本文選題：原代細(xì)胞培養(yǎng) + 鼻粘膜上皮細(xì)胞��； 參考：《中國人民解放軍軍醫(yī)進(jìn)修學(xué)院》2011年碩士論文

【摘要】：鼻粘膜上皮細(xì)胞具有屏障功能及離子、水轉(zhuǎn)運(yùn)功能等重要生理功能,上皮細(xì)胞對表層液體層離子及水正常轉(zhuǎn)運(yùn)是發(fā)揮粘液纖毛傳輸功能的重要基礎(chǔ)。電生理研究是探索鼻腔,鼻竇生理功能及發(fā)病機(jī)制重要研究手段,除了典型通道疾病囊性纖維化病,目前又有研究發(fā)現(xiàn)異常的離子及水的轉(zhuǎn)運(yùn)可能亦是鼻腔常見病鼻息肉、鼻竇炎發(fā)病機(jī)制之一。傳統(tǒng)的通道研究手段如膜片鉗技術(shù)主要針對單細(xì)胞研究,細(xì)胞分離時易受到機(jī)械及酶消化損傷,影響通道活性；而另一項(xiàng)電生理研究技術(shù)尤斯灌流室(Ussing chamber)技術(shù)主要應(yīng)用于培養(yǎng)在支持膜上的整片呼吸道上皮,并且無消化及機(jī)械損傷弊端,同時可根據(jù)實(shí)驗(yàn)設(shè)計要求靈活控制頂側(cè)膜及底側(cè)膜電解質(zhì)組成,因此受到越來越多學(xué)者的關(guān)注,不過本項(xiàng)技術(shù)需要在良好的細(xì)胞培養(yǎng)模型上完成。該模型要求細(xì)胞在支持膜上長期單層生長、高度分化、形成可靠的緊密連接等特點(diǎn),對培養(yǎng)技術(shù)要求較高,目前國內(nèi)尚無此類報道。 呼吸道粘膜培養(yǎng)技術(shù)雖然經(jīng)歷了多年發(fā)展,然而仍無統(tǒng)一方案�；A(chǔ)培養(yǎng)基配方眾多,其添加劑組成更是存在極大變異,結(jié)果差異較大。了解不同培養(yǎng)方案效果及適用范圍差異,建立用途廣泛的鼻粘膜上皮細(xì)胞體外模型,符合電生理研究要求特別是尤斯灌流室研究要求的模型,有重要的現(xiàn)實(shí)意義。本研究以建立此類鼻粘膜上皮體外模型為目標(biāo),從影響細(xì)胞分離、貼壁、增殖、分化多個要素入手,對比和優(yōu)化了培養(yǎng)方案,并從細(xì)胞形態(tài)、纖毛擺動頻率及電生理功能等方面評估所建立模型的可靠性,并對所培養(yǎng)的鼻息肉粘膜上皮的電生理特點(diǎn)做了初步觀察和研究。 建立滿足多種研究需要的,分化良好的人類鼻粘膜上皮細(xì)胞體外模型。方法： 取27例鼻息肉或正常鼻粘膜標(biāo)本,采取0.1％Protease XIV和0.001％DNase消化標(biāo)本,并對其中8例計數(shù)(血球計數(shù)板)。分別從培養(yǎng)基(含血清培養(yǎng)基、改良Wu方案[8]、BEGM方案(Clonetics)、改良Gray[9]方案(即ALI培養(yǎng)基,改良包括降低添加劑內(nèi)EGF濃度為0.01ng/ml,維持BEGM內(nèi)BPE濃度)及培養(yǎng)模式(氣—液界面、浸泡方式)兩個主要方面對比并優(yōu)化培養(yǎng)方案,建立了氣液界面下,ALI培養(yǎng)基培養(yǎng)方案。并通過形態(tài)、組成、纖毛分化、纖毛擺動頻率幾個方面證實(shí)該方案的可靠性。 ①.上皮細(xì)胞純度：取2例標(biāo)本,分4皿用含血清培養(yǎng)基培養(yǎng)3天,鼠抗人波形蛋白單克隆抗體標(biāo)記成纖維細(xì)胞,用Image-Pro Plus 6計算比率。 ②.血清及Wu方案對貼壁影響：取6例標(biāo)本,分別種植于3個96孔板內(nèi),每2例標(biāo)本為1組,分成1天、3天、5天組,每一大組內(nèi)分成5小組,分別為膠原預(yù)鋪+Wu方案組,膠原預(yù)鋪+Wu方案+10%FBS組,有血清(DMEM/F12+10%FBS)+膠原預(yù)鋪組,有血清無膠原預(yù)鋪組,空白對照組,每組8孔細(xì)胞,MTT法測量細(xì)胞含量,統(tǒng)計采取單因素方差分析。 ③.