脐血间充质干细胞体外分化为鼻黏膜纤毛上皮细胞的研究

发布时间：2018-02-04 08:47

本文关键词： 脐带血间充质干细胞鼻黏膜气液界面 FOXJ1 β-Tubulin MUC8 分化纤毛细胞　出处：《山西医科大学》2009年博士论文　论文类型：学位论文

【摘要】： 慢性鼻鼻窦炎(Ⅱ、Ⅲ型)鼻腔鼻窦手术后常须进行长期的随访治疗,以防术腔鼻黏膜瘢痕粘连及炎症复发。严重的黏膜病变还可能伴有免疫功能异常,如变应性鼻炎、哮喘、囊性纤维化、先天性纤毛不动综合征等。如何使病变或受损的鼻腔黏膜重新发挥正常功能是国内外鼻科学者关注的问题。近来研究表明小鼠胚胎干细胞和成人骨髓间充质干细胞可以分化成为纤毛细胞。但前者涉及伦理学,无法在人体进行研究,后者分化较差。脐带血含有丰富的造血干细胞/祖细胞(HSCs)和间充质干细胞(MSCs ),取材方便,来源广泛;受到胎盘屏障保护,其成分被病毒、细菌污染概率低;脐带血免疫系统较为原始,降低了移植后受体的排异反应;脐带血中含有丰富的干细胞,更为原始并具有有更强的分化能力;不涉及社会、法律及伦理方面的争议;脐带血不仅可以作为异体基因移植的供体,而且还可以低温保存数十年。因此,以脐血间充质干细胞作为组织修复材料具有广阔的前景。为了探索脐血间充质干细胞是否能向鼻黏膜上皮分化,进行了以下研究。第一部分体外脐血间充质干细胞培养体系的建立目的探讨脐血MSCs的分离、培养、纯化和扩增的方法及规律,研究其生物学特性,进一步探讨体外诱导脐血MSCs向纤毛上皮定向分化的可行性和诱导条件,为研究脐血MSCs作为种子细胞来治疗鼻科疾病提供理论依据和实验基础。方法取孕34～40周产妇自然分娩或剖宫产胎儿的脐血20～80 mL,分别采用羟乙基淀粉分离法和淋巴细胞分离液法分离单个核细胞。将DMEM/F12+10%胎牛血清加入离心管中,吹打制成单细胞悬液。以适当密度接种于胎牛血清(FBS)包被的6孔板中,置于37℃、饱和湿度,体积分数为5%CO2孵箱中培养。7 d首次全量换液,去掉未贴壁的悬浮细胞,以后每5天换液1次。待细胞长到80%汇合时,以1:1的0.25%胰酶/0.02EDTA混合液消化传代,每日在倒置相差显微镜下观察原代和传代细胞的生长情况和形态特征。流式细胞仪鉴定P3代细胞表面标志CD44、CD13、CD45、CD34,并取第3代细胞测定细胞周期。结果采用淋巴细胞分离液和羟乙基淀粉分离法均成功分离出MSCs。原代培养于2-4周时细胞可达80%汇合,此时可以传代。传至第3代时细胞形态较均一,折光性好。经流式细胞仪检测结果显示,P3代脐血间充质干细胞稳定地高表达CD13、CD44等表面抗原标记物,阳性率分别为86.18%、90.30%,弱表达CD34、CD45等造血细胞标志,表明MSCs是存在于脐带血中区别于造血细胞的一群非定向干/祖细胞,这与骨髓MSCs的表面抗原标志相一致。第3代脐血单个核细胞周期分析显示95.29%的细胞处在G0/G1期。18份脐血,都分离出不同量的单核细胞,只有3份孕34-37周和1份孕38周的的胎儿脐血得到足量可以传代的梭形细胞,占22%。结论早产胎儿脐血更易得到可以传代和纯化的干细胞,可能与其脐血中间充质干细胞量更多有关。但是足月胎儿脐血也可以获得一定数量纯化和具有增殖分化能力的干细胞。目前各种优化条件还在研究中,尚缺乏统一有效的方法。第二部分鼻黏膜上皮细胞的培养和鉴定目的建立分离细胞培养方案和气液界面培养法培养人鼻黏膜上皮细胞的培养体系。了解不同培养方法对纤毛上皮的影响。方法取全身麻醉下鼾症手术切取的健康下鼻甲黏膜,用含抗(氟康唑、庆大霉素) PBS溶液,在超净台中反复冲洗、浸泡,用0.1%胶原酶Ⅳ消化,分别接种到T25培养瓶进行鼻黏膜上皮分离细胞培养法培养;接种到鼠尾胶原I型包被Transwell支持膜的六孔培养板进行气液界面培养。在倒置相差显微镜下观察原代和传代细胞的生长情况和形态特征,行HE染色和PAS染色、免疫荧光染色、电镜及染色体检查。结果两种方法均成功培养了目的细胞。其中分离细胞培养法得到的细胞形态学上有典型卵石样排列,并夹杂有杯状细胞,抗人角蛋白CK-14阳性,说明其是上皮来源。抗β-Tubulin抗体与抗FOXJ1抗体均为阳性。扫描电镜和投射电镜均显示表面含有纤毛,但缺乏极性。P5代染色体检查正常,说明传代细胞可以保证染色体稳定,并进行相关试验。ALL法利用Transwell小室使细胞在模拟在体条件下生长,有助于表面纤毛的分化和颗粒的分泌,并且在形态学和免疫标志方面都得到证实。结论鼻黏膜细胞分离法可以得到纯度较高的鼻黏膜纤毛上皮细胞。ALL法不仅创造了与正常组织相似的微环境,而且应用鼠尾胶原蛋白Ⅰ型和适合气道上皮培养的支气管上皮培养基,是更适合纤毛分化的培养方法。第三部分脐血间充质干细胞分化为鼻黏膜纤毛上皮的研究目的探讨人脐带血间充质干细胞在气液界面培养,并用鼠尾胶原蛋白I型包被支持膜和支气管上皮专用培养基培养的条件下,诱导分化成纤毛细胞的可能性。方法用rAAv2-EGFP转染人脐带血间充质干细胞,用鼠尾胶原蛋白I型包被支持膜,使用支气管上皮细胞专用培养基,建立气液界面培养。分别于1周和2周后收集细胞,行RT-PCR法检测MUC8基因的表达。UCB-MSCs在膜上细胞生长2周后进行抗β-Tubulin抗体和FOXJ1免疫荧光染色。结果转染2小时后可见细胞发出绿色荧光。48小时消化后上流式细胞仪检测,阳性率达97.9%。RT-PCR结果显示,UCB-MSCs不表达MUC8mRNA,鼻黏膜上皮强表达。随诱导培养时间延长,1周时MUC8mRNA弱表达,培养2周后较前增强,2周时未检测到抗β-Tubulin抗体的阳性表达,而FOXJ1于转染的绿色荧光蛋白背景下可观测到红色荧光阳性表达。在共聚焦显微镜的组合像中可以看到,部分标记绿色荧光的MSCs核内可见红色FOXJ1阳性荧光表达。结论UCB-MSCs在气液界面培养,鼠尾胶原蛋白Ⅰ型包被支持膜,支气管上皮细胞无血清培养基培养,是体外分离、培养和扩增的脐带血间充质干细胞诱导成为表达鼻黏膜纤毛上皮标记的细胞的适宜分化条件。
[Abstract]:Chronic rhinosinusitis (II, III) after nasal surgery often have to be follow-up long-term treatment, to prevent the operation of nasal cavity scar and recurrent inflammation. Severe mucosal lesions may also be associated with immune dysfunction, such as allergic rhinitis, asthma, cystic fibrosis, congenital immotile cilia syndrome. How to make the diseased or damaged nasal mucosa to play normal function is the domestic and foreign science nasal concern. Recent studies have shown that mouse embryonic stem cells and adult bone marrow mesenchymal stem cells can differentiate into ciliated cells. But the former involves ethics research, can not be carried out on the human body, the differentiation of human umbilical cord blood contains the poor. Hematopoietic stem / progenitor cells (HSCs) and mesenchymal stem cells (MSCs), convenient, wide source; by placental barrier protection, its components are viruses, bacteria pollution probability is low; umbilical cord blood immune system is original First, reduce the rejection in recipients; rich cord blood contains stem cells that are more primitive and has a stronger ability of differentiation; do not involve social disputes, legal and ethical aspects; not only can be used as a donor allogeneic umbilical cord blood transplantation gene, but also can be stored for decades in low temperature. Therefore and in umbilical cord blood mesenchymal stem cells for tissue repair materials has broad prospects. In order to explore the human umbilical cord blood mesenchymal stem cells are able to nasal epithelial differentiation, the following research.
