摘要
目的:观察小鼠精原干细胞在成骨培养条件下的生物学特征,为探讨精原干细胞的多向分化能力提供实验依据。方法:实验于2006-03/2007-01在泸州医学院医学分子生物学实验室完成。①实验材料:生后6～8d左右昆明系小白鼠,雌雄均可,由泸州医学院动物科提供。②实验方法:取7～10d小白鼠双侧股骨和胫骨,制备骨髓基质饲养层。取6～8d雄性小鼠,脱颈椎处死后,无菌取出睾丸,进行精原干细胞分离、培养与鉴定。取培养3d的精原干细胞,分为两组:对照组用基本培养液(IMDM+100U/mL青霉素+100U/mL链霉素+10%胎牛血清)培养,实验组用条件培养液培养(基本培养液中添加50mmol/Lβ-甘油磷酸钠、10-6mol/L地塞米松、50mg/L维生素C、50nmol/L胰岛素、20nmol/L碱性成纤维细胞生长因子)培养。③实验评估:在倒置显微镜下观察细胞的形态学变化、紫外分光光度仪动态检测上清液中碱性磷酸酶活性变化、免疫荧光法检测细胞内Ⅰ型胶原的表达。结果:①细胞形态学特征变化:实验组精原干细胞在成骨条件培养液中较对照组细胞贴壁生长快,条件培养3～6d后见细胞生长迅速,呈集落样增长,细胞立体感增强,但未见明显细胞间桥;诱导培养15d见细胞增殖达高峰,增殖细胞的胞质间桥仍不明显。②Ⅰ型胶原免疫荧光检测结果:实验组细胞浆内出现绿色荧光,呈阳性反应,对照组细胞呈阴性反应。③培养上清液碱性磷酸酶活性变化:实验组与对照组培养上清液中的碱性磷酸酶活性开始很低,以后逐渐增强。实验组培养上清液的碱性磷酸酶活性每个时间段都明显高于对照组(P<0.05)。结论:小鼠精原干细胞在成骨诱导培养条件下能快速增殖,增殖过程中表现出成骨细胞的某些生物学特征,这为进一步研究精原干细胞的多向分化潜能提供实验参考。
AIM： To observe the biological characteristics of mouse spermatogonial stem cells （SSCs） cultured in conditions for osteoblasts, and provide the experimental evidences for exploring the multipotency of SSCs. METHODS： The experiment was conducted at the Laboratory of Molecular Biology in Luzhou Medical College from March 2006 to January 2007,①Empirical material： Kunming mice （female or male） of 6-8 days postpartum was offered from Animal Department of Luzhou Medical College. ②Empirical method： Bilateral thighbones and shinbones of mice aged 7-10 days old were isolated for preparing feeder layer of bone marrow stroma. The male mice of 6-8 days old were put to death by the breakdown of cervical cord, and testes were taken out to isolate, culture and identify the testis cells. SSCs were cultured for three days and then divided into two groups： Cells of control group were cultured with basal medium （IMDM ＋ 100 U/mL penicillin ＋ 100 U/mL Streptomycin ＋ 10% fetal calf serum）, while those of experiment group with conditional medium （basal medium ＋ 50 mmol/L β-glycerophosphate disodium ＋ 10^-6 mol/L Dexamethasone ＋ 50 mg/L ascorbic acid ＋ 50 nmol/L insulin ＋ 20 nmol/L basic fibroblast growth factor）. ③Empirical evaluation： The morphological changes were observed by inverted microscope, the dynamic changes of alkaline phosphatase （ALP） activity in supematant was detected by ultraviolet spectrophotometer, and the expression of collagen I was detected by immunofluorescence. RESULTS： ①Cellular morphological change： SSCs proliferated faster in the experiment group than that in the control group, cells grew rapidly in the conditional medium at 3-6 days, presented a colony-like manner. Three-dimensional feeling enhanced, and the cytoplasmic bridge was not obvious; Cellular proliferation peak occurred on the 15^th day, and the intercellular bridge of SSCs was still unclear. ②The result of collagen I by immunofluorescence detection： Cell staining for collagen I was positive in the experiment group, but negative in the control group. ③The activity of ALP in the supematant of two grdups was low at beginning and increased gradually, furthermore, the activity of the ALP were obviously higher in the experiment group than in the control group at every time （P 〈 0.05）. CONCLUSION： SSCs can proliferate rapidly and show some biological characteristics of osteoblast when cultured in conditions for osteoblast. This may give a suggestion for further studying the multipotency of the SSCs.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第33期6611-6614,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
四川省教育厅资助课题(川教函[2003407号-2003A055)~~
作者简介
胡红梅．女．1976年生．江西省抚州市人，汉族，2007年泸州医学院毕业．硕士，讲师，主要从事精原干细胞培养厦应用方向研究。huhongmei503@126.com
通讯作者：吴绍华．教授，泸州医学院组织胚胎学教研室．四川省泸州市646000wush8406@vipsine．com
