Fine Qualitative Study On The Structure Of Cervical Cancer And The Distribution Of The Nerve Fibers

Nowadays, cervical cancer is the second most common gynecologic malignancy after breast cancer, with nearly 50 million new cases and the death of about 23 million people each year, which is a serious threat to women’s lives and health.The new cases and the mortality each year in developing countries are both higher than in developed countries. Our country is one of the areas of high incidence of cervical cancer. Despite the persistent infection of HPV is known as the main cause of cervical cancer, many countries have carried out large-scale HPV vaccination to actively prevent the occurrence of cervical cancer after the successful launch of HPV vaccine, and cervix cytology combined detection of HPV infection have been widely applied in women’s screening for early detection and early treatment, however, the morbidity and mortality of cervical cancer in our county still maintain at a higher level, there is not an perfect cure method for cervical cancer so far, cervical cancer remains a major public health problem at present. Therefore, to explore the possible therapeutic direction, the further study on the pathogenesis of cervical cancer is very necessary.The development of tumor is closely related to the microenvironment, the continuous interaction of tumor cells and their surrounding environment promots the development and metastasis of tumor. Studies about tumor and micro environment mainly concentrated in the following several kinds of stromal elements, such as endothelial cells, inflammatory cells, immune factors and so on. In addition to the immune and inflammatory cells, basic tissue structure is also an important part of the microenvironment. About the study of the basic tissue structure of the tumor, many researches focused on the blood vessels and lymphatic vessels. Study confirmed that the formation of blood and lymphatic vessels are closely associated with the pathogenesis of tumors, which plays an important role to the development of tumor. This view has been accepted by domestic and foreign scholars generally, and it attracts people to focus on the function of basic tissue structure in tumor. However, less study focus on the other tissue structures.In the human body, nerve often accompany with vascular and lymphatic vessels. That perineural invasion (PIN) occurs in pancreatic cancer and other malignancies has been recognized by many scholars, however, the traditional view was that there is no nerve fiber in tumor tissue. Until the 21st century, many foreign researchers have confirmed the distribution of nerve fibers in bladder cancer, prostate cancer and other malignant tumors. Some studies have shown that there may also be neurogenesis in tumor. At the same time, the relationship of the neurotransmitters, neurotrophic factors and tumor biological behavior is also gradually known by people. Therefore, the studies above suggest us that the nerves exist in some tumor tissues, and it may have a certain relevance with the development of tumor.About the study of the basic tissue structure of cervical cancer, many researches focused on the blood vessels and lymphatic vessels, however, about the study of the nerve fibers in cervical cancer, only few reports on the PIN of cervical cancer, no research on the distribution of nerve fibers in cervical cancer tissue and the relation between the nerve fibers and the cancer tissue. So it is necessary for us to make a detailed qualitative research on the basic tissue structure of cervical cancer to comprehensive understand the tissue elements, and further exploring the distribution of nerve fibers to help to understand its relevance with the pathogenesis of cervical cancer.In our previous study, we have had the qualitative researches of organizational structures about the cardinal ligament, uterosacral ligament and paracolpium after hysterectomy respectively, including the qualitative and quantitative comparison study of the nerves, which provides a certain experimental experience and technology base for this study. This experiment is divided into three parts, research details:The first part:The qualitative research on the fine histological structure of the cervical cancerThis study was to qualitative analysis the basic organization structure of cervical cancer, including the inherent connective tissue, vascular tissue and nerve fibers. About the study of nerve fibers, we used the neural specificity antibody S-100 to mark the total nerve, which could detect whether there were nerve fibers in cancer tissues. Further study on the nature and type of the nerve fibers, we used the growth associated protein 43 antibody testing newborn nerve, special staining detecting autonomic nerve, and tyrosine hydroxylase TH antibodies, intestinal vascular active peptides VIP detecting sympathetic and parasympathetic nerve respectively.Objective:The aim was to investigate the fine structure of cervical cancer and realize the composition.Methods:33 cervical cancer tissues were collected from patients who had underwent radical hysterectomy in NanFang hospital during June to October in 2014. Every tissue was fixed up by 10% formalin, to be embedded by routine paraffine, to be sliced up. At last, HE staining, immunohistochemical staining and some other specific stain were used for qualitative research.Results:One:Staining results of the common structures in cervical cancer tissues1) HE staining results:33 specimens were visible in a large number