大鼠晶状体胚胎发育形态学变化及HIF-1α的表达

Morphological changes and expression of HIF-1α in rat lens during embryonic development

目的:观察大鼠胚胎发育不同时期晶状体的形态变化及缺氧诱导因子-1α(HIF-1α)的表达,探讨HIF-1α在大鼠晶状体胚胎发育过程中的作用。方法:清洁级Wistar孕鼠30只,分为6组,分别为胚胎第10,12,14,16,18,20d,每只孕鼠剖腹随机取2只胚鼠,每只胚鼠的一只眼以平行于视神经矢状轴的方向连续切片,常规HE染色,光镜下观察,采用免疫组织化学方法检测HIF-1α的阳性表达;另一只眼采用实时定量PCR方法检测晶状体组织中HIF-1αmRNA的阳性表达。结果:大鼠胚胎10d(E10),晶状体泡形成;胚胎12d(E12),前壁、后壁细胞分化,前壁细胞发育成上皮细胞;胚胎14d(E14),由后壁细胞发育来的原始纤维形成;胚胎16d(E16),上皮细胞增生活跃,次级纤维形成;胚胎20d(E20),晶状体基本发育成熟。免疫组织化学染色检测到HIF-1α在胚胎期晶状体中高表达,上皮细胞较纤维细胞表达强烈。E10至E16表达量升高,E16达到峰值,生发区、过渡区染色最强。E18之后表达量降低,E20最低。6组比较,P<0.01,差异有统计学意义。组与组间两两比较,除E10与E12之间,E14与E16之间,P>0.05,差异无统计学意义外,余各组间比较,P<0.05,差异有统计学意义。实时定量PCR显示,HIF-1αmRNA在胚胎期晶状体中持续表达,且随着胎龄不同变化,E10至E16,HIF-1αmRNA表达量升高,E16达到峰值,E18、E20下降。6组比较,P<0.01,差异有统计学意义。组与组间两两比较,除E10与E12之间比较,P>0.05,差异无统计学意义外,余各组间比较,P<0.05,差异有统计学意义。结论:在大鼠胚胎10d时,晶状体泡形成,发育开始,至出生前基本成熟。HIF-1α在晶状体胚胎发育过程中的阳性表达呈现时空变化。HIF-1α参与了晶状体的胚胎发育,且在此过程中起重要作用。

AIM： To investigate the morphological changes and the expression of hypoxia-inducible factor-1 alpha （ HIF-1α） subunit during embryonic development of rat lens and explore the role of HIF-1α in lens development process.METHODS： Thirty clean pregnant Wistar rats were divided into 6 embryon groups,10-d, 12-d, 14-d, 16-d, 18-d and 20-d embryo. Two embryons were randomized obtained from every pregnant rat. One of the eyeball samples that were parallel to sagittal axis of optic nerve were cut into serial sections, used HE staining and examined by light microscope. Expression of HIF- 1αprotein in lens was detected by immunohistochemistry. The positive expression of HIF-1α mRNA of the other eyeball samples was detected by real-time PCR. RESULTS=In the 10th d of embryo （El0）, the formation of lens vesicle were recognized under the light microscope. In the 12td of embryo （E12）, the anteriorly situated cells and posteriorly situated cells have already differentiated, The anteriorly situated cells were epithelium. In the 14th d of embryo （E14）, primary fibers which came from posteriorly situated cells were examined. In the 16th d of embryo （E16）, the lens epithelium undergoes extensive proliferation, and enlongate into the secondary fibers. In the 20th d of embryo （ E20 ）, the lens was maturation. By immunohistochemistry staining, the HIF-la was highly expressed in the lens embryonic development. The expression was gradually promoting from El0 to E16, thenreducing. The lens epithelium expressed more HIF-la than fibers. The highest mean density was at E16, the lowest at E20. The difference was significant among of the 6 groups （P〈0. 0001 ）. The El0 group was combined with the E12 group, the E14 group was combined with the E16 group, showing no significant difference （P〉0.05）. The other groups were compared with each other, finding significant difference （P〈0.05）. By the real-time PCR, HIF- la mRNA was highly expressed in the lens development, and was different at different time. The expression of HIF-la mRNA was increasing from El0 to E16, then descending at E18 and E20. The expression of HIF-la mRNA was the highest at E16, the lowest at E20. The difference was significant among of the 6 groups （ P〈 0. 0001 ）. The El0 group was combined with the E12 group, showing no significant difference （P〉0.05）. The other groups were compared with each other, finding significant difference （P〈0.05）. CONCLUSION ：The lens of Wistar rats differentiate from the El0 when the vesicle formed through the embryo phase. The lens is basic mature before birth. The HIF-la in the lens embryonic development is highly expressed and changeful. The varies of HIF-la expression is depend upon rat embryo development indicating that HIF- 1a might participate in the process of development of rat lens.