| Objective: Hypertensive disorders complicating pregnancy(HDCP),one of the most common obstetric diseases endangering the life of the fetus and the mother,accounting for 5%~10% of the total pregnancy,may occur during pregnancy,and also contribute to the further development of the original hypertension during pregnancy.They are mainly divided into: gestational hypertension,preeclampsia(mild and severe),eclampsia,chronic hypertension complicated with preeclampsia,and chronic hypertension complicated with pregnancy.The basic pathophysiological changes are spasm of small vessels,endothelial injury and local hemorrhage.With the progress of the HDCP and ion channel research,more and more evidence shows that the development of HDCP and ion channel connection,especially most close contact between potassium channels,ATP sensitive potassium channel(ATP-sensitive potassium channel,KATP)as one of the many potassium channels,By the structural subunits inward rectifier potassium channels(inwardly rectifying potassium channel,Kir)and adjust the base sulfonyl urea receptor(sulphonylurea receptor,SUR),widely exists in many kinds of excitable cells.Previous studies have shown that Kir6.1 and SUR2 B are expressed in human normal pregnancy uterine smooth muscle cells,but there are few studies on the expression of KATP channel in pathological pregnancy,such as gestational hypertension.This experiment uses the real-time fluorescent quantitative PCR(Real-time fluorescent PCR experiment policy,QPCR)and protein immunoblot technology(Western blotting)detection of KATP normal and severe preeclampsia in pregnant women,early hairstyle severe preeclampsia and late onset severe preeclampsia pregnant women,severe preeclampsia with fetal growth restriction and unincorporated fetal growth restriction pregnant placenta Chorionic plate artery(Placental Chorionic plate arteries,CPAs)Vascular Smooth Muscle cells(Vascular Smooth Muscle cell,VSMC)m RNA(messenger ribonucleic acid,m RNA)and protein expression level,analyze the severe preeclampsia group and normal group and late onset severe preeclampsia group and early hairstyle severe preeclampsia,severe preeclampsia with fetal growth restriction and unincorporated fetal growth restriction CPAs and differences of KATP expression in vascular smooth muscle in the pathological state of function change,explore CPAs KATP channels in vascular smooth muscle in the role of severe preeclampsia,in order to further reveal the pathogenesis of HDCP at the molecular level,To provide new ideas for clinical prevention and treatment.Methods:(1)selection of vaginal delivery or cesarean section admitted in our hospital obstetrics normal pregnant women CPAs blood vessels as the experimental group,positive control group normal pregnant uterine smooth muscle,distilled water as blank control group,the samples full grinding and extracting total RNA,reverse transcription for complementary DNA(complementary DNA and c DNA),the method of semi-quantitative PCR amplification c DNA,electrophoresis amplification product and imaging,direct contrast to the naked eye CPAs Kir6.1 KATP channels in vascular smooth muscle and SUR2 B expression or not.(2)the collection of normal pregnant women admitted in our hospital obstetrics postpartum placental chorionic plate arteries,15 cases in the same period of severe preeclampsia women postpartum placental chorionic plate arteries,15 cases(1)extracting total RNA and reverse transcriptase to c DNA,will glyceraldehyde phosphate dehydrogenase(glyceraldehyde phosphate dehydrogenase,GAPDH)as reference,QPCR,?Ct to get corresponding Ct value and calculated value(purpose gene within the Ct value and reference to the difference between the Ct value),??Ct value(experimental group gene Ct value-control group Ct value)? 