Longitudinal Muscle Bands Regeneration And Related Gene Expression Analysis In Apostichopus Japonicus

Apostichopus japonicus under adverse environmental conditions, such as water pollution,high water temperature, too dense or certain strong stimulation, will lead to body contraction,cloaca fracture and discharge some or all of the internal organs. This is a self-preservationmeasure against enemy and stress environment of Apostichopus japonicus.The Apostichopusjaponicus have strong ability of regeneration, that won’t die after remove some of the organsfrom the body, and can regenerate complete new organs. Because of its organ regeneration andrepair ability and its important position on evolution on the classification of organisms, study theregeneration mechanism of Apostichopus japonicus has high scientific research value, and hasimportant significance to the higher organisms’ cell proliferation, differentiation and tissue repairmechanism.(1) Using Apostichopus japonicus as test materials to refer to human surgical technique ofsuture, attempt to suture the surgical site, and established a kind of surgical suture method on seacucumber body wall. The results obtained the ideal effect, effectively solved the death problemcaused by the wound which is difficult to heal. Apostichopus japonicus were put into0.54mol/Lmagnesium sulfate to narcotize for1h in dissecting tray, then about a2cm incision along theabdomen distance anal1/3on longitudinal, deep to the body cavity which was cut by sterilizatedscalpel. Then Apostichopus japonicus were put back and well cultivated in the seawater withantibiotics after suture. The results showed that the wounds of experimental group basicly grewtogether at10d, and there was no distinction with normal tissue at20d. In the experimental group,the skin ulceration rate is10%and mortality is only5%. However,in control group(withoutsuture)，the time of wound healing delayed10days with the test group on average. In addition,the skin ulceration rate reaches as high as58.7%and the mortality is51.7%. This method can beapplied to the test study of and breeding production of the sea cucumber.(2) The LMBs at the back of abdominal cavity in sea cucumber, Apostichopas japonicus,were cut through traumatic abdomen with scissors. The surgically operated cucumbers wereraised in the sea water with antibiotics after the wound of abdomen was sutured. The changes ofmorphological and histological during muscle regeneration were studied using tissue slicemethod. There were0.5-1cm gaps around the broken muscle bands because of the contraction ofLMBs at the beginning of surgical operation. Ivory and flocculent tissue, named as muscleprecursor, was observed at the position of trauma at15day. And at30-45day, the muscleprecursor was thickening and made both ends of traumatic muscle connected together. Themuscle precursor was gradually changed into muscle bands with the half thickness of the normallongitudinal muscle at60-90day. At110day, the regenerated LMBs grew further, only with theslightly little diameter compared with the unwounded ones. And at150day, the longitudinal muscle bands had been regenerated completely and there were not any significant differenceswith the normal ones. Histological results showed that the muscle precursor occurred at15d wascomposed of connective tissue and individual muscle fibers. At30-45day, some channelsbetween thickening muscle precursor and the body wall could be observed, and there wereconnective tissue and body cavity epithelial cells in these channels. A large increase in musclefiber numbers could be founded in the connective tissue. At60-90d, the thickening muscleprecursor was mainly composed of muscle fibers, and could be considered as longitudinalmuscle bands, at the same time, the numbers of channel were further increasing. At110d, thenumbers of new muscle fibers increased greatly and the channel number decreased. And at150d,the LMBs had been regenerated completely and the channels were disappeared. It was suggestedthat the new muscle cells would be derived from the connective tissue cells in the body wall andthe body cavity epithelial cells.(3) Using Real time-PCR to analyse the quantitative gene expression of Apostichopusjaponicus EF1α gene, found the EF1α gene mRNA expression in the LMBs after traumatreatment increased significantly in every time point, the EF1α expression level increasedsignificantly in2d, the expression quantity is30493times than that of the control group (0d)(P<0.01). Other time points appear gradually rise trend, the30d reached the maximumexpression, and after40d the expression quantity keep basic stability, we analyse that it may bebecause the growth of muscle enter into steady stage. The expression of SRCR12gene in LMBsof Apostichopus japonicus after trauma treatment, found expression quantity of SRCR12mRNAin2d,20d,30d is significantly higher than0d, respectively46534,6386,58465times higherthan the control group, but in4d and10d expression difference was not significant. Theexpression quantities after40d were significantly higher than that of the control group (0d), andthe trend basically stable. Explain that SRCR12gene in Apostichopus japonicus after traumatreatment has an important role in immune response.This study respectively researched from the organization level and molecular level ofApostichopus japonicus LMBs regeneration process. The conventional paraffin sectiontechnology ensures the integrality of the organizational structure, and can be observed moreclearly and intuitively to Apostichopus japonicus LMBs regeneration process. Using RT-PCRtechnology to research the expression of EF1α gene and SRCR12in Apostichopus japonicusLMBs after trauma treatment, ascertained that they played an important role in immunity anddamage of Apostichopus japonicus, laid an important foundation for understanding the system ofimmune mechanism in Apostichopus japonicus. the results show that probe the molecularmechanism of EF1α gene and SRCR12gene in Apostichopus japonicus LMBs after traumatreatment from the level of gene expression, there are certain rules to follow in different stages ofpost-trauma. This study based on Apostichopus japonicus LMBs regeneration mechanismresearch, be of great significance to explore the molecular mechanism of Apostichopus japonicus after injury and improve the strategy of Apostichopus japonicus immune restoration mechanism.Also provides the theoretical basis for Apostichopus japonicus histopathological study, and laid afoundation for the further study of echinoderms and higher animals (especially humans) musclecell differentiation and tissue repair process mechanism.