of different degree of differentiation of cancer cells, a large number of blood vessels, including small veins and capillaries. In addition, abundant fibrous connective tissues could be found in the tissues.2) Fibrous connective tissue staining results:Improved Masson trichromatic staining showed that all the 33 cases were rich in dark green collagen fibers, while no smooth muscle fibers dyed to bright red. Collagen fibers were distributed around the nests in carcinoma stroma, some of which were in the artery.Aldehyde fuchsin staining showed that purple elastic fibers were found in the 33 cases. Elastic fibers were distributed around the nests in carcinoma stroma, most of which were in the artery.3) Vascular immunohistochemical staining results:Immunohistochemical staining of CD-34 antibody, the specific markers of vascular endothelial cells, showed that 33 cases of cervical cancer tissues contained rich brown blood vessels, and the invasion of the cancer cells could be found in part of blood vessels.4) Lymphatic histochemical staining resultsD2-40 antibody was used as the specific marker of lymph vessels. There were many brown lymphatic vessels distributed in the carcinoma stroma of cervical cancer tissues. Cancer invasion can be found in part of the lymphatic vessels.Two:Staining results of nerve fibers1) Total nerve immunohistochemical stainingImmunohistochemical staining showed that the positive number of S-100 staining were 14 in cervical cancer tissues, with the ratio is 42.42%.The nerve fibers were distributed in the tumor stroma.2) Neoneurogenesis immunohistochemical stainingImmunohistochemical staining of GAP-43 antibody, the specific marker of newborn nerve, showed that the positive number of newborn nerves was 11 cases, and the newborn nerves were distributed in the carcinoma stroma, the rate of S-100 positive proportion was 78.57%.3) Autonomic nerve immunohistochemical stainingSpecific staining of chromotropic acid 2R bright green method showed that cervical nerve myelin stained negative. Furthermore, TH and VIP antibody were used as the specific markers of sympathetic nerve and parasympathetic nerve respectively, and the results showed that the positive number of sympathetic and parasympathetic nerve was 7,5 cases respectively, and there were 3 cases for both the sympathetic and parasympathetic nerve positive, the rate of S-100 positive proportion was 50%, 35.71% respectively.Conclusions:In addition to the basic structure of the cancer cells, fibrous connective tissue, blood vessels and lymphatic vessels, cervical cancer also contained nerve fibers, and most of the nerve fibers were newborn nerves, the content of sympathetic nerves was slightly higher than parasympathetic nerves.The second part:Comparative Study of the distribution of nerve fibers among cervical cancer tissue, tumor-adjacent tissue and normal cervical tissue.Objective:To compare the distribution of nerve fibers in cervical cancer tissue, the tissue adjacent to carcinoma and normal cervical tissue, and to explore the characteristics of the distribution of nerve fibers in cervical cancer.Methods:65 specimens were collected into our study, including 11 specimens of the tissues adjacent to carcinoma from fresh cervical carcinoma after radical hysterectomy,21 specimens of normal cervical tissues from patients with uterine fibroids or the uterus gland disease after total hysterectomy in Nanfang hospital of Southern Medical University, and 33 specimens of cervical cancer tissues from the preliminary experiment. Every tissue was fixed up by 10% formalin, to be embedded by routine paraffine, to be sliced up. At last, S-100 antibody, newborn nerve GAP-43 antibody, sympathetic nerve TH and parasympathetic nerve VIP antibody were all used for immunohistochemical staining of nerve fibers.Place the slides under the microscope models to collected images. The positive numbers of nerves, newborn nerves, sympathetic and parasympathetic nerves were recorded and analiysised after Immune organization chemical dyeing and taking photos. We used statistics software SPSS13.0 to analysis data. If P<0.05 then the difference has statistical significance. R*C chi-square test were used for the statistical analysis of comparison of the positive rate of nerve fibers in three groups.Results:One:Nerve fiber dyeing results in the three groups(1) The results of the distribution of the total nerves in three groupsThe positive rate of the total nerve among cervical cancer tissues, the tissues adjacent to carcinoma and normal cervical tissues were 42.42%,72.73%,76.19% respectively. There was a significant difference of the distribution of the nerves in three groups (chi-square=7.135, P=0.028). The positive rate of nerves in cervical cancer tissues was lower than tissues adjacent to carcinoma and normal cervical tissues.(2) The results of the distribution of the newborn nerves in three groupsThe positive rate of the newborn nerves among cervical cancer tissues, the tissues adjacent to carcinoma and normal cervical tissues were 33.33%,9.09%,4.76% respectively. There was a significant difference of the distribution of the newborn nerves in three groups (chi-square= 7.532, P=0.023). The positive rate of newborn nerves in cervical cancer tissues was higher than tissues adjacent to carcinoma and normal cervical tissues.