Expressions And Significance Of Neuropeptide Y And Its Receptors In Cardinal Ligament And Anterior Vaginal Wall Tissue Of Pelvic Organ Prolapse

As the population aging, more and more people pay attention to pelvic floor dysfunction disease. It has been known as a disease of older, mainly including pelvic organ prolapse and stress urinary incontinence. POP and SUI often co-exist, and it has various etiology and pathogenesis. In recent years, the study of the PFD focused on imaging, collagen metabolism, bio-mechanics, estrogen and its receptor, etc., while the little nerve-muscle physiology. The normal vaginal wall is rich in nerve fibers, according to the mechanism of pelvic floor nerve injury, suggesting the changes in pelvic organ neurotransmitters may also be involved in the pathogenesis of PFD. As early as in the eighties, the study found that there were peptidergic nerve fibers within the vaginal wall, and in human reproductive system also found the existence of vasoactive intestinal peptide, neuropeptide Y, substance P, calcitonin generelated peptide, somatostatin, etc. Vasoactive intestinal peptide and neuropeptide Y is most abundant in all neuropeptide. In 1982, NPY was a polypeptide belongs to pancreatic polypeptide family which was first purified from porcine brain tissue by Tatemoto in the Swedish Karolinska Institute, which. In order to explore the relationship between the expression of neuropeptide Y and its receptor and the occurrence and development of POP, we use the method of detecting the main pelvic ligaments and vaginal tissue.1,Tissue samples and groupsThirty-two POP and seventeen NON-POP cardinal ligament and paries anterior vaginase tissue specimens were obtained from patients underwent surgical resection at Shengjing Hospital of China Medical University between 2008 January and 2009 October. Specimen collection were approved by the Ethics Committee and obtained informed consent by the patients themselves or their families, and signed informed consent. We used the diagnostic criteria of the POP-Q standards published by the International Continence Society in 1996. A total of 16 cases with degreeⅠandⅡof POP for A group; a total of 16 cases with degreeⅢandⅣof POP for B group. All the samples were divided into two parts. One was fixed by formalin and embedded in paraffin, and the other one was put into Eppendorf tube and preserved in liqud nitrogen.2,MethodsImmunohistochemistryImmunohistochemical staining was performed by the streptavidin-peroxidase (S-P) method. The sections were deparaffinized, water processed, treated by pepsin to expose antigen and incubated with primary antibody at 4.0℃overnight. The color was developed with diaminobenzidine and the sections were counterstained with hematoxylin. For the nonspecific reagent control, the primary antibodies were replaced by PBS. Results assessment:Use immunohistochemical semiquantitative method:choose 5 high power field randomly(×400) and we think that it is positive expression if it has buffy in intercellular substance.RT-PCR(1) Extraction of RNA:Take out of the specimens from liqud nitrogen, and extract RNA using the method of TRIzol(using 1ml TRIzol per 100mg tissue).(2)RT reaction:Put 1.25μl RNA which has been extracted into reverse transcription reaction liquid to undertake reverse transcription.(3)PCR amplificationNPY-Y1 primers:upstream primer 5'-CCA CTG ACC TCT ACA CCC AC-3 downstream primer 5'-CCA GCA GGC TCC AAA G-3'; NPY-Y2 primers:upstream primer 5'-AGG AGA CAG GAG CGA GTA-3', downstream primer 5'-AAG GTA AAG CGA GTT CAG-3'; within the frame of reference usingβ-actin, the upstream primer 5'-GTG GGG CGC CCC AGG CAC CA-3', downstream primer 5'-CTC CTT AAT GTC ACG CAC GAT TTC-3'. Procedure of PCR amplification was set according to that:94℃40s, anneal 40s,72℃40s,35 cycles。The anneal temperature ofβ- actin,NPY-Y1 and Y2 were 55.5℃,52.5℃,50.7℃. Before amplification, they were received in 94℃for 3min, and extended for 7min after amplification.