不同無血清培養(yǎng)基對細(xì)胞形態(tài)影響：取6例標(biāo)本分3組,共18皿細(xì)胞分別采取Wu方案,含血清培養(yǎng)基,BEGM在浸泡方式下培養(yǎng),觀察并記錄其早期貼壁及各不同培養(yǎng)時間細(xì)胞形態(tài)。 ④.基底細(xì)胞組成及分布：取3例標(biāo)本分6皿培養(yǎng),分含血清浸泡環(huán)境培養(yǎng)組,ALI培養(yǎng)基氣液界面培養(yǎng)組,培養(yǎng)1周,ALI培養(yǎng)組采取吸管吹打造成局部細(xì)胞損傷缺失次日CK14標(biāo)記基底細(xì)胞。 ⑤.ALI氣液界面培養(yǎng)細(xì)胞纖毛分化：取3例標(biāo)本分6皿培養(yǎng),觀察纖毛發(fā)生情況,并于培養(yǎng)21天后標(biāo)記β-Tubulin,用Image-Pro Plus 6計算纖毛覆蓋率。 ⑥.含血清培養(yǎng)基及Wu方案對纖毛擺動影響：取3例標(biāo)本,分6皿培養(yǎng),分為血清培養(yǎng)組及Wu方案組,浸泡培養(yǎng)連續(xù)1-9天測量纖毛擺動。 ⑦.AL1培養(yǎng)基氣液界面下培養(yǎng)細(xì)胞形態(tài)及纖毛擺動頻率影響：ALI培養(yǎng)基、氣液界面培養(yǎng)3例標(biāo)本6皿細(xì)胞,觀察細(xì)胞形態(tài)至培養(yǎng)60天,3皿細(xì)胞,每皿測5處纖毛擺動頻率,分別于培養(yǎng)14天及30天測纖毛擺動頻率,采取獨(dú)立樣本T檢驗(yàn)分析數(shù)據(jù)。 ⑧.掃描電鏡：1例標(biāo)本,用ALI培養(yǎng)基培養(yǎng)14天,掃描電鏡下觀察細(xì)胞形態(tài)及纖毛發(fā)生。 ①.獲得的上皮細(xì)胞純度：單次酶消化法獲取鼻粘膜上皮每次可獲得約4.2±1.8×106 (Mean±SEM, n=8)個活性良好的上皮細(xì)胞,波形蛋白陽性細(xì)胞(成纖維細(xì)胞)占4.6±0.5%(Mean±SEM, n=8). ②.血清及Wu方案對貼壁影響：單因素方差分析：第1天Wu方案十膠原組與含血清培養(yǎng)基無膠原組,含血清培養(yǎng)基+膠原組與含血清培養(yǎng)基無膠原組之間存在顯著性差異P0.05,第3天Wu方案與其余三組間存在顯著性差異P0.05,其余三組間無顯著性差異。第5天(DMEM/F12+10%FBS)結(jié)果與3天同。 ③.不同無血清培養(yǎng)基對細(xì)胞形態(tài)影響：Wu方案及含血清培養(yǎng)基培養(yǎng)的細(xì)胞呈扁平復(fù)層化改變,細(xì)胞極不規(guī)則,培養(yǎng)2周原有纖毛上皮消失,部分細(xì)胞死亡。BEGM培養(yǎng)基培養(yǎng)的細(xì)胞形態(tài)尚規(guī)則,培養(yǎng)2周仍無纖毛發(fā)生。 ④.基底細(xì)胞含量及分布：含血清培養(yǎng)基CK14標(biāo)記的基底細(xì)胞含量23±3%(Mean±SEM, n=6), ALI氣液界面CK14在腔面表層無表達(dá),在人為造成缺損周圍高表達(dá)。 ⑤.含血清培養(yǎng)基及Wu方案對纖毛擺動影響：析因設(shè)計的雙因素方差分析纖毛擺動頻率與培養(yǎng)基種類及培養(yǎng)時間均相關(guān),差異有統(tǒng)計學(xué)意義P0.01。含血清培養(yǎng)基纖毛擺動頻率高于Wu方案。 ⑥.ALI培養(yǎng)基氣液界面培養(yǎng),細(xì)胞形態(tài)、纖毛分化及纖毛擺動頻率：TransWell支持膜上細(xì)胞形態(tài)規(guī)則,排列整齊,未見復(fù)層化及空泡,培養(yǎng)14天可見纖毛發(fā)生,培養(yǎng)21天,纖毛覆蓋率達(dá)20.4±3.2%(Mean±SEM, n=12),培養(yǎng)至60日局部細(xì)胞增厚。培養(yǎng)14天,30天測得纖毛擺動頻率分別為8.8±1.7Hz,8.5±1.3 Hz (Mean±SEM, n=15)T檢驗(yàn)其差異無統(tǒng)計學(xué)意義(P0.05)。 ⑦.掃描電鏡：ALI培養(yǎng)基培養(yǎng)14天,細(xì)胞呈單層柱狀,表面可見大量微絨毛,部分細(xì)胞出現(xiàn)纖毛發(fā)生。 ①.酶消化法獲得的鼻粘膜上皮細(xì)胞純度高,細(xì)胞數(shù)量大,活性好。 ②.血清及膠原均有促進(jìn)細(xì)胞貼壁的作用,且二者有協(xié)同作用,可在培養(yǎng)早期加入血清協(xié)助細(xì)胞貼壁； ③.血清對上皮細(xì)胞生長具有抑制作用,且使培養(yǎng)的上皮細(xì)胞呈復(fù)層鱗狀化改變,不適合長期培養(yǎng)。 ④.Wu方案內(nèi)高濃度的Ca(1.05mM)與EGF (25ng/ml)在即使高濃度RA (50nM)存在的環(huán)境下對纖毛上皮分化亦不利,高濃度EGF對分化有抑制作用。 ⑤.我們所采取的ALI培養(yǎng)基以BEGM/DMEM (1:1)為基礎(chǔ)培養(yǎng)基,其內(nèi)與分化密切相關(guān)重要添加劑濃度分別為Ca0.96mM, RA50nM, EGF0.01ng/ml,可獲得分化良好的鼻粘膜上皮細(xì)胞,其形態(tài)及功能與體內(nèi)接近,且較長時間內(nèi)(60天)維持其正常形態(tài)及功能,進(jìn)一步說明良好的鼻粘膜上皮細(xì)胞分化至少需保證高濃度的Ca、RA及低濃度的EGF。 ⑥.分化良好的鼻粘膜上皮腔面無基底細(xì)胞,基底細(xì)胞可能在損傷修復(fù)過程中發(fā)揮重要作用。 目的： 利于尤斯灌流室對培養(yǎng)模型電生理功能進(jìn)行評估,對比不同培養(yǎng)時期其電生理功能特點(diǎn),并初步探索其離子轉(zhuǎn)運(yùn)機(jī)制及息肉粘膜電生理特點(diǎn)。 方法： 培養(yǎng)方法同第一部分,取4例鼻息肉標(biāo)本,種于16皿6.5mm Trans Well皿內(nèi),隨機(jī)分為兩個組培養(yǎng)組：14天、45天組各8例,原代鼻粘膜上皮細(xì)胞在培養(yǎng)4天內(nèi)完全匯合,形成氣液界面,兩組細(xì)胞分別于14天、45天用Ussing chamber做跨膜電勢PD、短路電流Isc測量,歐姆定律計算跨膜電阻RT；為測量各離子通道活性,按一定順序?qū)miloride, Forskolin, DIDS (4,4'-diiso-thiocyanostilbene-2,2'-disulfonic acid)加入頂側(cè)膜所在的小室內(nèi),待電流穩(wěn)定底側(cè)膜側(cè)加入Bumetanide。 結(jié)果： 培養(yǎng)14天、45天上皮細(xì)胞均形成了緊密連接,細(xì)胞形態(tài)上無明顯差異,14天、45天基礎(chǔ)Isc分別為44.7±2.8μA/cm2,18.1±1.9μA/cm2,Rt分別為347.7±28Ω/cm2,627.7±55Ω/cm2 (n=8,電阻及基礎(chǔ)Isc P值均0.05), Amiloride敏感的Na通道分別占基礎(chǔ)狀態(tài)下Isc的92+2%,85+3%,(n=8,P0.05)。各藥物敏感的通道活性在14天及45天均存在顯著性差異,P值均0.05,Forskolin激活CFTR同時對CaCC.NKCC亦有激活作用,阻斷NKCC后CFTR對C1-分泌明顯減少。 結(jié)論： ①.培養(yǎng)的人類原代鼻粘膜上皮細(xì)胞間形成緊密連接及高跨膜電阻,14天組及45天組均符合電生理研究要求,培養(yǎng)14天時細(xì)胞通道活性及對藥物敏感性高于培養(yǎng)45天。 ②.鼻粘膜上皮細(xì)胞頂側(cè)膜存在Amiloride敏感的Na+通道及CFTR(囊性纖維化轉(zhuǎn)運(yùn)因子)、CaCC (Ca依賴的CI通道)通道,底側(cè)膜存在Bumetanide敏感的NKCC (Na+-K+-2C1同向轉(zhuǎn)運(yùn)體); ③.所培養(yǎng)的鼻息肉上皮細(xì)胞Amiloride敏感的Na+通道占基礎(chǔ)電流極高比率或許與鼻息肉發(fā)病相關(guān) ④.頂側(cè)膜CFTR對Cl-分泌與底側(cè)膜NKCC (Na-K-2C1同向轉(zhuǎn)運(yùn)體)通道活性密切相關(guān)； ⑤.CFTR、CaCC、NKCC、ENaC之間存在密切的信息聯(lián)系,相互協(xié)調(diào)或拮抗,共同維持上皮細(xì)胞電生理穩(wěn)定及表層液體層容量。
[Abstract]:The epithelial cells of nasal mucosa have important physiological functions such as barrier function and ion, water transport function and so on. The normal transport of epithelial cells to surface liquid layer ions and water is an important basis for the transport of mucociliary transport. Electrophysiological study is an important research method to explore the physiological function and pathogenesis of nasal cavity and sinuses, except the typical channel disease sac. It is also found that abnormal ion and water transport may also be one of the pathogenesis of nasal polyps and nasosinusitis. Traditional methods such as patch clamp technique, such as patch clamp technique, are mainly aimed at single cell research. Cell separation is easily damaged by mechanical and enzyme elimination and affects channel activity; and another electrophysiology The technique of Ussing chamber is mainly applied to the cultivation of the entire respiratory epithelium on the support membrane, and the malpractice of no digestive and mechanical damage. At the same time, it can control the composition of the apical membrane and the bottom membrane electrolyte flexibly according to the design requirements. Therefore, the more and more scholars pay attention to it, but this technology needs to be good. A good cell culture model is completed. This model requires the cells to grow on the support membrane for a long time, highly differentiated, form a reliable close connection and so on. It has a high requirement for the culture technology, and there is no such report at home.