(3) The results of the distribution of the autonomic nerves in three groupsThe positive rate of the sympathetic nerves among cervical cancer tissues, the tissues adjacent to carcinoma and normal cervical tissues were 21.21%、54.55%、 66.67% respectively, and the positive rate of the parasympathetic nerves were15.15%、18.18%、14.29% respectively.1) There was a significant difference of the distribution of the sympathetic nerves among cervical cancer tissues, the tissues adjacent to carcinoma and normal cervical tissues (chi-square=11.841, P= 0.003). The positive rate of sympathetic nerves in cervical cancer tissues was lower than tissues adjacent to carcinoma and normal cervical tissues.2) There was no significant difference of the distribution of the parasympathetic nerves among cervical cancer tissues, the tissues adjacent to carcinoma and normal cervical tissues (chi-square= 0.087, P= 0.957).Two:The distribution results of newborn nerves and autonomic nerves accounted for the proportion of total positive nerve in three groups.(1) The distribution results of newborn nerves accounted for the proportion of total positive nerve in three groups.The proportion of the newborn nerves accounted for the total positive nerves among cervical cancer tissues, the tissues adjacent to carcinoma and normal cervical tissues were 78.57%,12.5%,6.25% respectively. There was a significant difference of the total nerve positive percentage of the newborn nerves in three groups (chi-square=19.474, P=0.000). The total nerve positive percentage of the newborn nerves in cervical cancer tissues was higher than tissues adjacent to carcinoma and normal cervical tissues.(2) The distribution results of autonomic nerves accounted for the proportion of total positive nerve in three groups.1) Most of the nerve fibers were autonomic nerves among the three groups, and the sympathetic nerves were significantly higher than the parasympathetic nerves.2) There was no significant difference of total nerve positive percentage of the sympathetic nerves among cervical cancer tissues, the tissues adjacent to carcinoma and normal cervical tissues(chi-square= 5.182,P= 0.075).3) There was no significant difference of nerve positive percentage of the parasympathetic nerves among cervical cancer tissues, the tissues adjacent to carcinoma and normal cervical tissues (chi-square= 1.117, P= 0.572).Conclutions:We could conclude the types of nerve fibers distributed in the cervical cancer tissues:1) Despite the positive rate of total nerves in cervical cancer tissues was lower than the tissues adjacent to carcinoma and normal cervical tissues, the positive rate of newborn nerves was obviously higher than that of other two groups, further prompting that the main nerves in cervical cancer tissues were newborn nerves, and the nerve fibers in cervical cancer was different from the rest groups.2) The distribution of autonomic nerves was almost the same among cervical cancer tissues, the tissues adjacent to carcinoma and normal cervical tissues. Besides, the sympathetic nerves were significantly higher than the parasympathetic nerves.The third part:study on the correlation between the nerve fibers in cervical cancer tissues and the clinical pathological parameters.ObjectiveThe aim was to study the distribution of nerve fibers in 139 cases of cervical cancer tissues, and to explore the relation between the nerve fibers and the clinical pathological parameters.Methods:139 specimens were collected, including 31 specimens of stage ⅠA-ⅡA cervical cancer tissues from the preliminary experiment and 108 specimens of stage ⅠA-ⅡA cervical cancer tissues from patients under radical hysterectomy in Anhui province people’s hospital. S-100 antibody and newborn nerve GAP-43 antibody and autonomic nerves antibodies were all used for immunohistochemical staining of nerve fibers.The positive number of total nerves, newborn nerves, sympathetic nerves and parasympathetic nerves were recorded and analiysised after Immune organization chemical dyeing and taking photos. We used statistics software SPSS13.0 to analysis data. If P<0.05 then the difference had statistical significance. Independent sample t test was used for the statistical analysis of the relation between the nerve fibers and patients’age. R*C chi-square test was used for the statistical analysis of the relation between the nerve fibers and other clinical pathological parameters.The binary classification Logistic regression analysis was used for the correlation between cervical nerve fibers in the cancer tisssues and clinical pathological parameters.Results:One:The general resultsThere were 80 cases total nerve staining positive among the 139 cases of cervical cancer specimens, with the positive rate was 57.55%;The number of positive newborn nerves was 48 cases, and the positive rate was 34.53%; There were 46 cases sympathetic nerve staining positive,18 cases parasympathetic nerve staining positive, and 11 cases both sympathetic and parasympathetic nerve staining positive, with the positive rate was 33.09% and 12.95% respectively.Two:The relation between the distribution of the nerve fibers in cervical cancer tissues and the clinic pathological parameters.