| Part I: The expression of Apelin in PCOS patientsContext:Apelin is a new adipokines recently discobered, it plays an important role in the process of energy metabolism and insulin resistance, such as improve glucose metabolism, lipid metabolism, obesity resistance. Apelin involved in the pathogenesis of hypertension, diabetes and atherosclerosis. But the role of Apelin in polycystic ovary syndrome that special energy metabolism is not clear now.Objective:Detected the Apelin levels, and learn the interrelated between Apelin and obesity, glucose metabolism, lipid metabolism. Further discussion the possible mechanisms of Apelin in the PCOS development, obesity, and energy metabolism.Design:Select 100 cases PCOS patients that to do health examination in People's Hospital of Zhengzhou and 100 cases of healthy women as the control group at the same time. Patients are parted into obesity group and non-obesity group in accordance with BMI?25kg/?, and obese PCOS group include 62 cases, non-obese PCOS group include 38 cases; Obesity control group include 50 cases, non-obesity control group of 50 cases. After all objects signed informed consent, then they were collected peripheral circulation serum 20 ml. Investigate serum Apelin level by Elisa; while investigate serum level of follicle stimulating hormone(FSH), luteinizing hormone(LH), estradiol(E2), progesterone(P), testosterone(T); electrochemical luminescence investigate fasting blood glucose(FBG), insulin(INs), total cholesterol, triglyceride(TG), triglyceride(TC), high-density lipoprotein(HDL) and low density lipoprotein(LDL)levels.Results:1. Apelin level in PCOS group is significantly higher than the normal control group(647.92±194.47)vs(478.26 ±178.4); Apelin level in obesity PCOS group is significantly higher than non-obesity PCOS group and obesity control group(698.78±177.25)vs(512.39±191.32)vs(537.54±201.65), the differences have statistically significant(P<0.05).2. All indicators have statistically difference in the level of BMI?WHR?LH? E2?T?FPG?GHB?FINS?HOMA-IR?TG?LDL?Apo B?TC/HDL?LDL/HDL between the PCOS group with the control group(P<0.05 or P<0.01); There have no statistically difference in FSH?P?TC?HDL?Apo A levels of each group(P>0.05).3. Compared obesity PCOS group with non-obesity group, there have statistically difference in the levels of LH?GHB%?TG?LDL?Apo B?TC/HDL?LDL/HDL. BMI?WHR?E2?T?FINS?HOMA-IR(P<0.05 or P<0.001). Levels have statistically difference between the obesity group with non obesity group however the women with PCOS or not(P<0.05, P<0.001).4. Serum Apelin level is a positive correlation with BMI?FINS and HOMA-IR; r values are 0.609?0.498?0.457(P<0.05); There have negatively correlated between Apelin with LH level, r value is-0.548(P<0.05). Other factors such as Age?FSH?E2?P?T?FPG?TC?TG?LDL-C?HDL-C?Apo B etc. have no correlation with Apelin's level. PCOS and BMI are significant factors affecting the level of Apelin in multiple linear regression analysis(P<0.05).Conclusion:1. The resume Apelin level is elevated significantly compared PCOS patients with non PCOS patients.2. Apelin level is directly regulated by the degree of obesity and IR index.3. Apelin may promote insulin secretion, participate in glucose regulation, and thus involved in the occurrence and development of PCOS.Part?: The expression of Apelin in PCOS granulosa cellsContext:Apelin is an important regulatory factor in the prosses of energy metabolism, and it is expressed in mouse and bovine ovarian granulosa cells, and presenced such as curve levels. Abnormal ovulation is an important clinical feature in PCOS patitents, and the granulosa cells are important parts of regulating ovulation occurs. It's cleared that growth differentiation factor 9 and Forkhead box O1-a are involved in the regulation of proliferation and apoptosis, which with PCOS women. But up to now, there have no report with the Apelin expression in human ovarian granulosa cells, also no report of correlation between Apelin with growth differentiation factor 9 and Forkhead box o1-a.Objective:Detect Apelin m RNA levels and protein expression in human original generation granulosa cells with PCOS, and mutual authentication with Apelin serum level in the Part ?; At the same time detected the m RNA and protein levels of growth differentiation factor 9 and Forkhead box o1-a, that is closely related with PCOS. At last explore the possible influence in follicular development prosses, such as Apelin.Design:Collect granular cells from Reproductive Center of First Affiliated Hospital of Zhengzhou University with IVF in PCOS patients as the experimental group, and collect IVF patients with tubal factor or male factor as the control group in the same time. Patients are parted into obesity group and non-obesity group with BMI?25kg/?. Detect m RNA and protein expression of Apelin, Foxo1 a and GDF-9 in the four groups with reberse transcription polymerase chain reaction and western bloting. Learn the expression trend and correlation between Apelin in granulosa cells with serum levels.Results:1. The expression of Apelin m RNA and positive protein are visible in each group;2. Apelin m RNA level and protein expression in the PCOS group is significantly higher than the control group; The expression in obesity PCOS group is higher than non-obesity PCOS group, whether or not with PCOS; and all differences have statistically significant(P<0.05).3. Foxo1 a m RNA level in obesity PCOS group is significantly higher than non-obesity PCOS group and control group; there have statistically difference between all groups(P<0.05), and it is significantly in obesity PCOS group.4. GDF-9 m RNA level in the PCOS group is significantly