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为了检测integrinβ1在出生前后大鼠中脑黑质的表达,并探索其在大鼠黑质神经细胞发育过程中的可能作用,本实验采用RT-PCR、Western blot和免疫组织化学染色方法,来测定胚龄(embryonic day,E)E16d、E18d、E20d的健康SD胎鼠及出生后1d(postnatal day1,P1)、P3d、P7d、P14d、P21d的健康仔鼠中脑黑质组织中integrinβ1的表达情况。RT-PCR和Western blot结果显示,在检测的各时间点,大鼠中脑黑质integrinβ1都有明显表达,且其mRNA和蛋白质水平变化一致。在E16时,integrinβ1mRNA和蛋白质出现高表达,此高表达一直持续到P7时;而至P14时,integrinβ1mRNA和蛋白质表达水平明显降低,且此低水平表达随发育而持续;P21时与P14时相比无明显差异。而免疫组织化学染色结果显示,在各时间点,大鼠中脑黑质一直有大量integrinβ1阳性细胞存在。在E16~P14各时间点,integrinβ1阳性细胞胞体逐渐增大,P21时与P14时相比,阳性细胞胞体大小无明显变化。而单位面积内integrinβ1阳性细胞数,在E16~P7各时间点无明显差异,但至P14时,单位面积内integrinβ1阳性细胞数则明显降低,且此降低随发育而持续,P21时与P14时相比无明显变化。以上结果表明,在大鼠中脑黑质发育过程中,integrinβ1在E16~P7期间持续高表达,而至P14~P21期间,其表达量明显降低。这种表达变化与大鼠发育期间黑质多巴胺(DA)能神经元的自然凋亡时程相一致,提示integrinβ1的表达与此期间黑质DA能神经元的凋亡有关。
In order to detect the expression of integrinβ1 in rat midbrain substantia nigra before and after birth and to explore its possible role in the development of rat substantia nigra neuronal cells, we used RT-PCR, Western blot and immunohistochemical staining The expression of integrinβ1 in substantia nigra of healthy offspring of embryonic day (E) E16d, E18d, E20d and postnatal day1 (P1), P3d, P7d, P14d, P21d . The results of RT-PCR and Western blot showed that at each time point, integrinβ1 in midbrain substantia nigra was obviously expressed and the mRNA and protein levels were consistent. At E16, the high expression of integrinβ1 mRNA and protein, which continued until P7; and by the time of P14, the level of integrinβ1 mRNA and protein expression was significantly reduced, and this low level of expression continued with the development; P21 when compared with P14 No significant difference. The results of immunohistochemical staining showed that at each time point, a large number of integrinβ1-positive cells existed in the substantia nigra of midbrain of rats. At each time point from E16 to P14, the integrinβ1-positive cell bodies increased gradually. Compared with P14, the cell bodies of positive cells did not change significantly at P21. However, the number of integrinβ1-positive cells per unit area had no significant difference at each time point of E16-P7, but by the time of P14, the number of integrinβ1-positive cells per unit area was significantly decreased, and this decrease persisted with development. No significant change than no change. The above results show that in the process of rat midbrain substantia nigra development, integrinβ1 continuously high expression during E16 ~ P7, and from P14 to P21, its expression was significantly reduced. This change in expression was consistent with the natural apoptotic process of dopaminergic neurons in substantia nigra during the development of rat, suggesting that the expression of integrinβ1 may be related to the apoptosis of dopaminergic neurons in substantia nigra during this period.