中国临床康复
中國臨床康複
중국림상강복
CHINESE JOURNAL OF CLINICAL REHABILITATION
2006年
33期
158-160,插图33-6
,共4页
何继银%劳杰%顾玉东%蒋良福%李继峰
何繼銀%勞傑%顧玉東%蔣良福%李繼峰
하계은%로걸%고옥동%장량복%리계봉
生物医学工程%生物相容性材料%许旺氏细胞/细胞学%周围神经%壳多糖/类似物和衍生物%显微镜检查,电子,扫描%显微镜检查,相差
生物醫學工程%生物相容性材料%許旺氏細胞/細胞學%週圍神經%殼多糖/類似物和衍生物%顯微鏡檢查,電子,掃描%顯微鏡檢查,相差
생물의학공정%생물상용성재료%허왕씨세포/세포학%주위신경%각다당/유사물화연생물%현미경검사,전자,소묘%현미경검사,상차
背景:新型组织工程材料和许旺细胞扩增后置入生物合成管内去修复周围神经的缺损,是人工生物材料管的两大进展.目的:以胶原几丁糖为支架,以激活态的许旺细胞为种子细胞,观察二者的亲和性以及激活态的许旺细胞在胶原几丁糖膜上的生长规律,为人工神经的预构做准备.设计:开放性实验.单位:复旦大学附属华山医院手外科.材料:实验于2003-07/2003-12在卫生部手功能重建重点实验室完成.选取清洁级雄性SD大鼠4只.胶原几丁糖膜(上海其胜生物材料技术研究所提供),许旺细胞激活液(自制).方法:大鼠麻醉后坐骨神经切断预变性7 d,再次麻醉后引颈处死,迅速切取双侧坐骨神经,置于含青霉素和链霉素的D-HANK'S液中,剔除神经外膜,剪碎成1 mm的小段,移入盛有质量浓度为5g/L的胰蛋白酶和0.6g/L的胶原酶的离心管中,每2 mL液体中加入激活液0.5 mL,复合酶分步消化法获激活态许旺细胞,以浓度为2×107 L-1激活态的许旺细胞200 μL接种于胶原几丁糖膜上和培养皿上,2周后通过相差显微镜和扫描电镜观察细胞生长情况,S-100染色鉴定细胞的纯化程度.主要观察指标:①绘制细胞生长曲线,确定体外倍增时间.②激活态许旺细胞倒置相差显微镜下观察结果.③激活态许旺细胞接种于胶原几丁糖膜上扫描电镜观察结果.结果:①体外倍增时间的确定:激活态的许旺细胞接种于胶原几丁糖膜和培养皿上时的浓度均是2×107 L-1,2周后细胞浓度分别达到30×107 L-1和20×107 L-1.根据DT=(t-t0)lg2/(lgn-lgn0)算出激活态许旺细胞在胶原几丁糖膜上的倍增时间为4 d.②激活态许旺细胞倒置相差显微镜下观察结果:接种于培养皿上的激活态许旺细胞24 h后大多数由圆球形变成长梭形,有突起,多为双极,也有的呈三极状;接种于膜上的激活态许旺外形上和培养皿中无明显差异,但膜上的激活态许旺细胞在相差显微镜下犹如"刻在沙地上的文字"一般.S-100染色见激活态许旺细胞成棕色,纯度达95%以上.③激活态许旺细胞接种于胶原几丁糖膜上扫描电镜观察结果:激活态许旺细胞多数生长于胶原几丁糖膜的凹陷中或紧贴膜表面,呈有规律的首尾相接贴附于膜上,胞体呈纺锤形,直径在4～6 μm,长度为60～80 μm,细胞呈梭形,有许多细小分枝.1周时膜的形态仍然完整.结论:胶原几丁糖膜和高度纯化的激活态许旺细胞有良好的亲和性,以其为工艺材料制成的组织工程支架很有可能在促进周围神经再生中发挥优势作用.
