Construction Of A System Of Human/rat Homology Small Interfering RNA For Collagen TypeⅠ And Its Inhibition Effects

Kidney fibrosis is the pathologic change of almost all Chronic Kidney Disease when it develops to later stage, which suggesting a pathologic process characterized as excessive accumulation of extracellular matrix (ECM). Collagen type I plays an important role in accumulation of extracellular matrix and among these Collagen,α1(I) with coding COL1A1 exerts the major biological functions. We are expecting that the usage of RNA interference technology in down-regulation of Collagen type I over expression can become the new method of slowing down kidney fibrosis process.Partâ… Design,screen and evaluation of human/rat homology small interfering RNA targeting collagen typeâ… Objective: Based on Collagen type Iα1(I) chain we designed and screened human-rat homology collagen type I (COL1A1-siRNA)and proved its efficacy.Methods: According to the known design principals, we design and choose the complete human-rat homology collagen type I siRNA sequence by utilizing RNA design software. Then we transfected the nonspecific siRNA with fluorescent into the rat mesangial cells (RMCs)to observe its transfection efficiency using microscope. We directly transfected collagen type I siRNA into the rat mesangial cells and used RT-PCR method to observe its inhibitory action on the collagen type I protein expression of rat mesangial cells . Results: The rat mesangial cells were able to transfect the nonspecific siRNA collagen,so it could be used as the vehicle of our siRNA experiment,the transfection efficiency was about 53.9%. We transfected collagen type I siRNA into the rat mesangial cells and compared them with blank and MOCK groups,each siRNA group showed specific 513bp COL1A1 gene strap, also, the gene strap showed decreased brightness when the siRNA concentration was increased . We can see that collagen type I siRNA down regulated the collagen type I expression and showed dosage dependency. Conclusion: The collagen type I siRNA we designed was complete human-rat homology siRNA sequence and all base pairs were totally matched. It can be successfully transfected into rat mesangial cells and provide sufficient inhibition on the mesangial expression of collagen type I.Partâ…¡Construction of vector of human/rat homology siRNA for collagen typeâ… Objective: Establish collagen type I siRNA expression plasmid to prolong its action duration, make it express more stably. Methods: We used the chosen collagen type I siRNA as target,synthesized 64 nt oligonucleotide first , after treated it with anneal and T4 polynucleotide kinase, we orientatingly linked it with the linearized plasmids pGPU6/GFP/Neo which has been treated with BamH I and PstI enzymic cleavage until it converted to competent cell, then performed PCR and sequencing evaluation. Finally we transfected the established plasmid into the rat mesangial cell and observed its inhibition action by using RT-PCR. Results: When compared with the electrophoresis strips from the blank and mock groups, each siRNA plasmid group showed specific 513bp COL1A1 gene strap, and the strap showed decreased brightness as the siRNA plasmid concentration was increased. We can see that collagen type I siRNA plasmid posseses the same ability to down -regulate collagen type I expression and its effect is significant with dosage dependency. Conclusion: Using plasmid vector method not only provided the same inhibition action on the formation of collagen type I, but also showed more stable expression and its effect was more significant.Partâ…¢The inhibition of human/rat homology Collagen Type I siRNA on the TGF-β₁-induced overexpression of Collagen Type IObjective: To observe the inhibition action of collagen type I siRNA on human TGF-β₁–induced expression of collagen type I under pathologic condition. Methods: We chose human TGF-β₁ -induced rat collagen type I expression on its mesangial cell and observed its expression effect, then we used collagen type I siRNA plasmid inhibited its expression. Results: By using TGF-β₁ induced collagen type I exerted an increased gene expression and showed a more significant effect when the TGF-β₁ dosage was increased. When compared with blank and MOCK groups, each stimulating factor strap that generated from the collagen type I ,which was first induced by stimulating factor TGF-β₁ and then transfected, increased the brightness gradually; while the transfected siRNA strap from the countergroups which was only induced by stimulating factor decreased the brightness slightly.Conclusion: Using our chosen siRNA vector in cell experiment,we proved that based on its specificity on collagen type I,siRNA was able to effectively inhibit the over expression level of collagen type I gene with an up regulation under pathologic condition;it also exerted inhibition action even under strong pathopoiesis,as a consequence, we established the foundation for future trials and found a better site for further animal experiments in slowing down kidney fibrosis process.