In vitro characteristics of pulmonary neuroendocrine cells isolated from rabbit fetal lung. I. Effects of culture media and nerve growth factor

Pulmonary neuroendocrine (NE) cells, dispersed throughout the airway mucosa as single cells and as innervated clusters (neuroepithelial bodies), were isolated from rabbit fetal lung and studied in short-term culture. The effects of culture media and nerve growth factor (NGF) on in vitro maintenance, differentation, and cell kinetics of isolated NE cells were examined. For demonstration of NE cells in intact lung, during cell separation and after culture, immunostaining for serotonin, formaldehyde-induced fluorescence method, histochemical reaction for acetylcholinesterase, and electron microscopy were used. The isolation procedure consisted of mechanical and enzymatic dissociation of lung tissue followed by separation of isolated cells on a discontinuous gradient of Percoll, resulting in 5- to 10-fold enrichment in NE cells. Cell fractions enriched in NE cells were cultured up to 7 days either in supplemented alpha-minimal essential medium with fetal bovine serum or in defined, hormone-supplemented, serum-free medium. NGF (2.5 S 5 to 50 ng/ml) was added to both serum-supplemented and serum-free media; cultures without NGF served as control. The number of serotonin-immunoreactive NE cells maintained in serum-supplemented medium (0.5% fetal bovine serum) increased significantly (p less than 0.05) on days 4 and 7 compared with cultures grown in serum-free medium. NE cells maintained in serum-supplemented medium incorporated [3H]thymidine and their labeling index was significantly increased (p less than 0.01) on day 7, whereas few or no NE cells were labeled in cultures grown in serum-free medium. NGF had no effect on the maintenance or kinetics of NE cells. Cultured NE cells formed elongated (unipolar or bipolar) neurite-like cytoplasmic processes with a button-like ending, regardless of the presence of NGF. Amine accumulated in perinuclear cytoplasm and in button-like endings. Staining for acetylcholinesterase (strongly positive in intact neuroepithelial bodies) was not detectable after separation into single cells, culture, or exposure to NGF. This study demonstrates that NE cells can be isolated from rabbit fetal lung and maintained in short-term culture. Low concentrations of fetal bovine serum enhanced the in vitro maintenance of NE cells, whereas NGF had no such effect. A feeder layer may be also important, since NE cells were closely associated with lung epithelial or fibroblast-like cells. The formation of neurite-like processes appears to be an expression of paracrine/paraneuron-type cell differentiation not mediated by NGF.(ABSTRACT TRUNCATED AT 400 WORDS)     

Different responses to mitogenic agents by Adult rat and human chromaffin cells in vitro

Adrenal medullary hyperplasia and pheochromocytomas occur frequently in laboratory rats, both in the courseof aging and in response to prolonged administration of a variety of drugs and other substances. In contrast, these lesions are rare in humans. Rat chromaffin cells proliferate throughout life, but the proliferative capacities of human chromaffin cells are unknown. To determine whether the difference in prevalence of adrenal lesions might be correlated with differences in cell proliferation, adrenal medullary cells from 3 patients undergoing radical nephrectomy were maintained in vitro for up to 2 weeks in control medium or in the presence of nerve growth factor (NGF) and/or tetradecanoyl phorbol acetate (TPA), an activator of protein kinase C. Both NGF and TPA are known mitogens for neonatal and adult rat chromaffin cells. At intervals, the cultures were pulsed for up to 36 hours with bromodeoxyuridine (BrdU) to label S-phase nuclei. They were then fixed and consecutively stained for BrdU and for tyrosine hydroxylase, to confirm that labeled cells were chromaffin cells. Cells from adult female F344 rats were similarly maintained. Human chromaffin cells labeled with BrdU were extremely rare (less than 0.1 %) under all culture conditions, and effects of NGF or TPA could not be demonstrated. Rat chromaffin cells showed little or no labeling with BrdU in control medium but, in contrast to their human counterparts studied, showed marked increases in the percentages of labeled cells in the presence of NGF (37% ± 3%), TPA (7% ± 1%), or both (31% ± 3%). The apparently lower responsiveness of human chromaffin cells to mitogenic signals, or responses to different types of signals, may contribute to the lower frequency of adrenal medullary hyperplasia and pheochromocytomas in humans compared to rats.     

Electron microscopic localization of acridine orange chromatin interaction products in rat pheochromocytoma PC12 cells.