揹景:新型組織工程材料和許旺細胞擴增後置入生物閤成管內去脩複週圍神經的缺損,是人工生物材料管的兩大進展.目的:以膠原幾丁糖為支架,以激活態的許旺細胞為種子細胞,觀察二者的親和性以及激活態的許旺細胞在膠原幾丁糖膜上的生長規律,為人工神經的預構做準備.設計:開放性實驗.單位:複旦大學附屬華山醫院手外科.材料:實驗于2003-07/2003-12在衛生部手功能重建重點實驗室完成.選取清潔級雄性SD大鼠4隻.膠原幾丁糖膜(上海其勝生物材料技術研究所提供),許旺細胞激活液(自製).方法:大鼠痳醉後坐骨神經切斷預變性7 d,再次痳醉後引頸處死,迅速切取雙側坐骨神經,置于含青黴素和鏈黴素的D-HANK'S液中,剔除神經外膜,剪碎成1 mm的小段,移入盛有質量濃度為5g/L的胰蛋白酶和0.6g/L的膠原酶的離心管中,每2 mL液體中加入激活液0.5 mL,複閤酶分步消化法穫激活態許旺細胞,以濃度為2×107 L-1激活態的許旺細胞200 μL接種于膠原幾丁糖膜上和培養皿上,2週後通過相差顯微鏡和掃描電鏡觀察細胞生長情況,S-100染色鑒定細胞的純化程度.主要觀察指標:①繪製細胞生長麯線,確定體外倍增時間.②激活態許旺細胞倒置相差顯微鏡下觀察結果.③激活態許旺細胞接種于膠原幾丁糖膜上掃描電鏡觀察結果.結果:①體外倍增時間的確定:激活態的許旺細胞接種于膠原幾丁糖膜和培養皿上時的濃度均是2×107 L-1,2週後細胞濃度分彆達到30×107 L-1和20×107 L-1.根據DT=(t-t0)lg2/(lgn-lgn0)算齣激活態許旺細胞在膠原幾丁糖膜上的倍增時間為4 d.②激活態許旺細胞倒置相差顯微鏡下觀察結果:接種于培養皿上的激活態許旺細胞24 h後大多數由圓毬形變成長梭形,有突起,多為雙極,也有的呈三極狀;接種于膜上的激活態許旺外形上和培養皿中無明顯差異,但膜上的激活態許旺細胞在相差顯微鏡下猶如"刻在沙地上的文字"一般.S-100染色見激活態許旺細胞成棕色,純度達95%以上.③激活態許旺細胞接種于膠原幾丁糖膜上掃描電鏡觀察結果:激活態許旺細胞多數生長于膠原幾丁糖膜的凹陷中或緊貼膜錶麵,呈有規律的首尾相接貼附于膜上,胞體呈紡錘形,直徑在4～6 μm,長度為60～80 μm,細胞呈梭形,有許多細小分枝.1週時膜的形態仍然完整.結論:膠原幾丁糖膜和高度純化的激活態許旺細胞有良好的親和性,以其為工藝材料製成的組織工程支架很有可能在促進週圍神經再生中髮揮優勢作用.
배경:신형조직공정재료화허왕세포확증후치입생물합성관내거수복주위신경적결손,시인공생물재료관적량대진전.목적:이효원궤정당위지가,이격활태적허왕세포위충자세포,관찰이자적친화성이급격활태적허왕세포재효원궤정당막상적생장규률,위인공신경적예구주준비.설계:개방성실험.단위:복단대학부속화산의원수외과.재료:실험우2003-07/2003-12재위생부수공능중건중점실험실완성.선취청길급웅성SD대서4지.효원궤정당막(상해기성생물재료기술연구소제공),허왕세포격활액(자제).방법:대서마취후좌골신경절단예변성7 d,재차마취후인경처사,신속절취쌍측좌골신경,치우함청매소화련매소적D-HANK'S액중,척제신경외막,전쇄성1 mm적소단,이입성유질량농도위5g/L적이단백매화0.6g/L적효원매적리심관중,매2 mL액체중가입격활액0.5 mL,복합매분보소화법획격활태허왕세포,이농도위2×107 L-1격활태적허왕세포200 μL접충우효원궤정당막상화배양명상,2주후통과상차현미경화소묘전경관찰세포생장정황,S-100염색감정세포적순화정도.주요관찰지표:①회제세포생장곡선,학정체외배증시간.②격활태허왕세포도치상차현미경하관찰결과.③격활태허왕세포접충우효원궤정당막상소묘전경관찰결과.결과:①체외배증시간적학정:격활태적허왕세포접충우효원궤정당막화배양명상시적농도균시2×107 L-1,2주후세포농도분별체도30×107 L-1화20×107 L-1.근거DT=(t-t0)lg2/(lgn-lgn0)산출격활태허왕세포재효원궤정당막상적배증시간위4 d.②격활태허왕세포도치상차현미경하관찰결과:접충우배양명상적격활태허왕세포24 h후대다수유원구형변성장사형,유돌기,다위쌍겁,야유적정삼겁상;접충우막상적격활태허왕외형상화배양명중무명현차이,단막상적격활태허왕세포재상차현미경하유여"각재사지상적문자"일반.S-100염색견격활태허왕세포성종색,순도체95%이상.③격활태허왕세포접충우효원궤정당막상소묘전경관찰결과:격활태허왕세포다수생장우효원궤정당막적요함중혹긴첩막표면,정유규률적수미상접첩부우막상,포체정방추형,직경재4～6 μm,장도위60～80 μm,세포정사형,유허다세소분지.1주시막적형태잉연완정.결론:효원궤정당막화고도순화적격활태허왕세포유량호적친화성,이기위공예재료제성적조직공정지가흔유가능재촉진주위신경재생중발휘우세작용.