(1) The relation between the distribution of the total nerves in cervical cancer tissues and the clinic pathological parameters.1) There was a significant difference between the distribution of the total nerves in cervical cancer tissues and the age (t=2.713, P=0.008).The ages of patients in total nerve positive groups were lower than the total nerve negative groups.2) There was a significant difference between the distribution of the total nerves in cervical cancer tissues and the pathologic types (chi-square= 6.279, P=0.0.012).The positive rate of the total nerves was much higher in cervical squamous cell carcinomas than in the cervical adenocarcinoma.3) There were no significant difference between all the FIGO stage, the pathologic grade, the cancer types, the cancer size, pelvic lymph node metastasis and the total nerves in cervical cancer tissues, with the P values were 0.452,0.798,0.810,0.640, 0.681 respectively.4) The age and pathological type were the influence factors of total nerves in cervical cancer. Both them were the protective factors, the greater the ages, the lower the positive rate of the total nerves in cervical cancer (B=-0.64, P=0.033). The positive rate of total nerves in cervical adenocarcinoma neural was lower than in squamous cell carcinomas (B=-1.571, P=0.013). And all the FIGO stage, pathologic grade, the types of cancer in general, cancer size and pelvic lymph node metastasis of cervical cancer had no significant correlation with the distribution of the total nerves.(2) The relation between the distribution of the newborn nerves in cervical cancer tissues and the clinic pathological parameters.1) There was no obvious difference between the distribution of the newborn nerves in cervical cancer tissues and the age (t=0.588, P=0.558).2) There were no significant difference between all the FIGO stage, the pathologic grade, the pathologic types, the cancer types, the cancer size, pelvic lymph node metastasis and the newborn nerves in cervical cancer tissues, with the P values were 0.163,0.673,0.308,0.565,0.771,0.539 respectively.3) The FIGO stage was the risk factor of the newborn nerves in cervical cancer. The higher the stages, the positive rate of the newborn nerves were higher in high stage cervical cancer(B=0.686, P=0.005). However, all the age, the pathologic grade, the pathologic types, the types of cancer in general, cancer size and pelvic lymph node metastasis of cervical cancer had no significant correlation with the distribution of the newborn nerves.(3) The relation between the distribution of the autonomic nerves in cervical cancer tissues and the clinic pathological parameters.①The relation between the distribution of the sympathetic nerves in cervical cancertissues and the clinic pathological parameters.1) There was a significant difference between the distribution of the sympathetic nerves in cervical cancer tissues and the age (t= 2.432, P= 0.016).The ages of patients in sympathetic nerve positive groups were lower than the sympathetic nerve negative groups.2) There was a significant difference between the distribution of the sympathetic nerves in cervical cancer tissues and the pathologic types (chi-square= 3.979, P =0.0.046).The positive rate of the sympathetic nerves was much higher in cervical squamous cell carcinomas than in the cervical adenocarcinoma.3) There were no significant difference between all the FIGO stage, the pathologic grade, the cancer types, the cancer size, pelvic lymph node metastasis and the sympathetic nerves in cervical cancer tissues, with the P values were 0.229,0.972, 0.548,0.709,0.837 respectively.4) The FIGO stage was the risk factor of the sympathetic nerves in cervical cancer. The higher the stages, the positive rate of the sympathetic nerves were higher in high stage cervical cancer(B=0.606, P=0.014). However, all the age, the pathologic grade, the pathologic types, the types of cancer in general, cancer size and pelvic lymph node metastasis of cervical cancer had no significant correlation with the distribution of the sympathetic nerves.② The relation between the distribution of the parasympathetic nerves in cervicalcancer tissues and the clinic pathological parameters.1) There was no obvious difference between the distribution of the parasympathetic nerves in cervical cancer tissues and the age (t= 0.366, P= 0.715).2) There were no significant difference between all the FIGO stage, the pathologic grade, the pathologic types, the cancer types, the cancer size, pelvic lymph node metastasis and the parasympathetic nerves in cervical cancer tissues, with the P values were0.081,0.681,0.354,0.631,0.258,0.538 respectively.3) The FIGO stage was the risk factor of the parasympathetic nerves in cervical cancer. The higher the stages, the positive rate of the parasympathetic nerves were higher in high stage cervical cancer(B=0.710, P=0.020). However, all the age, the pathologic grade, the pathologic types, the types of cancer in general, cancer size and pelvic lymph node metastasis of cervical cancer had no significant correlation with the distribution of the newborn parasympathetic nerves.Conclutions:We could conclude some characteristics of nerve fibers distributed in the cervical cancer tissues:1) The positive rate of the distribution of nerve fibers was higher in young patients with cervical cancer, give priority to with sympathetic nerve, no related with newborn nerve and parasympathetic nerve.2) The positive rate of nerve fibers in cervical squamous cell carcinomas was obviously higher than that of cervical adenocarcinoma, give priority to with sympathetic nerves, no related with newborn nerves and parasympathetic nerves.3)There were significant difference between the FIGO stage and the distribution of newborn nerves, sympathetic nerves and parasympathetic nerves. The higher the stages, the positive rate of the nerves were higher in high stage cervical cancer.4) The distribution of nerve fibers in cervical cancer tissues showed no obvious correlation with the pathologic grade, cancer types, cancer size and pelvic lymph node metastasis.