The purpose of the present study is to examine the distribution pattern of acridine orange (AO) binding to DNA in rat adrenal pheochromocytoma PC12 cells that respond to nerve growth factor (NGF). PC12 cells were incubated in a medium containing 100 ng/ml of NGF for 1-14 days for AO ultracytochemistry. Electron microscopic studies revealed that AO binds to DNA exclusively within the euchromatin portion of the cell nucleus. About 35% of the untreated PC12 cells showed characteristic electron-dense interaction products within the nuclei. The average numbers of AO chromatin interaction products per cell nucleus and per micron 2 nuclear area were 45 and 1.6, respectively. In the presence of NGF, cell proliferation was suppressed. The cells gradually extend neurite-like cell processes. Mean 3H-labeling index was 40.0 +/- 2.2% in untreated cells and 13.0 +/- 2.3% in PC12 cells with NGF for 6 days after incubation for 30 min. With increasing the cellular differentiation percentages of AO positive cells and average numbers of AO chromatin interaction products per cell nucleus and per micron 2 nuclear area showed a progressive decrease. The results of the present and previous studies suggest that AO chromatin interaction products may be indicative of cell proliferation and differentiation.     

Protein kinase C in human pheochromocytoma

Subtypes of protein kinase C were analyzed in adrenal and extra-adrenal pheochromocytoma of humans. Almost all protein kinase C of the adrenal tumor was type III, while the enzyme of the extra-adrenal tumor was separated into two major fractions corresponding to type II and type III by hydroxyapatite column chromatography. The extra-adrenal tumor but not the adrenal tumor spontaneously produced neurite-like processes when the cells were cultured in vitro. These results suggest that the high proportion of type II enzyme may reflect neuron-directed differentiation in human pheochromocytoma.     

Long-term depolarization changes morphological parameters of PC12 cells

It is well known that neuronal differentiation is strongly dependent on the intracellular level of free calcium ions ([Ca²⁺]_i). In the present study the morphological and intracellular free calcium concentration changes were compared on PC12 pheochromocytoma cells cultured in control conditions and in a medium with high KCl level. Culturing PC12 cells in a medium with 20–30 mM KCl deprived of nerve growth factor supported cell proliferation and rapid growth of small neurite-like processes. However, their lengths did not increase with prolongation of the time of culturing. During culturing with 40 mM KCl the growth of these processes became blocked; the cells stopped proliferating and showed signs of degeneration.Measurements of [Ca²⁺]_i level during the first days of PC12 cells culturing in a hyperpotassium medium indicate that such changes in this level could be an important factor in the induction of the observed morphological alterations; however, other effects induced by membrane depolarization may also be responsible for them.     

Ultrastructure of human pheochromocytoma cells cultured for long periods

We conducted ultrastructural analysis of human pheochromocytoma (PC) cells maintained in primary culture for about 10 months. The cells were first isolated by the enzymatic treatment of a surgically resected tissue specimen obtained from a 37-year-old man with PC, a condition which is characterized by elevated blood levels of adrenaline and noradrenaline. It was found that noradrenaline production in the medium continued until the 90th day of culture (1330 pg/ml). The production level decreased to 20 pg/ml on the 180th day, and to 18 pg/ml on the 300th day. Examination under a transmission electron microscope (TEM) at 4 weeks of culture revealed electron-dense granules (about 200 nm in size and, presumably, rich in catecholamines), which were also observed in the tumor cells from the original PC tissue. Neurite-like processes grew at around 1 week of culture, and were still maintained at 6 months of culture. But, after 6 months of culture, the neurite-like processes contained a rosary-like elevated structure, which was suggestive of cell degeneration, as determined by a plasma polymerization replica method and observed with a scanning electron microscope. When cells were examined under the TEM, fewer electron-dense granules were observed in the cell bodies, with more numerous lipofuscin-like granules and filaments. Thus, electron-dense granules, which, presumably, contain catecholamines, were seen in a long-term culture of human PC cells. These granules decreased in number in parallel with the decrease in catecholamine levels in the culture.     

