高温致神经管畸形相关基因Cx43的克隆及其致畸相关性研究

Objective To screen connexin 43 gene from the cDNA library of neural tube of golden hamster treated with hyperthermia. Discuss the relationship between connexin 43 gene and the NTDs caused by hyperthermia. Methods Based on the PCR method, 96-hole cell culture plate was used to screening the cDNA library of neural tube of golden hamster treated with hyperthermia. Using a pair of Cx43 specific primers,the positive hole was located using PCR. After selective amplification the positive plaques were identified. Then positive plaques were converted to plasmids, enzyme identified, and the sequence was analyzed to confirm the result. The cDNA fragment was extracted and labeled by DIG with random priming method. Then the probes were used to hybrid with total RNA of neural tube of heat-treated groups or control groups at E8.5d to affirm its abnormal expression in hyperthermia induced NTDs. Results The positive clone containing Cx43 cDNA fragment was obtained after five screenings. Results showed that the length of the cDNA insert was 1304bp and its sequence had the same homology with the CX43 of golden hamster in the gene bank. Northern blot showed that its expression in heat-treated groups was evidently higher than control groups. Conclusion Hyperthermia could not induce any mutations in Cx43 cDNA. The increasing expression of Cx43 is intimate with neural tube defects induced by hyperthermia.