The Effect Of Citral On The Temporal-Spatial Expression Of BAMBI In Chicken Embryos Lip Tissue

ObjectiveTo evoke the chicken embryos cleft lip locally by implanting the bead carried citral into the right side of nasal pit ectodermal tissue of the normal development of stage 20 chicken embryos, and then detect the influence of citral on temporal-spatial expression of BAMBI, to explore and illustrate the role of BAMBI in the process of the formation of chicken embryo lip development and lip tissue defect, which lays a foundation for further exploring the pathogenesis of human lip development and cleft lip, and provides a theoretical basis for the early discovery and early treatment of human cleft lip.Methods1. The establishment of a stable model of chick embryo cleft lipThrough the preliminary exploration of the subject group, we have already established a stable model of chick embryo cleft lip with the best concentration of citral 0.6g/ml to induce malformation. The study also uses the citral concentration of 0.6g/ml, the bead carried the best concentration of citral was implanted into the right side of nasal pit ectodermal tissue of the normal development of stage 20 chicken embryos to copy the stable model of chick embryo cleft lip.2. Digoxigenin labeled RNA probe preparation of BAMBI geneWe picked out the chicken embryos at stage 24, and then extracted the total RNA, Got the BAMBI cDNA with the RT-PCR method. The recovery of BAMBI cDNA was inserted into PGEM-T vector, then we obtained recombinant plasmid PGEM-T carrying BAMBI. The recombinant plasmid was linearized with restriction endonuclease. With the SP6 and T7 promoter of the plasmid, the digoxingenin-labeled antisense and sense probes of BAMBI were synthesized by in vitro transcription under the action of T7 RNA polymerase and SP6 RNA polymerase respectively.3. Detection the influence of citral on temporal-spatial expression of BAMBI in chick lip tissue with whole-mount in situ hybridization180 normal chicken embryos at stage 20 were randomly divided into blank control group, DMSO negative control group, antisense probe experiment group and sense probe experiment group, each group was allotted 40 chicken embryos. Blank control group was the normal development of embryo period with any treatment, DMSO negative control group was implanted the bead carring DMSO, antisense probe experiment group was implanted the bead carring the best concentration of citral, sense probe experiment group was also implanted the bead carring the best concentration of citral.The above-mentioned group after treatment, the chicken embryos were continued the incubation until stage21,22,23,24,25,26,27,28. And then each stage collected of 5 specimens. The influence of citral on temporal-spatial expression of BAMBI in chick lip tissue was detected with whole-mount in situ hybridization. Among them, blank control group, DMSO negative control group and antisense probe experiment group was detected by antisense probe of BAMBI, the sense probe experiment group was detected by sense probe of BAMBI.Results1. The establishment of stable model of chick embryo cleft lip results: according to the preliminary exploration of the research group, using the citral concentration of 0.6g/ml, we have copied the stable animal model of chick embryo cleft lip successfully.2. The preparation of RNA probe of chick embryo BAMBI gene results: the recombinant plasmid BAMBI/PGEM-T was successfully constructed, and the sense and antisense RNA probe were obtained by in vitro transcription. The RNA probe was as long as 858bp, the sense probemRNA was the same as the mRNA sequences of BAMBI, the base sequence of antisense probe and the mRNA base sequence of BAMBI was complementary, with digoxigenin labeled.3. Whole mount in situ hybridization results:changes on the temporal-spatial expression of BAMBI in chick lip tissue was detected successfully in blank control group, DMSO negative control group and antisense probe experiment group, the sense probe experiment group failed to detect.Among them, the expression of BAMBI was found in frontonasal prominence, lateral nasal process, maxillary process and mandibular process. BAMBI expression level was low of the antisense probe group during the same period compared with the blank group, DMSO group. And BAMBI expression level of treated side was lower than the untreated side.ConclusionsThis study shows that the use of citral can induce the chicken embryo lip 属性不符...