2-??Ct value(that is to says the experimental group was a multiple of the control group gene),Expression levels and differences of KATP channel Kir6.1 and SUR2 B subunit m RNA in two groups of CPAs Vascular Smooth Muscle cell were analyzed.;(2)to extract proteins of two groups of placental chorionic plate artery vascular smooth muscle,sodium dodecyl sulfate polyacrylamide gel electrophoresis(sodium dodecyl sulfate polyacrylamide gel electrophoresis,sds-page),turn to PVDF membrane,skim milk closed after incubation resistance and resistance,imaging and save the chemiluminescence imaging system,the image using the gray analysis Fion,respectively with GAPDH internal protein comparison,get the relative target protein expression and grayscale values.QPCR and Western blotting experimental results on mean ± standard deviation(x?±s)said,with SPSS 19.0 t test,P < 0.05 think the difference was statistically significant.(3)collected in our hospital obstetric termination of late onset severe preeclampsia patients with placenta chorionic plate arteries,15 cases in the same period to terminate the pregnancy early hairstyle severe preeclampsia patients with placenta chorionic plate arteries,15 cases and severe preeclampsia with fetal growth restriction and unincorporated fetal growth restriction of placental chorionic plate arteries all 15 cases,using the same method to QPCR and Western blotting,The m RNA and protein expressions of Kir6.1 and SUR2 B subunits of the KATP channel in CPAs vascular smooth muscle of late onset and early onset severe preeclampsia,severe preeclampsia combined with fetal growth restriction and uncombined with fetal growth restriction were compared and analyzed.Results:(1)for Kir6.1 subunit,the corresponding bands could be produced by electrophoresis in the control group with positive uterine smooth muscle,and the same size bands could be produced by electrophoresis in the placental chorionic lamella arterial smooth muscle group,but not in the distilled water group.For SUR2 B subunit,the corresponding bands can be electrophoresis in the uterine smooth muscle group,the same size bands can be electrophoresis in the placental chorionic lamella arterial smooth muscle group,but not in the distilled water group.(2)Analyse the results of normal pregnancy group and severe preeclampsia group,(1)QPCR results: Kir6.1 ?Ct value is 5.2808±0.34160 in 15 normal CPAs vascular smooth muscle,while the value is 5.0212±0.45322 in 15 severe preeclampsia CPAs vascular smooth muscle.Kir6.1 subunit m RNA has no statistical difference in two groups(P=0.089 >0.05).SUR2 B ?Ct value is 3.9898±0.3376 in 15 normal CPAs vascular smooth,while the value is 7.5576±1.4001 in 15 severe preeclampsia CPAs vascular smooth.SUR2 B subunit m RNA has statistical difference in two groups(P<0.001).The 2-??Ct value is 0.2091±0.3063,showing that compared with normal CPAs vascular smooth group,SUR2 B subunit m RNA in severe preeclampsia CPAs vascular smooth group is obviously reduced.(2)Western blotting results: Kir6.1/GAPHD gray value ratio is 0.7383±0.1781 in 15 normal CPAs vascular smooth,while the ratio is 0.7728±0.1060 in 15 severe preeclampsia CPAs vascular smooth.The protein expression level of Kir6.1 subunit has no statistical difference in two groups(P=0.719 >0.05).SUR2B/GAPDH gray value ratio is 0.7317±0.0497 in 15 normal CPAs vascular smooth,while the ratio is 0.4367±0.06327 in 15 severe preeclampsia CPAs vascular smooth.The protein expression level of SUR2 B subunit has statistical difference in two groups(P<0.001).(3)Analysing the results of lateonset severe preeclampsia group and early-onset severe preeclampsia group,(1)QPCR results: Kir6.1 ?Ct value is5.2517±0.4181 in 15 late-onset severe preeclampsia CPAs vascular smooth,while the value is 5.0917±0.5105 in 15 early-onset severe preeclampsia CPAs vascular smooth.Kir6.1 subunit m RNA has no statistical difference in two groups(P=0.356 >0.05).SUR2 B ?Ct value is 6.3931±0.4527 in 15 late-onset severe preeclampsia CPAs vascular smooth,while the value is 9.9218±0.3897 in 15 early-onset severe preeclampsia CPAs vascular smooth.SUR2 B subunit m RNA has statistical difference in two groups(P<0.001).The 2-??Ct value is 0.0959±0.0439,showing that compared with late-onset severe preeclampsia CPAs vascular smooth group,SUR2 B subunit m RNA in early-onset severe preeclampsia CPAs vascular smooth group is obviously reduced.