| Section I The expression of Midkine in rat ASMCsObjective To determine the expression of Midkine in rat airway smooth muscle cells(ASMCs)Methods In this study,cigarettes combined with lipopolysaccharide were used to establish a rat model of chronic obstructive pulmonary disease.After the COPD model was constructed,the lung tissue was taken for HE staining to observe the pathological changes of the lung tissue,and then the expression of Midkine was tested by immunohistochemistry.Rat ASMCs were cultured in vitro,and the lung injury model was established with different concentrations of lipopolysaccharide(LPS),which was known as LPS group.CCK-8 kit was used to detect the activity of ASMCs.The Half Maximal Inhibitory Concentration(IC50)of LPS on ASMCs was determined.Small interfering RNA(siRNA)related to Midkine gene and LPS were used to treat lung injury model induced by ASMCs at the same time,which was called MKsiRNA group.Negative control siRNA group was abbreviated as non-target siRNA group.The expression of Midkine in rat ASMCs of normal saline group(control group),LPS group,non-target siRNA group and MKsiRNA group immunofluorescence qPCR,siblo and cell detection were used.SPSS.20 statistical software package was used to analyze the data.T-test was used for the intergroup comparison.Results The results of HE staining of SD rat lung tissue showed that there were inflammatory cell infiltration,airway smooth muscle hyperplasia and hypertrophy in the COPD group.Immunohistochemistry showed that Midkine was highly expressed in the airway smooth muscle cells of lung tissue in the COPD group,while Midkine was lowly expressed in the airway smooth muscle cells of the control group.By using CCK-8 kit,it was found that the IC50 of LPS was 0.21mg/m L and ranged from 0.15 to 0.26mg/m L.Western blot showed ASMCs could produce Midkine after interfering with 0.1mg / m L,0.2 mg / m L,0.3 mg / m L LPS,while the control group showed low expression or no expression for Midkine.According to the analysis from WCIF Hmage J image analysis software,the expression of Midkine protein in LPS reference group after β-actin standardization(P < 0.01),and the gray value among LPS groups with different concentrations was slightly different,but there was no statistical significance(P > 0.05).Midkine protein bands could be detected by Western blot in control group,LPS group,non-target siRNA group and MKsiRNA group,among which the expression of Midkine in the control group was lower than that in LPS group,and that in MKsiRNA group was lower than that in LPS group.According to the analysis from WCIF Hmage J image analysis software,the expression of Midkine protein in LPS group was significantly higher than that in the reference group after β-actin standardization(P < 0.05);Expression of Midkine protein in non-target siRNA group was higher than that in MKsiRNA group(P < 0.05);The expression of Midkine protein in MKsiRNA group was significantly lower than LPS group(P < 0.05).By qPCR,it was found that the relative expression of Midkine mRNA in LPS group was higher than that in control group(P < 0.05)and MKsiRNA group(P <0.05).The relative expression of Midkine mRNA in non-target siRNA group was higher than that in MKsiRNA group(P < 0.05).The relative expression of Midkine mRNA in LPS group was slightly higher than that in non-target siRNA in group comparison(P > 0.05).Immunofluorescence Detection of Midkine expression in rat,LPS group was higher than that in control group(P < 0.01).The expression of Midkine in MKsiRNA group was lower than that in LPS group(P < 0.01).Conclusion Midkine is highly expressed in ASMCs of LPS group and non-target siRNA group,and low or no expression in ASMCs of MKsiRNA group and control group.Section Ⅱ Effect of Midkine with different concentrations and time on the proliferation,apoptosis of ASMCs.Objective Observing the effect of Midkine on proliferation,apoptosis of ASMCs.Methods MKsiRNA and lipopolysaccharide(LPS)were used to intervene ASMCs at the same time for 3 days.CCK-8 kit was used to detect the proliferation function of ASMCs.Recombinant Midkine with different concentrations was used to intervene the ASMCs of control group and LPS group,and the intervention time was2 d and 3d,respectively.CCK-8 kit was used to detect the proliferation function of ASMCs.Flow cytometry was used to detect the apoptosis of ASMC in the control group,LPS group,MKsiRNA group and non-target siRNA group.Results Through CCK-8 kit detection,the survival rate in LPS group was lower than that in control group after LPS intervention(P < 0.05),that in MKsiRNA group was lower than that in LPS group(P < 0.05).There was no significant difference in the survival rate of siRNA group compared with LPS group(P > 0.05).r MK had no significant effect on the survival rate of ASMCs in the control group,and there was no statistical significance(P > 0.05).r MK significantly increased the cell survival rate of ASMCs with the increase of concentration and