The first part of the culture system of human umbilical cord blood mesenchymal stem cells in vitro
Objective to investigate the separation of umbilical cord blood MSCs culture, purification and amplification methods and rules and study its biological characteristics, and to further explore the feasibility of umbilical cord blood MSCs induced differentiation of ciliated epithelium in vitro directional, provide theoretical and experimental basis for the study of umbilical cord blood MSCs as seed cells to treat nasal disease.
Methods 34~40 weeks pregnant maternal natural childbirth or cesarean section fetal umbilical cord blood of 20~80 mL, respectively by hydroxyethyl starch separation and lymphocyte isolation method to isolate mononuclear cells. DMEM/F12+10% fetal bovine serum into a centrifugal tube, and made into single cell suspension. With proper density inoculated in fetal bovine serum (FBS). 6 well plates were placed at 37 DEG C, saturated humidity, volume fraction of cultured.7 D for the first time was all changed 5%CO2 incubator, remove non adherent cell suspension, then was changed every 5 days for 1 times. When the cells grow to 80% confluence, with 1:1 / 0.02EDTA mixture of 0.25% trypsin digestion the daily passage under the inverted phase contrast microscope to observe the growth and morphology of primary and passaged cells. Flow cytometry was used to identify P3 cell surface markers CD44, CD13, CD45, CD34, and take the third generation cells by cell cycle.
The results of using lymphocyte separation liquid and hydroxyethyl starch separation method were successfully isolated from cultured MSCs. cells at 2-4 weeks up to 80% confluence, this can be passaged. At the third passage cells form a uniform refractive index. The results of flow cytometry showed that P3 generation of umbilical cord blood mesenchymal stem cells stably the expression of CD13, CD44 and other surface antigen markers, the positive rates were 86.18%, 90.30%, weak expression of CD34, CD45 and other hematopoietic cell markers, showed that MSCs is present in umbilical cord blood hematopoietic cells from a group of non directional stem / progenitor cells, which is consistent with the surface antigen MSCs in bone marrow of third generation of umbilical cord blood markers. Cell cycle analysis showed that 95.29% of the cells in G0/G1 phase.18 umbilical cord blood samples were isolated from mononuclear cells of different, only 3 copies at 34-37 weeks and 1 at 38 weeks of fetal umbilical cord blood can obtain enough passage of spindle cells, accounting for 22%.
Conclusion premature fetus umbilical cord blood can be obtained more easily cultured and purified stem cells, umbilical cord blood and mesenchymal stem cells. But more full-term fetal umbilical cord blood can be obtained and purified with a certain number of proliferation and differentiation of stem cells. The optimization is still under study conditions, still lacks a unified and effective way.
The culture and identification of the second part of nasal mucosa epithelial cells
Objective to establish a culture system for isolation of cell culture and culture of human nasal epithelial cells by gas liquid interface culture, and to understand the effect of different culture methods on ciliated epithelium.
