Primary Culture Of Chicken Myocardial Cells And Study Of The Effect Of MnSO₄ On The Expression And Regulation Of MnSOD Gene In Broiler Chicken Cadiocytes

MnSOD which is an important anti-oxidant in animals'body can catalyse toxical O2- ? generated by oxidative process into O2 and H2O2 so as to prevent many kinds relative diseases. MnSOD has an effect to resist oxidative stress and tumor. Broiler chickens grow fast and their metabolism is prosperity. More O2- ? were generated in metabolic process especially in those high metabolism organs like heart. MnSOD which is an important O2- ? scavenger plays a key point in the anti-oxidative stress system of broiler chickens. Mn which is a part of MnSOD plays an important role in the process of expression and regulation of MnSOD gene. The purpose of this research was to investigate the effect of MnSO4 on the expression and regulation of MnSOD gene in chicken cadiocytes. This paper has three parts: (1) primary culture of broiler chicken myocardial cells; (2) study on the effect of MnSO4 on the MnSOD activity in chicken cadiocytes; (3) study about the effect of MnSO4 on MnSOD mRNA in chicken cadiocytes.1 The cardiac muscle cell of broiler chickens was cultured by explant, enzyme-dispersed and differential attachment. Cultured cells were identified by immunocytochemical stain. Trypsin and collagenase were used to isolate cardiac muscle cells from cardiac muscles tissues. Cell survival rate was observed to find which enzyme was better for dispersing of cardiac muscles tissue. Cardiac muscles tissues were dispersed by 0.12% collagenase for 5,10,15min to determine which was the best one. Cadiocytes were culture in three kinds of medium: MDM,DMEM/F12,DMEM to find which was the best medium for cardiac muscle cell of broiler chickens. The result showed that the method of explant was convenient and simple but cardiac muscle cells harvest cycle was long and the purity of cadiocytes was relative lower. Meanwhile the way of enzyme-dispersed was complicated but the harvest cycle was short and the purity is relative high. A high purity of cadiocytes of broiler chickens was abstained by differential attachment and adding Brdu to the medium to suppressed fibroblastic cells. The result of immunocytochemical stain was indicated cultured cells were cardiac muscle cells. Collagenase was better than trypsinase for isolation of cadiocytes of broiler chickens and 0.12% collagenaseⅠto digest cardiac muscle at 37℃for 10min every time was a better way to isolate cadiocytes. MDM and DMEM/F12 were more suitable than DMEM for culturing cardiac muscle cells of broiler chickens.2 Broiler chickens which were fed Mn-unsupplemented corn-soybean meal basal diets to enhance the sensitivity to manganese at 1~7 d were killed to culture cardiac muscle cells. Cells were cultured 5~7d. When they were confluensed together, the culture medium was changed into the medium contained different concentration MnSO4(0.1,0.5,1.0,2.0mM/L) meanwhile the comparison group contained no MnSO4. Cells were harvested at 6,12,24,48h. MnSOD activity of cardiac muscle cells of broiler chickens were measured by MnSOD test box. The result showed that the MnSOD activity of 0.1,0.5,1.0mM/L had no significant difference with the comparison group but the MnSOD activity of 2mM/L was significant difference from other groups. The MnSOD activity of cardiac muscle cells of broiler chickens was little change during 6~24h after adding MnSO4 but its activity was significant increasing at 48h. Those data showed that MnSOD activity of cardiac muscle cells which treated by 2.0mM/L MnSO4 for 48h was increased most.3 Broiler chickens which were fed Mn-unsupplemented corn-soybean meal basal diets to enhance the sensitivity to manganese at 1~7 d were killed to culture cardiac muscle cells. Cells were cultured 5~7d. When they were confluensed together, the culture medium was changed into the medium contained different concentration MnSO4(0.1,0.5,1.0,2.0mM/L) meanwhile the comparison group contained no MnSO4. Total RNA was extracted from those cells at 6,12,24,48h after the medium was changed. And then the total RNA was purified. After that MnSOD mRNA was quantitated by fluorescent quantitation PCR. The result indicated that MnSOD mRNA level of 6,12,48h had no significant difference with each other but the MnSOD mRNA level of 24h which was significant higher than other groups was the highest group. The MnSOD mRNA of each group supplied with different concentration MnSO4 was higher than contrast group but there was no significant difference.