The effect of the controlled release of nerve growth factor from collagen gel on the efficiency of neural cell culture

In this study, we tested the hypothesis that the amount of nerve growth factor (NGF) required for pheochromocytoma (PC12) cell culture can be dramatically reduced by controlled release of NGF from a collagen gel coating on the culture surface. Cells were cultured on collagen gels loaded with various amounts of NGF. As a control, PC12 cells were cultured on collagen gels with daily addition of various amounts of NGF to the culture medium. After an initial 12h burst, NGF was steadily released from the gels for 4 days. Apoptotic activity and cell viability were determined using terminal uridine nick end labeling and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, respectively. Neuronal differentiation was determined using immunocytochemistry and Western blot analysis. Compared to 100 ng NGF daily addition (300 ng over 3 days), 1 ng NGF daily addition showed dramatically decreased cell viability and neuronal differentiation and increased apoptotic activity. In contrast, collagen gels loaded with 10 ng NGF yielded cell viability, apoptotic activity, and neuronal differentiation similar to those of culture with 100 ng NGF daily addition. Our method reduced the amount of NGF required for PC12 cell culture to 1/3th of that used in daily addition without affecting cell viability, apoptosis, or differentiation. This method could economize large-scale culture of stem cells by reducing the amount of costly growth factors needed.     

Characterization of Pheochromocytomas in a Mouse Strain with a Targeted Disruptive Mutation of the Neurofibromatosis Gene Nf1

Patients with neurofibromatosis type 1 (NF1) show an increased frequency of pheochromocytomas. TheNF1 gene encodes a GTPase-activating protein that controls the activity ofras proteins in intracellular signalling. A mouse strain with a knockout mutation of Nf1, the murine counterpart ofNF1, has recently been constructed. This mutation, designated Nf1ⁿ³¹, has been shown to be associated with the frequent development of pheochromocytomas in heterozygous animals. Pheochromocytomas are extremely rare in wild-type mice. We have characterized the tumors to assess their relevance as a model for human pheochromocytomas. The frequency of pheochromocytomas was determined in inbred compared to outbred mice carrying the Nf1ⁿ³¹ mutation. Paraffin sections of pheochromocytomas from seven mice were stained immunohistochemically for the catecholamine biosynthetic enzymes, tyrosine hydroxylase (TH) and phenylethanolamine-N-methyltransferase (PNMT) to infer their profiles of catecholamine synthesis, and for chromogranin A (CGA) to infer their content of secretory granules. Cultured cells from a representative tumor were studied in vitro to assess proliferation and neuronal differentiation. Pheochromocytomas arose in approx 15% of Nf1ⁿ³¹ mice with a mixed genetic background, but were absent in inbred mice. Approximately one-fourth of the tumors were bilateral. The tumors exhibited variable morphology. All included cells that appeared well differentiated and resembled normal chromaffin cells in that they expressed TH, PNMT, and CGA. Focal neuronal differentiation was also observed. In cell culture, the tumor cells ceased to proliferate and the majority underwent terminal differentiation into TH-positive cells with neuronal morphology. The phenotype of pheochromocytomas in mice with the Nf1ⁿ³¹ mutation resembles that of human pheochromocytomas, particularly with respect to their ability to produce epinephrine, as inferred from positive staining for PNMT. The tumors also resemble both normal and neoplastic human adrenal medulla with respect to their extensive differentiation into neuron-like cells in vitro. This change in phenotype may be related toras activation. These neoplasms may be valuable both as models for the pathobiology of adrenal medullary neoplasia, and as a source of epinephrine-producing pheochromocytoma cells lines, for which adequate models currently do not exist.     

NGF和GDNF基因重组逆转录病毒体外感染神经干细胞及其鉴定

目的 探讨神经干细胞（NSC）直接作为基因靶细胞能否被重组逆转录病毒感染以及感染后目的基因的表达。方法 分别用NGF和GDNF基因重组逆转录病毒上清液感染NSC2d;G418筛选后加入bFGF扩增培养;取感染后的NSC培养液（NGF／GDNF条件培养液）培养PC12细胞和中脑腹侧神经元;用免疫组织化学染色方法检测中脑腹侧多巴胺神经元的形态改变以及感染后NSC对目的基因的表达。结果 经G418筛选后,约50％感染NGF和GDNF基因重组逆转录病毒的NSC呈G418抗性;这些感染后的NSC开始分化,分裂球向四周长出放射状突起,部分细胞沿突起向外迁移生长;感染NGF基因的NSC呈星形,胞体较大,突起较粗,感染GDNF基因的NSC呈梭形,胞体较小,突起较长。经NGF条件培养液培养的PC12细胞,数量增多,突起明显变长。经GDNF条件培养液培养的中脑多巴胺神经元（TH阳性细胞）其胞体亦增大,突起伸长。大部分G418抗性的NSC出现NGF和GDNF免疫染色。结论 神经干细胞可直接作为基因靶细胞,能被NGF和GDNF基因重组逆转录病毒有效感染而表达和分泌有生物学活性的NGF和GDNF。     

