| Progesterone receptor M?mitochondrial progesterone receptor,PR-M?was a new truncated progesterone receptor found in 2002 by Saner KJ and others,which located in the nucleus of mitochondria,and compared with the traditional progesterone receptor PR-A PR-B,lacking of the structural area A/B in N-terminal and DNA binding region,only with the hinge region and ligand binding region,is a new truncated progesterone receptor.The mitochondrial localization signal,MLS,which contained in the 16 amino acids of PR-M N-terminal is related to its unique mitochondrial localization function.At present,PR-M may regulate the mitochondrial membrane potential?mitochondrial membrane,potential,MMP?to play its unique role.The study found that increased progesterone induced by primary culture and immortalized mitochondrial membrane potential of uterine smooth muscle cells,in a dose-dependent manner,and progesterone receptor antagonists can inhibit this reaction.It is suggested that progesterone may increase cell energy metabolism through nongenomic effects of PR-M,and the increase of intracellular energy resulting in abnormal cell proliferation.Progesterone induced PR-M to mitochondrial membrane potential increase in inhibition of breast cancer cells,apoptosis of primary and immortalized uterine smooth muscle cells.Myoma of uterus?leiomyoma?is the most common female pelvic benign tumor,30%-50% women of childbearing age,and the incidence increased year by year,is the main reason for clinical hysterectomy,the clinical manifestation of abnormal menstruation,infertility,and pain,seriously affect the quality of life of female patients.However,its etiology has not yet the machine is clear,to explore the pathogenesis and effective treatment has been a hot research experts at home and abroad.Gynecological and clinical studies reported in the literature are showed that progesterone plays an important role in the occurrence of uterine leiomyoma.Our previous study found that progesterone receptor PR-M in tumor tissue,the expression of PR-A and PR-B significantly higher than in adjacent normal tissues of uterine myoma of uterus smooth muscle expression,suggesting that PR-M play a positive role in leiomyoma growth process,progesterone in myoma tissues may At the same time,the genome and non genomic pathways play a dual role.Mifepristone?RU 486?is a compound structure similar to steroid hormones.This compound has a strong affinity for the progesterone receptor and glucocorticoid receptor,and combined with the androgen receptor to a lesser extent.Its path through different signal transduction,play genomic and non genomic effects.Initially as mifepristone early pregnancy abortion drug application,in recent years is also used in the treatment of diseases such as uterine leiomyoma,mifepristone for the treatment of uterine fibroids has good effect,less side effect,low recurrence rate advantages,but the mechanism is still unclear.PurposeBased on the collected specimens leiomyoma research group before the extraction of primary uterine leiomyoma cells as research material,to detect the expression of progesterone receptor Western blot and screened the PR-M positive cell lines,progesterone receptor RU486 inhibitors using different concentration gradient intervention changes respectively using CCK-8 method and the detection of PR-M positive group and PR-M negative group of uterus leiomyoma cell proliferation and apoptosis by flow cytometry,further study of PR-M in the occurrence and development of uterine leiomyoma,and explore its possible mechanism,so as to deepen the understanding of the role of the PR-M function,to provide new ideas and research directions for the diagnosis and treatment of Obstetrics and Gynecology progesterone related diseases.Materials and methods 1 research objects Selected from September 2015 to June 2016 in our hospital?the Third Affiliated Hospital of Zhengzhou University?for pure uterine myoma underwent laparoscopic hysterectomy or abdominal leiomyoma randomly in 11 cases of patients,aged 24 to 48 years old,the average?42±4.49?years old.All of the patients had no other hormone related complications.All the patients have not taken the steroids at least 6 months before the operation.The collected samples were confirmed by laparoscopy or laparotomy diagnosis,postoperative routine pathological examination.All specimens were collected after informed consent of patients and signedconsent,and approved by the hospital ethics committee approval.With the size of about 1cm3 in aseptic operation the number of tumor tissue blocks,placed in the temperature of 4 DEG containing 1% double antibody?mixture of Penicillin and streptomycin?DMEM-F12?FD?in the culture medium,taken back to the laboratory for further processing as soon as possible in the ice box.2 methods.?1?the separation of uterine leiomyoma,extraction,primary culture of uterine leiomyoma cells,and the cell morphology was observed by inverted phase contrast microscope,immunocytochemistry??