SD大鼠乳鼠皮层神经元细胞原代培养模型的建立及鉴定

摘要 目的：探讨SD大鼠乳鼠皮层神经元细胞原代培养方法,并鉴定其培养效果,以期建立一种生物学功能良好的体外细胞实验模型。方法：取出生24 h的SD大鼠乳鼠,分离出大脑皮层,在胰酶消化之前先进行离心,然后将胰酶消化后多次离心得到的细胞悬液接种于L-多聚赖氨酸包被的培养皿和共聚焦皿中,以加B27的Neurobasal-A培养基进行神经元细胞的原代培养,倒置显微镜下观察培养细胞的生长状态;通过免疫荧光组化的方法采用神经元标记物MAP-2进行神经元纯度的鉴定;在导入Fluo4-AM的原代神经元细胞,观察电刺激后胞内钙离子信号的变化,以验证神经元细胞的生理状态。结果：采用此方法培养的神经元细胞紧密贴壁、分散均匀、状态良好,神经元细胞周围突起相互连接形成网络;经MAP-2免疫荧光组化技术鉴定神经元的纯度达到95%以上;胞内钙离子信号的变化提示所培养的神经元具有良好的生物学功能。结论：该方法能获得纯度较高并且生物学功能良好的原代培养的SD大鼠乳鼠皮层神经元细胞。     

SD大鼠骨髓间充质干细胞分离培养及鉴定的实验研究

摘要 目的：探讨应用全骨髓贴壁法体外分离培养SD大鼠骨髓间充质干细胞（BMSCs)的可行性,研究其生物学特性,为骨组织工程提供种子细胞。方法：取SPF级5周龄健康SD大鼠2只,脱颈处死,分离双下肢股骨、胫骨,全骨髓贴壁法分离培养、纯化BMSCs;通过倒置显微镜观察原代、传代细胞生长情况、绘制生长、贴壁率曲线,研究其生物学特性;流式细胞仪检测表面标志物、诱导成成骨等方法进行鉴定。结果：应用全骨髓贴壁法可在体外分离出活性好、纯度高的BMSCs。倒置显微镜下可见原代细胞呈梭形、多角形,传代细胞形态均一呈纤维样;P3代BMSCs经流式细胞鉴定：CD44、CD90高表达,CD31、CD45低表达;定向诱导向成骨细胞分化,可见明显矿化结节。结论：证实应用全骨髓贴壁培养法体外可成功分离BMSCs,所分离培养、纯化的细胞生物学稳定,纯度高、活性好,具有多向分化潜能,能为骨组织工程、骨质疏松症和骨折不愈合疾病的研究提供种子细胞。     

人源诱导多能干细胞体外定向分化为功能性肝细胞的方法研究

摘要 目的：研究开发一种简易、快速在体外使多能诱导干细胞（induced pluripotent stem cells,iPSCs）定向分化为功能性肝样细胞的培养方法。方法：根据正常肝细胞在体内的发育规律,设计简化诱导方法使iPS细胞定向分化为内胚层细胞,应用qPCR和流式细胞术鉴定其纯度后进一步诱导分化为肝样细胞,并通过qPCR、ELISA、免疫荧光等技术鉴定肝细胞的性状和功能。结果：iPS细胞诱导7天后, OCT4和NANOG的表达水平显著下降,内胚层细胞相关基因CXCR4、FOXA2和HNF4A表达水平明显升高。内胚层细胞继续诱导培养15天后,肝细胞特异性标志基因ALB、TDO2、RBP4、G6PC和肝药酶基因CYPs等显著上调,同时产生高水平的白蛋白和尿素;PAS糖原染色为阳性,能主动摄取和释放吲哚菁绿,证实诱导成的肝样细胞具备正常肝细胞的部分功能。结论：该诱导方案能够在体外使iPS细胞遵循正常肝脏发育通路简易、高效地分化为功能性肝细胞。本研究为大量获得iPS来源的肝细胞及其在细胞疗法和药筛模型中的运用提供了可能性。     

