| Objective：Hypertensive disorder complicating pregnancy(HDCP) is a special disease which seriously endangers the health of mother andfetus in pregnancy. The basic pathological and physical change is systemicsmall artery spasm, placental hypo-perfusion, placental ischemia and placentalhypoxia. Large-conductance calcium-activated potassium channel (BKca) is anion channel that plays an important role in the regulation of the vascular smoothmuscle tension mechanism. Therefore, we proposed that BKca channel has thefunction of vasodilation in hypertensive disorders in pregnancy. The aim of thisstudy was to detect the expression of α-subunit (the gene encoding KCNMA1)and β1-subunit (the gene encoding KCNMB1) in placental arteriole smoothmuscle cells from hypertensive disorder complicating pregnancy and normaltension pregnancy and investigate whether BKca channel are involved indysfunctional relaxation of artery in the process of development fromhypertensive disorders of pregnancy. Methods:（1）reverse transcription-polymerase chain reaction experiments：①15patients from hypertensivedisorder complicating pregnancy，these patients as case group (HDCP group);and15normal patients as control group（NT group）; the placenta smallvascular tissue block as the object of study.②to extract total RNA fromplacental vascular smooth muscle，Synthesize cDNA and carry out PCR reaction.③to compare the mRNA level of the KCNMA1, KCNMB1genebetween the two groups by RT-PCR. The house keeping gene β-actin was usedas the control.(2) Western blot:①The research object is the same as theRT-PCR experiment.②First, total protein was extracted from placentavascular tissue sample and protein concentration was determined and theprotein samples were separated through polyacrylamide gel. Theelectrophoresis condition for stacking gel and separating gel were selectedsuitably. Then the proteins in the gel were transferred to membrane and thetransfer condition was400mA,2hours. Next, the membrane was blocked in5%dried skim milk to shake for1hour slowly.③The membrane was incubatedwith α-subunit, β1-subunit primary antibody（1:1000dilution）. Then themembrane was washed and incubated with HRP-goat-anti-rabbit-IgG secondaryantibody (1:2000dilution) for1hour at room temperature. The images ofα-subunit and β1-subunit specific bands were semi-quantitatively analyzedusing Quantity One software. The protein amount of α-subunit and β1-subunitwere normalized to β-actin. Data were expressed as means±SEM. Pairedsamples T tests for independent test were used for statistical analysis by theSPSS17.0software and the significant level was defined as P<0.05. Results：(1)The expression quantity of RT-PCR experiments for KCNMA1and KCNMB1gene in placental arteriole smooth muscle cells was as follows: The respectiveratio of KCNMA1/β-actin was1.0062±0.0776（n=15）in the HDCP group，and0.9938±0.1151in the NT group（n=15）. There was no significant differencesbetween the two groups （P=0.093＞0.05）; The respective ratio of KCNMB1/β-actin was0.4180±0.0197（n=15） in the HDCP group， and1.5722±0.0314in the NT group（n=15）, the KCNMB1gene mRNA levels ofHDCP Group were significantly lower（P=0.001<0.05）.(2) The change ofprotein expression of KCNMA1and KCNMB1in the HDCP group and HTgroup: The relative protein expression level of KCNMA1in the HDCP Groupwas1.0012±0.1698（n=15），and the NT group was1.0282±0.1806（n=15）.There was no significant differences between the two groups（P=0.091＞0.05）;the relative protein expression level of KCNMB1in the HDCP Group was0.4181±0.0808（n=15），and the NT group was1.6168±0.0126（n=15），theKCNMB1protein expression levels of HDCP Group were significantly lower（P=0.001<0.05）. Conclusions：(1) The mRNAexpression of KCNMB1, butnot KCNMA1, was reduced in placental arteriole smooth muscle cells fromhypertensive disorder complicating pregnancy.(2) As the observation inRT-PCR for gene expression in placental arteriole smooth muscle cells fromhypertensive disorder complicating pregnancy, the protein of KCNMA1wasnot altered while the KCNMB1protein level was reduced. |
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