(2)Western blotting results: Kir6.1/GAPHD gray value ratio is 0.7143±0.07639 in 15 late-onset severe preeclampsia CPAs vascular smooth,while the ratio is0.6859±0.0924 in 15 early-onset severe preeclampsia CPAs vascular smooth.The protein expression level of Kir6.1 subunit has no statistical difference in two groups(P=0.433>0.05).SUR2B/GAPDH gray value ratio is 0.4273±0.05377 in 15 late-onset severe preeclampsia CPAs vascular smooth,while the ratio is 0.2012±0.03784 in 15 early-onset severe preeclampsia CPAs vascular smooth.The protein expression level of SUR2B subunit has statistical difference in two groups(P<0.001).(4)Analysis of severe preeclampsia with fetal growth restriction and unincorporated fetal growth restriction placenta chorionic plate artery vascular smooth muscle groups.(1)QPCR results: 15 cases of severe preeclampsia with fetal growth restriction CPAs in vascular smooth muscle Kir6.1 subunits ? Ct value was 4.7257 ± 0.4392,15 cases of unincorporated fetal growth restriction CPAs in vascular smooth muscle Kir6.1 subunits ? Ct value was 4.6794 ± 0.3674,is similar between the two groups have no statistical significance(P = 0.757 > 0.05);15 cases severe preeclampsia with fetal growth restriction CPAs Vascular Smooth Muscle SUR2 B subunit ?Ct value was 9.8608±0.4154,15 cases severe preeclampsia without fetal growth restriction CPAs Vascular Smooth Muscle SUR2 B subunit ?Ct value was 6.6552±0.4393,The difference between the two groups was statistically significant(P<0.001),The 2-??Ct value is 0.1125±0.0422,It suggested that the SUR2 B subunit m RNA level in the CPAs vascular smooth muscle of the severe preeclampsia combined with fetal growth restriction group was lower than that of the non-combined fetal growth restriction group.(2)Western blotting results: the ratio of Kir6.1/GAPHD gray value in CPAs vascular smooth muscle of 15 cases of severe preeclampsia combined with fetal growth restriction was 0.7889 ± 0.04964,and the ratio of Kir6.1/GAPHD gray value in CPAs vascular smooth muscle of 15 cases of uncombined fetal growth restriction was 0.7847± 0.02936.The difference between the two groups was not statistically significant(P=0.875>0.05).The SUR2B/GAPHD gray scale value ratio in CPAs vascular smooth muscle of 15 cases of severe preeclampsia combined with fetal growth restriction was 0.2106± 0.0849,and the SUR2B/GAPHD gray scale value ratio in CPAs vascular smooth muscle of 15 cases of uncombined fetal growth restriction was 0.4004± 0.04185,the difference between the two groups was statistically significant(P<0.001).Conclusion:(1)according to the imaging strip diagram,the KATP channels Kir6.1 and SUR2 B subunits are expressed in CPAs vascular smooth muscle.(2)the normal pregnant women and severe preeclampsia in pregnant placenta chorionic plate artery vascular smooth muscle KATP channel Kir6.1 m RNA and protein expression of no significant difference,SUR2 B subunits m RNA and protein expression in severe preeclampsia in pregnant women than normal pregnant women decreased,prompt KATP channel SUR2 B the expression level of change may be associated with the development of severe preeclampsia.(3)compared with late onset severe preeclampsia,Kir6.1 KATP channel subunits m RNA and protein expression in early hairstyle severe preeclampsia placenta chorionic plate has no obvious difference in artery vascular smooth muscle,and SUR2 B subunits m RNA and protein expression are lower,indicates severe preeclampsia time before may associated with the degree of SUR2 B KATP channels on the decline.(4)Compared with severe preeclampsia unincorporated fetal growth restriction,Kir6.1 KATP channel subunits m RNA and protein expression in severe preeclampsia with fetal growth restriction has no obvious difference in placental chorionic plate artery vascular smooth muscle,and SUR2 B subunits m RNA and protein expression are lower,fetal growth restriction may be associated with the degree of SUR2 B KATP channels on the decline. |
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