多聚赖氨酸在悬浮细胞转染中的应用

目的:悬浮细胞的转染较贴壁细胞存在一定难度,用多聚赖氨酸包被的细胞培养板培养悬浮细胞使其贴壁,用脂质体2000按照贴壁细胞转染的方法转染悬浮细胞,提供一种高效的转染悬浮细胞的方法。方法:悬浮细胞Jurkat或CCRF-CEM培养于包被了0.1 mg/mL多聚赖氨酸的细胞培养板,16 h后洗掉未贴壁的细胞,用脂质体2000分别将pWPXLd质粒或靶向人ABL1基因的小干扰RNA(siRNA)转染细胞,24 h后于荧光显微镜下观察绿色荧光蛋白印迹鉴定siRNA的干扰效率。结果:pWPXLd成功转染2种细胞,siRNA成功抑制了ABL1的表达。结论:质粒和siRNA均能成功转染,提供了一种高效可行的转染悬浮细胞的方法。     

嗅球成鞘细胞的分离培养与鉴定

目的:探讨一种获取高纯度嗅球成鞘细胞(olfactory ensheathing cells,OECs)的方法.方法:从新生SD大鼠(3d)嗅球中迅速分离嗅神经层和嗅颗粒层,采用酶消化法分离细胞,差速贴壁法纯化细胞,接种于多聚赖氨酸包被的培养板内培养2d,采用NGFRp75和S100蛋白双标免疫组化、以Hoechst33342复染鉴定OECs的纯度.结果:OECs的纯度为(95.64±2.76)%.结论:本法是一种相对简便易行且经济、稳定、有效的OECs分离方法.     

新生BALB/c小鼠大脑皮质神经元细胞培养方法的建立

目的:经改良和优化,建立高纯度BALB/c小鼠大脑皮质神经元培养的方法.方法:采用L-多聚赖氨酸包被细胞培养板,取新生BALB/c小鼠(出生24 h内)大脑皮质组织,经0.25%胰酶消化后吹打成单个细胞,按1×106/孔接种于35 mm的六孔板中,用神经元细胞培养种植液培养6 h后换神经元细胞培养饲养液,培养40 h时...     

纺壳聚糖聚己内酯纳米纤维膜对鼠骨髓间充质干细胞成牙功能影响

摘要 目的：探讨纺壳聚糖聚己内酯纳米纤维膜对鼠骨髓间充质干细胞（bone marrow mesenchymal stem cell,BMSC）成牙功能影响。方法：从SD大鼠分离BMSCs,然后随机分为两组,一组与纺壳聚糖聚己内酯纳米纤维膜进行共培养（共培养组）;另一组不进行共培养,直接在细胞板内培养（对照组）。检测细胞增殖指数、细胞钙含量、碱性磷酸酶活性与成骨相关基因表达水平。结果：BMSCs细胞形态较为单一,呈纤维样、旋涡状梭形、生长,长期培养可见细胞成片生长并相互融合。与共培养24 h相比,共培养48 h后共培养组细胞增殖指数、钙含量、碱性磷酸酶活性以及O成骨相关基因CN、Col1、Runx2等相对表达水平均显著增加（P<0.05）。共培养24 h与48 h后,共培养组的细胞增殖指数、钙含量、碱性磷酸酶活性和骨钙素（OCN）、Ⅰ型胶原α1（Col1）、Runt相关转录因子2（Runx2）等成骨相关基因相对表达水平都显著高于对照组（P<0.05）。结论：纺壳聚糖聚己内酯纳米纤维膜在BMSCs中的应用能促进细胞增殖,提高碱性磷酸酶活性与钙含量,促进成骨相关基因的表达,从而发挥成牙功能。     

