中华神经医学杂志
中華神經醫學雜誌
중화신경의학잡지
CHINESE JOURNAL OF NEUROMEDICINE
2014年
11期
1101-1106
,共6页
高明勇%陶海鹰%卫爱林%宋建东%贺斌
高明勇%陶海鷹%衛愛林%宋建東%賀斌
고명용%도해응%위애림%송건동%하빈
功能化多肽水凝胶%细胞外基质%三维细胞培养%神经干细胞
功能化多肽水凝膠%細胞外基質%三維細胞培養%神經榦細胞
공능화다태수응효%세포외기질%삼유세포배양%신경간세포
Self-assembly poly-peptide hydrogel%Extracellular matrix%Three dimensional cell culture%Neural stem cell
目的 探讨一种整合精氨酸-甘氨酸-天冬氨酸(RGD)环肽的功能化多肽水凝胶RADA16-c(RGDfK)细胞外基质对神经干细胞(NSCs)增殖分化的调控影响,以期为涉及NSCs增殖分化调控因素的研究提供一种能高度模拟体内三维条件的体外仿生研究模型. 方法 收集新生1d龄SD大鼠脊髓组织,采用完全培养基培养脊髓源性NSCs,采用细胞免疫荧光染色观察NSCs形态并鉴定.实验分3组,即Ⅰ型胶原组、基本序列为16个氨基酸残基的两性多态水凝胶(RADA16)组和RADA16-c(RGDfK)组,分别选择Ⅰ型胶原、RADA16及RADA16-c(RGDfK)[由ac-(RADA)4-CONH2和ac-(RADA)4-GG-cyclo(RGDfK)按摩尔比3∶1物理混合而成]这3种水凝胶,采用AR2000EX型流变仪测量水凝胶的弹性模量,建立水凝胶细胞外基质与脊髓源性NSCs三维共培养模型,通过噻唑蓝(MTT)法检测不同水凝胶细胞外基质体系中NSCs的增殖活性,及细胞免疫化学染色和激光共聚焦观测技术观察NSCs的分化表达. 结果 (1)RADA16及RADA16-c(RGDfK)水凝胶弹性模量在生理条件下分别为(0.42±0.07) kPa及(0.47±0.09) kPa,2者间差异无统计学意义(P＞0.05);而Ⅰ型胶原弹性模量为(0.87±0.12) kPa,明显强于其他2组,差异有统计学意义(P＜0.05).(2)RADA16-(cRGDfK)细胞外基质中神经球形态细胞团均匀分布,克隆球直径相当,介于200～300 μm.RADA16组和RADA16-(cRGDfK)组较Ⅰ型胶原组具有更好的促细胞增殖特性,其中RADA16-(cRGDfK)组对NSCs体外增殖的支持能力最优,与其他2组比较差异具有统计学意义(P＜0.05).(3)RADA16组和RADA16-c(RGDfK)组在血清诱导环境下可见大量神经前体细胞分化为神经胶质细胞,神经元分化比例较低,分别为17.6％±3.1％及19.0％±3.6％,与Ⅰ型胶原组神经元分化比例(10.6％±2.3％)差异均有统计学意义(P＜0.05). 结论 功能化多肽水凝胶RADA16-c(RGDfK)细胞外基质具有优良的神经细胞相容性和细胞黏附性,对体外脊髓源性NSCs的增殖分化有良好的促进作用,可作为神经细胞组织工程研究的新型载体.
目的 探討一種整閤精氨痠-甘氨痠-天鼕氨痠(RGD)環肽的功能化多肽水凝膠RADA16-c(RGDfK)細胞外基質對神經榦細胞(NSCs)增殖分化的調控影響,以期為涉及NSCs增殖分化調控因素的研究提供一種能高度模擬體內三維條件的體外倣生研究模型. 方法 收集新生1d齡SD大鼠脊髓組織,採用完全培養基培養脊髓源性NSCs,採用細胞免疫熒光染色觀察NSCs形態併鑒定.實驗分3組,即Ⅰ型膠原組、基本序列為16箇氨基痠殘基的兩性多態水凝膠(RADA16)組和RADA16-c(RGDfK)組,分彆選擇Ⅰ型膠原、RADA16及RADA16-c(RGDfK)[由ac-(RADA)4-CONH2和ac-(RADA)4-GG-cyclo(RGDfK)按摩爾比3∶1物理混閤而成]這3種水凝膠,採用AR2000EX型流變儀測量水凝膠的彈性模量,建立水凝膠細胞外基質與脊髓源性NSCs三維共培養模型,通過噻唑藍(MTT)法檢測不同水凝膠細胞外基質體繫中NSCs的增殖活性,及細胞免疫化學染色和激光共聚焦觀測技術觀察NSCs的分化錶達. 結果 (1)RADA16及RADA16-c(RGDfK)水凝膠彈性模量在生理條件下分彆為(0.42±0.07) kPa及(0.47±0.09) kPa,2者間差異無統計學意義(P＞0.05);而Ⅰ型膠原彈性模量為(0.87±0.12) kPa,明顯彊于其他2組,差異有統計學意義(P＜0.05).(2)RADA16-(cRGDfK)細胞外基質中神經毬形態細胞糰均勻分佈,剋隆毬直徑相噹,介于200～300 μm.RADA16組和RADA16-(cRGDfK)組較Ⅰ型膠原組具有更好的促細胞增殖特性,其中RADA16-(cRGDfK)組對NSCs體外增殖的支持能力最優,與其他2組比較差異具有統計學意義(P＜0.05).(3)RADA16組和RADA16-c(RGDfK)組在血清誘導環境下可見大量神經前體細胞分化為神經膠質細胞,神經元分化比例較低,分彆為17.6％±3.1％及19.0％±3.6％,與Ⅰ型膠原組神經元分化比例(10.6％±2.3％)差異均有統計學意義(P＜0.05). 結論 功能化多肽水凝膠RADA16-c(RGDfK)細胞外基質具有優良的神經細胞相容性和細胞黏附性,對體外脊髓源性NSCs的增殖分化有良好的促進作用,可作為神經細胞組織工程研究的新型載體.
