Morphologic And Immunohistochemical Study On The Development Of Nasopharynx In Mice And Human

Part Ⅰ A morphologic comparison of the nasopharyngeal development in mice and human fetusesBackgroundHuman nasopharynx is a part of the respiratory tract. It is an irregular cube below the body of sphenoid and clivus, composed by six parts:posterior wall, ventral wall, roof wall, anterior wall and two lateral walls. Nasopharynx has four distinct layers:mucous layer, submucosa, muscularis and outer membrane. There is a clear basement membrane which divides the mucosa into epithelium and lamina propria. Common disorders in nasopharynx include chronic diseases such as chronic pharyngitis, benign tumors such as pharyngeal papilloma and adenoma, malignant tumors such as nasopharyngeal carcinoma.There are discordant opinions on the nasopharyngeal morphology so far. For example, differernt opinions exist on the appearance of squamouse epithelium. According to some authors, it appeared only at the adult age as a result of metaplasia. For others, on the contrary, it would be present in the newborn, as a normal type of epithelium. The research in this field is impeded first of all by the difficculty in obtaining human material. Mouse is the best animal models in life science research. Research showed that99%genes of the mouse are identical with human. There are many similarities in embryonic development of mouse and human. The gestation time of mouse is short, and with high level of prolificacy. It can provide mouse material easily for the researcher.Current studies of nasopharyngeal development in human fetuses are very few and no one was doing any research of nasopharyngeal development in mouse fetuses. This paper compared the anatomy and histology of nasopharyngeal development in mice and human, providing references for the study on pathogenesis of diseases in nasopharynx.Materials and MethodsHuman tissues were available from23fetuses of9-28weeks gestational age. And the specimens were obtained from legal abortion or induced abortion with prostaglandin. The gestational ages were determined by the last catamenia time before pregnancy, and verified by measuring the full length of the fetus and observation of the morphology of fetal hands and feet. No limitations to genders. C57BL/6J mouse tissues were available from33fetuses of embryonic day9.5-19.5. Three fetuses each embryonic day were uesd in the research. Mice mated at4-6PM daily and were predicted fertilization by checking the pessary and the microscopic smear8AM the next day. The time we sure the mice fertilized was embryonic day0.5. The whole nasopharynx and soft palate were collected within4hours after abortion and fixed in Bouin’s stationary liquid, dehydrated through graded ethanols, transparence with dimethylbenzene and embedded in paraffin.4-6μm thick sections were cut and mounted on glass slides. The sections were stained with H.E. From nasal cavity to oropharynx, we took a section of every50microns.10visual fields in the each wall were selected in the slice. It was taken full wall if it was less than10visual fields. The thickness and morphology of epithelium, layers of cells and the growth of submucosal gland were examined under microscopy after H.E staining.Results The human nasopharynx had been become a defined region in week9of intra-uterine life. It was an irregular cube with a120-degree arc-shaped posterior wall. There were many foils, crypts and lymphoid tissue in nasopharynx. The mouse nasopharynx becomes a defined region at E15D. It was an arc lumen which was approximately straight line. There was not crypt or lymphoid tissue in nasopharynx.The embryonic development of nasopharyngeal mucosa in human was similar to the mouse. Usually, the differentiation of nasopharyngeal mucosa was regular, but the development of epithelial cells in different parts of the nasopharynx was at different stage. The development of nasopharyngeal mucosa in the proximal segment was significantly earlier than that in the distal. The epithelial cells in posterior wall and lateral wall were the earliest developing area of nasopharynx and the cells in roof wall were the slowest ones. Nevertheless, the development of nasopharyngeal mucosa in human was earlier than mice. The human nasopharynx became a defined region at a quarter (E9W) of intra-uterine life, but it was about3/4length of pregnancy (E15D) in mouse. The pseudostratified ciliated columnar epithelium of mouse was fully differentiated at birth, but it achieved terminal differentiation in the middle of pregnancy (E21W) in human. It was the same for some tissue of nasopharyngeal development, such as submucosal gland. The submucosal gland of human began to form in week11of intra-uterine life, and formed bubble-like structure in week17of intra-uterine life. We could definitely tell the difference of two types of glands at the moment. Most of the glands had developed after embryonic week21. The submucosal gland of mouse began to form in E17D. And it had not been fully differentiated at birth.Most of mucosas in human nasopharynx were pseudostratified ciliated columnar epithelium, with a small amount of stratified squamous epithelium and transitional epithelium during week13-28. Most of the transitional epithelium was located in lateral wall, posterior wall and the border of posterior wall and roof wall. All of the mucosa in anterior wall was stratified squamous epithelium. There were two types of transitional epithelium in nasopharynx. Squamous metaplasia was found in the superficial or deep layer cells of transitional epithelium. All of