中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2013年
4期
352-357
,共6页
陈侠%李磊%鲜光军%王薇%朱晓燕%谢琳
陳俠%李磊%鮮光軍%王薇%硃曉燕%謝琳
진협%리뢰%선광군%왕미%주효연%사림
核酸适配子%纳米载体%壳聚糖%Tenon囊成纤维细胞%转化生长因子%生物靶向治疗
覈痠適配子%納米載體%殼聚糖%Tenon囊成纖維細胞%轉化生長因子%生物靶嚮治療
핵산괄배자%납미재체%각취당%Tenon낭성섬유세포%전화생장인자%생물파향치료
Aptamer%Nano-carrier%Chitosan%Tenon capsule fibroblast%Transforming growth factor%Biologically targeted therapy
背景 本课题组前期已筛选得到了靶向转化生长因子-β(TGF-β)Ⅱ型受体(TβRⅡ)的核酸适配子S58,并证明S58可抑制TGF-β介导的人Tenon囊成纤维细胞(HTFs)转分化作用.壳聚糖纳米微粒(CS-NP)已被证实可作为药物载体,但其包埋S58后抑制HTFs增生的有效性和安全性值得关注. 目的 制备CS(S58)-NP,研究其生物学特性,并观察CS-NP对S58的保护及缓释作用.方法 本研究所用细胞由本课题组前期原代培养的HTFs传代而来,取第4~10代对数生长期的细胞用于实验.以S58为实验对象,通过离子交联法制备出不同浓度CS-NP与S58表面电荷比(N/P比)的CS(S58)-NP.应用Zeta粒径分析仪检测CS(S58)-NP粒径及其Zeta电位;用扫描电子显微镜观察粒径的大小、形态及分布情况;采用琼脂糖凝胶电泳法检测CS-NP与S58的结合情况,并评估CS(S58)-NP对核酸酶的抵抗能力;用紫外分光光度计检测不同时间点缓释液PBS中S58的含量,以判断CS(S58)-NP缓释S58的情况;利用乳酸脱氢酶(LDH)释放实验检测CS(S58)-NP对HTFs的细胞毒性. 结果 制备的CS(S58)-NP粒径分布于130~ 270 nm之间,其Zeta电位分布范围为+16 ~ +28 mV;扫描电子显微镜下可观察到CS(S58)-NP呈球形,分布均匀,粒径在300 nm以内.当CS-NP与S58 N/P比例为10、20、30、40时,CS(S58)-NP平均包封率分别为88.9%、89.3%、91.7%、90.5%;加入DNase Ⅰ组与无DNase Ⅰ组比较,S58经CS-NP包裹后能够明显抵御DNase Ⅰ的酶解作用;体外缓释实验发现,CS-NP在PBS中持续缓慢释放S58,随着时间的延长,S58累计释放量增加,以24 ~ 36 h释放速率最高,96 h S58累计释放量为100%.随着CS(S58)-NP浓度的增加,HTFs上清液中LDH相对释放率逐渐增加,差异有统计学意义(F=588.018,P=0.000).50 nmol/L CS(S58)-NP处理HTFs 48 h时LDH相对释放率最大,为(12.853±0.375)%. 结论 CS-NP对S58具有保护及缓释作用,其浓度在50 nmol/L以下时对HTFs的毒性作用小,具有良好的生物相容性,可用于核酸适配子的载体研究.CS(S58)-NP可用于抑制TGF-β介导的HTFs转分化研究.
揹景 本課題組前期已篩選得到瞭靶嚮轉化生長因子-β(TGF-β)Ⅱ型受體(TβRⅡ)的覈痠適配子S58,併證明S58可抑製TGF-β介導的人Tenon囊成纖維細胞(HTFs)轉分化作用.殼聚糖納米微粒(CS-NP)已被證實可作為藥物載體,但其包埋S58後抑製HTFs增生的有效性和安全性值得關註. 目的 製備CS(S58)-NP,研究其生物學特性,併觀察CS-NP對S58的保護及緩釋作用.方法 本研究所用細胞由本課題組前期原代培養的HTFs傳代而來,取第4~10代對數生長期的細胞用于實驗.以S58為實驗對象,通過離子交聯法製備齣不同濃度CS-NP與S58錶麵電荷比(N/P比)的CS(S58)-NP.應用Zeta粒徑分析儀檢測CS(S58)-NP粒徑及其Zeta電位;用掃描電子顯微鏡觀察粒徑的大小、形態及分佈情況;採用瓊脂糖凝膠電泳法檢測CS-NP與S58的結閤情況,併評估CS(S58)-NP對覈痠酶的牴抗能力;用紫外分光光度計檢測不同時間點緩釋液PBS中S58的含量,以判斷CS(S58)-NP緩釋S58的情況;利用乳痠脫氫酶(LDH)釋放實驗檢測CS(S58)-NP對HTFs的細胞毒性. 結果 製備的CS(S58)-NP粒徑分佈于130~ 270 nm之間,其Zeta電位分佈範圍為+16 ~ +28 mV;掃描電子顯微鏡下可觀察到CS(S58)-NP呈毬形,分佈均勻,粒徑在300 nm以內.噹CS-NP與S58 N/P比例為10、20、30、40時,CS(S58)-NP平均包封率分彆為88.9%、89.3%、91.7%、90.5%;加入DNase Ⅰ組與無DNase Ⅰ組比較,S58經CS-NP包裹後能夠明顯牴禦DNase Ⅰ的酶解作用;體外緩釋實驗髮現,CS-NP在PBS中持續緩慢釋放S58,隨著時間的延長,S58纍計釋放量增加,以24 ~ 36 h釋放速率最高,96 h S58纍計釋放量為100%.隨著CS(S58)-NP濃度的增加,HTFs上清液中LDH相對釋放率逐漸增加,差異有統計學意義(F=588.018,P=0.000).50 nmol/L CS(S58)-NP處理HTFs 48 h時LDH相對釋放率最大,為(12.853±0.375)%. 結論 CS-NP對S58具有保護及緩釋作用,其濃度在50 nmol/L以下時對HTFs的毒性作用小,具有良好的生物相容性,可用于覈痠適配子的載體研究.CS(S58)-NP可用于抑製TGF-β介導的HTFs轉分化研究.
