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我用下面的方法，取原代的牙周膜干细胞，整个操作完成后观察不到细胞。（我在筛网过滤后加入了培养基终止消化，1500转离心了10分钟，离心管底未见细胞）9 b9 K8 ?6 O5 J
收集临床上正常人（12-18岁）拔除的牙周健康、无龋的新鲜第三磨牙，（拔除后立即在双抗PBS液和IMDM液中反复清洗牙齿，或双抗PBS浸泡10-20min），刮取根中1/3牙周膜组织，移入离心管内，加入3mg/ml的I型胶原和4mg/ml Dispase 轻轻震荡，37℃下消化1h，通过70μm筛网，获得单个离散的细胞，调整细胞密度为1*104/ml 接种于5ml培养基（ 含200ml/L胎牛血清，100μmol/L L-抗坏血酸，2mmol/L L-谷胺酰胺，100U/ml青霉素，100μg／ml链霉素的DMEM培养基），37OC、50ml／L CO2的孵箱中培养-----《人牙周膜干细胞的分离培养鉴定和体外诱导分化的实验性研究》 高秦
$ w, F* D1 d3 D Normal impacted third molars (n=25) were collected from 16 individuals aged 19–29 years at the Dental Clinic of the National Institute of Dental and Craniofacial Research, USA, following approved guidelines set by the National Institutes of Health Office of Human Subjects Research. PDL was gently separated from the surface of the root and then digested in a solution of 3 mg/mL collagenase type I (Worthington Biochem, Freehold, NJ, USA) and 4 mg/mL dispase (Roche, Mannheim, Germany) for 1 h at 37oC. PDL samples from different individuals were pooled and single-cell suspensions were obtained by passing the cells through a 70μm strainer (Falcon, BD Labware, Franklin Lakes, NJ, USA).To identify putative stem cells, single-cell suspensions (1*104 cells) were seeded into 10-cm culture dishes (Costar, Cambridge, MA, USA) with alphamodification of Eagle’s medium (GIBCO BRL, Grand Island, NY, USA) supplemented with 15% fetal calf serum (Equitech-Bio Inc, Kerrville, TX, USA), 100μmol/L ascorbic acid 2-phosphate (WAKO, Tokyo, Japan), 2 mmol/L glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Biofluids, Rockville, MD, USA), and then incubated at 37ºC in 5% carbon dioxide.-----《Investigation of multipotent postnatal stem cells from human periodontal ligament》 Byoung-Moo Seo* z! k% G' [4 l
我用的是临床现拔的牙，用的I型胶原酶是Sigma分装的，100mg 350元。中性蛋白酶是国产的，100g，60元。细胞筛200目的（公司说就是70μm那种），65元一个。
3 S' q9 o, F+ E3 o1 T) U& K前辈们帮我看看，是什么地方出来问题，我取了两次原代了，什么细胞都看不到的。 |
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发表于 2010-10-25 19:40