the nasopharyngeal mucosa in mouse fetus was pseudostratified ciliated columnar epithelium at E19.5D. At the13th week, the goblet cells had been observed on the lateral wall and the border of lateral wall and roof wall. But the goblet cells had not been observed in the nasopharyngx of mice all of the intra-uterine life.Conclusions1. The nasopharyngeal differentiation of human has been completed at21th week, but the epithelial differentiation of mouse has not been completed until birth. Compared to human, the development of nasopharynx in mouse is later.2. The nasopharynx of mouse fetus looks distinctly different from human in anatomy and histology. The differences of anatomy and histology of nasopharyngx in mice and human fetuses may be lead to different susceptibilities to some diseases.3. The transitional epithelia are characterized by variation in the size and shape. And squamous metaplasia is found in some transitional epithelia. It is show that the differentiation of transitional epithelia may be controlled jointly by ectoderm and endoderm. The location of transitional epithelium is the boundary between ectoderm and endoderm. In addition, the transitional epithelia may have the characteristics of stem cells. Part Ⅱ Immunohistochemical study on the expression of nesetin during the development of nasopharynx in human fetusesBackgroundRecent studies have revealed the existence of stem cells in various human tissues. Although there are a lot of debates about the definition of stem cells, but they are widely seen as a group of clone cells possessing two special properties: self-renewal and potency of multiple differentiations. The pluripotent stem cells can proliferate and keep multi differentiation potential. By asymmetric cell division, stem cells produce one copy of the parent and a second cell that is programmed to turn into another cell type. The epithelia of nasopharynx are constantly being renewed of lifelong. It is based on the proliferation and mult-differentiation of stem cells. Nasopharynx with the outside world is the body’s arteries, having an important defense, breathing, swallowing, voice resonance and other functions. Nasopharynx and in vitro environment are the same. The structure of it is vulnerable to be attacked by external pathogenic factors. So the epithelia of nasopharynx are often at the stage of small lesion. After injury, the stem cells will speed up celP division and enhance the recovery of injured mucosa.The existence in the nasopharynx of a ’universal’ plunpotent cell has long been. speculated upon. Many studies directed at understanding the growth properties of specific subsets of cells constituting the nasopharyngeal epithelium. But stem cell research in the nasopharynx has progressed rather slowly due to the anatomical and functional complexities associated with numerous distinct cell types. A definitive progenitor-progeny relationship in the normal steady developing state of human fetal nasopharynx has not yet been fully elucidated. And there are no operative methods to identify the nasopharynx epithelial stem cells till now. It has become one of the maximum drawbacks in clinical application of stem cells to cure the nasopharyngeal diseases.At present, the discovery and identification of stem cells are mainly by using specific markers. But up to now, there is not any research relevant special marker of nasopharyngeal stem cell. Nestin was described as a neuronal stem cell/progenitor cell marker during central nervous system. It is also expressed in immature or progenitor cells in non-neuronal cells in normal tissues. Previous research had shown that nestin was expressed in nasopharyngeal carcinoma. Recent studies suggest that, during multi stages carcarcinogenesis, epithelial cells could abnormally express some proteins that often existing only in embryo development other than in the well differentiated tissues. The proteins could be clinically used as molecular markers for early tumor diagnosis, therapy and evaluating the prognosis. Nestin may be one of the stem cell markers in nasopharynx.Special attention is focused on the changes of nestin during the development, and identification nestin as a marker of nasopharyngeal stem cells. Data from the research provides a good start for understanding the relationship of nestin between nasopharyngeal development and stem cell marker of nasopharynx, providing histological reference for the further studies on regulating mechanism of nasopharyngeal development.Materials and MethodsHuman tissues were available from23fetuses of9-28weeks gestational age (see Part I for this). The SP method of immunohistochemical analysis was performed to examine the expression of nestin in nasopharynx, using mouse anti-human nestin monoclonal antibody diluted at1:800. Nestin positive slides was used as positive control.ResultsThere was no nestin detected in the mucosa of nasopharynx during9-28weeks gestational age. Nestin was detected in cytoplasm of a few immature-looking gland cells, nearly all primary myoblasts and capillary endothelial cells. The expression of nestin was showed dynamic changes during9-28weeks gestational age. The distribution of nestin was detected in the primary myoblasts and capillary endothelial cells of nasopharynx as early as E11W. There were few nestin positive cells at that time. Along with the growth of nasopharynx, nestin positive cells rate obviously rose. Nestin was detected in a few immature-looking gland cells as early as13weeks of gestation. Strongly positive nestin reaction was shown in the primary myoblasts during9-28weeks gestational