取材:取宰杀12h以内猪的胸5到腰6脊椎,用刀分离出椎间盘,然后切开纤维环取出髓核,髓核大小约为1*1*0.6cm。用含10K IU/ml蛋白酶抑制剂的PBS缓冲液漂洗髓核3次,每次30min,去除血液,贴附的脂肪碎片和其他杂质。
(2)脱细胞步骤如下:
①、在含15K IU/ml蛋白酶抑制剂的PBS缓冲液中,低温(4℃)摇床100rpm震荡36小时,然后用液氮冻融3个循环(-80℃/37℃);
②、在含1.2M NaCl的PBS缓冲液中,1:1加入青霉素和链霉素(20KIU/ml,20g/ml)混合抗菌液,低温(4℃)摇床100rpm震荡36小时;
③、在浓度为0.08%的Triton X-100的PBS缓冲液中,1:1加入青霉素和链霉素(20KIU/ml,20g/ml)混合抗菌液,低温(4℃)摇床100rpm震荡36小时;
④、在浓度为0.3%的含SDS的PBS缓冲液中,1:1加入青霉素和链霉素(20KIU/ml,20g/ml)混合抗菌液,低温(4℃)摇床100rpm震荡36小时;
⑤、在浓度为1.5mg/ml的含DNA酶的PBS缓冲液中,1:1加入青霉素和链霉素(20KIU/ml,20g/ml)混合抗菌液,37o C摇床100rpm震荡20小时。其余参考实施例1的方法进行,得到天然组织来源的髓核脱细胞材料。
实施例3:脱细胞髓核材料的制备和再生修复研究
(1)取材:取宰杀12h以内羊的胸5到腰6脊椎,用刀分离出椎间盘,然后切开纤维环取出髓核,髓核大小约为0.6*0.6*0.6cm。用含10K IU/ml蛋白酶抑制剂的PBS缓冲液漂洗髓核3次,每次30min,去除血液,贴附的脂肪碎片和其他杂质。
(2)脱细胞步骤如下:
①、在含20K IU/ml蛋白酶抑制剂的PBS缓冲液中,低温(4℃)摇床100rpm震荡48小时,然后用液氮冻融3个循环(-80℃/37℃);
②、在含1.5M NaCl的PBS缓冲液中,1:1加入青霉素和链霉素(20KIU/ml,20g/ml)混合抗菌液,低温(4℃)摇床100rpm震荡48小时;
③、在浓度为0.1%的Triton X-100的PBS缓冲液中,1:1加入青霉素和链霉素(20KIU/ml,20g/ml)混合抗菌液,低温(4℃)摇床100rpm震荡48小时;
④、在浓度为0.5%的含SDS的PBS缓冲液中,1:1加入青霉素和链霉素(20KIU/ml,20g/ml)混合抗菌液,低温(4℃)摇床100rpm震荡48小时;
⑤、在浓度为2mg/ml的含DNA酶的PBS缓冲液中,1:1加入青霉素和链霉素(20KIU/ml,20g/ml)混合抗菌液,37℃摇床100rpm震荡24小时。其余参考实施例1的方法进行,得到天然组织来源的髓核脱细胞材料。
实施例4:脱细胞髓核材料的制备和再生修复研究
(1)取材:取宰杀12h以内牛的胸5到腰6脊椎,用刀分离出椎间盘,然后切开纤维环取出髓核,髓核大小约为1.5*1.5*1cm。用含10K IU/ml蛋白酶抑制剂的PBS缓冲液漂洗髓核3次,每次30min,去除血液,贴附的脂肪碎片和其他杂质。
(2)脱细胞步骤如下:
①、在含20K IU/ml蛋白酶抑制剂的PBS缓冲液中,低温(4℃)摇床100rpm震荡30小时,然后用液氮冻融3个循环(-80℃/37℃);
②、在含1.5M NaCl的PBS缓冲液中,1:1加入青霉素和链霉素(20KIU/ml,20g/ml)混合抗菌液,低温(4℃)摇床100rpm震荡24小时;