Although the technique of respiratory tract mucosa culture has been developing for many years, there is still no unified plan. There are many formulas for the basic culture medium, and there are great variations in the composition of its additives. The difference of the results is great. It is of great practical significance to require the model of the study of the yusa perfusion room. This study aims at establishing such an in vitro model of the nasal epithelium, and compares and optimizes the culture scheme from the factors that affect cell separation, adherence, proliferation and differentiation, and comments on the morphology of the cell, the frequency of cilium swinging and the electrophysiological function. The reliability of the established model was evaluated, and the electrophysiological characteristics of the mucosal epithelium of the cultured nasal polyps were preliminarily observed and studied.
Objective: to establish a well differentiated human nasal epithelial cell model in vitro to meet various research needs.
27 cases of nasal polyps or normal nasal mucosa were collected, and 0.1%Protease XIV and 0.001%DNase digestive specimens were taken, and 8 cases were counted (blood cell count plate). The improved Gray[9] scheme (ALI medium, Clonetics) was improved from the culture medium (containing the serum medium, the improved Wu scheme [8], Clonetics), and the improvement included reducing the EGF concentration in the additive as 0.01ng/m. L, maintaining the BPE concentration in BEGM) and the culture mode (gas liquid interface, soaking mode), two main aspects are compared and optimized, and the culture scheme of ALI medium is established under the gas liquid interface, and the reliability of the scheme is proved by the form, the composition, the cilium differentiation and the cilium oscillating frequency.
1. Epithelial cell purity: 2 specimens were collected and 4 dishes were cultured with serum containing medium for 3 days. The McAb anti human vimentin monoclonal antibody was labeled as fibroblast, and the ratio was calculated with Image-Pro Plus 6.
2. Effect of serum and Wu regimen on adherence: 6 specimens were planted in 3 96 orifice plates, each 2 specimens were divided into 1 groups, divided into 1 days, 3 days and 5 days, each group was divided into 5 groups, which were collagen pre paved +Wu program group, collagen prepaved +Wu scheme +10%FBS group, serum (DMEM/F12+10%FBS) + collagen pre placement group, and serum free pre placement group, empty sera group, empty White control group, each group of 8 pore cells, MTT method to measure cell content, statistical analysis by one-way ANOVA.
3. The effect of different serum-free medium on cell morphology: 6 specimens were divided into 3 groups. A total of 18 Petri dish cells were treated with Wu, serum containing medium and BEGM in immersion mode, and the early adherence and different culture time of cell morphology were observed and recorded.
The composition and distribution of basal cells: 3 specimens were divided into 6 Petri dishes, divided into serum immersion environment culture group, ALI culture medium gas liquid interface culture group, culture for 1 weeks, and ALI culture group adopted tube blowing to build up CK14 labeled basal cells after the absence of local cell injury.
(5).ALI gas liquid interface culture cell cilium differentiation: 3 specimens were divided into 6 Petri dishes to observe cilium occurrence and to mark beta -Tubulin after 21 days of culture, and ciliary coverage was calculated with Image-Pro Plus 6.
The influence of serum culture medium and Wu scheme on cilium swinging: 3 specimens were collected and divided into 6 Petri dishes and divided into serum culture group and Wu program group. The cilium swinging was measured for 1-9 days.
.AL1 culture medium air liquid interface culture cell morphology and cilium oscillating frequency influence: ALI medium, gas liquid interface culture 3 specimens of 6 Petri dish cells, observe cell morphology to 60 days, 3 Petri dish cells, each dish measured 5 cilium oscillating frequency, respectively 14 days and 30 days to measure cilium oscillating frequency, take independent sample T test analysis data.