Adrenergic Differentiation and Ret Expression in Rat Pheochromocytomas

Pheochromocytomas are catecholamine-producing tumors of the adult adrenal medulla. They are rare in humans and most other species but common in laboratory rats. However, the relevance of rat pheochromocytomas as a model for their human counterparts is uncertain. Previous studies of spontaneous and drug-induced rat pheochromocytomas and the PC12 pheochromocytoma cell line suggested a distinctive noradrenergic phenotype, possibly reflecting origin from a progenitor not present in the adult human adrenal. In this study, we studied 31 pheochromocytomas derived from test and control male and female rats in toxicologic studies for expression of the epinephrine-synthesizing enzyme phenylethanolamine-N-methyltransferase (PNMT) and the receptor tyrosine kinase Ret. PNMT, which defines adrenergic chromaffin cells, is frequently expressed in human pheochromocytomas, often in tumors that also overexpress RET. We also tested for the expression of the cell cycle checkpoint protein p27^Kip1, which recently was reported absent in pheochromocytomas from a strain of rats with a hereditary mixed multiple endocrine neoplasia (MEN)-like syndrome. Using immunoblots, we demonstrated PNMT expression in almost 50% of the 31 tumors, although often at lower levels than in normal rat adrenal medulla. The majority of tumors overexpressed Ret. There was no apparent correlation between PNMT and Ret. However, in this study, PNMT expression was strongly associated with tumors arising in female rats, while overexpression of Ret did not show a sex predilection. Robust expression of p27^Kip1 was seen in all tumors from the toxicologic studies and also in a small sample of pheochromocytomas from Long–Evans rats, which were reported to have a mixed MEN-like syndrome in the 1980s. The present results show that rat pheochromocytomas have greater phenotypic diversity than previously believed and greater similarity to their human counterparts with respect to these two important markers. Loss of p27^Kip1 does not appear to account for the high frequency of pheochromocytomas in commonly utilized rat strains.     

人参环氧炔醇对体外培养RSC96细胞的实验观察

目的研究人参环氧炔醇(Panaxydol,PND)对体外培养RSC96细胞神经营养因子及髓鞘蛋白表达的影响并探讨其机制。方法用含不同血清浓度的培养基培养RSC96细胞,MTT检测其增殖能力,以获取最适宜RSC96细胞体外生长的血清浓度。在适宜的血清培养基中加入PND(10μmol/L)处理RSC96细胞,以RT-PCR、western blot及ELISA检测RSC96细胞NGF和BDNF的表达。另外,预先在培养基中加入钙离子通道阻滞剂尼非地平探讨PND可能的作用途径。结果培养基血清浓度为4mmol/L时,RSC96细胞生长形态最接近于原代Schwann细胞。PND增强RSC96细胞表达和释放NGF和BDNF(P0.05)。尼非地平的使用,则削弱了PND对RSC96细胞的作用(P0.05)。结论在适宜的血清培养基中,体外培养的RSC96细胞可替代原代Schwann细胞,作为研究药物对它的影响及机制的细胞模型。PND增强RSC96细胞的生物活性的作用机制可能通过Ca2+信号途径介导。     

Rodent and primate adrenal medullary cells in vitro: phenotypic plasticity in response to coculture with C6 glioma cells or NGF