-SMA alpha monoclonal antibody?identified as uterine leiomyoma cells;?2?Detection the expression of progesterone receptor PR-M?PR-A and PR-B by immunoblotting?Western Blotting?,and screened the PR-M positive cell lines?i.e.,the expression of PR-M,not expression of PR-A/B cell line?as the study group,PR-M negative cell lines?i.e.not the expression of PR-M,expression of PR-A/B cell line as control?group of breast cancer cell line T-47D?American Type Culture purchased from Collection?as a positive control of screening cells?T47D is also expressed by literature confirmed PR-A/B and PR-M cell lines,because of the lack of stage specific antibody against PR-M,the positive cell lines were used to confirm the screening results??3?different concentrations of RU-486(blank control,1×10-6mol/L,1×10-5mol/L,1×10-4mol/L)intervention in two groups of cells,cell proliferation was detected by CCK-8 method and calculate the inhibitory rate of cell proliferation(after adding CCK-8 reagent of each cell in the absorbance at a wavelength of 450nm?A?were tested,each repeated three,set of four holes.The proliferation inhibition ratio is calculation as the control group and the experimental group and the difference between the control group and blank control group difference.?4?Different concentrations of RU-486(blank control,1×10-6mol/L,1×10-5mol/L,1×10-4mol/L)were used to intervene in the cells of the two groups.The apoptosis rate was detected by flow cytometry and the apoptosis rate was calculatedResult 1.of 11 cases of uterine leiomyoma cells cultured successfully in 10 cases,including 1 cases with small cell,abandoned;the success rate of 91%,close to the literature.Identification of uterine leiomyoma cells: spindle most cells within 24 h after inoculation and adherent was gradually extended the basic rules and slow growth,3 days cell growth speed,a week or so to enter the logarithmic phase,and dense at a spiral,"peak valley" showed typical microscopic phenomenon,by immunohistochemical method for identification of uterine leiomyoma cells?see Figure 1?.2.Western blot was used to detect the expression of progesterone receptor PR-A/B and PR-M in uterine leiomyoma cells.The specimens with obvious PR-M expression were selected as the study group and the specimens of PR-A/B were used as negative control group 3.Effects of RU-486 on proliferation of uterine leiomyoma cells in two groups: inverted microscope,RU-486 48 h after the cell shrinkage deformation,and with the increase of RU-486 concentration,two group cell proliferation inhibition rate increased gradually,PR-M positive observation group and blank control group with significant difference?F=8.27,P < 0.05?;PR-M negative group and blank control group had significant difference?F=9.14,P < 0.05?.The proliferation inhibition rate increased in a dose dependent manner?P < 0.05?.PR-M positive observation group the inhibition rate of less than PR-M negative group,compared with the same concentration of effect between the 22 groups,the difference was statistically significant?P < 0.05?.4.RU-486 on the two groups of myoma cell apoptosis: PR-M positive observation group and the control group,the difference was statistically significant?F= 7.44,P < 0.05?;PR-M negative group and blank control group had significant difference?F=8.32,P < 0.05?.With the increase of drug concentration cell apoptosis rate gradually increase?P < 0.05?.When the RU-486 concentration is more than 1 * 10 6mol/L,the apoptosis rate of PR-M positive group was less than the PR-M negative group,there was statistical significance between the 22 groups of the same concentration difference?P < 0.05?.Conclusion1.PR-M may play an important role in the development of uterine leiomyoma by inhibiting the apoptosis of uterine leiomyoma cells and promoting the proliferation of uterine leiomyoma cells2.the role of PR-M may be inhibited by progesterone receptor inhibitors... |
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