安徽医科大学学报
安徽醫科大學學報
안휘의과대학학보
ACTA UNIVERSITY MEDICINALIS ANHUI
2015年
1期
20-24
,共5页
张硕%杜怡斌%杜公文%张辉%余涛%方家刘%高维陆%尹宗生
張碩%杜怡斌%杜公文%張輝%餘濤%方傢劉%高維陸%尹宗生
장석%두이빈%두공문%장휘%여도%방가류%고유륙%윤종생
NSCs%EPCs%增殖%分化%微环境%脊髓损伤
NSCs%EPCs%增殖%分化%微環境%脊髓損傷
NSCs%EPCs%증식%분화%미배경%척수손상
neural stem cells%endothelial progenitor cells%proliferation%differentiation%niche%spinal cord injury
目的：观察骨髓源性内皮祖细胞( EPCs)对脊髓源性神经干细胞( NSCs)增殖分化的影响。方法通过密度梯度离心法获取骨髓血单个核细胞,以 EBM-2进行诱导培养EPCs并进行免疫细胞化学染色鉴定,成熟的方法获取及鉴定SD大鼠的脊髓NSCs,1×105/ml第3代NSCs置于Tran-swell小室下层与1×105/ml上层原代EPCs进行体外1∶1共培养,以单纯第3代的NSCs培养为对照,培养7 d,双盲法分别计数各组在相差显微镜下神经球形成的数目,并用目镜测微尺测量神经球的平均直径,通过5%血清诱导培养NSCs 7 d后,行β-微管蛋白-Ⅲ免疫荧光染色,Hoechst细胞核染色后在显微镜下计算神经元/细胞总数得出百分率。结果骨髓源性EPCs与脊髓源性NSCs共培养组神经球平均数目为(22．27±3．85)个,平均直径为(61．70±7．21)μm,诱导培养后分化为神经元的平均百分率为(46．10±3．70)%,与对照组比较差异均有统计学意义( P<0．01)。结论骨髓源性EPCs能促进脊髓源性NSCs增殖及其向神经元分化。
目的：觀察骨髓源性內皮祖細胞( EPCs)對脊髓源性神經榦細胞( NSCs)增殖分化的影響。方法通過密度梯度離心法穫取骨髓血單箇覈細胞,以 EBM-2進行誘導培養EPCs併進行免疫細胞化學染色鑒定,成熟的方法穫取及鑒定SD大鼠的脊髓NSCs,1×105/ml第3代NSCs置于Tran-swell小室下層與1×105/ml上層原代EPCs進行體外1∶1共培養,以單純第3代的NSCs培養為對照,培養7 d,雙盲法分彆計數各組在相差顯微鏡下神經毬形成的數目,併用目鏡測微呎測量神經毬的平均直徑,通過5%血清誘導培養NSCs 7 d後,行β-微管蛋白-Ⅲ免疫熒光染色,Hoechst細胞覈染色後在顯微鏡下計算神經元/細胞總數得齣百分率。結果骨髓源性EPCs與脊髓源性NSCs共培養組神經毬平均數目為(22．27±3．85)箇,平均直徑為(61．70±7．21)μm,誘導培養後分化為神經元的平均百分率為(46．10±3．70)%,與對照組比較差異均有統計學意義( P<0．01)。結論骨髓源性EPCs能促進脊髓源性NSCs增殖及其嚮神經元分化。
목적：관찰골수원성내피조세포( EPCs)대척수원성신경간세포( NSCs)증식분화적영향。방법통과밀도제도리심법획취골수혈단개핵세포,이 EBM-2진행유도배양EPCs병진행면역세포화학염색감정,성숙적방법획취급감정SD대서적척수NSCs,1×105/ml제3대NSCs치우Tran-swell소실하층여1×105/ml상층원대EPCs진행체외1∶1공배양,이단순제3대적NSCs배양위대조,배양7 d,쌍맹법분별계수각조재상차현미경하신경구형성적수목,병용목경측미척측량신경구적평균직경,통과5%혈청유도배양NSCs 7 d후,행β-미관단백-Ⅲ면역형광염색,Hoechst세포핵염색후재현미경하계산신경원/세포총수득출백분솔。결과골수원성EPCs여척수원성NSCs공배양조신경구평균수목위(22．27±3．85)개,평균직경위(61．70±7．21)μm,유도배양후분화위신경원적평균백분솔위(46．10±3．70)%,여대조조비교차이균유통계학의의( P<0．01)。결론골수원성EPCs능촉진척수원성NSCs증식급기향신경원분화。
Objective To investigate the effects of bone marrow-derived endothelial progenitor cells( EPCs) on the proliferation and differentiation of spinal cord-derived neural stem cells( NSCs) . Methods Bone marrow mononu-clear cells were isolated by density gradient centrifugation methods and EPCs were cultured by EBM-2 basal medi-um, identified by fluorescent immunocytochemistry. Spinal cord-derived NSCs were isolated, cultured and identi-fied by the mature methods. 1 × 105/ml tertiary NSCs were plated on the base of culture wells, the upper transwell compartment was seeded with 1 × 105/ml primary EPCs, EPCs and NSCs (1 ∶ 1) were co-cultured in vitro, set the untreated tertiary NSCs as a control group. 7 days after co-culture, the number and diameter of neurospheres were calculated and measured with the double blind method. After that, NSCs were maintained for 7 days in DMEM/F12+ 5 % serum medium, and immunochemically stained with neuronal specific marker β-tubulin-Ⅲ, ratio of posi-tive cells to total cells was calculated. Results The average number of neurospheres in co-culture group was (22. 27 ± 3. 85) and the average diameter was(61. 70 ± 7. 21)μm. The percentage of neurons immunostaining was (46. 10 ±3. 70)%, differences are statistical significance compared with the control group (P<0. 01). Conclu-sion Bone marrow-derived EPCs promote the proliferation of spinal cord-derived NSCs and induce the differentia-tion of spinal cord-derived neural stem cells into neurons.
