Study On Supplemented Jia Wei Dan Shen Yin Combined With Bmscs Transplantation's Protective Effect On The Rat Cardiomyocytes With IRI And Its Mechanism

objective:1. To discuss Supplemented Jia Wei Dan Shen Yin combined with BMSCs transplantation's protective effect on the rat cardiomyocytes in the IRI.2. To initially discuss Supplemented Jia Wei Dan Shen Yin's inducing effect in vivo on the directed differentiation of BMSCs.3. To discuss the effect mechanism of Supplemented Jia Wei Dan Shen Yin combined with BMSCs transplantation's protective effect on myocardial ischemia-reperfusion injury through the p38MAPK pathway.Method:1. The whole bone marrow adherent method and the density gradient centrifugation method were adopted for BMSCs separation and culture.2. The ischemic-reperfusion injury rat model was adopted and310male SD rats were randomly divided into5groups:sham operation group as Group A; model group as Group B; stem cell transplantation group as Group C; Supplemented Jia Wei Dan Shen Yin with small dose group (equivalent dose) as Group D; Supplemented Jia Wei Dan Shen Yin with large dose group (4times as the equivalent dose) as Group E. Observation was made respectively in the three time points of3d,7d and14d after reperfusion. To observe the changes of myocardial enzyme level in rats, the deposition of myocardial interstitial collagen type Ⅰ, Ⅲ and Desmin's expression with immunohistochemical method and myocardial cells'ultra structure with transmission electron microscopy.3. TGF-β1level in the rat serum was detected by radioimmunoassay, the expression of p38MAPK mRNA by PCR and the protein expression of p38MAPK by Western-blot detection.Result:1. Under optical microscope, most P3cells are spindle-shaped, polygon shaped, or arranged in fibrous shape, consistent in the shape, manifesting with the swirl-like growth. The cells have good homogeneity, high purity and meet the experimental requirements after checked by FACScan flow cytometry.2. When blood flow in the left coronary artery was blocked, visible ST segment elevation and T wave height were observed in the leader Ⅱ of ECG; pathological Q waves emerged in the ECG and some rats had ventricular arrhythmias after the reperfusion began; with the prolongation of the reperfusion time, ST segment depression occurred with the pathological Q waves deepening.3. Serum myocardial enzyme level decreases in different model groups with the time flowing. On the3rd day after reperfusion, compared with A group, serum myocardial enzyme levels in other groups all increased remarkably and the difference were statistically significant (P<0.01). Compared with B group, serum myocardial enzyme levels in C group, D group and E group were relatively lower and the difference were statistically significant (P<0.05). On the7th and14th day after reperfusion, serum myocardial enzyme levels in C group, D group and E group were remarkably lower than other model groups and the difference were statistically significant (P<0.01).4. Compared with the sham A group, immunohistochemical results showed that on the3rd day after reperfusion, the expression of myocardial interstitial Ⅰ, Ⅲ collagen in other groups were increased but the difference of I collagen was not statistically significant among the other four groups (P>0.05), the difference of Ⅲ collagen was statistically significant among the other four groups (P<0.05). On the7th day after reperfusion, IOD in each group all increased, compared with the A group, every group was different and the difference were statistically significant(P<0.01&P<0.05); compared with B group, IOD of Ⅲ collagen in C group,D group or E group was different and the difference were statistically significant(P<0.01&P<0.05); IOD of I collagen in D group was different and the difference were statistically significant(P <0.05).Compared with the value detected on the3rd days, IOD in the B group and C group increased significantly and the difference were statistically significant(P<0.01&P<0.05). On the14th day after reperfusion, IOD in the B group increased while IOD in the other groups declined compared with the value detected on the7th day after reperfusion, compared with B group and C group, IOD in D group. E group declined and the difference were statistically significant (P<0.01&P<0.05).5. Immunohistochemical results showed:on the3rd day after reperfusion, compared with A group, the expression of Desmin in other groups were decreased and the difference were statistically significant among the other four groups (P<0.01). On the7th day and14th day after reperfusion, IOD in B group was decreased, but the difference has no significant, IOD in C group,D group and E group increase, compared with the values of IOD on3rd day, difference in D group was statistically significant(p<0.05). on the7rd day after reperfusion, compared with A group, the values of IOD in every model group was decreased and the difference were statistically significant (P<0.05&P<0.01); compared with B group, the values of IOD in D group,E group was higher than it in B group and the difference were statistically significant(P<0.05); On the 14rd day after reperfusion, the values of IOD in B group,C group was lower than it in A group and the difference were statistically significant (P<.05&P<0.01); the values of IOD in C group,D group,E group was higher than it in B group and the difference were statistically significant (P<0.05&P<0.01); the values of IOD in D group was higher than it in C group and the difference were statistically significant (P<0.05)6. Rat's myocardial ultra structure under electron microscope showed that the damage of myocardial cells in B group were the most serious while the C group the next, myocardial cells in D group and E group were gradually repaired with the time points flowing, the effect of D group was relatively better, but the difference was not significant compared with the E group.7. To detect the level of TGF-β1in rat serum by radioimmunoassay, the results showed that on the3rd day after reperfusion, compared with A group, the levels in B group and C group decreased, compared with A group the level in the model group was different and the difference was statistically significant(P<0.05), while the levels in D group and E group increased and the difference were statistically significant (P<0.01). On the7th day after reperfusion, the levels of TGF-β1in B group and C group increased, and the difference were statistically significant (P<0.05) compared with the levels detected on the3rd day after reperfusion, while the levels of TGF-β1in D group and E group decreased and the difference had no significant (P<0.05) compared with the levels detected on the3rd day after reperfusion. On the14th day after reperfusion, the levels of TGF-β1in B group continued to increase while the level of TGF-β1in D group continued to decrease, and the difference were statistically significant (P<0.05) compared with the levels detected on the7th day after reperfusion.8. To detect the expression of p38MAPK mRNA with PCR assay. On the3th and7th day after reperfusion, the values of2-△Ct in D group and E group both increased, compared with other groups, the difference were statistically significant (P<0.01); The increasing amplitude of E group was higher than D group and the difference was statistically significant (P <0.01);On the14th day, the values of2-△Ct in D group and E group decreased, but still higher than other groups, and the difference was statistically significant (P<.01&P<0.05).