饱和脂肪酸诱导肝细胞系建立代谢相关脂肪性肝病细胞损伤模型

摘要 目的：探讨不同脂肪酸对肝细胞系脂质积累、细胞损伤的影响,选择合适诱导试剂及肝细胞系建立一种具有严重细胞损伤及炎症反应的晚期代谢相关脂肪性肝病（MAFLD）体外细胞模型。方法：以油酸（OA）或棕榈酸（PA）或其混合物分别处理HepG2和LO2细胞,以CCK8检测细胞存活率;以油红O染色及甘油三酯酶法检测细胞脂质积累程度;以qRT-PCR检测凋亡相关蛋白、纤维化相关蛋白、自噬相关蛋白、炎症因子的mRNA表达水平。结果：0.25 mmol/LPA作用HepG2细胞24 h可显著诱导甘油三酯（TG）和脂质积累,但对LO2细胞无明显影响;0.25 mmol/L PA处理两种细胞系可诱导显著的细胞损伤及炎症,OA可缓解PA对细胞的损伤作用。结论：利用PA处理HepG2细胞可引起一定程度的脂质积累,诱导显著的细胞损伤及炎症,是合适的MAFLD体外细胞模型。     

探究壮方柔肝化纤颗粒对肝纤维化大鼠肝组织病理影响机制

摘要 目的：探究壮方柔肝化纤颗粒对肝纤维化大鼠肝组织病理影响机制。方法：选取80只雄性Wistar大鼠随机将其平均分成正常对照组、病理模型组、柔肝化纤颗粒低、中、高剂量组,每组各16只。四氯化碳复合因素造模,观察记录大鼠肝脏形态,采用HE染色、Masson染色观察大鼠肝组织和胶原纤维变化,检测血清丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）指标数值,分析大鼠肝组织中谷胱甘肽过氧化物酶（GSH-Px）、羟脯氨酸（HYP）、超氧化物歧化酶（SOD）与丙二醛（MDA）的含量。结果：柔肝化纤颗粒低、中、高剂量组大鼠肝细胞变性、坏死及纤维化组织增生程度均较病理模型组明显减轻。柔肝化纤颗粒低、中、高剂量组血清ALT、AST指标数值与肝组织中HYP、MDA指标水平较病理模型组呈现剂量依赖性降低（P＜0.001）,而柔肝化纤颗粒高剂量组GSH-Px、SOD的指标含量显著高于病理模型组,差异均有统计学意义（P＜0.001）。结论：柔肝化纤颗粒可有效改善肝纤维化大鼠的肝细胞损害情况,减轻其肝组织炎症程度,对肝纤维化大鼠有一定的保肝护肝与抗肝纤维化作用,其机制可能与提高GSH-Px与SOD的水平、降低HYP与MDA的含量、降低氧化应激水平、调节脂质代谢、减少机体胶原蛋白合成等相关。     

大鼠脑微血管内皮细胞的分离与原代培养

为了建立大鼠脑微血管内皮细胞体外培养模型,探索纯度较高的大鼠脑微血管内皮细胞分离和原代培养的方法并进行形态学观察。采用2～3周龄的SD大鼠,解剖得到大脑皮质,两次酶消化及牛血清白蛋白或葡聚糖和Percoll梯度离心获得较纯的脑微血管段后,接种于涂布基质的培养皿进行原代培养;培养的细胞采用相差显微镜形态学观察、透射电镜观察及Ⅷ因子相关抗原免疫组化检测鉴定。结果发现,培养12h即可见细胞从贴壁的脑微血管段周围长出,细胞呈短梭形,区域性单层生长,5～7天内皮细胞融合,内皮细胞纯度达90％以上;内皮细胞的贴壁和生长有赖于所涂布的基质,纤连蛋白／Ⅳ型胶原优于鼠尾胶和明胶;Ⅷ因子相关抗原免疫组化检测内皮细胞表达阳性,透射电镜观察可见相邻内皮细胞间存在紧密连接结构。提示该方法能成功进行纯度较高的大鼠脑微血管内皮细胞原代培养,可用于脑微血管内皮的生理、生化及药理学研究,亦可用于构建大鼠血脑屏障模型。     