age. The expression of nestin in glands reached the pinnacle in17th week and began to decrease after that.21th weeks later, the expression of nestin in glands was almost unseeable.Conclusions1. The timel7th week in the embryogenesis may be the the most active period for the nasopharynx to grow and differentiate. At that time, the transitional epithelium distinctly separates into two groups (see Part Ⅰ for this), and the number of nestin positive cells in submucosal gland reach the pinnacle. It is the most intensive period of mucosa and glands development.2. Nestin is one of the nasopharyngeal stem cells. It is closely related to the development of nasopharynx as it is detected in some organization. But it doesn’t prove the relation between nestin and the development of mucosa. Part Ⅲ The hyperplasia of pharynx and larynx in nestin transgenic miceBackgroundNestin is a class Ⅵ intermediate filament protein. It was originally described as a neuronal stem cell/progenitor cell marker during central nervous system (CNS) development. Nestin is expressed in dividing cells during the early stages of development in the CNS, peripheral nervous system, and other tissues. With differentiation, nestin is downregulated and replaced by tissuespecific IF proteins. Nestin is also expressed in immature or progenitor cells in non-neuronal cells in normal tissues. In adult, nestin-expressing cells are restricted to defined locations, where they may function as a cellular reserve that is capable of proliferation, differentiation, and migration after reactivation such as hair follicle cells, pancreatic stellate cells and angiogenic endothelial cells. Furthermore, the expression of nestin in several tumors was reported to be closely correlated with aggressive growth, metastasis, and poor prognosis.Evidence from in vitro experiments suggests that nestin plays a role in promoting cell survival and proliferation. Nestin combining with glucocorticoid receptor increased the activity of PI3K. It caused a series of enzymatic reactions that induced the progenitor cells to proliferate. Nestin overexpression had a neuroprotective role in ST15A cells injury induced by H2O2through an antioxidant mechanism. Data from Sahlgren suggest that as a signal, nestin protected cell from oxidative damage through Cdk5/P35cascade. But some research found that nestin had nothing to do with proliferation. After the expression of nestin was inhibited successfully by RNA interference induced by siRNA expressing vector, the growth of neuroblastoma and astrocytoma cells was inhibited in vitro. But the expression of ki67did not reduce.At the same time, the precise role of nestin in vivo was not clear until now. Nestin expression substantially reduced in nestin knockout mice. However, in nestin+/-genotype heterozygous mice, the lower expression of nestin did not cause a visible phenotype. It shows that nestin expression levels may not affect the development and survival of mice. In homozygous mice (nestin-/-), there was overt and lethal defects in the development of neural tube. Nestin missing could cause a large number of neural tube cells to apoptosis, which had nothing to do with the cytoskeletal function of nestin. Nestin was required for the proper self-renewal of neural stem cells. But it had no effect on the growth and proliferation of neural stem cells. Nestin was also expressed outside the nervous system in mouse. But the knockout mice contained no obvious abnormalities in the other parts where the nestin-positive cells were found. The function of nestin in vivo is complex.To date, no report in nestin over-expression transgenic mice has been found. In order to explore the role of nestin in vivo, we produced nestin overexpression transgenic animal models. This article explored the effect of over-expressing exogenous nestin on the development of pharynx and larynx in mouse.Materials and MethodsMouse tissues were available from nestin transgenic mice fetuses of14.5-19.5days gestational age and adult mice at12month. No limitations to genders. The mouse DNA was extracted from transgenic mice organization and the genotype of mice were detected by multiplex polymerase chain reaction (PCR). The mice were divided into controlled group and experiment group by genotype. This study adopted nestin transgenic mice as experiment group and littermates as control group. There were3fetuses each group.The whole pharynx and larynx together with surrounding tissue was collected and fixed in Bouin’s stationary liquid (see Part I for this). The morphology of pharynx and larynx were examined under microscopy after H.E staining. Immunohistochemical staining was performed by streptavidin-perosidase method on the tissue sections. Sections were incubated overnight at4℃with a1:800dilution of anti-nestin monoclona antibody.Statistical analysis was computed using SPSS13software. The data are presented as mean±standard deviation (SD). All data were analyzed by chi-square test. The P values smaller than0.05were considered statistically significant.Results The expression of nestin was detected in most of the mucosal cells of the pharynx, larynx, trachea and other parts of transgenic mice fetuses. There were about28%transgenic mice formation neoplasms in pharynx and larynx. The indexes of experiment group changed significantly as compared with those of control group (P=0.008).ConclusionIt is showed that abnormal nestin overexpression can cause neoplasms in pharynx and larynx. Nestin gene may be concerned with regulation the proliferation and process death of epithelial cell in pharynx and larynx.