عنوان المقالة:تخليق/توليد خطوط الخلايا العصبية الخالدة/المستمدة من تجمع الخلايا العصبية بالنواة المقوسة والمنطقة القبل بصرية الوسطى في الأبقار. Generation of Immortalized Neuronal Cell Lines Derived from Bovine Hypothalamic Arcuate Nucleus and Medial Preoptic Area.
فوكو ماتسودا - يوتا سيتومي - أحمد سعد أحمد حسانين - ماساهيرو كاتو - شوتشي ماتسوياما - كوجي كيمورا - ساتوشي أوكرا
المؤلفون بالإنجليزي
FFuko Matsuda, Yuta Suetomi, Ahmed S. Hassaneen, Masahiro Kato, Shuichi Matsuyama, Koji Kimura, Satoshi Ohkurar .
الملخص الانجليزي
Neurons essential for reproductive regulation, such as GnRH and kisspeptin neurons, are mainly located in the hypothalamic arcuate
nucleus (ARC) or medial preopticarea (mPOA) in mammals. However, the precise functions of these neurons are not fully examinedat a
cellular level, especially in domestic animals including cattle. Therefore, hypothalamic neurosecretory cell lines for analyzing cellular
mechanisms that control domestic animal reproduction are required. In this study, we tried to generate neuronal cell lines derived from either
the ARC or mPOA of cattle. A hypothalamus was obtained from a 6-month-old Japanese Black steer. The brain was obtained from thesteer
under deep anesthesia, and then the ARC and mPOA tissues were dissected out. Each tissue was dispersed using papain and cultured on polyL-lysine-coated plates with Neurobasal-A medium containing fetal bovine serum, B-27 supplement, L-glutamine and basic fibroblast growth
factor. After 24 hours from the start of culture, cells were washed with phosphate buffered saline and added new medium and cultured for
additional 24 hours. Then lentiviral vector containing SV40 large T antigen and neomycin resistance gene was added to the primary cultured
hypothalamic cells. Seventy-two hours after the infection, culture medium was changed and the next day G418 was added to selectcells in
which the lentiviral genes were inserted. After the 2 weeks selection by G418, we performed cell cloning of the obtained cell population. Next,
we examined gene expressions ofthe cell clones by RT-PCR. Total RNA was extracted and then cDNA was synthesized from each cellclone,
and expression of neuronal (neuron specific enolase, NSE) or glial (glial fibrillary acidic protein, GFAP) markers was evaluated by PCR. From
the expression pattern of these markers, we determined neuron-derived cell clones. Subsets of the bovine ARC and mPOA tissues were also
provided for RT-PCR analyses as positive controls for NSE and GFAP expressions. After the infection of lentivirus, primary cultured bovine
hypothalamic cells acquired strong proliferative activity, suggesting that these cells were immortalized by insertion of SV40 large T antigen.
We obtained more than 50 cell clones from both ARC- and mPOA-derived immortalized cell populations. By RT-PCR analyses, we confirmed
that both NSE and GFAP were expressed inbovine ARC and mPOA tissues. As for immortalized cell clones, wefound NSE-positive and
GFAP-negative cell clones, which were defined as neuron-derived cell lines. In summary, we succeeded to obtain a number of neuronal cell
lines derived from bovine ARC and mPOA. Further expressional analysis including neuropeptides such as GnRH, kisspeptin, neurokinin B and
dynorphin A will determine neuronal cell lines appropriate for examining central mechanism regulating ruminant reproduction in vitro.
أحمد سعد أحمد حسانين | Ahmed Saad Ahmed Hassaneen | 7158