The Extra-corporeal Cytology Research On The Rregulatory Role Of PGE2 In Rat NR8383 Macrophage Promoting HUVECs Migration And Its Ability To Form Vessels

Embryo implantation is the initial and key step for successful pregnancy.Human embryo implantation usually occurs in the 6-8 days after ovulation during this period is called peri-implantation or implantation window, this is key speed limit steps for pregnancy occurrence and initiating. Numbers of variables decide the success of implantation,among which sound endometrial receptivity is a key element. Studies show that the development of endometrial is divided into three phases:pre-receptivity stage, receptivity stage and non-receptivity stage. Only in receptivity endometrial accepts the embryo implantation. The abnormality of endometrial receptivity is the main cause for the failure of implantation. There are many factors affecting endometrial receptivity, has not been fully clarified till now. Studies demonstrated that the physiological angiogenesis process of the reproductive system in peri-implantation plays a critical role in the establishment of endometrial receptivity.Angiogenesis refers to the entire process of forming and development of capillary vessels in existing vessels, which is an important feature and prerequisite for tissue regeneration. Specific steps include:vascular endothelial cells migrating to area created by cracking of the original vessels, multiplication of capillary sprouts, growth and reconstruction to form a new arterial or venous. Angiogenesis in adult occurrs in pathological conditions, such as the inflammation and immune response, neoplastic development and transfer, as well as damage repair process. Notably, human physiological angiogenesis exists only in normal reproductive system of female in the childbearing ages, its throughout the entire cycle of ovarian and endometrial cycle, reflected in the formation of ovary metarion vascular networks and the creation of micro environment for the formation of endometrial vessel, whcih is essential for development of the female reproductive system and maintenance for endocrine, reproductive functions.Studies show that the peak of endometrial local vessel formation is an obvious signnal of peri-implantation stage. When blocks in r endometrios vessel formation happened, implantation rate is significantly lower, and fetal rate of abortion increases obviously, which indicates the formation of endometrial vessel lay an important role in implantation and pregnancy maintenance progesterone created after ovulation is the main source for progesterone,p4,one of the most significant histology characteristics is the formation of a rich blood vessels of the mesh and its blood flow velocity is the highest in all glandular, whose role is to facilitate the transformation of endometriosis, to enhanced endometrial recepticvity, to facilitate implantation and maintenance of pregnancy at the early stages. The formation of metoarion vascular networks after ovulation is closely related with secretion of progesterone. The study found that when the formation of metoarion vascular network was abnormal, progesterone levels decrease significantly, the implantation has failed. As a result, angiogenesis of the reproductive system in peri-implantation period plays a key role in successful implantation and pregnancy maintenance, while the regulatory mechanism in this stage has not yet been fully verified, which worthy of further study.Studies demonstrated that the female reproductive system partial macrophage is vital in the maintenance of normal female reproductive activities. Inperi-implantation stage, the quantity of endometrial partial macrophage increases significantly, and will collecting around the point of implantation, indicating they are closely related to this process. Further researches found that macrophage distribution in peri-implantation stage is closely related with the distribution of new vessels, indicating endometrial macrophage is important for endometrial angiogenesis in peri-implantation period. Peri-ovulation period also exists the raise of macrophage 5,304,722 and obvious metoarion collection. Some foreign scholars knocking out macrophage in the ovary of mice in their ovulation period, found that the the implantation has failed, and by IHC the researchers observed that the formation of metoarion vascular network was abnormal, the structure of vessels were incomplete and the progesterone level has also significantly reduced. The results of the study indicated that macrophage by participating in the formation of metoarion vascular network after ovulation plays a key role in the success of implantation. Numerous researches show that macrophage can produce a variety of cytokines promoting angiogenesis, of which the most important factor is vascular endothelial growth factor (VEGF). VEGF is one of the most important factors promoting angiogenesis, it is also significant in physiopathological process such as the occurrence, development and transformation of tumor and damage repair. Macrophage is in one of the important sources for VEGF, macrophage participate in the angiogenesis process by secreting