lower than the control group; there have statistically difference of protein expression between the each groups, and PCOS obesity group is the lowest(P<0.05).5. Apelin is positively correlated with Foxo1 a m RNA level, and it is negative related with GDF-9 m RNA level.Conclusion:1. The levels of Apelin mRNA and protein expression in PCOS patients are increasing significantly in PCOS granulosa cells; the levels of GDF-9 m RNA and protein expression are lower in PCOS patients.2. Apelin level is closely related with Foxo1 a and GDF-9. It may participate in abnormal follicular development process with PCOS.Part ? Effects of Apelin on proliferation and apoptosis of the human ovary granulosa cellsContext:Apelin involved proliferation and differentiation regulation in a variety of in human cells, and it plays an active role in terms of resistance to obesity and IR. Obesity promoted abnormal follicle development, proliferation and apoptosis of granulosa cells is the inevitable result of follicular development or atresia. The results show that Apelin with high expression in human PCOS granulosa cells in the part ? and ?. Apelin is an important factor and relevance with GDF-9 and Foxo1 a, two regulating proliferation and apoptosis of granulosa cells clearly in relevance. But it not clear that the effect of Apelin with proliferation and apoptosis in human granulosa cells.Objective:Realize the effect of Apelin with proliferation and apoptosis in granulosa cells, and the possible regulatory role of Apelin in follicular.Design:With the same material as the second part, we collected follicular fluid that undergoing IVF due to tubal factor or male factor infertility and extracted ovarian granulosa cells and made their in primary culture, when cells were adherent grown, according to different adding medium grouping: the group which add Apelin medium is experimental group( low concentration group: Apelin10-8 mol/L, high concentration group: Apelin10-10mol/L). The control group is add the same volume of serum-free DMEM medium / F12(equal volume serum-free DMEM/F12 nutrient solution), culture was continued after 12 h, 24 h, 48 h later, MTT-OD and Annexin V-FITC / PI methods were used to detect the effect of Apelin with proliferation and apoptosis in granulosa cells.Results:1.Apelin can promote cell proliferation, MTT-OD value was detected after 12h?24h?48h later:Apelin low concentration group:(0.327±0.033)vs(0.562±0.046)vs(0.669±0.018);Apelin high concentration group:(0.283±0.015)vs(0.544±0.038)vs(0.567±0.051);control group:(0.149±0.017)vs(0.295±0.011)vs(0.321±0.024);MMT-OD value of experimental group and control group have statistically difference at the same time node(P<0.05);MMT-OD value of 48 h in low concentration group is the highest group(P<0.05).2.Apelin inhibits cell apoptosis, the apoptosis rate of 12 h, 24 h and 48h:low concentration group(8.21±0.53)% vs(11.41±1.01)% vs( 7.12±0.89)%;high concentration group(10.15±0.73)% vs(14.86±1.47%)vs(18.64±1.76)%;control group(12.94±1.29)% vs(21.93±1.65)% vs(23.64±1.76)%;There have statistically difference between the each group in the same time(P<0.05);the 48 h later apoptosis rate is the lowest in low concentration group(P<0.05).Conclusion:Apelin has the effect on proliferation and apoptosis of human ovary granulosa cells, and the concentration of Apelin10-8mol/L is the most statistically when cells were cultivated 48 h late.Part ? The role of Apelin in the regulation of expression in human granulosa cells and the underlying molecular mechanismContext:PI3K/Akt has been shown to paly key roles in the reglation of ovulation. Apelin is important molecule that regulates many ovarian functions under normal physiological and pathological conditions. GDF-9?Foxo1a and Bcl-2 were expressed in human granulose cells. However, to date, whether they will be regulated by Apelin's level, which in turn contribute to the process of ovulation, remains unknown.Objective:To investigate the effect of Foxo1a?GDF-9 and Bcl-2 expression by Apelin in human granulosa cells. And elaborate the regulatory role of Apelin in PCOS disease outcome process.Design:With the same material as the second part, we collected follicular fluid that undergoing IVF due to tubal factor or male factor infertility. We extracted ovarian granulosa cells and made their in primary culture. We observed phosphorylation of Akt protein by Apelin after 24 hours, using the technology by Apelin-si RNA and PI3 K inhibitor LY294002 and HIMO that Akt's signal blocker that cells be transfected with the intervention process. Then we detected MTT-OD value after 12 h and 24 h and 48 h later. And learn the treatment of proliferation and apoptosis for post-intervention granulosa cells by Apelin.Results:1. Apelin can activate the channel signal Akt protein kinase,and phosphorylate it, at its peak after 30 minutes later; Apelin-si RNA group and PI3 K signal blocker LY294002 Group and HIMO group Akt signal blocker all can inhibit Akt kinase phosphorylate.2. Apelin-si RNA could transfected with the intervention process for cells, the maximum transfection efficiency of 60% at the time 48 hours later.3. Apelin-si RNA group, blocker LY294002 group, blocker HIMO group could inhibit the proliferation and apoptosis for granulosa cells.4. When Apelin's action was blocked, the expression of Foxo1 a up-regulated, the expression of GDF-9 and Bcl-2 were down-regulated.Conclusion:Apelin contral downstream gene of Foxo1a?GDF-9 and BCL-2 in PI3K/Akt signaling pathways, and it effect proliferation or apoptosis for granulosa cells, and participates abnormal follicular development in PCOS women. |
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