人鼻粘膜上皮细胞体外培养方案优化及离子转运功能研究

发布时间：2018-05-08 07:02

  本文选题：原代细胞培养 + 鼻粘膜上皮细胞　； 参考：《中国人民解放军军医进修学院》2011年硕士论文

【摘要】：鼻粘膜上皮细胞具有屏障功能及离子、水转运功能等重要生理功能,上皮细胞对表层液体层离子及水正常转运是发挥粘液纤毛传输功能的重要基础。电生理研究是探索鼻腔,鼻窦生理功能及发病机制重要研究手段,除了典型通道疾病囊性纤维化病,目前又有研究发现异常的离子及水的转运可能亦是鼻腔常见病鼻息肉、鼻窦炎发病机制之一。传统的通道研究手段如膜片钳技术主要针对单细胞研究,细胞分离时易受到机械及酶消化损伤,影响通道活性；而另一项电生理研究技术尤斯灌流室(Ussing chamber)技术主要应用于培养在支持膜上的整片呼吸道上皮,并且无消化及机械损伤弊端,同时可根据实验设计要求灵活控制顶侧膜及底侧膜电解质组成,因此受到越来越多学者的关注,不过本项技术需要在良好的细胞培养模型上完成。该模型要求细胞在支持膜上长期单层生长、高度分化、形成可靠的紧密连接等特点,对培养技术要求较高,目前国内尚无此类报道。 呼吸道粘膜培养技术虽然经历了多年发展,然而仍无统一方案。基础培养基配方众多,其添加剂组成更是存在极大变异,结果差异较大。了解不同培养方案效果及适用范围差异,建立用途广泛的鼻粘膜上皮细胞体外模型,符合电生理研究要求特别是尤斯灌流室研究要求的模型,有重要的现实意义。本研究以建立此类鼻粘膜上皮体外模型为目标,从影响细胞分离、贴壁、增殖、分化多个要素入手,对比和优化了培养方案,并从细胞形态、纤毛摆动频率及电生理功能等方面评估所建立模型的可靠性,并对所培养的鼻息肉粘膜上皮的电生理特点做了初步观察和研究。 建立满足多种研究需要的,分化良好的人类鼻粘膜上皮细胞体外模型。方法： 取27例鼻息肉或正常鼻粘膜标本,采取0.1％Protease XIV和0.001％DNase消化标本,并对其中8例计数(血球计数板)。分别从培养基(含血清培养基、改良Wu方案[8]、BEGM方案(Clonetics)、改良Gray[9]方案(即ALI培养基,改良包括降低添加剂内EGF浓度为0.01ng/ml,维持BEGM内BPE浓度)及培养模式(气—液界面、浸泡方式)两个主要方面对比并优化培养方案,建立了气液界面下,ALI培养基培养方案。并通过形态、组成、纤毛分化、纤毛摆动频率几个方面证实该方案的可靠性。 ①.上皮细胞纯度：取2例标本,分4皿用含血清培养基培养3天,鼠抗人波形蛋白单克隆抗体标记成纤维细胞,用Image-Pro Plus 6计算比率。 ②.血清及Wu方案对贴壁影响：取6例标本,分别种植于3个96孔板内,每2例标本为1组,分成1天、3天、5天组,每一大组内分成5小组,分别为胶原预铺+Wu方案组,胶原预铺+Wu方案+10%FBS组,有血清(DMEM/F12+10%FBS)+胶原预铺组,有血清无胶原预铺组,空白对照组,每组8孔细胞,MTT法测量细胞含量,统计采取单因素方差分析。 ③.不同无血清培养基对细胞形态影响：取6例标本分3组,共18皿细胞分别采取Wu方案,含血清培养基,BEGM在浸泡方式下培养,观察并记录其早期贴壁及各不同培养时间细胞形态。 ④.基底细胞组成及分布：取3例标本分6皿培养,分含血清浸泡环境培养组,ALI培养基气液界面培养组,培养1周,ALI培养组采取吸管吹打造成局部细胞损伤缺失次日CK14标记基底细胞。 ⑤.ALI气液界面培养细胞纤毛分化：取3例标本分6皿培养,观察纤毛发生情况,并于培养21天后标记β-Tubulin,用Image-Pro Plus 6计算纤毛覆盖率。 ⑥.含血清培养基及Wu方案对纤毛摆动影响：取3例标本,分6皿培养,分为血清培养组及Wu方案组,浸泡培养连续1-9天测量纤毛摆动。 ⑦.AL1培养基气液界面下培养细胞形态及纤毛摆动频率影响：ALI培养基、气液界面培养3例标本6皿细胞,观察细胞形态至培养60天,3皿细胞,每皿测5处纤毛摆动频率,分别于培养14天及30天测纤毛摆动频率,采取独立样本T检验分析数据。 ⑧.扫描电镜：1例标本,用ALI培养基培养14天,扫描电镜下观察细胞形态及纤毛发生。 ①.获得的上皮细胞纯度：单次酶消化法获取鼻粘膜上皮每次可获得约4.2±1.8×106 (Mean±SEM, n=8)个活性良好的上皮细胞,波形蛋白阳性细胞(成纤维细胞)占4.6±0.5%(Mean±SEM, n=8). ②.血清及Wu方案对贴壁影响：单因素方差分析：第1天Wu方案十胶原组与含血清培养基无胶原组,含血清培养基+胶原组与含血清培养基无胶原组之间存在显著性差异P0.05,第3天Wu方案与其余三组间存在显著性差异P0.05,其余三组间无显著性差异。第5天(DMEM/F12+10%FBS)结果与3天同。 ③.