Extended primary culture of human hepatocytes in a collagen gel sandwich system

Summary To develop a strategy for extended primary culture of human hepatocytes, we placed human hepatocytes between two layers of collagen gel, called a “collagen gel sandwich.” Maintenance of hepatocellular functions in this system was compared with that of identical hepatocyte preparations cultured on dry-collagen coated dishes or co-cultured with rat liver epithelial cells. Human hepatocytes in a collagen gel sandwich (five separate cultures) survived for more than 4 wk, with the longest period of culture being 78 d. They maintained polygonal morphology with bile canaliculuslike structures and high levels of albumin secretion throughout the period of culture. In contrast, hepatocytes on dry-collagen became feature-less, and albumin secretion could not be detected after 14 d of culture. This loss of albumin secretion was partially recovered by overlaying one layer of collagen gel. Ethoxyresorufin O-deethylase activity, associated with cytochrome P450 1A2, was detected basally up to 29 d in collagen gel sandwich culture. These activities were induced four- to eightfold after induction with dibenz(a,h)anthracene. Cocultures also maintained basal activity up to 29 d. However, their inducibility was lower than that of hepatocytes in collagen gel sandwich. No ethoxyresorufin O-deethylase activity was detected in hepatocytes cultured on dry-collagen at 7 d. Thus, the collagen gel sandwich system preserves differentiated morphology and functions of human hepatocytes in primary culture for a prolonged period of time. This system is a promising model for studying human hepatocellular function, including protein synthesis and drug metabolism in vitro.     

Crude liver membrane fractions as substrate preserve liver-specific functions in long-term,serum-free rat hepatocyte cultures

Summary Over time, rat hepatocytes cultured on collagen lose the capacity to express liver-specific functions. The influence on this degradation process of an alternative substratum—crude membrane fractions prepared from the liver of the same rat strain—was investigated. Freshly isolated rat hepatocytes were cultured in serum-free Williams E medium supplemented with aprotinin, selenium, dexamethasone, and insulin in flasks coated with a mixture of rat liver crude membrane fractions:collagen type I (100:1). The cells adhered firmly, exhibiting minimal spreading and remaining grouped in columns or in cell islands, and retained their liver-specific functions for more than 1 wk. Hepatocytes secreted substantially higher amounts of albumin than cells cultured on collagen-coated dishes, and on Days 1 and 9 in culture the total P-450 content was 72 and 40%, respectively, of that of freshly isolated cells. On Day 6, the 7-ethoxyresorufin-O-de-ethylase and the aldrin epoxidase activities were still more than 50% that of freshly isolated hepatocytes. Exposure to phenobarbital on Days 3 to 6 increased the total cytochrome P-450 content twofold; exposure to 3-methylcholanthrene increased the activity of the corresponding cytochrome P-450 isoforms to 20 times that observed in untreated cultures and 6 times that observed in freshly isolated cells. Thus, given the ease with which they are prepared, the use of crude membrane fractions combined with culture medium supplemented with aprotinin and selenium can facilitate the preparation of reproducible cultures suitable for long-term in vitro pharmacotoxicologic studies using rat hepatocytes.     

Liposome Internalization by Isolated Rat Hepatocytes

Abstract

We studied the in vitro interaction and the endocytic process of peroxidase-loaded liposomes with isolated rat hepatocytes maintained in suspension culture. We report morphological (both at light and electron microscopy) and biochemical evidence that cationic egg PC (egg phosphatidylcholine)-liposomes are taken up by isolated rat hepatocytes via an endocytic pathway. The incubation of 2.5 mM liposomal lipids for at least 4 hours was not cytotoxic to the cells. The uptake of peroxidase-fluoresceine isothiocyanate conjugate-liposomes was not distributed homogeneously among the hepatocyte population. However the hepatocytes which have apparently internalized greater amount of probe were not damaged since cell shape and integrity of the membrane are retained as evaluated by conventional light microscopy. Therefore the fusion between liposomes and hepatocytes seems to be dependent on the viability of the isolated hepatocytes.     