③、在浓度为0.1%的Triton X-100的PBS缓冲液中,1:1加入青霉素和链霉素(20KIU/ml,20g/ml)混合抗菌液,低温(4℃)摇床100rpm震荡24小时;
④、在浓度为0.1%的含SDS的PBS缓冲液中,1:1加入青霉素和链霉素(20KIU/ml,20g/ml)混合抗菌液,低温(4℃)摇床100rpm震荡36小时;
⑤、在浓度为2mg/ml的含DNA酶的PBS缓冲液中,1:1加入青霉素和链霉素(20KIU/ml,20g/ml)混合抗菌液,37℃摇床100rpm震荡24小时。其余参考实施例1的方法进行,得到天然组织来源的髓核脱细胞材料。
实施例5:脱细胞髓核材料的制备和再生修复研究
取宰杀12h以内牛的胸5到腰6脊椎,骤④在浓度为0.1%的Triton X-200的PBS缓冲液中,1:1加入青霉素和链霉素(20KIU/ml,20g/ml)混合抗菌液,低温(4℃)摇床100rpm震荡24小时。其余参考实施例1的方法进行,得到天然组织来源的髓核脱细胞材料。
对实施例2-5所得髓核脱细胞材料分别进行组织学评价、抗原成分定量检测和结构检测,结果与实施例1类似,这表明可通过上述多种方法制得多种组织来源,多种组织大小的脱细胞髓核材料,并且对该材料进行组织学评价、抗原成分定量检测检测均说明材料已经完全去除细胞成分,无明显抗原成分残留。同时用上述方法获得的髓核脱细胞材料均具有极好的细胞相容性。该髓核脱细胞材料可以作为临床上促进退变髓核修复再生的,安全可靠、有效、快速的生物材料。
Claims (1)
1.一种天然组织来源的髓核脱细胞材料的制备方法,其特征在于,该方法具体包括以下步骤:
(1)取宰杀12h以内任意哺乳动物胸5到腰6脊椎,用刀分离出椎间盘,然后切开纤维环取出髓核;用含蛋白酶抑制剂的PBS缓冲液漂洗髓核3次,每次30min,去除血液,贴附的脂肪碎片和其他杂质;
(2)在含蛋白酶抑制剂的PBS缓冲液中,在4℃温度下用摇床100rpm震荡24-48小时;然后用液氮冻融3个循环;
(3)在含NaCl的PBS缓冲液中,加入混合抗菌液,在4℃温度下用摇床100rpm震荡24-48小时;
(4)在含TritonX的PBS缓冲液中,加入混合抗菌液,在4℃温度下用摇床100rpm震荡24-48小时;
(5)在含SDS的PBS缓冲液中,加入混合抗菌液,在4℃温度下用摇床100rpm震荡24-48小时;
(6)在含DNA酶的PBS缓冲液中,加入混合抗菌液,37℃摇床120rpm震荡12-24小时;
步骤(2)-(6)中,每个步骤完成后均用生理盐水冲洗8小时;
所述含蛋白酶抑制剂的PBS缓冲液,蛋白酶抑制剂浓度为10-20KIU/mL;
所述含NaCl的PBS缓冲液为浓度1M-1.5M的NaCl的PBS缓冲液;
所述含TritonX的PBS缓冲液为浓度0.05%-0.1%的TritonX-100或Triton X-200的PBS缓冲液;
所述含SDS的PBS缓冲液为浓度0.1%-0.5%的SDS的PBS缓冲液;
所述含DNA酶的PBS缓冲液为浓度为1-2mg/mLDNA酶PBS缓冲液;
所述混合抗菌液中青霉素和链霉素的浓度分别为20KIU/mL,20g/mL;青霉素和链霉素的比例为1:1,加入的混合抗菌液与原溶液体积比为1:1。
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