| Objective:Hypertensive disorder complicating pregnancy?HDCP?is a pregnancy-specific disorder,endangering the health of maternal-child.Its basic pathological changes are small systemic vascular spasm.Large conductance calcium-activated potassium channel?BKca?is a key ion channel to regulate vasomotor function.The BKca channel is composed of?-subunit and?1-subunit.There are?1-?4 four subtypes,among them?1-subunit mainly express in smooth muscle tissue,especially vascular smooth muscle.This experiment uses HE staining to determine the material is the uterine small artery which meets the requirements of experiment,then using Real-time fluorescence quantitative PCR?QPCR?and Western blotting to study uterine arterial smooth muscle cells in BKca channel?-subunit and?1-subunit mRNA and protein amount of difference between the level of expression,which come from the normotensive pregnant women and with hypertensive disorder complicating pregnancy of pregnant.So as to analyze the relationship between the change of the BKca channel?-subunit and the?1-subunit and the pathogenesis of hypertensive disorder complicating pregnancy,and to investigate the role of the BKca channel?-subunit and the?1-subunit in hypertensive disorder complicating pregnancy.Methods:?1?HE staining test:?1?Collected during the October 2014 to August 2015 in the obstetrics department of Affiliated Hospital of Luzhou Medical College underwent cesarean section operation to terminate the pregnancy and with hypertensive disorder complicating pregnancy?excluding chronic hypertension complicated with preeclampsia and chronic hypertension complicated with pregnancy?patients'uterine small artery as case group,namely the HDCP group,have a total of 40 cases.In the same time,collected normal blood pressure and because of scar uterus,pelvic stenosis or social factors for cesarean section operation to terminate pregnancy pregnants'uterine small artery as control group,namely NP?normal pregnancy?group,have a total of 40 cases,too;?2?The uterine small arteries are isolated in the laboratory and taking out a small part of the artery fixed with formaldehyde;?3?Conventional paraffin embedded,4?m thick sections,with xylene dewaxing,the various levels of ethanol to wash,dyeing,dehydration,transparent,sealing,observing with microscope of whether it is a small artery,photography and comparison the differences of NP group and HDCP group.?Real-time fluorescence quantitative PCR experiment?QPCR?:?1?Each of the 20 cases in the case group and the control group,which were confirmed by HE staining,for this experiment.?2?Extraction of uterine artery vascular smooth muscle total RNA,total RNA concentration was measured by UV spectrophotometer,using reverse transcription polymerase chain reaction?ImPCR?technique to extract the total RNA reverse transcription into cDNA,with GAPDH as the reference gene,SYBR Green I real-time fluorescence quantitative PCR reaction,the corresponding Ct value,calculated?Ct?is equal to the target gene Ct value subtract reference gene Ct value?,??Ct value?is equal to?Ct value of gene in HDCP group subtract?Ct value of gene in NP group?,and 2-??Ct?Means that how many multiples HDCP group gene is NP group gene?.Statistical analysis was performed using t test on SPSS19 software,comparison between NP group and HDCP group gene BKca?and BKca?1mRNA change.?3?All PCR products of the target gene and reference gene using1.5%agarose gel electrophoresis and image preservation.?Western blotting?Western blotting experiment?:?1?The subjects were the case group and the control group,which were confirmed by HE staining,exclusion for QPCR subjects,each have 20 cases;?2?Mash organization in liquid nitrogen,plus the right amount of lysate extraction,extract the total protein of uterine artery vascular smooth muscle and determinate the protein concentration;?3?transfer to PVDF film after SDS-PAGE electrophoresis,antibody incubation,using Bio-Rad gel imaging system to image and preserve them,measurement the gray value with Quantity-One software,using GAPDH as internal control,correction of the relative expression of the target protein,Statistical analysis was performed with SPSS19 software t-test,the changes of relative expression level of BKca?and BKca?1 in NP group and HDCP group were compared.The results of the experiments are expressed in mean±standard deviation?x?±s?,P<0.05,in that the difference was statistically significant.Results:?1?40 cases NP group and 40cases HDCP group were confirmed the tissue are uterine arteries and meet the experimental requirements,24 cases of the HDCP group,among them,5 cases of mild preeclampsia,18 cases of severe preeclampsia,1 cases of eclampsia,?accounted for 60%of the HDCP group?,were seen with acute atherosclerotic necrosis.?2?The result of QPCR experiment assay.In HDCP group,1 case failed experiment,the tissue volume of 2 cases were too little for 3 times repeated experiments,so we excluded them.In NP group,there were 1 case of experimental failure,the tissue of the samples of 1 case was only two experiments,1 case's Ct value are too large than the others.So they were exclused.Therefore,HDCP group and NP group each have 17 cases to obtain the effective data.HDCP group BKca?-subunit?Ct value is 1.7671±0.7071?n=17?,NP group BKca?-subunit?Ct value is 1.7549±0.8423?n=17?,there is no significant difference between the two groups in the comparison of BKca?mRNA,?P=0.964>0.05?;HDCP group BKca?1-subunit?Ct value is1.8820±0.4256?n=17?,NP group BKca?1-subunit?Ct value is1.4515±0.2280?n=17?,there is significant difference between the two groups in the comparison of BKca?1 mRNA,?P=0.001<0.05?;2-??Ct??Ct value of gene BKca?1 is 0.7677±0.1987,therefore,the level of HDCP gene BKca?1 mRNA was lower than that of NP group;It can be seen from the PCR amplified products after electrophoresis gel image strips,no obvious tail,irregular phenomenon,this experiment no other interference of the two polymer,HDCP group and NP group have the expression of BKca?mRNA and BKca?1mRNA,and there is no significant difference in the expression level of BKca?mRNA in the two groups,but,the expression of BKca?1 mRNA in HDCP group was significantly decreased.?3?The result of Western blotting experiment assay:In HDCP group,1 cases were not enough to be used in the experiment,1cases had very little gray value,and the remaining 18 cases were effective.In NP group,20 cases were successful,but there are 2 cases'gray value were far greater than the other specimens,so they were ruled out.HDCP group BKca?/GAPDH gray value ratio is 0.1115±0.0487?n=18?,NP group BKca?/GAPDH gray value ratio is 0.1004±0.0306?n=18?,there is no significant difference in the expression level of BKca?protein between the two groups,?P=0.416>0.05?;HDCP group BKca?1/GAPDH gray value ratio is 0.4904±0.1899?n=18?,NP group BKca?1/GAPDH gray value ratio is 0.8009±0.3250?n=18?,there is a significant difference in the expression level of BKca?1 protein between the two groups,?P=0.002<0.05?;the expression level of BKca?1 protein in HDCP group is lower than that in NP group.Conclusion:?1?In the condition of hypertensive disorder complicating pregnancy excluding chronic hypertension,the acute atherosclerosis changes maybe happen in the vascular smooth muscle of human uterine artery.?2?In the condition of hypertensive disorder complicating pregnancy excluding chronic hypertension,there is no significant change in the human uterine artery vascular smooth muscle cells the mRNA and protein levels of ion channel BKca?-subunit encoding.?3?In the condition of hypertensive disorder complicating pregnancy excluding chronic hypertension,in human uterine artery vascular smooth muscle cells the mRNA and protein levels of ion channel BKca?1-subunit encoding are significantly reduced.This suggests that ion channel BKca activity changes may play a role in the pathogenesis of hypertensive disorder complicating pregnancy,?1-subunit downregulation changes may may be a key point to cause the dysfunction of the systemic small vessel of hypertensive disorder complicating pregnancy. |
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