维甲酸对先天性马蹄内翻足相关致病基因表达影响的研究

Affection of retinoic acid on the expression of pathogenic genes related to congenital clubfoot

目的研究维甲酸对于先天性马蹄内翻足相关基因表达的影响。方法选取的SD大鼠交配后，分为对照组和实验组。对照组随机选取孕SD大鼠2只，于怀孕10d时以橄榄油灌胃一次。实验组选取孕SD大鼠6只，于怀孕10d时以140mg／kg的维甲酸剂量（40mg／ml溶于橄榄油中）灌胃一次。于孕16～18d待孕鼠分娩。对照组孕鼠所产幼鼠为对照组，实验组孕鼠所产幼鼠根据是否出现后肢畸形分为畸形组及非畸形组。取两组新生幼鼠后肢软骨及肌肉组织，应用RT-PCR方法测定组织中HoxA9、TBX3、CASP10基因表达情况，并与对照组正常新生幼鼠比较。结果2只对照组SD孕大鼠共产仔22只，未发现畸形或死胎。6只实验组SD孕大鼠中有一只在灌胃后2d死亡，余下可利用5只孕鼠共产仔49只，其中死胎6只，无肛无尾畸形1只，后肢出现类马蹄内翻足畸形的共30只，未出现下肢畸形共19只。畸形出现率为61．2％（30／49），并以此将实验组分为畸形组与非畸形组。荧光RT-PCR结果：实验组中畸形组在HoxA9及TBX-3基因的平均拷贝数分别为（7．00±0．52、4．55±0．52）明显低于对照组（7．92±0．47、4．99±1．02）和非畸形组（8．07±0．28、4．94±0．24），P〈0．001，P〈0．05；而在CASP10基因的平均拷贝数4．34±0．35则明显大于对照组3．51±0．42和非畸形组3．29±0．50（P〈0．001）。而对照组与非畸形组在各个基因的拷贝数比较上则无明显差异（P〉0．05）。结论维甲酸能成功诱导出SD大鼠新生幼鼠类马蹄内翻足畸形的模型；维甲酸对于SD大鼠的TBX-3、HoxA9、CASP10基因的表达均有影响；维甲酸可能与通过影响TBX-3、HoxA9、CASP10基因的表达导致SD新生大鼠产生类马蹄内翻足样畸形。

Objective To investigate the affection of retinoic acid （RA） on the expression of the genes related to congenital club foot. Methods Six SD rats were fed with RA （140 mg/kg） olive oil once on 10th day of pregnant by intragastric-administration, two rats in control group were fed with olive oil. The offsprings of the experimental group were divided into deformity group and nowdeformity group according to the deformity on hind limbs, the copy numbers of mRNA of HoxA9, TBX 3, CASP10 in the offspring were detected by real-time fluorescent quantitative RT PCR. Results There were totally 22 offspring from control group without deformities or stillbirths. One of the experimental pregnancy rats died after the intragastric-administration. The remaining five pregnancy rats delivered 49 newborn,including six fetal death and one anorectal malformation without tail, and the number of analogical clubfoot deformity of hind limbs was 30. The average gene copy number of HoxA9 and TBX-3 in the deformity group （7. 00 ±0. 52 and 4. 55 ± 0. 52） was significantly lower than those of control group （7.92± 0.47 and 4. 99±1.02） and non deformity group （8.07± 0.28 and 4. 94 ± 0. 24）. And the average copy number of CASP10 in the deformity group was significantly greater than that in control group and nowdeformity group （4. 34 ± 0. 35 vs. 3. 51 ± 0. 42 and 3.29 ± 0. 50,P〈0. 001）. Meanwhile, there was no significant difference of the average gene copy number between the control group and nowdeformity group （P〈0. 05）. Conclusions Retinoic acid can successfully induce analogical clubfoot deformity in SD neonatal rat. RA may lead to analogical clubfoot deformity in SD newborn rat by affecting the expression of TBX 3, HoxA9, CASP10.