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作者王效（2019）在《大鼠腰椎软骨终板干细胞的获取及脱细胞软骨基质组织支架的制备》一文中研究指出：目的:分离及鉴定大鼠腰椎软骨终板干细胞,并制备脱软骨细胞基质,探究脱软骨细胞基质的生物相容性及对大鼠腰椎软骨终板干细胞的分化影响;将脱细胞软骨基质联合壳聚糖经冷冻干燥法制备组织支架,探究软骨干细胞在支架上的增殖与凋亡。方法:分离大鼠腰椎软骨终板,使用酶联合消化获得原代软骨细胞;将软骨细胞按照一定的密度接种培养后,结晶紫染色,计算软骨干细胞集落形成的百分比;胰酶消化集落并进行扩增培养,加入成骨,成脂肪及成软骨诱导培养液诱导相应的天数后,使用茜素红,油红O及番红染色,观察其多向分化能力;流式细胞仪检测干细胞表面阳性标记物CD44和CD90及阴性标记物CD45的表达情况。将家猪软骨粉碎后,胰酶联合核酶TritonX-100处理得到脱细胞软骨基质,DAPI染色观察软骨组织脱细胞效果;电镜扫描观察脱细胞基质的形态;傅里叶红外光谱仪分析脱细胞基质的组成成分;将脱细胞基质溶于醋酸后,制备成脱细胞基质膜,观察其大体特点;将获得的终板软骨干细胞种植于膜上,倒置相差显微镜下观察软骨干细胞生长形态,并用CCK-8检测软骨干细胞在基质膜上的增殖情况,与空白板进行对照;引入细胞相对增值率（RGR）的概念,建立新的从细胞学评价其生物相容性的评价系统;微量注射器吸取1ml脱细胞基质溶液、醋酸,分别注射到SD大鼠皮下,观察皮丘周围是否出现红肿,分别测量注射时及注射后12h、24h、36h各组大鼠的体温和皮丘直径的变化,从活体方面评价其生物相容性。另外,取P3代终板软骨干细胞种植于铺有软骨基质的孔板,然后进行成骨、成脂肪及成软骨分化诱导,2周后,茜素红、油红O及番红染色,同时Real-Time PCR分别检测成骨、成脂肪及成软骨特征性基因Runx2、Col-2a1和PPAR?在各组中的相对表达量,分析干细胞在软骨基质上的分化倾向性。最后,将获得的脱细胞软骨基质与壳聚糖按照一定的比例混合后,冷冻干燥后,电镜下扫描,观察软骨干细胞在脱细胞软骨基质组织支架的增殖与凋亡情况。结果:通过不同细胞密度接种培养终板软骨细胞,在培养3到5天可见细胞集落形成,且集落内细胞的形态呈梭形。计算集落形成率,其中100个/cm2组（4.87%±0.30%）,200个/cm2组（4.12%±0.42%）,300个/cm2组（2.43%±0.12%）,经单因素方差分析,P=0.0002,结果有统计学意义。软骨干细胞经多向分化诱导后能被茜素红、油红O及番红染色,说明其具有成骨、成脂肪及成软骨分化能力。其表面表达CD44、CD90、CD45比例分别为90.75%、99.7%和1.36%。经RTCA检测,EPSCs增殖能力强于EPCs。制备的脱细胞软骨基质肉眼呈白色糜状,其膜呈白色透明状,电镜下为交错排列的胶原纤维组成,其具有良好的生物相容性。脱细胞软骨基质能够促进软骨干细胞向成骨方向分化,抑制成软骨及成脂肪方向分化;Real-Time PCR检测成骨基因Runx2在DEPM组较其他两组呈较高表达,而Col-2a1和PPAR?基因表达较低。制备的脱细胞软骨基质联合壳聚糖支架肉眼呈乳白色“海绵”状多空结构,软骨干细胞在其上具有良好的增殖能力,发生较少的凋亡。结论:软骨组织中存在具有多向分化能力、单克隆集落形成能力及CD44及CD90高表达的软骨干细胞,其增殖能力远强于软骨细胞;制备的脱细胞软骨基质促进软骨干细胞成骨方向分化,且具有良好的生物相容性;混合壳聚糖后,制备的冷冻干支架细胞毒性较小,符合组织工程支架的选材标准。
Abstract
mu de :fen li ji jian ding da shu yao chui ruan gu zhong ban gan xi bao ,bing zhi bei tuo ruan gu xi bao ji zhi ,tan jiu tuo ruan gu xi bao ji zhi de sheng wu xiang rong xing ji dui da shu yao chui ruan gu zhong ban gan xi bao de fen hua ying xiang ;jiang tuo xi bao ruan gu ji zhi lian ge ke ju tang jing leng dong gan zao fa zhi bei zu zhi zhi jia ,tan jiu ruan gu gan xi bao zai zhi jia shang de zeng shi yu diao wang 。fang fa :fen li da shu yao chui ruan gu zhong ban ,shi yong mei lian ge xiao hua huo de yuan dai ruan gu xi bao ;jiang ruan gu xi bao an zhao yi ding de mi du jie chong pei yang hou ,jie jing zi ran se ,ji suan ruan gu gan xi bao ji la xing cheng de bai fen bi ;yi mei xiao hua ji la bing jin hang kuo zeng pei yang ,jia ru cheng gu ,cheng zhi fang ji cheng ruan gu you dao pei yang ye you dao xiang ying de tian shu hou ,shi yong qian su gong ,you gong Oji fan gong ran se ,guan cha ji duo xiang fen hua neng li ;liu shi xi bao yi jian ce gan xi bao biao mian yang xing biao ji wu CD44he CD90ji yin xing biao ji wu CD45de biao da qing kuang 。jiang jia zhu ruan gu fen sui hou ,yi mei lian ge he mei TritonX-100chu li de dao tuo xi bao ruan gu ji zhi ,DAPIran se guan cha ruan gu zu zhi tuo xi bao xiao guo ;dian jing sao miao guan cha tuo xi bao ji zhi de xing tai ;fu li xie gong wai guang pu yi fen xi tuo xi bao ji zhi de zu cheng cheng fen ;jiang tuo xi bao ji zhi rong yu cu suan hou ,zhi bei cheng tuo xi bao ji zhi mo ,guan cha ji da ti te dian ;jiang huo de de zhong ban ruan gu gan xi bao chong zhi yu mo shang ,dao zhi xiang cha xian wei jing xia guan cha ruan gu gan xi bao sheng chang xing tai ,bing yong CCK-8jian ce ruan gu gan xi bao zai ji