cytokines such as VEGF which can promote the formation of vessels.Many studies suggested that partial macrophage of female reproductive system by participating in angiongenesis plays a key role in the success of implantation. However its regulatory mechanisms are still not fully clarified.In peri-implantation period, the local micro environment of female reproductive system partial is undergoing significant changes, multiple factors including estrogen and progesterone all rising. Among them, we found that prostaglandins E2, PEG 2, may bean important factor regulating the reproductive system partial macrophage promoting angiogenesis. PGE2 are vital for normal female reproductive activities, and in peri-implantation period its level in endometrial and ovaries levelsrise. And in the surface of macrophage surface, there are specific receptors for the expressions PGE2. All these demonstrated that the rise of PGE2 in peri-implantation period may interact with the reproductive system partial macrophage thus to adjust macrophage role in promoting angiogenesis. However relevant studies on mechanism were not reported yet.This studies on the use of extracorporeal cytology testing methods, treating mouse NR8383 macrophage with different concentration of PGE2 handling large, using fluorescent quantitative PCR test methods, observing the whether the messenger express level is changed in different concentration of PGE2 handling the RAT NR8383 macrophage VEGF mRNA, to reveal PGE2 can affect macrophage VEGF within the expression of mRNA level so as to affect macrophage promoting angigenesis. Then we used transwell small room cells migration experiments and matrigel experiment to observe whether under different concentrations PGE2, NR8383 macrophage’s ability in promoting HUVECS migration and angiogenesis changes, to dissccusswhether PGE2 can affect macrophage’s capacity in promoting angiogenesis. Lastly, we using PGE2 receptors specificity inhibitors AH6809 PGE2 EP2-specificity inhibitors) and AH23848 PGE2 (EP4-specificity inhibitors) to handle the RAT NR8383 macrophage, respectively, as well as Quantitative fluorescent PCR technology transwell small room cells migration experiments and matrigel experiment, to observe whether PGE2 receptors specificity inhibitor can impede PGE2 regulating macrophage’s role in angiogenesis. The above study are key to further clarify the regulatory mechanism of female reproductive system partial micro environment of macrophage promoting angiogenesis in peri-implantation period, and provides a theoretical basis for furthur study in this mechanism.Chapter one PGE2 impact NR8383 macrophage VEGF MRNA expressionPurposeIn order to explore whether PGE2 which are important to normal female reproductive system functions in peri-implantation period play a pivotal role in the regulating PGE2 female reproductive system partial macrophage signal elements, we pick rat macrophage NR8383 in extracorporeal cytology experiment, handling them with a 0/0.1 nmol/1 PGE2/1 nmol/1 PGE2 respectively, by detecting the expression level of the RAT NR8383 VEGF mRNA within macropahge, to verify that PGE2 can affect NR8383 macrophage’s ability to secret VEGF.MethodIn logarithmic phase rat NR8383 macrophage, process them withOnmol/1 Pge2,0.1nmol/L PGE2,lnmol/L PGE2 solution respectivelyfor 5 days. After processing, place the rat NR8383 macrophage in incubator at 37℃,5%CO2. After 5 days, collect ratNR8383 macrophage processed with different PGE2 level processing, 1000r/min,5 minutes centrifugal, Remove supernate, and then rinse with the PBS, again after 5 minutes of 1000r/min centrifugal, add trizol and then reset rat NR8383 macrophage, next extract the RNA of theseNR8383 macrophage, use PCR to detect the expression level of VEGF mRNA in NR8383 macrophage. All the data record with average ± standard deviation (χ±S). Using one-was ANONA among various groups, P<0.5there are statistical differences.Results1.PGE2 can promote VEGE mRNA expression in rat NR8383 macrophageThe expression of VEGF mRNA was higher in PGE2-stimulating group than the group without PGE2 stimulating(P<0.000).2. PGE2’s ability of promoting the expression of rat NR8383 macrophage VEGF mRNA increases with the concentration of PGE2.The expression of VEGF mRNA was higher in O.lnmol/L PGE2-stimulating group than the group without PGE2 stimulation(P<0.05);No significant difference was observed on the expression of VEGF mRNA between the O.lnmol/L PGE2-stimulating group and 1nnmol/L PGE2-stimulating group (P>0.05)ConclusionsThe results show that after processing with PGE2, the expression of VEGF mRNA in macrophage increased significantly. Also, further study found that, PGE2’s ability of promoting the expression of rat NR8383 macrophage VEGF mRNA are positively correlated with the concentrationofPGE2, with PGE2 concentration increasing,the VEGF mRNA expression level of Rat NR8383 macrophage also raised.Therefore, the experimental results indicates PGE2 can enhance the RAT NR8383 macrophage secret VEGF, making it possible to enhance its capacity of promoting angiogenesis.Chapter Two PGE2 affects the NR8383 macrophage’s ability in promoting HUVECs migrationPurposeAs