目的:构建Ⅰ型钠通道(Nav1.1)与绿色荧光蛋白(GFP)融合表达载体及其突变载体。方法:利用In-Fusion技术将SCN1A基因亚克隆到绿色荧光蛋白真核细胞融合表达载体(p Ac GFP1-C In-Fusion Ready Linear Vector)。PCR扩增SCN1A基因(与线性载体对应两端有15个相同碱基),In-Fusion技术进行融合即得到p CMV-GFP-C-SCN1A。将其转染HEK293T细胞,Western blot检测Nav1.1的表达。定点诱变试剂盒对其进行定点诱变。结果:1.成功构建Nav1.1与GFP融合表达载体p CMV-GFP-C-SCN1A;2.DNA测序表明:在预期位点已经发生突变,SCN1A基因第190位色氨酸密码子(TGG)突变为终止密码子(TGA)。结论:Nav1.1与GFP融合表达载体及其突变载体的构建成功,为进一步研究该突变位点导致Nav1.1功能的改变奠定了基础。
AIM:To explore the correlation between the retinal nerve fiber layer(RNFL) thickness by using optical coherence tomography(OCT) and by histological measurements in normal adult rats and optic nerve transected rats.·METHODS:The RNFL thickness of 36 rats was scanned in a circle 3.46mm far from the optic disc by OCT.The two experimental groups were the normal group(n =20 rats) and the optic nerve transected group(n =16 rats).The latter group included 4 groups(n =4 /group)surviving for 1 day,3,5 and 7 days.Then the RNFL thickness of the same retina area was also measured by NF-200 immunohistochemical staining method.Linear regression was used to analyze the correlation between the data obtained from these two methods.·RESULTS:The RNFL thickness of normal right eyes around optic disc by OCT was 72.35 ±5.71μm and that ofthe left eyes was 72.65 ±5.88μm(P =0.074).The RNFL thickness of the corresponding histological section by immunohistochemistry was 37.54 ±4.05μm(right eyes)and 37.38 ±4.23μm(left eyes)(P =0.059).There was a good correlation between the RNFL thickness measured by OCT and that measured by histology(R2=0.8131).After optic nerve transection,the trend of the RNFL thickness was thinner with the prolonged survival time.The correlation of the thickness detected by the above two methods was approximately(R2=0.8265).Value of the RNFL thickness in rats around optic disc measured by OCT was obviously higher than that measured by common histological measurement in normal adult rats and optic nerve transected rats.·CONCLUSION:The RNFL thickness measured by OCT has a strong correlation with that measured by histological method.Through OCT scanning,we found that the thickness of RNFL gradually becomes thinner in a time-dependent manner.