zhi mo shang de zeng shi qing kuang ,yu kong bai ban jin hang dui zhao ;yin ru xi bao xiang dui zeng zhi lv （RGR）de gai nian ,jian li xin de cong xi bao xue ping jia ji sheng wu xiang rong xing de ping jia ji tong ;wei liang zhu she qi xi qu 1mltuo xi bao ji zhi rong ye 、cu suan ,fen bie zhu she dao SDda shu pi xia ,guan cha pi qiu zhou wei shi fou chu xian gong zhong ,fen bie ce liang zhu she shi ji zhu she hou 12h、24h、36hge zu da shu de ti wen he pi qiu zhi jing de bian hua ,cong huo ti fang mian ping jia ji sheng wu xiang rong xing 。ling wai ,qu P3dai zhong ban ruan gu gan xi bao chong zhi yu pu you ruan gu ji zhi de kong ban ,ran hou jin hang cheng gu 、cheng zhi fang ji cheng ruan gu fen hua you dao ,2zhou hou ,qian su gong 、you gong Oji fan gong ran se ,tong shi Real-Time PCRfen bie jian ce cheng gu 、cheng zhi fang ji cheng ruan gu te zheng xing ji yin Runx2、Col-2a1he PPAR?zai ge zu zhong de xiang dui biao da liang ,fen xi gan xi bao zai ruan gu ji zhi shang de fen hua qing xiang xing 。zui hou ,jiang huo de de tuo xi bao ruan gu ji zhi yu ke ju tang an zhao yi ding de bi li hun ge hou ,leng dong gan zao hou ,dian jing xia sao miao ,guan cha ruan gu gan xi bao zai tuo xi bao ruan gu ji zhi zu zhi zhi jia de zeng shi yu diao wang qing kuang 。jie guo :tong guo bu tong xi bao mi du jie chong pei yang zhong ban ruan gu xi bao ,zai pei yang 3dao 5tian ke jian xi bao ji la xing cheng ,ju ji la nei xi bao de xing tai cheng suo xing 。ji suan ji la xing cheng lv ,ji zhong 100ge /cm2zu （4.87%±0.30%）,200ge /cm2zu （4.12%±0.42%）,300ge /cm2zu （2.43%±0.12%）,jing chan yin su fang cha fen xi ,P=0.0002,jie guo you tong ji xue yi yi 。ruan gu gan xi bao jing duo xiang fen hua you dao hou neng bei qian su gong 、you gong Oji fan gong ran se ,shui ming ji ju you cheng gu 、cheng zhi fang ji cheng ruan gu fen hua neng li 。ji biao mian biao da CD44、CD90、CD45bi li fen bie wei 90.75%、99.7%he 1.36%。jing RTCAjian ce ,EPSCszeng shi neng li jiang yu EPCs。zhi bei de tuo xi bao ruan gu ji zhi rou yan cheng bai se mei zhuang ,ji mo cheng bai se tou ming zhuang ,dian jing xia wei jiao cuo pai lie de jiao yuan qian wei zu cheng ,ji ju you liang hao de sheng wu xiang rong xing 。tuo xi bao ruan gu ji zhi neng gou cu jin ruan gu gan xi bao xiang cheng gu fang xiang fen hua ,yi zhi cheng ruan gu ji cheng zhi fang fang xiang fen hua ;Real-Time PCRjian ce cheng gu ji yin Runx2zai DEPMzu jiao ji ta liang zu cheng jiao gao biao da ,er Col-2a1he PPAR?ji yin biao da jiao di 。zhi bei de tuo xi bao ruan gu ji zhi lian ge ke ju tang zhi jia rou yan cheng ru bai se “hai mian ”zhuang duo kong jie gou ,ruan gu gan xi bao zai ji shang ju you liang hao de zeng shi neng li ,fa sheng jiao shao de diao wang 。jie lun :ruan gu zu zhi zhong cun zai ju you duo xiang fen hua neng li 、chan ke long ji la xing cheng neng li ji CD44ji CD90gao biao da de ruan gu gan xi bao ,ji zeng shi neng li yuan jiang yu ruan gu xi bao ;zhi bei de tuo xi bao ruan gu ji zhi cu jin ruan gu gan xi bao cheng gu fang xiang fen hua ,ju ju you liang hao de sheng wu xiang rong xing ;hun ge ke ju tang hou ,zhi bei de leng dong gan zhi jia xi bao du xing jiao xiao ,fu ge zu zhi gong cheng zhi jia de shua cai biao zhun 。
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