Study On Building A Cell Primary Culture System Of Pheretima Aspergillum

Speciality:Ch i nese MedicineAuthor:L i n XiaohuaTutor:Prof. Li WeiPheretima aspergillum (E.Perrier), collected as one of the four original animals under the category of Lumbricus in Pharmacopoeiao of People’s Repubilc of China 2010, is a member of the family Megascolecidae, Pheretima Division. Mainly originating in Guangdong Province and Guangxi Province, it is usually called Lumbricus Kwang. On account of the elaborate processing and exact curative effect of Lumbricus Kwang, it is commonly recognized as with the best quality. Lumbricus is salty in flavor and cooling in nature. Lumbricus acts on the meridians of the liver, the spleen and the bladder. It helps clear heat and calm endogenous wind, unclog meridians, relieve cough and asthma, and can be used as diuretics. The medicine is also used in the treatment of the following diseases: acute hot diseases and epilepsy, Qi deficiency and stagnancy of Qi and blood, hemiplegia, asthma caused by heat of the lung, dysuria, and numbness caused by heat.At present, Lumbricus Kwang and other earthworm medicines, mostly from wild resources, are of quite unstable quality on account of different environmental conditions, ways of collecting and technology of processing. The quality problem particularly manifests in the heavy metal excess, which is still difficult to control. According to pertinent reports these years, with the unique body structure and habits of life, Lumbricus shows the feature of heavy metal enrichment of kinetics.Previous researches of this subject showed that Pheretima aspergillum are strongly tolerant to heavy metals and of heavy metal enrichment, however, what both the mechanisms are still remains unknown. In order to study the mechanism of enrichment at a molecular level, a cell culture system of Pheretima aspergillum has to be built in the first place. After that, through experimental induction of gene expression of Lumbricus cells in vitro under circumstances of different heavy metals, gene transcription and expression can be tested to identify which specific gene tends to be of heavy metal enrichment. Consequently, the mechanism of heavy metal enrichment can be explained at cellular and molecular levels. To the end, this study is to build a cell primary culture system of Pheretima aspergillum in order to obtain considerable sterile cell groups of high consistency, which is also an essential support for realizing the gene study at cellular and molecular levels. The project is also for further studying the mechanism of heavy metal enrichment of Pheretima aspergillum, and providing experimental evidence for establishing strategies to lower or eliminate heavy metal excess in Lumbricus Kwang. All the above work are of great importance for uprooting problems of heavy metal excess in Lumbricus medicine, promoting sustainable development of Chinese medicine, as well as assuring the safety of its clinical use.To better accomplish the project, we have mainly done the following work.1. Literature ReviewReferring to amounts of pertinent domestic and foreign literature, we have systematically summarized both the study achievement and problems of our predecessors. Development of ancient studies nowadays, conditions of contemporary studies and technology of animal cell culture have been specifically discussed, on which this study objects and contents are based.2. The Study on Sterilization Method of Cell Primary Culture of Pheretima aspergillumThrough the examination on the disinfection method of cell primary culture of Pheretima aspergillum, the best method has been selected and a primary cell experimental culture system has been established. With the L9(34) Orthogonal Test, the disinfection effects on the cells caused by compound antibiotic solution of different concentration has been examined and compared. It came out with a solution group of the best disinfection effect:Benzylpenicillin Sodium 200 U/mL+Streptomycin Sulfate 400μg/mL+ Gentamycin Sulfate 300 U/mL+Amphotericin B 5μg/mL. After a 10-day use of the solution, the cells got no contamination, remained active and adherent to cell walls. It proved that compound disinfection was the best, as well as simple and convenient, method to culture primary cell of Pheretima aspergillum.3. The Study on the Method of Dissociating Somatic Cells of Pheretima aspergillum In order to select the best method of cell dissociation, different approaches, such as mechanical methods (including methods of explanting and homogenizing), enzymolysis method, were examined. Effects of different approaches were compared, the best one from which were also optimized. It turned out that the enzymolysis method was much more effective than the mechanical