배경 본과제조전기이사선득도료파향전화생장인자-β(TGF-β)Ⅱ형수체(TβRⅡ)적핵산괄배자S58,병증명S58가억제TGF-β개도적인Tenon낭성섬유세포(HTFs)전분화작용.각취당납미미립(CS-NP)이피증실가작위약물재체,단기포매S58후억제HTFs증생적유효성화안전성치득관주. 목적 제비CS(S58)-NP,연구기생물학특성,병관찰CS-NP대S58적보호급완석작용.방법 본연구소용세포유본과제조전기원대배양적HTFs전대이래,취제4~10대대수생장기적세포용우실험.이S58위실험대상,통과리자교련법제비출불동농도CS-NP여S58표면전하비(N/P비)적CS(S58)-NP.응용Zeta립경분석의검측CS(S58)-NP립경급기Zeta전위;용소묘전자현미경관찰립경적대소、형태급분포정황;채용경지당응효전영법검측CS-NP여S58적결합정황,병평고CS(S58)-NP대핵산매적저항능력;용자외분광광도계검측불동시간점완석액PBS중S58적함량,이판단CS(S58)-NP완석S58적정황;이용유산탈경매(LDH)석방실험검측CS(S58)-NP대HTFs적세포독성. 결과 제비적CS(S58)-NP립경분포우130~ 270 nm지간,기Zeta전위분포범위위+16 ~ +28 mV;소묘전자현미경하가관찰도CS(S58)-NP정구형,분포균균,립경재300 nm이내.당CS-NP여S58 N/P비례위10、20、30、40시,CS(S58)-NP평균포봉솔분별위88.9%、89.3%、91.7%、90.5%;가입DNase Ⅰ조여무DNase Ⅰ조비교,S58경CS-NP포과후능구명현저어DNase Ⅰ적매해작용;체외완석실험발현,CS-NP재PBS중지속완만석방S58,수착시간적연장,S58루계석방량증가,이24 ~ 36 h석방속솔최고,96 h S58루계석방량위100%.수착CS(S58)-NP농도적증가,HTFs상청액중LDH상대석방솔축점증가,차이유통계학의의(F=588.018,P=0.000).50 nmol/L CS(S58)-NP처리HTFs 48 h시LDH상대석방솔최대,위(12.853±0.375)%. 결론 CS-NP대S58구유보호급완석작용,기농도재50 nmol/L이하시대HTFs적독성작용소,구유량호적생물상용성,가용우핵산괄배자적재체연구.CS(S58)-NP가용우억제TGF-β개도적HTFs전분화연구.
Background Our previous study demonstrated that the aptamer S58 specifically targeted transforming growth factor-β receptor Ⅱ (TβRⅡ) and inhibited the transdifferentiation of human Tenon capsule fibroblasts (HTFs) mediated by transforming growth factor-β (TGF-β).Chitosan-nanoparticles (CS-NP) are good drug carriers,but the efficacy and safety of CS-NP/aptamer complexes deserve attention.Objective The aim of this study was to synthesize a novel CS-NP/aptamer complex called CS (S58)-NP and investigate its properties and applicability.Methods Human Tenon capsule tissue was obtained from patients during strabismus surgery,and HTFs were cultured and passaged using the explant culture method.The fourth to tenth generations of cells were used in the experiment.Different concentrations of CS-NP were used to prepare the CS(S58)-NP by the ionic cross-linking method with a surface charge rate (N/P) for S58 of 10,20,30 or 40.The particle size and Zeta potential were measured by the Zeta analyzer.The shape and distribution of CS (S58)-NP particles were examined under the scanning electron microscope.The binding of CS-NP with S58 and resistance of CS (S58)-NP to DNase Ⅰ were examined by agarose gel eletrophoresis.The release rate of S58 from CS (S58)-NP in PBS was quantitatively analyzed by a ultraviolet spectrophotometer.The cytotoxicity of CS(S58)-NP to HTFs was evaluated by detecting the production of lactate dehydrogenase (LDH).Results The Zeta analyzer showed that the particle size of CS (S58)-NP was 130-270 nm and its electric potential ranged from + 16 to +28 mV.The CS (S58)-NP particles appeared spherical with an even distribution under the scanning electron microscope.The mean encapsulation efficiency of CS(S58)-NP was 88.9%,89.3%,91.7% or 90.5%,respectively,when the N/P was 10,20,30 or 40.After being encapsuled by CS-NP,S58 could resist the degradation from DNase I.Its total releasing level in PBS increased with the lapse of time,with a maximum releasing speed at 24 to 36 hours.The total releasing level reached 100% at 96 hours.With increaseing concentrations of CS(S58)-NP,the relative releasing level of LDH in HTFs suspension gradually elevated with a significant difference among the groups (F =588.018,P =0.000),with the highest released LDH level at 50 nmol/L of CS(S58)-NP (12.853% ±0.375%).Conclusions CS-NP provides a protective and slow-releasing effect on the S58 aptamer.CS (S58)-NP shows a good biocompatibility with HTFs with a low cytotoxicity at a concentration of <50 nmol/L.CS(S58)-NP could be used to inhibit TGF-β induced transdifferentiation of HTFs in the future.