EFFECT OF ENZYMATICALLY DIGESTED HUMAN AMNIOTIC BASEMENT MEMBRANE ON THE GROWTH OF CHICK EMBRYONIC FOREBRAIN NEURONS / 解剖学报

ABSTRACT

Enzymatic digestion of the human amniotic basement membrane (HABM) using heparinase Ⅰ, heparinase Ⅱ, collagenase and chondroitinase ABC enabled us to study the supportiveness of HABM for forebrain neuronal growth. HABM-coated 24-well plates were enzymatically digested for 1 hr. at 37℃ before dissociated neurons from the forebrain of E8 chick embryo were seeded. The neurons were cultured for 20hr. in RPMI 1640 medium containing 20mmol/L HEPES and 60％ fetal calf serum before being quantified for neuronal growth by an automatic colorimetric microassay. The assay, utilizing a tetrazolium derivative named MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide] which can be converted by viable cells to form a formazan product, can be measured with an Elisa reader. The results revealed that heparinase Ⅰ, collagenase, and chondroitinase ABC had significantly removed from the HABM the attachment sites for neurons, and that heparinase Ⅱ had significantly increased the sites for attachment and/or enhanced the survival growth of neurons.