本文關(guān)鍵詞： 臍帶血 間充質(zhì)干細(xì)胞 鼻黏膜 氣液界面 FOXJ1 β-Tubulin MUC8 分化 纖毛細(xì)胞　出處：《山西醫(yī)科大學(xué)》2009年博士論文　論文類型：學(xué)位論文

【摘要】： 慢性鼻鼻竇炎(Ⅱ、Ⅲ型)鼻腔鼻竇手術(shù)后常須進(jìn)行長期的隨訪治療,以防術(shù)腔鼻黏膜瘢痕粘連及炎癥復(fù)發(fā)。嚴(yán)重的黏膜病變還可能伴有免疫功能異常,如變應(yīng)性鼻炎、哮喘、囊性纖維化、先天性纖毛不動(dòng)綜合征等。如何使病變或受損的鼻腔黏膜重新發(fā)揮正常功能是國內(nèi)外鼻科學(xué)者關(guān)注的問題。近來研究表明小鼠胚胎干細(xì)胞和成人骨髓間充質(zhì)干細(xì)胞可以分化成為纖毛細(xì)胞。但前者涉及倫理學(xué),無法在人體進(jìn)行研究,后者分化較差。臍帶血含有豐富的造血干細(xì)胞/祖細(xì)胞(HSCs)和間充質(zhì)干細(xì)胞(MSCs ),取材方便,來源廣泛;受到胎盤屏障保護(hù),其成分被病毒、細(xì)菌污染概率低;臍帶血免疫系統(tǒng)較為原始,降低了移植后受體的排異反應(yīng);臍帶血中含有豐富的干細(xì)胞,更為原始并具有有更強(qiáng)的分化能力;不涉及社會(huì)、法律及倫理方面的爭(zhēng)議;臍帶血不僅可以作為異體基因移植的供體,而且還可以低溫保存數(shù)十年。因此,以臍血間充質(zhì)干細(xì)胞作為組織修復(fù)材料具有廣闊的前景。為了探索臍血間充質(zhì)干細(xì)胞是否能向鼻黏膜上皮分化,進(jìn)行了以下研究。 第一部分體外臍血間充質(zhì)干細(xì)胞培養(yǎng)體系的建立 目的探討臍血MSCs的分離、培養(yǎng)、純化和擴(kuò)增的方法及規(guī)律,研究其生物學(xué)特性,進(jìn)一步探討體外誘導(dǎo)臍血MSCs向纖毛上皮定向分化的可行性和誘導(dǎo)條件,為研究臍血MSCs作為種子細(xì)胞來治療鼻科疾病提供理論依據(jù)和實(shí)驗(yàn)基礎(chǔ)。 方法取孕34～40周產(chǎn)婦自然分娩或剖宮產(chǎn)胎兒的臍血20～80 mL,分別采用羥乙基淀粉分離法和淋巴細(xì)胞分離液法分離單個(gè)核細(xì)胞。將DMEM/F12+10%胎牛血清加入離心管中,吹打制成單細(xì)胞懸液。以適當(dāng)密度接種于胎牛血清(FBS)包被的6孔板中,置于37℃、飽和濕度,體積分?jǐn)?shù)為5%CO2孵箱中培養(yǎng)。7 d首次全量換液,去掉未貼壁的懸浮細(xì)胞,以后每5天換液1次。待細(xì)胞長到80%匯合時(shí),以1:1的0.25%胰酶/0.02EDTA混合液消化傳代,每日在倒置相差顯微鏡下觀察原代和傳代細(xì)胞的生長情況和形態(tài)特征。流式細(xì)胞儀鑒定P3代細(xì)胞表面標(biāo)志CD44、CD13、CD45、CD34,并取第3代細(xì)胞測(cè)定細(xì)胞周期。 結(jié)果采用淋巴細(xì)胞分離液和羥乙基淀粉分離法均成功分離出MSCs。原代培養(yǎng)于2-4周時(shí)細(xì)胞可達(dá)80%匯合,此時(shí)可以傳代。傳至第3代時(shí)細(xì)胞形態(tài)較均一,折光性好。經(jīng)流式細(xì)胞儀檢測(cè)結(jié)果顯示,P3代臍血間充質(zhì)干細(xì)胞穩(wěn)定地高表達(dá)CD13、CD44等表面抗原標(biāo)記物,陽性率分別為86.18%、90.30%,弱表達(dá)CD34、CD45等造血細(xì)胞標(biāo)志,表明MSCs是存在于臍帶血中區(qū)別于造血細(xì)胞的一群非定向干/祖細(xì)胞,這與骨髓MSCs的表面抗原標(biāo)志相一致。第3代臍血單個(gè)核細(xì)胞周期分析顯示95.29%的細(xì)胞處在G0/G1期。18份臍血,都分離出不同量的單核細(xì)胞,只有3份孕34-37周和1份孕38周的的胎兒臍血得到足量可以傳代的梭形細(xì)胞,占22%。 結(jié)論早產(chǎn)胎兒臍血更易得到可以傳代和純化的干細(xì)胞,可能與其臍血中間充質(zhì)干細(xì)胞量更多有關(guān)。但是足月胎兒臍血也可以獲得一定數(shù)量純化和具有增殖分化能力的干細(xì)胞。目前各種優(yōu)化條件還在研究中,尚缺乏統(tǒng)一有效的方法。 第二部分鼻黏膜上皮細(xì)胞的培養(yǎng)和鑒定 目的建立分離細(xì)胞培養(yǎng)方案和氣液界面培養(yǎng)法培養(yǎng)人鼻黏膜上皮細(xì)胞的培養(yǎng)體系。