The β-catenin SiRNA Regulatory Mechanism Of Wnt/β-catenin Signaling On Renal Tubular Epithelial Cell’s Transdifferentiation

[Objection] To research the correlation between the Wnt/ β-catenin signaling and renal interstitium fibrosis in unilateral ureteral obstructions mice,providing theoretical basis for chronic kidney disease.[Methods] 1、According to the design principles of sh RNA, si RNA targeting β-catenin gene was constructed into lentivirus vector,the lentiviral titer was detected by the method of 293 T cells transfection; 2、12 healthy male SD mice were randomly divided into 4 groups(n=3),negative lentivirus were injected in different doses, 1×105 ifu、1×106 ifu、1×107ifu and 1.5×107ifu respectively, the renal tissue were collected to be maked of frozen section after 3 days so as to observe the efficiency of infection in those four groups, and then to group the best injection dose for the subsequent experiment. 3、128 healthy male SD mice were randomly divided into 4 groups:①unilateral ureteral obstructions group(UUO,N=32)was operated by ligation of left urter;②Sham operated group(Sham,N=32) was underwnt a sham procedure and manipulation of the left ureter without ligation but isolation. ③β-catenin slience group(N = 32)was injected by lentivirus under renal capsule on the basis of UUO;④negative slience group(N=32) was injected by negative lentivirus. 8 mice of different groups were sacrificed on the 3 th,7 th,10 th and 14 th day. The renal tissue were collected for the following purpose: ① Morphological and fibrosis changes of renal tissue were observed by HE and Masson stain; ②Immuno-histochemistry method was performed to investigate the location of α-SMA and β-catenin; ③Exploring the expression ofα-SMA and β-catenin by Western blot; ④ Using RT-PCR to quantify the expression of α-SMA m RNA and β-catenin m RNA.[Results](1) β-catenin si RNA lentiviruses were packaged and concentrated successfully. The lentiviral titer was measured to be 2×108TU/ml;(2)Frozen section: a titer of 1×107ifu had a high efficiency of infection with the negative lentivirus in rats;(3)HE and Masson stain: In UUO groups, renal pathology showed a different degree of inflammatory cell infiltration, expansion and deformation of renal tubules, edema and broadening of renal interstitial, with tubules occlusion or atrophy in the end, accompanide by a different levels of interstitial fibrosis. The sham groups showed no obvious changes at the same time. Changes in the negative slience groups were roughly the same as UUO groups, after the silence of β-catenin, renal structure and the degree of interstitial fibrosis were amelioratived in the later;(4) Immuno-histochemistry: Compared with sham groups, amounts of renal tubular epithelial cells which expressed α-SMA and β-catenin were increased in UUO groups and negative slience groups;after the slience of β-catenin, those two target protein were reduced began in 10 th.(5)The expression of α-SMA and β-catenin began to increase and were much high than that in Sham group(P<0.05); Compared with negative slience group, the protein expression of β-catenin and α-SMA in β-catenin si RNA group were decreased in 10 th,14th(P<0.05),and their m RNA expression were decreased begin with 14 th and 10 th respectively(P<0.05).[Conclusion](1) Wnt/β- catenin signaling involve d in the transdifferentiation of renal tubular epithelial cells;(2) Specific β- catenin si RNA can down-regulate the expression of β- catenin in renal tissue of UUO rats effectively;(3)β- catenin si RNA alleviated the transdifferentiation of renal tubular epithelial cells in the model of UUO rats.