移植用小鼠胶质前体细胞的培养及其移植后形态和功能初探

Culture of mouse glial progenitors for transplantation and their morphology and function

目的完善体外分离培养移植用胶质前体细胞的方法,并运用双光子显微镜在活体动物水平,对小鼠大脑皮层移植的胶质细胞进行单细胞水平的在体功能研究。方法体外分离培养小鼠胚胎神经干细胞,并将其诱导为胶质前体细胞及星形胶质细胞。分别用Nestin、A2B5以及GFAP对体外培养的神经干细胞、胶质前体细胞以及星形胶质细胞进行形态学鉴定。体外给予ATP及毒胡萝卜素(Thapsigargin),对体外分化的星形胶质细胞进行功能学鉴定。将以上诱导得到的胶质前体细胞移植入成年小鼠大脑皮层,并在移植12周后对其进行形态学观察。另外,应用双光子活体钙成像技术,观察移植胶质细胞对ATP刺激的反应。结果 1体外诱导神经干细胞成为胶质前体细胞(A2B5阳性)的比率达到(87.4±3.4)%,继续将其诱导为成熟星形胶质细胞(GFAP阳性)的比率达到(83.4±3.3)%。2体外诱导而来的星形胶质细胞可对ATP及毒胡萝卜素产生钙反应。3移植进入成年小鼠皮层的胶质前体细胞,在移植12周后仍可存活,并可分化为成熟的星形胶质细胞。4双光子活体钙成像实验证实,ATP可诱导活体小鼠皮层内移植细胞产生钙信号。结论神经干细胞来源的胶质前体细胞在移植进入成年小鼠皮层内可分化为成熟星形胶质细胞,并至少存活12周。另外,胶质前体细胞在移植4周后,便可在活体动物大脑皮层内具备功能活动。

Objective To improve a method for isolating and culturing glial progenitors for transplantation, and study in vivo function of the engrafted glial progenitors in cortex in single cell level by two- photon imaging. Mothocls Neural stem cells （NSCs） were isolated from the embryonic cortex, and cultured in vitro. Then, the Cells were induced into glial progenitors and astrocytes. In order to detect the cellular morphology, Nestin, AzBsand GFAP as markers of NSCs, glial progenitors and astrocytes, respectively, were detected. In order to detect the cellular function, calcium responses of the astrocytes to ATP and thapsigargin were recorded by confocal imaging. The glial progenitors were engrafted into the cerebral cortex of adult mice, and the morphology of the engrafted cells was observed by immunostaining after 12 weeks. In vivo two-photon imaging was adopted to observe calcium signals of the engrafted cells stimulated with ATP after 4 weeks. Results The percentage of glial progenitors （Az Bs-positive cells） derived from the NSCs accounted for （87.4 ± 3.4） %, while that of astrocytes （ GFAP-positive cells） derived from the glial progenitors aelounted for （83.4 ± 3.3）%. The astrocytes derived from the NSCs could respond to ATP and thapsigargin. The engrafted glial progenitors could survive and differentiate into the astrocytes after 12 weeks. In vivo two-photon imaging showed that ATP could induce the engrafted cells in the cortex to generate calcium signals after 4 weeks. Conclusion The engrafted glial progenitors derived from the NSCs can survive for at least 12 weeks and differentiate into the astrocytes in the cortex. In addition, the engrafted glial progenitors function in the cortex after 4 weeks.