Summary In order to maintain a chronic supply of growth factor for medulla cells in vitro, chromaffin cells from rat, African green monkeys and man were co-cultured with C₆ glioma cells, which secrete growth factors that sustain sympathetic neurons in vitro. The response of chromaffin cells to coculture was compared to treatment of medullary cells with nerve growth factor (NGF) alone. Dispersed chromaffin cell preparations were obtained by a trypsin-collagenase procedure, and subjected to differential plating on collagen-coated surfaces. With both human and monkey tissue, non-chromaffin cells did attach to the culture plates and an enriched chromaffin cell population could be replated. Rat adrenal medulla cells survived very poorly in vitro and were not enriched in this procedure. Cultured human and monkey chromaffin cells survived as epithelial cells (50%) and showed neuritic outgrowth on 55 to 66% of the cells after eight days when treated with nerve growth factor (NGF). These cells showed strong catecholamine histofluorescence, tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DBH) immunoreactivity. In contrast, only ten percent of adult rat chromaffin cells survived in culture, although NGF treatment rescued an additional 20% of the cells and induced neuritic outgrowth after one week in vitro. C₆ glioma cells were treated with mitomycin C bromodeoxyuridine to inhibit mitosis and were plated with the various medulla cells in a one to one ratio. Both human and monkey chromaffin cells expressed extensive and enhanced neuritic arborization within eight days of co-culture, (64–82% respectively) and exhibited intimate contact with the glioma cells as seen at the ultrastructural level. Importantly, survival of adult rat adrenal medulla cells was enhanced to 50% or more with 40% of the cells extending neurites when co-cultured with glioma cells for seven days. Chromaffin cells from all three species reacted for TH, DBH and PNMT in co-culture and were histofluorescent. The majority of these cells were also immunoreactive for serotonin and enkephalin, while only 37% of chromaffin cells indicated the presence of NPY. These data indicate that adrenal medulla can be maintained in vitro as the neuronal phenotype when co-cultured with growth factor producing cells and that this strategy may be useful for in vivo transplantation studies.     

Establishment,characterization and fibre outgrowth of isolated bovine adrenal medullary cells in long-term cultures

Medullary cells were enzymatically dissociated from adult bovine adrenal medullae by a perfusion technique using collagenase and maintained in culture for up to 4 weeks. The perfusion technique was a simplified modification of that described by Livett & Co-Workers (Fenwick, Fajdiga, Howe & Livett, 1978) and included cyclical perfusions of cortex-free medullae via the central vein at 37 C for up to 90 min with three 30 ml batches of 0.5% collagenase-containing phosphate buffered saline or Hanks's balanced salt solution and a final trituration step of the minced tissue. The cells were cultured on collagen-coated glass coverslips in modified Rose chambers, on collagen-coated Petri dishes or in Falcon flasks, studied by phase contrast and electron microscopy, catecholamine cytochemistry and assayed for adrenaline and noradrenaline.When grown on collagen about 50% of the catecholamine-storing cells extended short, broad processes as judged by catecholamine cytochemistry using the glyoxylic acid method. These processes rarely grew longer than 60 μm and many of them were seen to retract after 1 week in culture. Process formation was more pronounced and increased with time, when cells were grown on plastic surfaces. Fifteen to 20% of these cells formed long varicose axons after 18 days in culture. Fibre outgrowth from bovine chromaffin cells was also observed, when pieces of medullary tissue were transplanted on to the sympathetically denervated iris in the anterior eye chamber of Nunu mice.Addition of either nerve growth factor (10–1000 ng/ml 2.5S nerve growth factor) or antibodies to nerve growth factor (1.5 μg/ml) did not affect fibre outgrowth in vitro.Ultrastructural examinations revealed that the vast majority of cells in our cultures were typical chromaffin cells. Using the different densities of primary and secondary amine storing granular vesicles as a criterion for distinguishing noradrenaline- and adrenaline-containing cells we found a significant decrease in the number of adrenaline-containing cells with time. By 3 weeks virtually all chromaffin cells contained storage vesicles typical of noradrenaline. Chromaffin storage vesicles appeared to be more densely packed and the amount of rough endoplasmic reticulum was increased in cells treated with nerve growth factor.Results obtained by biochemical analyses indicated that chromaffin cells lost about 50% of their original amine content and stored equal amounts of noradrenaline and adrenaline from day 9 onwards compared to an initial noradrenaline/adrenaline ratio of approximately 1:6. Administration of nerve growth factor did not significantly alter the proportion and levels of adrenaline and noradrenaline in cultured bovine chromaffin cells.This investigation reveals that the capacity of bovine catecholamine-storing cells to form neurite-like processes differs considerably from that which corresponding cells from young rat adrenal medullae exhibit in vitro (Unsicker, Krisch, Otten & Thoenen, 1978 b). Chromaffin cells from adult bovine adrenal medullae kept in culture retain a large number of features typical of differentiated cells over a considerable length of time. Thus they may be profitably used to study long-term effects of drugs and interactions with other cells at morphological and biochemical levels.     