Long-term culture of functional hepatocytes on chemically modified collagen gels

For long-term maintenance of functional hepatocytes in primary culture, a new culture system with chemically modified type-I collagen gel was developed. Isolated hepatocytes spread as flat cells and rapidly lost their viability and functions when cultured on native collagen gel. In contrast, they survived for several weeks when cultured on collagen gels that had been modified by treatment with sodium-borohydride (NaBH₄) or by digestion with pepsin, which resulted in destruction of crosslinking of collagen fibers and marked decrease in meachanical strength of the gels. These long-lived cells were round and aggregated and maintained high levels of various differentiated liver functions including albumin secretion and activities of tyrosine aminotransferase and P450. Moreover on collagen gels modified by treatment with NaBH₄ or pepsin, the cell showed less DNA synthesis in response to mitogenic stimulation than cells cultures on gel containing native collagen. Interestingly, crosslinking of these chemically modified gels with D-ribose resulted in changes in various phenotypes of hepatocytes cultures on them including shape, longevity, and functions expressed when the cells were cultured on native collagen gel, suggesting that the effect of modification of the collagen gel is reversible. Thus the structure of collagen gels, probably due to the degree of crosslinking, seems to affect the morphology, maintenance of differentiated functions, and growth of primary cultured hepatocytes.     

Primary rat hepatocyte culture on 3D nanofibrous polymer scaffolds for toxicology and pharmaceutical research

Primary rat hepatocytes are a widely used experimental model to estimate drug metabolism and toxicity. In currently used two‐dimensional (2D) cell culture systems, typical problems like morphological changes and the loss of liver cell‐specific functions occur. We hypothesize that the use of polymer scaffolds could overcome these problems and support the establishment of three‐dimensional (3D) culture systems in pharmaceutical research. Isolated primary rat hepatocytes were cultured on collagen‐coated nanofibrous scaffolds for 7 days. Cell loading efficiency was quantified via DNA content measurement. Cell viability and presence of liver‐cell‐specific functions (albumin secretion, glycogen storage capacity) were evaluated. The activity of liver‐specific factors was analyzed by immunofluorescent staining. RNA was isolated to establish quantitative real‐time PCR. Our results indicate that primary rat hepatocytes cultured on nanofibrous scaffolds revealed high viability and well‐preserved glycogen storage. Albumin secretion was existent during the entire culture period. Hepatocytes remain HNF‐4 positive, indicating highly preserved cell differentiation. Aggregated hepatocytes re‐established positive signaling for Connexin 32, a marker for differentiated hepatocyte interaction. ZO‐1‐positive hepatocytes were detected indicating formation of tight junctions. Expression of cytochrome isoenzymes was inducible. Altogether the data suggest that nanofibrous scaffolds provide a good in vitro microenvironment for neo tissue regeneration of primary rat hepatocytes. Biotechnol. Bioeng. 2011; 108:141–150. © 2010 Wiley Periodicals, Inc.     

Conditions of the primary culture for rat hepatocytes and plasminogen activator release