Methods general anesthesia snoring surgery cut the health of the inferior turbinate mucosa with anti (fluconazole, gentamicin) PBS solution, soaking in clean, Taichung repeatedly washed by 0.1% collagenase IV digestion, were inoculated into T25 culture flask were isolated and cultured epithelial cells of nasal mucosa; inoculation to rat tail collagen I the package type supported by Transwell film board six well culture air2liquid culture. To observe the growth and morphology of primary and passaged cells under the inverted microscope, HE staining and PAS staining, immunofluorescence staining, electron microscopy and staining examination.
The results of the two methods were successfully cultured cells. The objective cell separation method to get the morphology of the cells have typical pebble like arrangement, and mixed with goblet cells, human anti keratin CK-14 positive, indicating its epithelial origin. Anti beta -Tubulin antibody and anti FOXJ1 antibody were positive. Scanning electron microscopy and transmission electron microscopy were the display surface contain cilia, but the lack of polar.P5 chromosome examination was normal, that cell can guarantee the stability of chromosome, and related experiments were carried out using the.ALL method to Transwell cell growth in vivo cells under simulated conditions, the secretion of differentiation and contribute to the cilia on the surface of the particles, and have been confirmed in morphology and immunophenotype.
The nasal mucociliary epithelial cells.ALL conclusion nasal mucosa cell separation method can obtain high purity not only creates a microenvironment similar to normal tissue, bronchial epithelium and application of rat tail collagen type I and airway epithelial culture, is the culture method is more suitable for the differentiation of cilia.
Study on the differentiation of umbilical cord blood mesenchymal stem cells into nasal ciliated epithelium in the third part
Objective to explore the possibility of inducing human umbilical cord blood mesenchymal stem cells to differentiate into ciliated cells under the condition of culture at the gas-liquid interface and culture with rat tail collagen I coated support membrane and bronchial epithelial specific culture medium.
RAAv2-EGFP transfection of human umbilical cord blood mesenchymal stem cells, with rat tail collagen type I coated membrane support, the use of bronchial epithelial cells in special culture medium, the establishment of the gas-liquid interface culture. In 1 weeks and 2 weeks were collected and the expression of.UCB-MSCs RT-PCR MUC8 gene was detected in the cell membrane on the growth of 2 weeks after the anti beta -Tubulin antibody and FOXJ1 immunofluorescence staining.
The cells from flow cytometry to detect green fluorescent.48 hours after digestion for 2 hours after the transfection, the positive rate of 97.9%.RT-PCR showed that the expression of UCB-MSCs MUC8mRNA, which the expression of the nasal mucosa. With time prolonging induction at 1 weeks, the weak expression of MUC8mRNA, after 2 weeks of culture than before enhancement, 2 weeks is not detected the positive expression of anti -Tubulin antibodies to beta, and green fluorescent protein in transfected FOXJ1 background can be observed. The positive expression of red fluorescence in combination with confocal microscopy as can be seen, part of green fluorescence in the MSCs nucleus visible red FOXJ1 positive expression of fluorescence.
Conclusion UCB-MSCs in the gas-liquid interface culture, rat tail collagen type coated support membrane, serum culture medium without bronchial epithelial cells, in vitro isolation, culture and amplification of umbilical cord blood mesenchymal stem cells induced into suitable differentiation conditions expression in nasal mucosa cilia labeled cells.

【学位授予单位】：山西医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R329

【引证文献】