摘要
目的:探索川芎嗪联合丹参对大鼠慢性移植肾肾病(Chronic allograft nephropathy,CAN)的治疗作用,揭示其与CAN之间的关系,进一步发现其延缓大鼠CAN的作用机制,为临床上延长移植肾的存活时间提供新的治疗途径。
方法:
1.大鼠模型的建立:F344近交系大鼠和Lewis近交系大鼠作为供、受体,取供体的左肾作为供肾,在裸眼直视下行供体下腔静脉与受体肾静脉的端端吻合,供体腹主动脉与受体腹主动脉行端侧吻合,进行原位异体肾移植。
2.将125只肾移植大鼠分成5组,每组25只,分别为A组环孢素组,B组川芎嗪联合环胞素组,C组丹参联合环胞素组,D组川芎嗪、丹参联合环胞素组,E组为空白组。给药途径:环孢素灌胃,丹参、川芎嗪腹腔注射。手术后连续用药10天。期间观察大鼠的生活状态,分别于移植术后2,4,6,8,12周处死大鼠,通过检测HE、PAS以及Masson染色观察各组大鼠移植肾组织病理学变化。
3.采用免疫组织化学和实时荧光定量PCR检测TGF-β1的蛋白和mRNA在移植肾的表达及其定位情况,揭示川芎嗪联合丹参延缓大鼠CAN的作用及机制。
结果
1.大鼠CAN模型的建立
通过手术训练技术成熟后共进行140例手术,手术成功率96.43%;手术总时间约150min,其中取肾约20min,进行移植术约70min。通过对移植肾病理学动态检查,移植肾术后4周开始出现CAN的病理改变,12周可见典型慢性排斥反应病变。从而建立大鼠CAN模型。
2.肾移植术后移植肾组织病理学改变
肾移植术后E组肾移植大鼠存活均未超过2周;A组4周最早出现CAN病理改变,并随病程进展移植肾病变逐渐加重,12周可见典型的CAN病变;而B、C组移植肾出现的时间较A组出现晚,D组出现时间最晚,且病变相对较轻。A组与其它各组相比移植肾病理变化Banff总分差异有统计学意义(P<0.05),与D组间的差异更显著(P<0.01);B、C组之间Banff总分差异无统计学意义(P>0.05)。
3.免疫组化及实时荧光定量PCR检测TGF-β1的蛋白和mRNA在移植肾的表达情况
移植术后4周TGF-β1阳性细胞数开始增多,其中以A组增高最明显,B、C组次之,D组最低;随着移植术后时间的推移,各组TGF-β1的表达进一步增强,12周时以A组表达增强明显,D组最低。各组间进行统计学分析,A组与其余各组比较均有统计学意义(P<0.05),其中与D组间的差异更显著(P<0.01);B、C组之间相比较无统计学意义(P>0.05)。
通过对移植术后大鼠移植肾各组各时相实时荧光定量PCR检测TGF-β1mRNA的表达发现,A组TGF-β1mRNA的表达较其余各组明显增高(P<0.05),与D组相比差异具有显著性(P<0.01);B组和C组相比,TGF-β1mRNA表达的相对比值数无统计学意义(P>0.05)。
结论:
1.F344大鼠和Lewis大鼠作为供、受体进行原位异体肾移植,该方法手术操作简单、直观,简化了显微外科技术的设备要求,建立了大鼠原位异体肾移植模型,且能达到建立CAN模型的要求。
2.川芎嗪、丹参联合应用安全、可靠,副作用较小,对大鼠CAN的进程有延缓作用。
3.川芎嗪联合丹参可能通过下调TGF-β1的表达,从而发挥抗CAN纤维化作用,延缓慢性移植肾肾病过程。
Objective:To explore the therapeutical effect of Tetramethylpyrazine and Dan-ShenRoot on Chronic allograft nephropathy of rat , reveal the relationship with CAN , then discovering mechanism of action that delaying the proceeding of CAN of rat,which provide a new way of therapy to extend survival time of transplanting kidney.
Methods:
1.We used Fisher rats as donors and Lewis rats as recipients to establish the model of CAN.The left kidney of the donors was removed,the inferiorvena of the donor was anastomosed end-to-end with the left renal vein of the recipient, and the donor’s abdominal aorta was anastomosed end-to-side with that of the recipient with the unaided eyes to carry out renal allograft in prime position.
2.125 rats with transplanting kidney were divided into five groups: Ciclosporin group, TMP associating with Ciclosporin group, Dan-ShenRoot ssociating with Ciclosporin group, TMP and Dan-ShenRoot ssociating with Ciclosporin group,black group; every group for 25 rats. Route of administration: Ciclosporin was intragastric administration, TMP and Dan-ShenRoot were intraperitoneal injection. Medication was used for 10 days after postop. During the postop periods, observed the life of rats, all rats were sacrificed at the 2nd,4th,6th,8th and 12th, examined the changes of HE、PSA、Masson staining was apllied to transplanting kidney of five groups.
3.Transplanting kidney were detected by real-time PCR and immunohistochemistry methods were apllied to detect TGF-β1 mRNA and protein expression in the transplanting kidney, revealed the mechanism of action that delaying the proceeding of CAN of rat.
Results:
1.The model of CAN of rat was established
It was marched 140 pou operation after technique techniqued, the ratio of operation was reached 96.43%.Cumulative time of operation was 150 min, recipe kidney was 20 min, transplantation was 70 min. The pathological changes of the transplanted kidney began to appear 4 weeks after transplantation ,then the chronic rejection could be seen after 12 weeks.
2.Histopathological changes of Transplanting kidney
The rats of black group was died in two weeks. Group A appearance the histopathological changes of CAN since 4th week, it is thus evident that typical histopathological changes of CAN at 12th week.But Group B、C occurrenced were late,Group D was later. There was significant difference between group A and others (p<0.05); between group A and D has predominance difference(P<0.01);there was no significant difference between group B and group C (p>0.05).
3.TGF-β1 mRNA and protein expression in transplanting kidney detected by real-time PCR and immunohistochemistry
4 weeks after transplantion, masculine cells with TGF-β1 began to increase,which of group A increased obviously,group B and C were lower,group D lowest.The expression of TGF-β1 enhanced by the time of transplantation.At 12 week,A group enhanced obviously,D group was lowest.Then we made statistics analyse during every group,group A compared with other groups it had statistical significance(P<0.05),the difference between group A and D was more significant(P<0.01); It had no statistical significance that group B compared with group C (P>0.05).
Through the expreesiom of TGF-β1 detected by real-time PCR in transplanted kidney of rats after transplantion,we found it group A increased obviously than other groups(P<0.05), the difference between group A and D was more significant(P<0.01); It had no statistical significance that group B compared with group C (P>0.05).
Conclusion:
1. This method was simple and direct-viewing,and reduced microsurgery equipment,which successfully established rat renal transplantation model of chronic allograft nephropathy and can achieve the requirement of establishing CAN models.
2. The together use of TMP and Dan-ShenRoot was safe、reliable and had lower side effect,then slowed the proceeding of CAN.
3. TMP and Dan-ShenRoot used together can decline the expression of TGF-β1 ,then it could play effect of anti-fibrous degeneration of CAN,and slow the proceeding of CAN.
引文
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