The conditions of primary culture for rat hepatocytes was investigated on the releasing effect of Plasminogen Activator (PA). The culture method using Collagen Coated Dish (CCD-method) which is currently available and the ordinary culture method using Plastic Culture Dish (PCD-method) were employed for that purpose in a comparative way. The effect of the addition of some supplements, that is FN, Aprotinin, EGF were also investigated. The following results were obtained. The dissociated rat hepatocytes formed a monolayer with pavementlike morphology at 24-48 hours after seeding. No difference was observed in the morphology of hepatocytes during the culture period between the two methods, although CCD-method allowed 120 hours culture, whereas PCD-method allowed 72 hours. The PA activity was demonstrated on the hepatocytes by either culture method according to the fibrinolysis autography. The cultured hepatocytes released PA into the medium continuously as long as the viability and morphology of the cells were maintained in good state. The PA activity reached the maximum after 96 hours culture in CCD-method, whereas it reached the maximum after 48 hours in PCD-method. The addition of Aprotinin to the culture medium was not necessary for PA release in CCD-method in contrast to PCD-method. When EGF was discontinued in the culture medium, the release of PA was reduced in association with the occurring of morphological disintegration of hepatocytes.     

L-proline is an essential amino acid for hepatocyte growth in culture

For improvement of the culture conditions of adult rat hepatocytes in primary culture in collagen coated dishes, effects of various commercial culture media on the induction of replicative DNA synthesis of the cells stimulated by insulin plus epidermal growth factor were studied. Proline-deficient media, such as Leibovitz's L-15, Eagle's minimal essential medium and Dulbecco's modified minimal essential medium, did not induce DNA synthesis in hepatocytes, whereas proline-rich media, such as Williams medium E, McCoy's 5A and Ham's F-12, induced markedly hepatocyte proliferation. Moreover, when the proline-deficient media were supplemented with L-proline, the cells synthesized DNA in response to the two hormones. Cis-4-hydroxyl-L-proline strongly inhibited the induction of DNA synthesis, without affecting protein synthesis of the cells or showing any cytotoxicity. This inhibition was recovered completely by adding excess proline to the medium. Addition of other amino acids not present in the medium did not promote DNA synthesis. These findings indicate that L-proline is essential for induction of hepatocyte proliferation in culture, through its affect on synthesis of intracellular collagen.     

Long-term culture of primary rat hepatocytes with high albumin secretion using membrane-supported collagen sandwich

Primary cultured rat hepatocytes in a membrane-supported collagen sandwich maintained their normal cell morphology and high level of albumin secretion for over 56 days. It was found that the existence of an upper layer of collagen gel is crucial for long-term culture and that the transference of cellular nutrients between the culture media and hepatocytes from both the upper and the lower sides of gel layers promotes albumin secretion. These facts suggest that the membrane-supported collagen sandwich mimics well thein vivo environment of hepatocytes. This method has great potential for the long-term culture of primary cells.     

Primary culture of rat hepatocytes using membrane-supported collagen sandwich with hormone-free medium

Summary Primary culture of rat hepatocytes in hormone-free medium using membrane-supported collagen sandwich maintained their cellular morphology and expressed albumin secretion for about 3 weeks in vitro. It was reconfirmed that mimicking the cellular environment in vivo was effective for cellular maintenance.     

Effect of hydrocortisone and nicotinamide on gamma glutamyltransferase in primary cultures of rat hepatocytes

Summary Isolated rat hepatocytes cultured on collagen coated plates exhibit a gradual fetal phenotypic change during time in culture. The fetal liver marker gamma glutamyltransferase (GGT) was used to follow this change. Inasmuch as a significant overgrowth of nonparenchymal liver derived cells is seen frequently in primary cultures of hepatocytes, a technique was utilized that corrects for the presence of nonparenchymal cells. In media supplemented with either hydrocortisone (10⁻⁵ M) or nicotinamide (25 mM) the original epithelial morphology of hepatocytes was preserved for a longer period of time than in unsupplemented media. Hepatocytes in unsupplemented media exhibited an increase in GGT specific activity over time. Hydrocortisone (10⁻⁵ M) induced an increase in GGT activity compared to controls. Nicotinamide (25 mM) inhibited the increase in GGT activity compared to the unsupplemented hepatocytes. Our results indicate that GGT is regulated by hydrocortisone and nicotinamide. This study was supported by NIH Grant CA30241-01.