The ret-Activating Ligand GDNF Is Differentiative and Not Mitogenic for Normal and Neoplastic Human Chromaffin Cells In Vitro

Activating mutations of the receptor tyrosine kinase, ret, are associated with multiple endocrine neoplasia type 2A (MEN 2A). However, the mechanisms leading to tumor development are unclear. Glial-derived neurotrophic factor (GDNF) activates wild-type ret via interaction with a second receptor, GFR a-l. We have utilized GDNF to stimulate normal and neoplastic chromaffin cells in order to ask whether ret activation is mitogenic. Cells from three normal adult adrenal medullas, one sporadic pheochromocytoma, and three MEN-2A pheochromocytomas were labeled with bromodeoxyuridine (BrdU) for 12 d in the presence or absence of GDNF or nerve growth factor (NGF), which is known to stimulate neurite outgrowth, but not proliferation in human chromaffin and pheochromocytoma cell cultures. Responses to GDNF and NGF were comparable, except for two MEN-2A pheochromocytomas that responded minimally to GDNF and robustly to NGF. These tumors responded to GDNF biochemically, as measured by phosphorylation of mitogen-activated protein kineses, despite their weak morphological responses. Our findings suggest that activation of ret may not be sufficient to produce chromaffin cell hyperplasia or neoplasia directly by stimulating cell proliferation. However the possibility that altered cell-cell or cell-substrate interactions might cause responses to become differ entiative rather than proliferative in vitro has not been ruled out. We also demonstrate, for the first time, that at least some human pheochromocytomas with an MEN-2A ret mutation respond to a normal ret ligand. This responsiveness could be mediated by a remaining normal ret allele or by other mechanisms.     

Immunocytochemical localization of the intermediate filament protein peripherin in adult mouse adrenal chromaffin cells in culture

Peripherin is the main intermediate filament protein in sympathetic neurons. Immunoreactivity to peripherin was studied in mouse adrenal chromaffin cells after 6 days in culture, and compared to immunoreactivity to tyrosine hydroxylase used as a general marker of chromaffin cells in culture. Most of the cells immunoreactive to tyrosine hydroxylase were rounded, with a glandular phenotype and a few of them had processes. The cells reactive to peripherin only constituted a small proportion of the chromaffin cells (2%), and most of them sent out processes. However, not all the cells with processes were reactive for peripherin. These results did not change in the presence of nerve growth factor. The discussion focuses on the significance of the sub-population of cells reactive to peripherin. We suggest that these cells resemble the small granule chromaffin cells, regarded as an intermediate cell type between glandular cells and neurons. The cells that expressed peripherin here are compared to those selected to form the PC12 clone. The presence of peripherin in only a few of the cells sending out neurite-like processes is discussed in relation to the expression of other neurofilament proteins in developing cells and to the influence of non-chromaffin cells.     

Theret-activating ligand GDNF is differentiative and not mitogenic for normal and neoplastic human chromaffin cells in vitro

Activating mutations of the receptor tyrosine kinase,ret, are associated with multiple endocrine neoplasia type 2A (MEN-2A). However, the mechanisms leading to tumor development are unclear. Glial-derived neurotrophic factor (GDNF) activates wild-typeret via interaction with a second receptor, GFR α-1. We have utilized GDNF to stimulate normal and neoplastic chromaffin cells in order to ask whetherret activation is mitogenic. Cells from three normal adult adrenal medullas, one sporadic pheochromocytoma, and three MEN-2A pheochromocytomas were labeled with bromodeoxyuridine (BrdU) for 12 d in the presence or absence of GDNF or nerve growth factor (NGF), which is known to stimulate neurite outgrowth, but not proliferation in human chromaffin and pheochromocytoma cell cultures. Responses to GDNF and NGF were comparable, except for two MEN-2A pheochromocytomas that responded minimally to GDNF and robustly to NGF. These tumors responded to GDNF biochemically, as measured by phosphorylation of mitogen-activated protein kineses, despite their weak morphological responses. Our findings suggest that activation ofret may not be sufficient to produce chromaffin cell hyperplasia or neoplasia directly by stimulating cell proliferation. However, the possibility that altered cell-cell or cell-substrate interactions might cause responses to become differentiative rather than proliferative in vitro has not been ruled out. We also demonstrate, for the first time, that at least some human pheochromocytomas with an MEN-2Aret mutation respond to a normalret ligand. This responsiveness could be mediated by a remaining normalret allele or by other mechanisms.     