shown previously, PGE2 can increase the expression of VEGF mRNA in RAT NR8383 macrophage, maybe by increasing its secretion, thereby enhancing the RAT NR8383 macrophage’s ability in promoting angiogenesis. Thus, We used extracorporeal cytology experiment -transwell, processing HUVECs with different concentration of PGE2 processed NR8383 rat macrophage cultivation supernate, observeing the number of HUVECs migration, verify whether PGE2 can enhance the RAT NR8383 macrophage promoting HUVECs migration, further argued PGE2 can enhance the RAT NR8383 macrophage promoting angiogenesis.MethodSelect the logarithm phase rat NR8383 macrophage, resuspend the cells with 10% of fetal bovine serum RPMI-1640, adjust the cell density to 1 X105/ML, processing RAT NR8383 macrophagefor five days with0,0.1nmol/1 PGE2 lnmol/1 PGE2, respectively.5 days later, centrifugal with 2000 r/min for 5 min, remove debris, extract supernate for experiments. Select the logarithm phase HUVECs, resuspend with 10% fetal bovine serum RPMI-1640 culture, adjust the cell density to 1 X106/ml in transwell small room cell culture board upper room add 100 μ L HUVECs suspension; placing different PGE2 concentration processed rat NR8383 macrophage into the lower room, place the board in incubator and cultivate for 12h. After 12h, remove the transwell room, with buds gently wipe the surface cells with cutton buds; fix with 10% formaldehyde for 30 min; wash with PBS for 2 times,5 mins for each time; Crystal purple dyeing for 30 min, reinse repeatedly with clean water, dry; remove Polycarbonate membrane, neutral gum package. Under the microscope (X 200) count cells migrated to the lower surface of the membrane, each film random count 5 horizons, use the average as the chemotaxis number. Using one-was ANONA among various groups, P<0.5 there are statistical differences.Results1.After PGE2 processing, the RAT NR8383 macrophage’s ability in promoting HUVECS migration was enhanced.The numbers of migrating HUVECs in O.lnmol/L PGE2-stimulating group and lnnmol/L PGE2-stimulating group were higher than the number of group without PGE2 stimulation(P<0.000).The results showed that PGE2 could enhance the ability of NR8383 stimulating HUVECs migration.2.PGE2’s ability in enhancing rat NR8383 macrophage promoting HUVEC migration is positively correlated with PGE2 concentration PGE2.The numbers of migrating HUVECs were higher in 0.1nmol/L PGE2-stimulating group than the numbers of group without PGE2 stimulation(P<0.000); The numbers of migrating HUVECs were higher in lnmol/L PGE2-stimulating group than the numbers of lnmol/L PGE2-stimulating group(P<0.05).The results showed that the ability of PGE2 enhancing NR8383 to stimulate HUVECs migration was related to its concentration.ConclusionsTranswell experimental results indicated that after processing with PGE2, rat NR8383 macrophage’s ability of promoting HUVECs migration has been markedly strengthened, and such kind of ability increasing with the concentration of the PGE2 rising. Research findings suggest PGE2 can enhance the RAT NR8383 macrophage promote intravascular skin cells collection, which is a key step for angiogenesis.This further indicates PGE2 canenhance the RAT NR8383 macrophage’s ability in promoting angigenesis.Chapter Three PGE2 affects NR8383 ability in promoting HUVEC cells forming vesselsPurposeThe above study found that canPGE2 enhances rat NR8383 macrophage’s ability in promoting HUVECs migration. The results also indicate PGE2 might enhance thecollection of vascular endothelial cells so as to improve the macrophage’s ability in promoting angiogenesis. However Angiogenesis is a complex process in which the nascent intravascular skin cells forming vessel-like structure is a crucial step. We therefore introduce further matrigel matrix experiment, process ratNR8383 macrophage with different PGE2 concentration, extract varied supernate coming from different processing and observethe size of HUVECs forming vessles. So as to further verify theregulatory role of PGE2 in rat NR8383 macrophage promoting angiogenesis.MethodBefore the experiment,pre-cooling the materials may contact with matrigel such as tip head, nuturiton board and so on. Place them in the refrigerator at -20℃.Then put the matrigel to 2-8℃ refrigerator overnight to melt. When the matrix turn into liquid, render 60 μ L to each of 96 holes in the cultivation board, place in 37℃ so as to form matrix. Using the above method obtained different NR3838 cells cultivation supernate to resuspend HUVECs, inoculation 96 hole which has already been laid out by matrix,1.5～2×104 cells for each hole, each section 3 replicas of holes. Place the board into incubator for another 6 h. Remove the cell culture board, observe under the Inverted microscope (X4) and take photographs, use image-pro plus software analysis the size of formed vessel. Using one-was ANONA among various groups, P<0.05 there are statistical differences.Conclusion1.PGE2 can enhance NR8383 rat macrophage promoting HUVECs ability of forming vessels.The area of tube formation of HUVECs in O.lnmol/L PGE2-stimulating group and 1nnmol/L PGE2-stimulating group were higher than the area of group without