bFGF联合EGF对人肠神经嵴前体细胞增殖及分化能力的影响

Effects of EGF and bFGF on proliferation and differentiation of human enteric neural crest progenitors in vitro

目的观察表皮生长因子（EGF）、碱性成纤维细胞生长因子（bFGF）对人肠神经嵴前体细胞（hENCP）在体外增殖和分化能力的影响，对人ENCP的体外分离培养、传代以及分化特点等基础研究进行探索，为巨结肠患儿自体细胞移植治疗提供实验依据。方法收集胎龄〉8周非药物流产胎儿，消化后制成单细胞悬液，体外培养3、5、7d后分别观察hENCP在不同生长因子作用下形成的克隆球数量、体积。MTT法观察hENCP在1周内0D值变化曲线。荧光免疫组织化学法p75NTR（鉴定hENCP）、peripherin（鉴定肠神经元）、GFAP（鉴定胶质细胞）抗体染色。结果能够从胎儿肠壁分离出少量细胞，经体外培养增殖形成克隆球。bFGF联合EGF培养下hENCP的克隆球数量、体积在各个时间点均明显大于单独添加EGF（P〈0．01）或bFGF培养的克隆球（P〈0．01）。bFGF＋EGF联合培养的hENCPOD值在各时间点均高于其他各组。克隆球中含有p75NTR免疫阳性细胞（hENCP），并在体外血清诱导下可分化为peripherin免疫阳性细胞（肠神经元）、GFAP免疫阳性细胞（神经胶质细胞）。bFGF组分化为神经元的百分率明显高于其他各组（P〈0．01），EGF组分化为神经胶质细胞的百分率明显高于其他各组（P〈0．01）。结论人胚肠壁中存在具有自我更新能力的ENCP，其在体外可分化为神经元和神经胶质细胞。EGF＋bFGF对hENCP有明显的促进增殖作用，bFGF、EGF对体外定向诱导hENCP分化细胞类型具有不同促进作用。

Objective To isolate and culture human enteric neural crest progenitors （hENCP） from human enteric muscular layer in vitro and observe the effects of growth factors EGF and bFGF effect on the proliferation of HENCP so as to provide experimental rationales for therapy of Hirschsprung＇s disease using autonomous cells. Methods The gut muscular layer in non-drug aborted fetus was stripped from the muscular layer under microscope. After digestion, single cells were seeded into serum free medium with bFGF or bFGF ＋ EGF to observe the size and amount of neurospheres at Days 3, 5, 7 and observe the curve of optic density （OD） value in 1 week by methyl thiazolyl tetrazolium （MTT）. After an addition of 10% fetal bovine sera, neurospheres and differentiated cells were identified with immunofluorescent staining of p75NTR, peripherin and GFAP. Results Few cells could be separated from interstitial muscle in human embryo gut and formed neurospheres in simplified serum free medium. There were significant differences between proliferation and number of hENCP cultured with bFGF＋ EGF and bFGF （P〈0. 01） or EGF （P〈0. 01）. The staining of neurospheres and their derivatives indicated neurospheres could generate neurons, gliat cells under serum induction in vitro. The percentage of differentiation into neuron in bFGF group was significantly higher than that in other groups （P〈0. 01 ）. The percentage of differentiation into glial cells in EGF group was significantly higher than that in other groups （P〈0. 01）. Conclusions With the capacities of self-renew and proliferation, hENCP exists in human embryonic gut. After isolation and culturing in vitro, hENCP can differentiate into neurons and glial cells essential for enteric nerve system. EGF ＋ bFGF may significantly promote the proliferation of hENCP. And EGF and bFGF may affect cell types differentiated from hENCP.