| Part oneObjective To establish a stable and reliable Wistar-SD rat cervical heterotopic heart transplantation model.Methods Make a neck incision on the receptor rats after anesthesia.Remove the left submandibular gland, separation carotid artery and external carotid vein. Evert blood vessels over the cuff. Ligature all blood vessels that in and out the heart of donor rats, other than the aorta and pulmonary artery after heparinization. Remove the heart after, using the cuff to connect the aortic with the carotid artery and the pulmonary artery with external jugular vein respectively. Homologous control group:operate on SD to SD rats; Untreatment group:operate on Wistar to SD rats.Results Preliminary experiments include 25 couples,16 cases complete the heart transplantation successfully, the success rate is about 64%, without immu-nosuppressant, the heart survive as long as 8 days. In formal experiment,85 couples of rats were used,77 cases complete the heart transplantation successfully, the success rate is about 90.6%, the average operation time is 103.0±5.0 min. Rats revival soon after the operation, the heart beat strong, pathological examination shows the untreatment group have a serious immunologic rejection while the homologous control group is slight.Conclusion Using an improvement cuff of homemade, we complete the cervical heterotopic heart transplantation model in rats successfully. This method is suitable for most laboratory because of its high success rate without using a microscope.Part twoObjective To explore the oral tolerance dose of Halofuginone in rats, and preliminary research on the relationship with the expression of IL-17 and secretion.Methods Establish cervical heterotopic heart transplantation models, and the donors are Wistar rats, receptors are SD rats, Divided into 4 groups, each group three couples. Untreatment group:the day preoperative to 5th day after operation, oral administration the same volume of physiological saline; Low-dose group:the day preoperative to 5th day after operation, oral administration halofuginone 10 mg/kg; Medium dose group:the day preoperative to 5th day after operation, oral administration halofuginone 20 mg/kg; High dose group:the day preoperative to 5th day after operation, oral administration halofuginone 30 mg/kg. Observe the heart beats and the general situation of the receptor rats postoperative. Harvest the heart of each group in the 5th day. Detection of IL-17 mRNA expression level using Quantitative Real-time PCR.Results The postoperative receptor rats was responsive to stimuli and the heart beat normally. In the 5th day, the untreatment group donor heart turn to dark red or black, low dose and medium dose group donor heart was red only with a small amount of focal necrosis. Two of the high dose group dead in the 3th day. Quantitative Real-time PCR detection shows the level of IL-17 mRNA of medium dose group was the lowest(P<0.05).Conclusion Halofuginone can cause the inhibition of the acute rejection in heart transplantation in rats The dose of 20 mg/kg in the experiments, the rats can tolerance and have the best treatment effect of all group. High dose of halofuginone (30 mg/kg), inhibit normal phvsiological activities of the rats severely and lead to its death easilv. Medium dose group:the day preoperative to 5th day after operation, oral administration halofuginone 20 mg/kg; High dose group:the day preoperative to 5th day after operation, oral administration halofuginone 30 mg/kg. Observe the heart beats and the general situation of the receptor rats postoperative. Harvest the heart of each group in the 5th day. Detection of IL-17 mRNA expression level using Quantitative Real-time PCR.Part threeObjective Treat the heart transplantation with halofuginone and tacrolimus, to research the effect of halofuginone on T helper cells 17 (TH17) and its related factors and explore the mechanism of action.Methods Establish cervical heterotopic heart transplantation models, the donors and receptors are all SD rats in homologous control group. The other group, the donors are Wistar rats, receptors are SD rats. All rats are divided into 5 groups, each group 17 couples.Untreatment group:the day preoperative to 7th day after operation, oral administration the same volume of physiological saline; Halofuginone group:the day preoperative to 7th day after operation, oral administration halofuginone 20 mg/kg; Tacrolimus group:the day preoperative to 7th day after operation, oral administration Tacrolimus 1mg/kg; Halofuginone+Tacrolimus group:the day preoperative to 7th day after operation, oral administration halofuginone20 mg/kg, the lth to 7th day after operation. Harvest 5 hearts in each group in the 3th,5th,7th day. Detection of pathology and molecular biology, draw blood from the inferior vena cava about 5 ml, uses a centrifuge to separate serum for serologic test.Results In the homologous control group, the expression of IL-17 and the cytokines associated are always low, the untreatment group was the opposite. Halofuginone and tacrolimus treatment showed a certain inhibition of IL-17, particularly depress RORyt which is the key nuclear factor of TH17. At the same time, halofuginone can restrain the TH1 related cytokine IFN-y, and can significantly inhibit the expression of TGF-β, but the effect to IL-4, the TH2 related cytokine factor, can’t compare with tacrolimus.Conclusion Halofuginone can significantly reduce the IL-17 and the expression of the key nuclear factor of TH17, the impact is deeper than tacrolimus. The effect of halofuginone on TH1/TH2/Treg cells related cytokines is weaker than tacrolimus, while the detection of pathology are similar. So we think TH17 cells play a leading role in the acute rejection after heart transplantation, halofuginone can effectively suppress the rejection of this period, and RORyt may be an important targets in the process. |
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