不同无血清培养基对细胞形态影响：Wu方案及含血清培养基培养的细胞呈扁平复层化改变,细胞极不规则,培养2周原有纤毛上皮消失,部分细胞死亡。BEGM培养基培养的细胞形态尚规则,培养2周仍无纤毛发生。 ④.基底细胞含量及分布：含血清培养基CK14标记的基底细胞含量23±3%(Mean±SEM, n=6), ALI气液界面CK14在腔面表层无表达,在人为造成缺损周围高表达。 ⑤.含血清培养基及Wu方案对纤毛摆动影响：析因设计的双因素方差分析纤毛摆动频率与培养基种类及培养时间均相关,差异有统计学意义P0.01。含血清培养基纤毛摆动频率高于Wu方案。 ⑥.ALI培养基气液界面培养,细胞形态、纤毛分化及纤毛摆动频率：TransWell支持膜上细胞形态规则,排列整齐,未见复层化及空泡,培养14天可见纤毛发生,培养21天,纤毛覆盖率达20.4±3.2%(Mean±SEM, n=12),培养至60日局部细胞增厚。培养14天,30天测得纤毛摆动频率分别为8.8±1.7Hz,8.5±1.3 Hz (Mean±SEM, n=15)T检验其差异无统计学意义(P0.05)。 ⑦.扫描电镜：ALI培养基培养14天,细胞呈单层柱状,表面可见大量微绒毛,部分细胞出现纤毛发生。 ①.酶消化法获得的鼻粘膜上皮细胞纯度高,细胞数量大,活性好。 ②.血清及胶原均有促进细胞贴壁的作用,且二者有协同作用,可在培养早期加入血清协助细胞贴壁； ③.血清对上皮细胞生长具有抑制作用,且使培养的上皮细胞呈复层鳞状化改变,不适合长期培养。 ④.Wu方案内高浓度的Ca(1.05mM)与EGF (25ng/ml)在即使高浓度RA (50nM)存在的环境下对纤毛上皮分化亦不利,高浓度EGF对分化有抑制作用。 ⑤.我们所采取的ALI培养基以BEGM/DMEM (1:1)为基础培养基,其内与分化密切相关重要添加剂浓度分别为Ca0.96mM, RA50nM, EGF0.01ng/ml,可获得分化良好的鼻粘膜上皮细胞,其形态及功能与体内接近,且较长时间内(60天)维持其正常形态及功能,进一步说明良好的鼻粘膜上皮细胞分化至少需保证高浓度的Ca、RA及低浓度的EGF。 ⑥.分化良好的鼻粘膜上皮腔面无基底细胞,基底细胞可能在损伤修复过程中发挥重要作用。 目的： 利于尤斯灌流室对培养模型电生理功能进行评估,对比不同培养时期其电生理功能特点,并初步探索其离子转运机制及息肉粘膜电生理特点。 方法： 培养方法同第一部分,取4例鼻息肉标本,种于16皿6.5mm Trans Well皿内,随机分为两个组培养组：14天、45天组各8例,原代鼻粘膜上皮细胞在培养4天内完全汇合,形成气液界面,两组细胞分别于14天、45天用Ussing chamber做跨膜电势PD、短路电流Isc测量,欧姆定律计算跨膜电阻RT；为测量各离子通道活性,按一定顺序将Amiloride, Forskolin, DIDS (4,4'-diiso-thiocyanostilbene-2,2'-disulfonic acid)加入顶侧膜所在的小室内,待电流稳定底侧膜侧加入Bumetanide。 结果： 培养14天、45天上皮细胞均形成了紧密连接,细胞形态上无明显差异,14天、45天基础Isc分别为44.7±2.8μA/cm2,18.1±1.9μA/cm2,Rt分别为347.7±28Ω/cm2,627.7±55Ω/cm2 (n=8,电阻及基础Isc P值均0.05), Amiloride敏感的Na通道分别占基础状态下Isc的92+2%,85+3%,(n=8,P0.05)。各药物敏感的通道活性在14天及45天均存在显著性差异,P值均0.05,Forskolin激活CFTR同时对CaCC.NKCC亦有激活作用,阻断NKCC后CFTR对C1-分泌明显减少。 结论： ①.培养的人类原代鼻粘膜上皮细胞间形成紧密连接及高跨膜电阻,14天组及45天组均符合电生理研究要求,培养14天时细胞通道活性及对药物敏感性高于培养45天。 ②.鼻粘膜上皮细胞顶侧膜存在Amiloride敏感的Na+通道及CFTR(囊性纤维化转运因子)、CaCC (Ca依赖的CI通道)通道,底侧膜存在Bumetanide敏感的NKCC (Na+-K+-2C1同向转运体); ③.所培养的鼻息肉上皮细胞Amiloride敏感的Na+通道占基础电流极高比率或许与鼻息肉发病相关 ④.顶侧膜CFTR对Cl-分泌与底侧膜NKCC (Na-K-2C1同向转运体)通道活性密切相关； ⑤.CFTR、CaCC、NKCC、ENaC之间存在密切的信息联系,相互协调或拮抗,共同维持上皮细胞电生理稳定及表层液体层容量。
[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锛，

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