목적 탐토일충정합정안산-감안산-천동안산(RGD)배태적공능화다태수응효RADA16-c(RGDfK)세포외기질대신경간세포(NSCs)증식분화적조공영향,이기위섭급NSCs증식분화조공인소적연구제공일충능고도모의체내삼유조건적체외방생연구모형. 방법 수집신생1d령SD대서척수조직,채용완전배양기배양척수원성NSCs,채용세포면역형광염색관찰NSCs형태병감정.실험분3조,즉Ⅰ형효원조、기본서렬위16개안기산잔기적량성다태수응효(RADA16)조화RADA16-c(RGDfK)조,분별선택Ⅰ형효원、RADA16급RADA16-c(RGDfK)[유ac-(RADA)4-CONH2화ac-(RADA)4-GG-cyclo(RGDfK)안마이비3∶1물리혼합이성]저3충수응효,채용AR2000EX형류변의측량수응효적탄성모량,건립수응효세포외기질여척수원성NSCs삼유공배양모형,통과새서람(MTT)법검측불동수응효세포외기질체계중NSCs적증식활성,급세포면역화학염색화격광공취초관측기술관찰NSCs적분화표체. 결과 (1)RADA16급RADA16-c(RGDfK)수응효탄성모량재생리조건하분별위(0.42±0.07) kPa급(0.47±0.09) kPa,2자간차이무통계학의의(P＞0.05);이Ⅰ형효원탄성모량위(0.87±0.12) kPa,명현강우기타2조,차이유통계학의의(P＜0.05).(2)RADA16-(cRGDfK)세포외기질중신경구형태세포단균균분포,극륭구직경상당,개우200～300 μm.RADA16조화RADA16-(cRGDfK)조교Ⅰ형효원조구유경호적촉세포증식특성,기중RADA16-(cRGDfK)조대NSCs체외증식적지지능력최우,여기타2조비교차이구유통계학의의(P＜0.05).(3)RADA16조화RADA16-c(RGDfK)조재혈청유도배경하가견대량신경전체세포분화위신경효질세포,신경원분화비례교저,분별위17.6％±3.1％급19.0％±3.6％,여Ⅰ형효원조신경원분화비례(10.6％±2.3％)차이균유통계학의의(P＜0.05). 결론 공능화다태수응효RADA16-c(RGDfK)세포외기질구유우량적신경세포상용성화세포점부성,대체외척수원성NSCs적증식분화유량호적촉진작용,가작위신경세포조직공정연구적신형재체.
Objective In vitro model with three dimensional cell culture provides the appealing biomimetic platform to probe the biological characteristics of multiple stem cells,which serves as an important in vitro tools to investigate regulating factors controlling the proliferation and differentiation of neural stem cells (NSCs).The present study aims to reconstruct an integrated poly-peptide hydrogel made extracellular matrix (ECM) enhanced with cyclo-RGD molecular [c(RGDfK)] for the exploration of NSCs bio-characteristics.Methods Spinal cords from one-d-old SD rats were collected and spinal-derived NSCs were induced in the complete medium; immunofluorescence staining was employed to observe the NSCs morphology and identify NSCs.Three hydrogel including type Ⅰ collagen,self-assembly poly-peptide nanofiber hydrogel (SAPNH) of RADA16 and RADA16-c(RGDfK) were employed to serve as culturing ECM of spinal-derived NSCs to mimic the ex vivo 3-D culturing.With the theological analysis,cyto-morphological observation was performed,NSCs proliferation was observed by MTT assay,and cell immunochemistty and confocal microscopy were employed to detect the NSCs differentiation.Results SAPNH born appropriate elastic module conducive to the cellular adhesion and proliferation of neural cells (RADA16 and RADA16-c (RGDfK)=(0.42±0.07) kPa and (0.47 ±0.09) kPa,without significant difference (P＞0.05); however,the elastic module in the type Ⅰ collagen group was (0.87±0.12) kPa,which was significantly stronger than the two groups (P＜0.05).Uniform distribution of neuron-shape cells was noted in the extracellular matrix ofRADA16-c (RGDfK) cells,with almost the diameter of cell sphere (200-300 μm); cells in the RADA16 and RADA16-c (RGDfK) had better growth characteristics than the other two groups; RADA16-c(RGDfK) had significantly better cellular adhesion and proliferation of neural cells as compared with RADA16 and collagen groups (P＜0.05).A large number of neural precursor cells differentiated into neurogliocytes was noted in the RADA16 group and RADA16-c (RGDfK) group,with low proportion of neuronal differentiation (17.6％ ±3.1％ and 19.0％±3.6％,respectively); this proportion of neuronal differentiation was significantly higher than that in the type Ⅰ collagen group (10.6％±2.3％,P＜0.05).Conclusions The functionalized SAPNH enhanced with c(RGDfK) presents the excellent biocompatibility and promotes the adhesion and proliferation of spinal NSCs.Serving as the engineered cellular vector,functionalized SAPNH has laid a solid foundation for the studies of neural regeneration and repair with novel neuro-engineering techniques in the subsequent researches.
