| Objective: To investigate the effect of the solution of miR-200 a and miR-29 c on the expression of miR-200 a and miR-29 c in culture of HK-2 cell line.Methods: 1.30 male SPF grade 3-month-old rats were used to extract the serum containing kidney and kidney.Thirty rats were randomly divided into two groups: normal rat serum group and Shennao rat serum group.Adult and kidney network dosage of 30g/d,according to "experimental animals and people according to the body surface area equivalent amount conversion ratio" can calculate the equivalent dose of rats.The rats were 10 times the dose of the human body and about 27 g / d / kg of Shenxin Granules.Normal rats were given the same amount of normal saline every day.Gavage of the 7th day after administration of liver 90 min,the rat abdominal aorta blood 10 ml.Centrifuge the supernatant,56 ? water bath 30 min inactivated complement,0.22 um,filter filter sterilization,-80 ? refrigerator.Culture and treatment of HK-2 cells HK-2 cells were cultured in DMEM / F12 medium containing 5% fetal bovine serum at 37 C and 5% CO2.In the experiment,the cells were inoculated into the 6-well plate at the appropriate concentration.When the cells were about 70%-80% fused,they were cultured in serum-free DMEM / F12 medium for 24 hours to synchronize the cells.3.Experimental grouping,EMT induction and subsequent culture of TGF-?1 induced HK-2 cell EMT to form mesenchymal cells.The patients were divided into four groups: blank control group,TGF-?1 group(concentration 5ng / ml),TGF-?1 + 5% normal rat serum group,TGF-?1 + 5% drug serum group,72 h.4.Morphological changes of cells were observed by inverted phase contrast microscope.HK-2 cells were seeded in 105 ml / ml of 60% in the 6 empty plates of the sterile coverslip and replaced with serum-free DMEM / F12 The culture medium was cultured for 24 hours to synchronize the cells.According to the above-mentioned grouping scheme,the corresponding drugs were added and incubated at 37 ? for 5 hours in 72% incubated with 5% CO2.The morphological changes of EMT were observed by inverted phase contrast microscope.5.Western Blot detection of ?-SMA,E-cadherin and other changes.6.Detection of miR-200 a and miR-29 c expression The first c DNA strand was synthesized according to Takara's reverse transcription system kit,and c DNA was used as template and U6 small nuclear RNA was used as internal reference for SYBR Green quantitative PCR amplification.Each RNA sample was subjected to three replicates,and the relative expression was calculated using the 2-?? Ct method.The changes of expression of miR-200 a and miR-29 c were analyzed,and the mechanism of the effect of radix saliva on renal interstitial fibrosis was analyzed.Result: The expression of ?-SMA in HK-2 cells was significantly higher than that in control group(P <0.05),and the expression of E-cadherin was significantly decreased(P <0.01)The expression of miR-200 a and miR-29 c was significantly lower than that of the control group(group A)(P <0.05).After 5 h of normal rat serum,the expression of ?-SMA,E-cadherin and(P <0.05).The expression of miR-200 a and miR-29 c was significantly lower than that of group B(P <0.05).The expression of miR-200 a and miR-29 c was significantly lower than that of group B(P <0.05)(P <0.05).The expression of miR-200 a and miR-29 c was significantly higher than that of group B(P <0.05).Conclusion: And the amount of miR-200 a and miR-29 c in HK-2 cells were delayed and the process of renal interstitial fibrosis was delayed. |
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