谷氨酸影响星形胶质细胞表达神经生长因子的实验研究

本文利用双向ＥＬＩＳＡ 法定量检测体外培养乳鼠大脑皮质星形胶质细胞在不同浓度的谷氨酸条件培养基中ＮＧＦ的表达分泌量,观察谷氨酸的调节作用,并利用氯胺酮或ＥＧＴＡ 限制谷氨酸ＮＭ ＤＡ 型受体介导的钙离子内流,观察谷氨酸调节作用的改变。结果表明：体外培养的星状胶质细胞２４ ｈ 能表达分泌ＮＧＦ ３．１２３ ｐｇ／１０５ 细胞;１０ μｍ ｏｌ／Ｌ～１ ｍ ｍ ｏｌ／Ｌ的谷氨酸能显著促进ＮＧＦ表达,其中以１００ μｍ ｏｌ／Ｌ的谷氨酸作用为最强,而１０ ｍ ｍ ｏｌ／Ｌ 的谷氨酸反而起抑制作用;限制谷氨酸ＮＭＤＡ 型受体介导的钙离子内流,能消除谷氨酸的这种调节作用。结果提示：谷氨酸可因其浓度不同促进或抑制星形胶质细胞表达ＮＧＦ,这种调节作用可能通过其ＮＭ ＤＡ 受体介导的钙离子内流来实现。     

Controlled release of nerve growth factor from fibrin gel

Nerve growth factor (NGF) is known to promote the axonal regeneration in injured nerve system. Delivery of NGF for a long period in a controlled manner may enhance the regeneration efficacy. In this study, we investigated whether NGF can be released from fibrin gel for a long period in a controlled manner. We also investigated whether sustained delivery of NGF using fibrin gel can enhance the efficacy of NGF in vitro. The addition of heparin to fibrin gel decreased the rate of NGF release from the fibrin gel. As the concentrations of thrombin and fibrinogen in fibrin gel increased, the NGF release rate decreased significantly, and the initial release burst decreased. NGF was released for up to 14 days in vitro. The bioactivity of NGF released from fibrin gel was assessed by morphological changes of pheochromocytoma (PC12) cells cultured in the presence of NGF-containing fibrin gel. NGF released from fibrin gel exhibited significantly higher degrees of PC12 cell viability and differentiation than NGF added in a free form daily into the culture medium. This study demonstrates that fibrin gel can release NGF in a sustained, controlled manner and in a bioactive form.     

Laminin nanofiber meshes that mimic morphological properties and bioactivity of basement membranes

The basement membrane protein, laminin I, has been used broadly as a planar two-dimensional film or in a three-dimensional form as a reconstituted basement membrane gel such as Matrigel to support cellular attachment, growth, and differentiation in vitro. In basement membranes in vivo, laminin exhibits a fibrillar morphology, highlighting the electrospinning process as an ideal method to recreate such fibrous substrates in vitro. Electrospinning was employed to fabricate meshes of murine laminin I nanofibers (LNFs) with fiber size, geometry, and porosity of authentic basement membranes. Purified laminin I was solubilized and electrospun in parametric studies of fiber diameters as a function of polymer solution concentration, collecting distance, and flow rate. Resulting fiber diameters ranged from 90 to 300 nm with mesh morphologies containing beads. Unlike previously described nanofibers (NFs) synthesized from proteins such as collagen, meshes of LNFs retain their structural features when wetted and do not require fixation by chemical crosslinking, which often destroys cell attachment and other biological activity. The LNF meshes maintained their geometry for at least 2 days in culture without chemical crosslinking. PC12 cells extended neurites without nerve growth factor stimulation on LNF substrates. Additionally, LNFs significantly enhance both the rate and quantity of attachment of human adipose stem cells (ASCs) compared to laminin films. ASCs were viable and maintained attachment to LNF meshes in serum-free media for at least 3 days in culture and extended neurite-like processes after 24 h in serum-free media conditions without media additives to induce differentiation. LNF meshes are a novel substrate for cell studies in vitro, whose properties may be an excellent scaffold material for delivering cells in tissue engineering applications in vivo.