treatment time in the LPS group(P< 0.05).By flow cytometry detection,it was found that the apoptosis rate increased with the increase of LPS concentration after the intervention of different concentrations of LPS on ASMCs(P < 0.05).The apoptosis rate of ASMC treated with r MK slightly down,but no statistically significant(P > 0.05).The apoptosis rate of LPS group was significantly higher than that of control group(P < 0.01),that in MKsiRNA group was higher than that LPS group(P < 0.05),and that MKsiRNA group was much higher than the reference group(P < 0.05).The apoptosis rate of r MK group was lower than that of LPS group(0.2mg/m L)after treatment with different concentrations(P < 0.05),but the apoptosis rate in r MK group with different concentrations was not statistically significant(P > 0.05).Among them,the apoptosis rate in r MK with 1ng /m L group was much higher than that of reference group(P < 0.01),the apoptosis rate in r MK with 10 ng / m L group was higher than that in control group(P < 0.05)The apoptosis rate in r MK with 100 ng / m L group was higher than that the reference group.(P < 0.05).Conclusion Midkine can promote the proliferation of ASMCs and reduce apoptosis in lung injury induced by LPS.When Midkine was blocked,the proliferation effect of ASMCs decreased and apoptosis of ASMCs increased.Section Ⅲ The effect of Midkine on the Notch 2 gene.Objective To observe the expression of Notch 2 in ASMCs of control,LPS and MKsiRNA groups.To observe the effect of Notch on the proliferation and apoptosis of ASMC in Control group,LPS group,MKsiRNA group and LY411575 group.Methods The lung tissues of the SD rat COPD model and the control group were taken for immunohistochemistry experiments to detect the expression of Notch2.CCK-8 kit was used to detect the IC50 of Notch inhibitor(LY411575)to ASMC,and the curve was drawn.The mRNA and protein of the control group,LPS group,MKsiRNA group and LY411575 group were extracted and the expression of Notch 2 in each group.It was detected by qPCR,Western blot and immunofluorescence.CCK-8 kit was used to detect the cell activity of control group,LPS group,MKsiRNA group and LY411575 group.Flow cytometry was used to detect the apoptosis of ASMCs in the control group,LPS group,MKsiRNA group and LY411575 group.SPSS.20 statistical software package was used to analyze the data.T-test was used for the intergroup comparison,.Results Immunohistochemical experiments showed that Notch2 was highly expressed in the airway smooth muscle cells of lung tissue in the COPD group,while Notch2 was lowly expressed in the airway smooth muscle cells of the control group.By using CCK-8 kit,it was found that the IC50 of LY411575 was 0.03mg/m L and ranged from 0.024 to 0.035 mg/m L.Western blot showed that Notch 2 protein bands could be detected in the control group,LPS group,MKsiRNA group and LY411575 group,and the expression of Notch 2 protein in the control group,LY411575 group and MKsiRNA group was lower than that in LPS group.According to the analysis from WCIF Hmage J image analysis software,the expression of Notch 2 protein in LPS group is better than that in the control group(P < 0.05),MKsiRNA group(P < 0.05),LY411575 group(P <0.05)after β-actin standardization.By qPCR assay,relative expression of Notch 2 mRNA in LPS group was higher than that in control group(P < 0.05),MKsiRNA group(P < 0.05)and LY411575group(P < 0.05).The relative expression of Notch2 mRNA in LPS group was slightly higher than that in non-target siRNA group,but there was no statistical significance(P > 0.05).The relative expression of Notch2 mRNA in the non-target siRNA group was higher than that in the MKsiRNA group(P < 0.05).By immunofluorescence assay,the expression of Notch 2 in LPS group was higher than that in control group(P < 0.05).The expression of Notch2 in LPS group was slightly lower than that in non-target siRNA group,but it’s no statistically significant(P > 0.05).Expression of Notch2 in LPS group was higher than that in MKsiRNA group(P < 0.05).The expression of Notch2 in LPS group was higher than that in LY411575 group(P < 0.05).By using CCK-8 kit,the cell survival rate in LPS group was lower than that in control group(P < 0.05).The cell survival rate in MKsiRNA group was lower than that in LPS group(P < 0.05).The cell survival rate in LY411575 group was lower than that in LPS group(P < 0.05).By flow cytometry detection,it was found that the apoptosis rate in LPS group was significantly higher than that in control group(P < 0.01).The apoptosis rate in MKsiRNA group was significantly higher than that in LPS group(P < 0.01).The apoptosis rate in LY411575 group was significantly higher than that in LPS group(P< 0.01).Conclusion The main effect of Midkine on the function of ASMCs is mainly achieved through Midkine / Notch 2 signaling pathway.Inhibition of Midkine expression or Notch 2 expression can inhibit the proliferation of ASMC and promote apoptosis. |
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