【作者】 王启凤；

【导师】 王锋；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2003， 硕士

【摘要】 本文以SD大鼠、黇鹿、牧羊犬的皮肤组织为材料进行皮肤成纤维细胞和皮肤类干细胞培养，比较了采集皮肤组织样本时进行不同消毒处理的消毒效果，以及不同原代培养方法在获得传代细胞时间及培养效果的不同；同时对皮肤成纤维细胞和皮肤类干细胞的部分生物学特性进行了研究，为组织工程研究和人工皮肤研制提供基础资料，为充分开发、利用及保存特种动物资源提供借鉴资料。研究结果如下： 1．消毒处理及培养方法对SD大鼠尾尖皮肤成纤维细胞原代培养的影响 本试验采用不同消毒方法对SD大鼠尾尖皮肤组织进行原代成纤维细胞培养消毒处理，结果表明：采样前联合使用1％新洁尔灭与碘酒对术部皮肤消毒，采样后对皮肤组织块用1‰的新洁尔灭浸泡5min或高浓度双抗液(8×10⁴IU·ml^-1 Penicillin+1×10⁵IU·ml^-1 Streptomycin)浸泡10min可获得彻底消毒的效果，细胞生长良好。采样前联合使用75％乙醇与碘酒对术部皮肤消毒，采样后使用70％乙醇进行浸泡30s消毒不彻底，浸泡5min虽可达到无菌，但细胞生长不良；采用高浓度双抗浸泡10min可灭菌，但细胞生长不良。采用不同培养方法对SD大鼠尾尖皮肤成纤维细胞进行原代培养，结果表明：组织块法培养在第7-10天有细胞长出，第15～20天可获得传代细胞；0.25％胰蛋白酶消化30min后进行组织块法培养，培养第2～5天有细胞游离并生长、第10～13天时可传代培养；0.25％胰蛋白酶消化分散细胞法培养效果不稳定，虽能获得大量分散细胞，但细胞贴壁性差，生长慢。 2．成年黇鹿耳皮肤成纤维细胞的分离培养及生物学特性研究 本试验对1头黇鹿的耳缘皮肤组织采用0.1％胶原酶（Ⅰ型）消化组织块法进行原代培养，反复传代纯化，获得了成纤维细胞系，并进行了生物学特性观察和常规冷冻保存。对F12代细胞染色体进行了常规核型分析，结果表明该黇鹿的染色体数目为68，常染色体中第1和第7对染色体为M型，其余常染色体为A型；X染色体是最大的A染色体，Y染色体是最小的M染色体；核型式为4（M）+62（A），XY（A，M）；在进行核型分析时，利用Photoshop软件处理传统核型分析中的照片放大、剪切、排列等工作，在一定程度上提高了准确性。对冷冻保存的部分细胞进行了解冻培养，解冻存活率和贴壁率分别为61.04％、38.85％。 3．牧羊犬耳皮肤类干细胞的分离与培养 本试验以牧羊犬耳皮肤组织为材料，采用0.1％胶原酶消化组织法进行原代培养，获得了皮肤类干细胞和细胞集落，建立了牧羊犬皮肤类干细胞系，并部分生物学特性进行了研究，对各代细胞进行了常规冷冻保存。皮肤类干细胞在培养过程中发生了不同程度的分化，表现在以下两方面：①细胞集落有多种表现形式如鸟巢状、岛状、L几启凤:翅J鹿和牧羊犬皮肤细胞的分离与培养研究山脊状等;②出现了多种特殊细胞，如毛发状细胞、多角型细胞、神经样细胞、内胚层样细胞等。血清可促进原代皮肤类干细胞生长及细胞集落的形成.皮肤类干细胞在不同饲养层及培养基质上的生长行为不同，细胞集落在姑鹿成纤维细胞饲养层形成快，存在时间短，混合传代共培养无细胞集落重新出现;SD大鼠心肌细胞饲养层能使皮肤类千细胞长时间保持未分化状态，细胞集落存在时间最长达25天;明胶能促进皮肤类干细胞贴壁生长。皮肤类干细胞集落传代后可重新出现，且退化时不漂浮;贴鹿成纤维细胞集落传代培养后无细胞集落重新出现，且退化时漂浮到培养液中。 本研究创新之处在于改进了动物皮肤组织采样消毒方法，建立了小样组织酶消化组织块培养法;建立了姑鹿皮肤成纤维细胞系和牧羊犬皮肤类干细胞系。更多还原

【Abstract】 To establish a good method and culture system to isolate skin stem-like cells and expand it in vitro so as to lay a foundation for exploring the proliferation and differentiation mechanism of skin stem cells. In this paper, fibroblasts and stem-like cells have beenharvested from the skin tissues of SD rat Dama and Shepherd. The results were as follows:1. Effect of disinfection and culture methods on primary culture of fibroblast from trail tissue of SD rat1% Benzalkonium Bromide Solution(BBS) and Iodine were used as disinfectants before the skin tissues harvest. The skin samples from trail SD rat were incubated by 1% BBS or antibiotic solution ( 1x105IU ml-1 Penicillin + 8x104IU ml-1 Streptomycin ) for 5 minutes or lOminutes at room temperature, respectively, could prevent bacteria and mycotic’ infections. After the disinfectants were scoured off by Dulbecco’s Phosphate Buffered Saline (D-PBS), Skin samples were cut into small bits (lmm3) and incubated in 0.25% trypsin for 30 minutes at 37C. Then Dulbecco’s Modified Eagle Medium(high glucose, DMEM) with 10%(v/v) New Cattle Serum(NCS) were added to stop digestion action of trypsin. Skin samples were explanted into culture flask to culture at 37C, 5%CO2. Fibroblasts were obtained after 10-13 days,, while it took 15-20 days without trypsinization digestion.2. Establishment of fibroblasts cell line from ear skin tissue of adult Dama and studies on some biological characteristicsFibroblasts from ear skin tissue of adult Dama were harvested by the method as before, 0.25% trypsin was displaced by 0.1% collagenase ( I type), purificated by seeding and digesting times. Some biological characteristics of the cell lines including the morphology of cell, the growth rate, were studied. Cryopreservation of fibroblasts in different passages was also studied, the average vitality and the attachment percentage were 61.04% and 38.35%, respectively. The chromosome karyotype was analyzed by conventional cytogenetics technique, the chromosome number of 12th passages was 2n=68 and classified 33 pairs. Among the autosomes, the shape of 1th and 7th pairs were metacentric, others wereacrocentric. X chromosome was the largest acrocentric chromosome and Y was the smallestmetacentric chromosome in all chromosomes.3. Isolation and culture of skin stem-like cells from ear skin tissue of ShepherdSkin stem-like cell colonies were obtained by explant culture. The colonies had some characteristics of embryonic stem cells, such as the ability to be continuously subcultured (passage 6, while fibroblasts colonies didn’t), show a generalized plasticity. In vitro, these cells can be induced to be hair-like cells, polygon-like cell and neuron-like cells, et al. The cell colonies had some different morphologies, including column-like island-like, nest-like, chine-like, crater-like, et al. The skin stem-like cell colonies had different behaviors on fibroblasts of Dama and cardiomyocytes of SD rat, the latter made them keeping the same behavior for a long time.更多还原