Scanning electron microscopy: 1 specimens were cultured in ALI medium for 14 days. The cell morphology and cilia were observed under scanning electron microscope.
(1) the purity of epithelial cells obtained: a single enzyme digestion method can obtain approximately 4.2 + 1.8 * 106 (Mean + SEM, n=8) epithelial cells each time, and vimentin positive cells (fibroblasts) account for 4.6 + 0.5% (Mean + SEM, n=8).
2. The effect of serum and Wu regimen on adherence: single factor analysis of variance: first days Wu program ten collagen group and serum containing medium without collagen group, serum culture medium + collagen group and serum containing collagen without collagen group, there was significant difference between P0.05, third days Wu scheme and the remaining three groups of significant differences between P0.05, the rest of the three groups did not show Sexual differences. The fifth day (DMEM/F12+10%FBS) result is the same as the 3 day.
3. The effects of different serum-free medium on the cell morphology: the Wu scheme and the cells in the serum culture medium were flattened and stratified, the cells were very irregular, the ciliated epithelium disappeared in 2 weeks, and the cell morphology of the.BEGM culture medium was still regular, and there was no cilium in the culture for 2 weeks.
The content and distribution of basal cells: the content of basal cells marked by CK14 containing serum culture medium was 23 + 3% (Mean + SEM, n=6), and the CK14 in ALI gas-liquid interface was not expressed on the surface of the cavity surface, and was highly expressed around the artificial defect.
The effect of serum culture medium and Wu scheme on cilium swinging: double factor analysis of variance analysis of factorial design, the frequency of cilium swinging was related to the type of culture medium and the time of culture. The difference was statistically significant in P0.01., the frequency of cilium swinging was higher than that of Wu.
(6).ALI culture medium gas liquid interface culture, cell morphology, cilium differentiation and cilium oscillating frequency: TransWell support membrane cell morphology rules, orderly arrangement, no stratification and vacuoles, culture 14 days can be seen cilia, culture 21 days, cilium coverage rate of 20.4 + 3.2% (Mean + SEM, n=12), culture to 60 days local cell thickening. Culture 14 days, 30 The cilia swing frequency was 8.8 + 1.7Hz, 8.5 + 1.3 Hz (Mean + SEM, n=15) T test showed no significant difference (P0.05).
Scanning electron microscopy: ALI medium for 14 days, the cells showed a single columnar surface, a large number of microvilli were seen on the surface, and cilia occurred in some cells.
1. The purity of nasal epithelial cells obtained by enzymatic digestion is high, the number of cells is large and the activity is good.
Serum and collagen can promote cell adherence, and the two have synergistic effect.
Serum inhibits the growth of epithelial cells, and makes the epithelial cells of the cultured cells become stratified, which is not suitable for long-term culture.
(4) the high concentration of Ca (1.05mM) and EGF (25ng/ml) in the.Wu scheme are also disadvantageous to the differentiation of ciliated epithelium in the presence of high concentration RA (50nM), and the high concentration EGF can inhibit the differentiation of the ciliated epithelium.
5. The ALI medium we adopted was based on BEGM/DMEM (1:1) as the basal medium. The concentration of important additives in the medium was Ca0.96mM, RA50nM, EGF0.01ng/ml, and the well differentiated nasal epithelial cells were obtained. The morphology and function were close to the body, and the normal form and function were maintained for a long time (60 days). One step indicates that good differentiation of nasal epithelial cells requires at least a high concentration of Ca, RA and low concentration of EGF..
There is no basal cell in the nasal mucosa epithelium with good differentiation. Basal cells may play an important role in the process of injury repair.
Objective:
It is beneficial to evaluate the electrophysiological function of the culture model, compare the electrophysiological function of different culture period, and explore the mechanism of ion transport and the electrophysiological characteristics of polyp mucous membrane.
Method錛，

本文編號：1860427