摘要
目的:观察钾通道开放剂米诺地尔在不同浓度下对体外培养人外根鞘细胞增殖活性的影响。方法:实验于2005-04/11在解放军第四军医大学西京医院烧伤实验室完成。①实验材料:所有毛囊标本均来自解放军第四军医大学西京医院美容整形术后,供体为健康成年男性,年龄28～49岁,局部头皮无秃发、感染及其他皮肤疾病,均对本实验知情同意。以同一供体的毛囊标本为一个批次,至少取3批以上标本进行培养。米诺地尔(Sigma公司,美国,批号M20001001)。②实验方法:将头皮剪成0.3～0.5cm宽的皮条,再将真皮层剪去少许,消毒后以Dispase酶消化过夜,16～18h后揭去头皮表皮,拔出毛囊,使用无血清DMEM培养基清洗分离的毛囊。将毛囊置于胰酶+乙二胺四乙酸中消化8min,血清中止消化后离心,再加无血清DMEM培养基洗涤,然后置于预先铺Ⅰ型胶原作基质的角质形成细胞无血清培养基中培养,至外根鞘细胞长出并接近融合时按1∶传代。电子天平称取米诺地尔21mg,加入角质形成细胞无血清培养基20mL溶解,用稀释法分别配制成30,0.01,0.05,0.1,0.5,1.0mmol/L6个梯度浓度。③实验评估:取第2代外根鞘细胞,胰酶消化后计数,用相应培养基稀释成2×107L-1的细胞悬液,接种于96孔板。1d后细胞贴壁生长,分别换用含有0,0.01,0.05,0.1,0.5,1.0mmol/L的米诺地尔培养基,第4天加入四氮噻唑蓝在酶联免疫检测仪上检测吸光度值。结果:0,0.01,0.05,0.1,0.5,1.0mmol/L米诺地尔吸光度值分别为0.501±0.019,0.774±0.038,0.819±0.040,0.418±0.040,0.329±0.026,0.220±0.031。结论:①角质形成细胞无血清培养基有助于人外根鞘细胞的生长和传代,是较合适的培养基。②0.01～0.05mmol/L米诺地尔能够促进外根鞘细胞的增殖,而0.1mmol/L以上浓度反而抑制其生长。
AIM： To observe the influence of minoxidil at different concentration on proliferation capability of human outer root sheath cells in vitro. METHODS： The experiment had been carried out from April to November 2005 at Burn Laboratory of Xijing Hospital in Fourth Military Medical University of Chinese PLA. ① All specimen ware come from normal adult males whose had aesthetic and plastic surgery operation at Xijing Hospital in Fourth Military Medical University of Chinese PLA. Aged of these males ranged from 28 to 49 years. The partly scalp was no baldness, injection and other disease of skin. All of them knew the fact of the experiment. We took hair follicular specimen from the same donator as one group. At least three groups specimen were cultured. Minoxidil was purchased from Sigma Company, American, No. M20001001. The scalp was cut off 0.3-0.5 cm wide by scissors, the dermis was also cut off a bit and then was digested by Dispase enzyme after sterilization. The epidermis of scalp was removed after 16-18 hours. Hair follicle was pulled out and washed by DMEM serum free medium. It was centrifugated after had been digested by pancreatin and EDTA for 8 minutes. And was cultured in keratinocyte serum free medium which spread with Ⅰ collagen as matrix in advance after had been washed by DMEM serum free medium. The passage was undertaking in accord with 1：3 ratios when the outer root sheath cells ware came out and mixed together. Minoxidil was taken 21 mg by electronic balance, dissolved in 20 mL of keratinocyte serum free medium. A total of six groups were made including 0,0.01,0.05,0.1,0.5 and 1.0 mmol/L concentrations of minoxidil by the method of dilution. ③The second passage of outer root sheath cells were counted after digestion of pancreatin, ware diluted 2×10^7 L^-1 of cell suspension by corresponding medium and then inoculated in 96-well plate. Different concentration of minoxidil （0,0.01,0.05,0.1,0.5,1.0 mmol/L） were added in the medium when outer root sheath cells were growing and absorbance （A） were detected by the method of MTT at the 4^th day. RESULTS： A of 0,0.01,0.05,0.1,0.5,1.0 mmol/L minoxidil were 0.501±0.019,0.774±0.038,0.819±0.040,0.418±0.040, 0.329±0.026,0.220±0.031, respectively. CONCLUSION： ①Keratinocyte serum free medium is good for the culture of human outer root sheath cells. ②The concentration range of minoxidil （0.01-0.05 mmol/L） stimulates proliferation of outer root sheath cells, while 0.1 mmol/L and more higher concentration inhibit proliferation of outer root sheath cells.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第28期5461-5464,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
