Study On Primary Culture And Identification Of Pheretima Aspergillum Muscle Cell

Objective:Considering the main medicinal parts of earthworm is the body wall muscle tissue, this study attempts to establish the muscle cell primary culture system of Pheretima aspergillum. In order to get a quantity, high consistency and the stable genetic material of sterile cell group. Finally it can lay the foundation for muscle cell line establishment of Pheretima aspergillum, in order to provide the material to achieve the following studies at the cellular and molecular level, as well as to provide referential methods for similar animal cell culture of invertebrates.Method:1.The sample collection and identification of Pheretima aspergillumCollecting the living samples random in the wild, and identify. Choosing the strong and viable one, which have obvious reproductive bandand, and the average wet weight is about 5-8 g each healthy earthworm.2.Screening the dissociation method of Pheretima aspergillum muscle tissueTissue cultivation and enzyme digestion are the most basic method of primary culture. By comparing the two methods of muscle tissue dissociation for Pheretima aspergillu. Screening the one which can gain the large number and activity muscle cells. Through the two factors completely random design experiment method, screening optimum digestion conditions. Cell counting with trypan blue dying and CCK-8 were used to evaluate the growth of cells and activity. The obtained data are processed according to the statistical methods.3.The screening and optimization of muscle cell culture conditions of Pheretima aspergillumAccording to the preliminary experiment, we select the influencing key factors of the muscle cell cultures. By screening the conditions of muscle cell culture, such as cell concentration, serum concentration, basal medium type and cell growth facters, which would influence its in-vitro culture, and then optimized this conditions. Selecting the most appropriate growth conditions for cell culture. Through drawing the growth curve and CCK-8 test is to compare and evaluate the cell activity. The obtained data are processed according to the statistical methods.4. The identification of Pheretima aspergillum muscle cellBy using transmission electron microscopy (sem) method, with the help of ultrastru-crural identification of the muscle tissue and cultured muscle cells, to prove that the cells derived from the Pheretima aspergillum muscle tissue.Result:1.Identified, all the samples we collected in the wild are Megascolecidae Pheretima aspergillum(E.Perrier).2. With the living cells count method and cell counting kit-8, the best dissociation method has been chosen for the muscle tissue of Pheretima aspergillum, that is enzyme digestion method. While the enzyme type is Collagenase I, the enzyme concentration is 1 mg/mL, the time of enzyme digestion is 24 h. In this conditions, we can get the most cells as well as the most activity.3.With the living cells counting and the state of muscle cell culture, the best cell concentration of earthworm is about 2×105 per/mL, Grace’s Insect Cell Culture Medium as the basic medium, adding the serum concentration of 15% is relatively appropriate. Compared with free cell growth factor-EGF adding, cell proliferation of the sample with EGF had no significant difference.4. By transmission electron microscopy (sem) identification, cell ultrastructure showed that the nuclei is long fusiform, located at the edge or near the end of the muscle cells.The main organelles are mitochondria and sarcoplasmic reticulum vesicles around the muscle cells cytoplasm.There are two types of the muscle cell, circular muscle and longitudinal muscle cells. The culturing muscle cells and muscle tissue of Pheretima aspergillum has the same structure features, thus it proves that the cells derived from muscle tissue of Pheretima aspergillum(E.Perrier). Conelusion:The study established a set of muscle cell primary culture methods of Pheretima aspergillum (E.Perrier), as well as identified the source of the muscle cell. It can get the large number of cells and the better activity through this method. While the cell proliferation is slow, can live for 30 days. The results have important sense to the establishment of the muscle cell line. Becase a cell line not only can provide the necessary carrier for active substances of earthworm for the future of gene function component analysis, gene expression regulation mechanism and the related research of metabolic activity regularity, but also provide samples for a large scale and long time study in laboratory, to some extent, can avoid difficulties of the experiment material sources. In addition, the research of muscle cell proliferation and the cell lines establishment remains to be further exploration and practice.