BACKGROUND: New-type tissue engineering materials and post-proliferation Schwann cells are implanted into biosynthesis tube for repairing peripheral nerve defect, which are two great developments in the field of artificial biomaterial tube.OBJECTIVE: Taking chitosan-collagen as scaffold, activated Schwann cells as seed cells, we are in attempt to observe the affinity between them as well as growth rule of activated Schwann cells on Chitosan-collagen, so as to provide basis for pre-construction of artificial nerve.DESIGN: Open experiment.SETTING: Department of Hand Surgery, Huashan Hospital Affiliated to Fudan University.MATERIALS: This experiment was conducted at the Key Laboratory of Hand Function Reconstruction, Ministry of Public Health from July 2003 to December 2003. Four male SD rats, of clean degree, were used in this experiment. Chitosan-collagen film was made in Qisheng Biomaterial Technique Institute, Shanghai, Schwann cells activator solution was made in our laboratory (self-made).METHODS: After rats were anaesthenia, the sciatic nerve was cut off to perform predegeneration for 7 days. Another anaesthenia later, the rats were euthanized. Both sides of sorciatic nerves were cut off quickly and put in the D-HANK's solution containing penicillin and streptomycin.Epineurium was eliminated and chipped into 1 mm pieces, then put in the centrifuge tube containing 5 g/L trypsinase and 0.6 g/L collagenase. 0.5 mL activator solution every 2 mL liquid was added and the activated Schwann cells were harvested with the way of two-step enzymolysis. 2×107 L-1 activated Schwann cells in 200 μL were inoculated to Chitosan-collagen film and Petri dish . Two weeks later, cellular growth was observed under phase contrast microscope and scanning electron microscope. Cellular purity was identified with S-100 staining.MAIN OUTCOME MEASURES: ① Drawing cell growth curve and confirming in vitro doubling time. ②Observation of activated Schwanri cells under an inverted phase contrast microscope. ③ Observation of activated Schwann cells inoculated on Chitosan-collagen film under scanning electron microscope.RESULTS: ① Confirmation of in vitro doubling time: Concentration of activated Schwann cells inoculated on both Chitosan-collagen and Petri dish was 2×107 L-1, the final concentration was up to 3.0×108 L-1 and 2.0×108 L-1 respectively 2 weeks later. Doubling time of activated Schwann cells cultured on Chitosan-collagen film was 4 days calculated according to DT=(t-t0) lg2/(lgn-lgn0). ②Observation of activated Schwanncells under an inverted microscope: 24 hours later, the activated Schwann cells inoculated to Petri dish mostly changed from spherical to long shuttle-shape,mutation appeared and most were two-pole shape, fewer were three-pole shape; Morphologically, there was no significant difference between activated Schwann cells inoculated on Chitosan-collagen film and on Petri dish. Activated Schwann cells inoculated to Chitosan-collagen film were like "words cayed on the sand" under phase contrast microscope and the purity was over 95%. ③ Observation of activated Schwann cells inoculated to Chitosan-collagen under scanning electron microscope: Most of activated Schwann cells grew in the introcession of Chitosan-collagen or closely to surface of Chitosan-collagen, presenting regular head-to-end connection and adhesion to Chitosan-collagen film. The cell body was fusiform,with diameter of 4-6 μm, 60-80 μm in length. Cells were shuttle-shape with some small branches. Morphology of Chitosan-collagen film was still complete at week 1.CONCLUSION: There exists great affinity between Chitosan-collagen film and high-purity activated Schwann cell; so tissue-engineering scaffold made of the two components probably promote peripheral nerve regeneration.