了解不同培養(yǎng)方法對(duì)纖毛上皮的影響。 方法取全身麻醉下鼾癥手術(shù)切取的健康下鼻甲黏膜,用含抗(氟康唑、慶大霉素) PBS溶液,在超凈臺(tái)中反復(fù)沖洗、浸泡,用0.1%膠原酶Ⅳ消化,分別接種到T25培養(yǎng)瓶進(jìn)行鼻黏膜上皮分離細(xì)胞培養(yǎng)法培養(yǎng);接種到鼠尾膠原I型包被Transwell支持膜的六孔培養(yǎng)板進(jìn)行氣液界面培養(yǎng)。在倒置相差顯微鏡下觀察原代和傳代細(xì)胞的生長情況和形態(tài)特征,行HE染色和PAS染色、免疫熒光染色、電鏡及染色體檢查。 結(jié)果兩種方法均成功培養(yǎng)了目的細(xì)胞。其中分離細(xì)胞培養(yǎng)法得到的細(xì)胞形態(tài)學(xué)上有典型卵石樣排列,并夾雜有杯狀細(xì)胞,抗人角蛋白CK-14陽性,說明其是上皮來源。抗β-Tubulin抗體與抗FOXJ1抗體均為陽性。掃描電鏡和投射電鏡均顯示表面含有纖毛,但缺乏極性。P5代染色體檢查正常,說明傳代細(xì)胞可以保證染色體穩(wěn)定,并進(jìn)行相關(guān)試驗(yàn)。ALL法利用Transwell小室使細(xì)胞在模擬在體條件下生長,有助于表面纖毛的分化和顆粒的分泌,并且在形態(tài)學(xué)和免疫標(biāo)志方面都得到證實(shí)。 結(jié)論鼻黏膜細(xì)胞分離法可以得到純度較高的鼻黏膜纖毛上皮細(xì)胞。ALL法不僅創(chuàng)造了與正常組織相似的微環(huán)境,而且應(yīng)用鼠尾膠原蛋白Ⅰ型和適合氣道上皮培養(yǎng)的支氣管上皮培養(yǎng)基,是更適合纖毛分化的培養(yǎng)方法。 第三部分臍血間充質(zhì)干細(xì)胞分化為鼻黏膜纖毛上皮的研究 目的探討人臍帶血間充質(zhì)干細(xì)胞在氣液界面培養(yǎng),并用鼠尾膠原蛋白I型包被支持膜和支氣管上皮專用培養(yǎng)基培養(yǎng)的條件下,誘導(dǎo)分化成纖毛細(xì)胞的可能性。 方法用rAAv2-EGFP轉(zhuǎn)染人臍帶血間充質(zhì)干細(xì)胞,用鼠尾膠原蛋白I型包被支持膜,使用支氣管上皮細(xì)胞專用培養(yǎng)基,建立氣液界面培養(yǎng)。分別于1周和2周后收集細(xì)胞,行RT-PCR法檢測(cè)MUC8基因的表達(dá)。UCB-MSCs在膜上細(xì)胞生長2周后進(jìn)行抗β-Tubulin抗體和FOXJ1免疫熒光染色。 結(jié)果轉(zhuǎn)染2小時(shí)后可見細(xì)胞發(fā)出綠色熒光。48小時(shí)消化后上流式細(xì)胞儀檢測(cè),陽性率達(dá)97.9%。RT-PCR結(jié)果顯示,UCB-MSCs不表達(dá)MUC8mRNA,鼻黏膜上皮強(qiáng)表達(dá)。隨誘導(dǎo)培養(yǎng)時(shí)間延長,1周時(shí)MUC8mRNA弱表達(dá),培養(yǎng)2周后較前增強(qiáng),2周時(shí)未檢測(cè)到抗β-Tubulin抗體的陽性表達(dá),而FOXJ1于轉(zhuǎn)染的綠色熒光蛋白背景下可觀測(cè)到紅色熒光陽性表達(dá)。在共聚焦顯微鏡的組合像中可以看到,部分標(biāo)記綠色熒光的MSCs核內(nèi)可見紅色FOXJ1陽性熒光表達(dá)。 結(jié)論UCB-MSCs在氣液界面培養(yǎng),鼠尾膠原蛋白Ⅰ型包被支持膜,支氣管上皮細(xì)胞無血清培養(yǎng)基培養(yǎng),是體外分離、培養(yǎng)和擴(kuò)增的臍帶血間充質(zhì)干細(xì)胞誘導(dǎo)成為表達(dá)鼻黏膜纖毛上皮標(biāo)記的細(xì)胞的適宜分化條件。
[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.

【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 蔣濤;基于“腎主骨生髓”理論的補(bǔ)腎中藥聯(lián)合BMP-2誘導(dǎo)人臍血MSCs體外成骨分化的研究[D];南京中醫(yī)藥大學(xué);2012年

相關(guān)碩士學(xué)位論文 前1條

1 魯俊山;補(bǔ)腎中藥聯(lián)合BMP-2對(duì)人臍血間充質(zhì)干細(xì)胞體外增殖及成骨活性影響的實(shí)驗(yàn)研究[D];南京中醫(yī)藥大學(xué);2012年

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本文編號(hào)：1489844