methods. When the hydrolysis temperature reached 25℃, with the use of enzyme solution:Trypsin 1g/L+CollagenaseⅠ1g/L, hydrolyzing for 30min, the largest numbers of active cells were obtained. After being cultured for 10 days, the cells remained adherent to cell walls. It proved that using the method of enzymolysis method to dissociate explants of Pheretima aspergillum, sufficient cells of high purity and strong vitality could be obtained, which has laid a solid foundation for building a cell primary culture system of Pheretima aspergillum.4. Comparing the Cell Culture in Different Parts of Pheretima aspergillumConditions of adherence to cell walls in different parts of Pheretima aspergillum were compared so as to select the cells of faster proliferation and stronger vitality. In order to better identify cells in different parts, the morphology of crop cells, prostate cells, hemocytes, yellow cells, intestinal cells and integumentary cells were all observed. Through the comparison of how well the crop cells, prostate cells, intestinal cells and integumentary cells adhered to cell walls. It proved that there was no sign of growth in crop cells and prostate cells. Nevertheless, the number of intestinal cells and ingumentary cells both grew, with an apparent sign that cells from the intestines grew much faster and adhered more easily to cell walls. When the inoculation density reached 2×105 cell/mL, the cells began to adhere to cell walls 7 days later. On account of this outcome, we not only shifted our focus on the optimization of culturing intestinal cells, but also became much more determined to accomplish the study on the mechanism of heavy metal enrichment in Pheretima aspergillum. During some pre-related tests, we’ve ever found out that the intestinal wall was the main part where heavy metals were absorbed and where metallothionein expressed. There’s no doubt that the successful culture of intestinal cells has provided significant support for the study objects.5. Identifying and Optimizing Conditions of Intestinal Epithelial Cell Culture of Pheretima aspergillumDuring the study, how the conditions, such as cell concentration, serum concentration, basal medium type and culture temperature, of intestinal epithelial cell culture, would influence its in-vitro culture were systematically examined. Furthermore, conditions of in-vitro culture were optimized, and the origins of cells were also that the best inoculation density of intestinal epithelial cells was:2×1cell/mL or 5×105 cell/mL. It was more suitable to apply the Schneider’s Drosophila Medium, adding serum with the concentration of 13% when culturing. Compared to 37℃, temperature of 25℃was much more suitable for the growth of Pheretima aspergillum cells. At the end, by using the transmission electron microscopy, intestinal epithelial cells of Pheretima aspergillum were observed and identified. This test achieved the aim to optimize the culture conditions of intestinal epithelial cells, which has laid a foundation for building cell models in vitro and studying the mechanism of heavy metal enrichment.6. Study on Cd2+ Tolerance of Pheretima aspergillum Epithelial CellsThrough comparing how well the Pheretima aspergillum epithelial cells survived in different concentration of Cd2+, Cd2+ tolerance of these cells have been preliminarily studied. The results showed that the cells remained adherent to cell walls when the Cd2+ concentration reached 100mg/L. It suggested that Pheretima aspergillum epithelial cells were more tolerant towards heavy metals than the living bodies. That’s to say, heavy-metal-tolerant gene expression of the epithelial cells were more active, which also provided an experimental basis for the further study on MT gene transcription and regulation using the intestinal epithelial cells.7. Conclusions and ProspectsIn this paper, the cell primary culture of Pheretima aspergillum has been systematically studied; methods of disinfection, methods of cell dissociation, culture sites, culture conditons were all examined in depth; both the cell primary culture system and models of cells in vitro have basically been built, which laid a foundation for further testing and confirming how heavy metal enrichment of specific genes would transcript and express. On account of the above work, the mechanism of heavy metal enrichment could be explained at cellular and molecular levels. At the same time, heavy metal tolerance of Pheretima aspergillum epithelial cells to was also basically studied, which proved that the cells were more tolerant to Cd2+at the cellular level. Hereafter, the mechanism of heavy metal enrichment of Pheretima aspergillum, and the mechanism of how MT Protein transcripts and regulates will be further discussed and studied.