PGE2 stimulation(P<0.000).The results showed that PGE2 could enhance the ability of NR8383 stimulating HUVECs’ tube formation.2.PGE2’s ability of enhancing NR8383 rat macrophage promoting HUVECs capability of forming vessels increases with the concentration of PGE2 used in processing.The area of tube formation of HUVECs were higher in O.lnmol/L PGE2-stimulating group than the area of group without PGE2 stimulation(P<0.000); The areawas higher in 0.1nmol/L PGE2-stimulating group than the area of lnmol/L PGE2-stimulating group(P<0.05).The results showed that the ability of PGE2 enhancing NR8383 to stimulate HUVECs’ tube formation was related to its concentration.Discussion:Matrigel experiment results indicate, PGE2 processed NR8383 rat macrophage supernate can significantly promoting HUVECS froming vessels, the size of formed vessels increased significantly; and such kind of promoting ability of rat NR8383 PGE2 macrophages increases with the rise of PGE2 concentration. The results of the further confirmed that PGE2 might affect the process of VEC forming tubular structure, to enhance rat NR8383 macrophage capacity of promoting angiogenesis. This further prompting PGE2 is akey regulatory factor in the process of macrophage promoting angiogenesis. It is worthwhile for us to further explore the specific molecular signaling mechanism.Chapter Four PGE2 specificity receptors antagonist suppresses PGE2 regulatoryfunction in rat NR8383 macrophage enhancing HUVECs promoting angiogenesisPurposeThe above-mentioned related Experiment results show that PGE2 can act on NR8383 PGE2 rat macrophage, improve the expression of VEGF expression in NR8383 rat macrophage and can significantly strengthen NR8383 rat macrophage promoting HUVECS migration and the capacity of forming vessels and to enhance NR8383 rat macrophage promoting angiogenesis. In order to further verify the interaction between PGE2 and rat NR8383 macrophage is the direct cause of enhancing rat NR8383 macrophage ability of promoting angiogenesis, we chose PGE2 EP2-specificity antagonist ---H6809 and PGE2 EP4-specificity antagonist--AH23848 NR8383 to process rat macrophage NR8383 by observing the expression level of VEGF mRNA and the effect of processed rat NR8383 macrophagesupernate on HUVECS migration and forming vessels to further confirmed that PGE2 by acting on the surface specificity receptor of rat NR8383 rat macrophage, to enhanced NR8383rat macrophage ability in promoting angiogenesis.MethodSelect the logarithm phase rat NR8383macrophage, useNR8383 rat macrophage processed withl nmol/1 PGE2as a control group, lnmol/1 Pge2+10nmol/1 PGE2 EP2-inhibitors Ah6809+10nmol/1 PGE2 EP4-inhibitors AH23848 processed rat NR8383 macrophage as a experimental group. After 5 days of cultivation. Use PCR to test the expression level of VEGF mRNA in rat NR8383 macrophage; and use transwell cell migration experiments and matrigel vessel forming experiment to detect rat NR8383 macrophage’s ability in promoting HUVECs capability of collection and forming vessels. Specific experimental methods are described above.Results:1.PGE2 specificity receptors antagonist can inhibit PGE2 enhancing the expression of VEGF mRNA in rat NR8383 macrophage.The expression of VEGF mRNA was less in PGE2-stimulating group with AH6809 and AH23848 than the PGE2 stimulating group (P<0.000).The result showed that AH6809 and AH23848 can inhibit PGE2’s ability of stimulating VEGF mRNA expression in NR8383.2. PGE2 specificity receptors antagonist can inhibit PGE2’s ability of enhancing rat NR8383 macrophage promoting HUVECs migrationThe migrating numbers of HUVECs was less in PGE2-stimulating group with AH6809 and AH23848 than the PGE2 stimulating group (P<0.000). The result showed that AH6809 and AH23848 can inhibit PGE2’s ability of stimulating HUVECs migration through NR8383.3. PGE2 specificity receptors antagonist can inhibit PGE2’s ability of enhancing rat NR8383 macrophage promoting HUVECs capability of forming vessels.The area of tube formation was less in PGE2-stimulating group with AH6809 and AH23848 than the PGE2 stimulating group (P<0.000). The result showed that AH6809 and AH23848 can inhibit PGE2’s ability of stimulating HUVECs’ tube formation through NR8383.DiscussionThe results demonstrate that, when processing rat NR8383 macrophage with PGE2 specificity receptors antagonist,cansignificantly inhabit the effect of improving VEGF mRNA expression of rat NR8383 macrophage, and significantly suppress the ability of PGE2 enhance rat NR8383 macrophage promoting HUVECs ability to form vessels, inhibit PGE2 promote NR8383 rat macrophage promote angiogenesis effect. Therefore the findings confirmed that tthere are interactions betwseen PGE2 and rat NR8383 macrophage by directly act on the according surface recepter of the later, PGE2 enhanced the effect of rat NR8383 macrophage promoting angiogenesis. Further prompting, PGE2 is a key factor in regulating the process of macrophage promoting angiogenesis, which provide ffundemental theroretical basis for the follow-up researches on whether PGE2 is a key regulatory factorin partial macrophagepromoting angiogenesis activities in female reproductive system.