• 한국임상수의학회지
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• 제22권4호
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• pp.377-381
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• 2005

We tried to extract bone morphogenetic protein (BMP) from the freeze-dried bovine cortical bone (FBCB) for bone graft, which were defatted with chloroform-methanol for 20 days, freeze-dried at $-80^{\circ}C$ for 7 days and sterilized by ethylene oxide gas. Two kg of FBCB were pulverized in a wheel mill to $0.5-2.0mm^3$ cubic in size. The bone particles were demineralized in 0.6N HCI for 10 days at chloroform-methanol$4^{\circ}C$ and defatted with chloroform-methanol for 6 hours at room temperature, which was going to be defatting and demineralized cortical bone (DDM). For extracting BMP, DDM was agitated continuously through 72 hours with magnetic stirrer at $4^{\circ}C$ into 12 times of volume of 6 M guanidine hydrochloride (Gdn-HCl) solution containing proteinase inhibitors to protect BMP such as 2mM N-ethylaleimide, 1mM iodoacetic acid, 1mM phenylmethylsulfonyl fluoride and a sterilizer, 1mM sodium azide. The extraction procedure was repeated for three times. All extracted solution was centrifuged at 10,000 rpm for 30 min and then, the supernatant was dialyzed with 12 times of volume of deionized water at $4^{\circ}C$ for 24-72 hours, which cut off below 6,000-8,000 molecular weight. The dialyzed specimen contained crude-BMP was centrifuged, freeze-dried, and weighted. Through these processing, we could obtained $84.9\%$ as FBCB, $17.8\%$ as DDM and $0.71\%$ as crude-BMP from the wet cortical bone without cancellous bone, marrow and muscles. The crude-BMP were obtained $68.3\%$ from the first extraction, $29.6\%$ from secondary and $2.1\%$ from tertiary, respectively. It was suggested that high yield of crude-BMP migth be explained by three-time repetition of the extraction processing for crude-BMP with Gdn-Hcl sol.

• 한국산림과학회지
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• 제103권1호
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• pp.72-79
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• 2014

낙엽송의 성숙배로부터 최대 부정아 유도는 LP배지에 1.0 mg/L zeatin이 첨가된 처리구(76.1%)였으며, 2 mm 이상의 신초생장을 위한 효과적인 처리구는 Litvay(LM)배지에 0.5 mg/L zeatin(75.2%)을 첨가하거나, 혹은 Quoirin & Lepoivre(LP)배지에 2.0 mg/L zeatin(70.2%)에서 각각 나타났다. 배지의 염류함량을 반으로 줄인 부정아 유도 시험에서는 1/2LP배지에 1.0(83.3%) 혹은 2.0 mg/L(81.7%) zeatin을 첨가한 처리구에서 높은 유도율을 보였으나, 2 mm 이상의 신초생장을 위한 효과적인 처리구는 1/2LM배지에 1.0 mg/L zeatin(66.7%)에서였다. 배지의 염류함량을 반으로 줄인 부정아 유도 시험에서는 1/2LP배지에 1.0(83.3%) 혹은 2.0 mg/L(81.7%) zeatin을 첨가한 처리구에서 높은 유도율을 보였으나, 2 mm 이상의 신초생장을 위한 효과적인 처리구는 1/2LM배지에 1.0 mg/L zeatin(66.7%)에서였다. 그리고 1.0 mg/L zeatin처리구에서 2주배양 후 1.0 mg/L thidiazuron(TDZ) 처리구로 2주간 배양할 때 가장 높은 52.9%의 부정아 유도율을 보였으나 평균 부정아 유도수는 낮게 나타났다. 부정아유도를 위한 에틸렌활성제 혹은 억제제 첨가시험에서는 최대 부정아 유도율은 1.0 mg/L zeatin+2.0 mg/L MGBG(methylglyoxal bis-[guanylhydrazone])가 첨가된 처리구(34.6%) 였으며, 평균 최대 부정아 유도수는 1.5개를 보인 1.0 mg/L zeatin+2.0 mg/L $CoCl_2$ 처리구에서 나타났다.