The Research On Component, Function And In Vitro Culture Of Hemocytes From The Mitten Crab, Eriochier Sinensis

As an undertaker in cellular immunity and a provider in humoral immunity,hemocytes play important roles in crustacean immune defense. Different types ofhemocytes are responsible for various denfense mechanisms including phagocytosis,encapsulation, melanization, cytotoxicity, cell-cell communication and activation ofproPO system. Chinese mitten crab, Eriocheir sinensis, is an economically importantfreshwater species for aquaculture in China and was chosen as our experimental subject.To investigate the hemocyte components, classification and role in innate immunity, westudied the morphology and enzyme content of hemocytes, together with the change ofTHC, DHC during the infection of Aeromonas hydrophilia; To investigate the optimalculture conditions about medium, serum, pH, temperature and Osmolarity, a primaryhemocytes culture was performed in vitro; We developed the survival ofsemigranulocytes and hyalinocytes up to15d by separating different type of hemocytes.Our work may lay a foundation for the further researches in the cytogenesis, mechanismof innate immunity and establishment of a cell line. The results are listed below:1. Four types of hemocytes, i.e., granulocytes, intermediate granulocytes,semigranuloctyes and hyalinocytes could be distinguished, according to cellmorphology, cytochemical assays and cytoenzymatic assays. granulocytes, with thegreatest size, roundish or spindle shape and containing acidophilic granules same asintermediate granulocytes; semigranulocytes, with a greatest amount, usually withspindle morphology and containing a large number of basophilic granules; hyalinocyteshave the smallest size and lack any granules inside the cytoplasm. Only granulocytesshowed positivity to four enzymes. We found THC various during the infection ofAeromonas hydrophilia, it peaked after6hours and recoverd gradually. The dectectionof DHC in injection group showed that the frequence of hyalinocytes increasedsignificantly in3hous while semigranulocytes maintained a stable amount. Other twotypes of hemocytes decreased in different degree. Granulocytes decreasd dramaticallyand got the lowest frequence at12h. We confirmed that granulocytes have highenzymatic activitiy and hyalinocytes represent strong anti-bacteria action.2. The effects of several frequently-used cell culture media (L-15,2×L-15,3×L-15, M199and RMPI-1640) on cell morphology and viability of hemocytes from the mittencrab，Eriocheir sinensis were studied, in order to screen an optimum culture medium forcrab hemocytes. Furthermore different proportions of fetal bovine serum (FBS)(0%,5%,10%, and15%) as a supplement were added into anoptimum culture medium toresearch the effects of serum on hemocyte culture. The results showed that:3×L-15medium could support the best survival of hemocytes in in vitro culture. The hemocytesremained a maximum number and intact morphology relatively，and the viability ofhemocytes after96h culture was more than60%in3×L-15medium. On the contrary，the other media did not support well of the hemocyte culture, with the viability decreaseto lower than50%and obvious cell morphology changes within12h. In addition，thesupplement of FBS had adverse effect on cell morphology and viability. These resultsshow that3×L-15medium without FBS is adequate for E.sinensis primary hemocyteculture.3. The effect of different pH (6.4,6.8,7.2,7.6and8.0), temperature (26,28and30℃) and osmolarity (500,700,900,1100and1300mOsM kg-1) on the morphologyand survival rate of the hemocytes were studied to standardized an optimal method. Wealso compared the effect of different medium (L-15and3×L-15) with osmolarity in1100and1300mOsM kg-1respectively on hemocytes culture. Furthermore, differenttype of hemocytes were separated successfully by a percoll step gradient centrifugationand cell morphology and survival rate of three types of hemocytes in the optimalconditions were observed and analyzed respectively. Our results showed that3×L-15medium,1100mOsM kg-1, pH6.8, and28℃were the optimal conditions for primaryhemocytes culture. Hemocytes at these conditions retained a better morphology andhigher survival rate: hemocytes remained a survival rate＞60%after5days and＞40%after7days. Three cell fractions enriched hyalinocytes, semigranulocytes andgranulocytes respectively were separated preliminarily. Single cultured hyalinocytes andsemigranlocytes could maintain survival rate＞50%after15days but granulocytescould only remain a survival rate about26%after5days.