(4)Electrophoresis5μl production of PCR was underwent separation in 2% Agarose Gel Electrophoresis. Then take a picture under ultraviolet lamp, and analyse the picture.3,Statistical analysisThe statistical package SPSS 13.0 was used for all analyses. The chi-square test was used to determine the correlation between NPY expression and clinical pathological factors. The comparison of NPY-Y1 and Y2 mRNA among group A, B and control group was analized by analysis of variance. Values of P<0.05 were considered statistically significant. The relationship between NPY-Y1 and Y2 mRNA expression in cardinal ligament and paries anterior vaginase tissue was analized by pearson test.Results1.Expression of NPY in the cardinal ligament and paries anterior vaginase tissueIn patients of each group, the cardinal ligaments are mainly constituted by smooth muscle cells, fibroblasts and collagen ingredients, containing a considerable number of blood vessels and nerves. Vaginal wall is constituted by mucous layer, muscular layer and adventitia pose from outside to inside under microscope. Mucosa is composed of epithelium and lamina propria, and the epithelium is stratified pinacocyte which keratinized not obviously while the lamina propria is mainly composed of connective tissue, in which we can see smooth muscle tissues, small blood vessels, capillaries and small nerve tissue structure. NPY express in both the cardinal ligament and paries anterior vaginase tissue in all patients. Observed under the microscope, NPY mainly expressed in the smooth muscle cells, collagen composition and fiber cells, with relationship to blood vessels. The positive expression rate of NPY in cardinal ligament and paries anterior vaginase tissue of POP group is 25.0%,34.4%, lower than the control group 76.5%,70.6%(P=0.001,0.020). In cardinal ligament and paries anterior vaginase tissue of the POP group, the expression of NPY in group B prolapsed is less than group A, respectively,12.5%VS50.0%,25.0%VS62.5%, There was a significant difference according statistical analysis of two groups (P=0.024,0.035). 2. Expression of NPY-Y1 and Y2 mRNA in the cardinal ligament and paries anterior vaginase tissueIn RT-PCR, we found that NPY-Y1 and Y2 mRNA were expressed in the cardinal ligament and paries anterior vaginase tissue.In the cardinal ligaments, the NPY-Y1 mRNA expression level in POP group has the tendency to increase contrast to the control group, and the difference was statistically significant (P=0.003). The difference between group A and B isn't statistically significant (P=0.342). The difference between group A and control group as well as between group B and control group are statistically significant (P=0.016, P =0.001). The NPY-Y2 mRNA expression level in POP group has the tendency to increase contrast to the control group, but the difference is not statistically significant (P=0.170). The differences between group A and B, as well as between group A and control group between group B and control group are not statistically significant (P= 0.492, P=0.240, P=0.065).In the paries anterior vaginase tissue, the NPY-Y1 mRNA expression level in POP group has the tendency to increase contrast to the control group, and the difference was statistically significant (P=0.012). The difference between group A and B isn't statistically significant (P=0.861). The difference between group A and control group as well as between group B and control group are statistically significant (P=0.013, P =0.008). The NPY-Y2 mRNA expression level in POP group has the tendency to increase contrast to the control group, and the difference was not statistically significant (P=0.002). The differences between group A and B, as well as between group A and control group are not statistically significant (P=0.096, P=0.559). The difference between group B and control group is statistically significant (P=0.002).3. The relationship between NPY-Y1 and NPY-Y2 mRNA expressionWhile analizing the relationship between NPY-Y1 and Y2 mRNA with pearson test in POP group, results showed that:in the cardinal ligament tissue, r=0.415, P= 0.018, prompted that NPY-Y1 and Y2 mRNA expression in the POP group was positively related, with relevant higher degree. In the paries anterior vaginase tissue r=-0.216, P=0.235, prompted that NPY-Y1 and Y2 mRNA expression in the POP group had no correlation. Conclusions1,In POP group, the expression of NPY in both cardinal ligament and paries anterior vaginase tissue may have a relationship with the genesis and development of POP.2,In POP group, NPY-Y1 in cardinal ligament may have contributed to the occurrence of uterine prolapse.3,In POP group, NPY-Y1 and Y2 in paries anterior vaginase tissue may jointly promot the occurrence of uterine prolapse.4,In POP group, NPY-Y1 and Y2 in cardinal ligament may have synergistic effect in the genesis and development of POP, the main ligament in patients with uterine prolapse in NPY-Y1 and Y2 may be the occurrence and development of uterine prolapse.